TR-518: Triethanolamine (CASRN 102-71-6) in B6C3F1 Mice (Dermal Studies)

NTP TECHNICAL REPORT ON

THE

TOXICOLOGY AND

CARCINOGENESIS STUDIES OF

TRIETHANOLAMINE

(CAS NO. 102-71-6)

IN B6C3F

MICE

(DERMAL STUDY)

NTP TR 518

MAY 2004

NTP TECHNICAL REPORT

ON THE

TOXICOLOGY AND CARCINOGENESIS

STUDIES OF TRIETHANOLAMINE

(CAS NO. 102-71-6)

IN B6C3F

MICE

(DERMAL STUDY)

NATIONAL TOXICOLOGY PROGRAM

P.O. Box 12233

Research Triangle Park, NC 27709

May 2004

NTP TR 518

NIH Publication No. 04-4452

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

National Institutes of Health

FOREWORD

The National Toxicology Program (NTP) is made up of four charter agencies of the U.S. Department of Health and

Human Services (DHHS): the National Cancer Institute (NCI), National Institutes of Health; the National Institute

of Environmental Health Sciences (NIEHS), National Institutes of Health; the National Center for Toxicological

Research (NCTR), Food and Drug Administration; and the National Institute for Occupational Safety and Health

(NIOSH), Centers for Disease Control and Prevention. In July 1981, the Carcinogenesis Bioassay Testing

Program, NCI, was transferred to the NIEHS. The NTP coordinates the relevant programs, staff, and resources

from these Public Health Service agencies relating to basic and applied research and to biological assay

development and validation.

The NTP develops, evaluates, and disseminates scientific information about potentially toxic and hazardous

chemicals. This knowledge is used for protecting the health of the American people and for the primary

prevention of disease.

The studies described in this Technical Report were performed under the direction of the NIEHS and were

conducted in compliance with NTP laboratory health and safety requirements and must meet or exceed all

applicable federal, state, and local health and safety regulations. Animal care and use were in accordance with the

Public Health Service Policy on Humane Care and Use of Animals. The prechronic and chronic studies were

conducted in compliance with Food and Drug Administration (FDA) Good Laboratory Practice Regulations, and

all aspects of the chronic studies were subjected to retrospective quality assurance audits before being presented for

public review.

These studies are designed and conducted to characterize and evaluate the toxicologic potential, including

carcinogenic activity, of selected chemicals in laboratory animals (usually two species, rats and mice). Chemicals

selected for NTP toxicology and carcinogenesis studies are chosen primarily on the bases of human exposure, level

of production, and chemical structure. The interpretive conclusions presented in this Technical Report are based

only on the results of these NTP studies. Extrapolation of these results to other species and quantitative risk

analyses for humans require wider analyses beyond the purview of these studies. Selection per se is not an

indicator of a chemical’s carcinogenic potential.

Details about ongoing and completed NTP studies are available at the NTP’s World Wide Web site:

http://ntp-server.niehs.nih.gov. Abstracts of all NTP Technical Reports and full versions of the most recent reports

and other publications are available from the NIEHS’ Environmental Health Perspectives (EHP)

http://ehp.niehs.nih.gov (866-541-3841 or 919-653-2590). In addition, printed copies of these reports are available

from EHP as supplies last. A listing of all the NTP Technical Reports printed since 1982 appears at the end of this

Technical Report.

NTP TECHNICAL REPORT

ON THE

TOXICOLOGY AND CARCINOGENESIS

STUDIES OF TRIETHANOLAMINE

(CAS NO. 102-71-6)

IN B6C3F

MICE

(DERMAL STUDY)

NATIONAL TOXICOLOGY PROGRAM

P.O. Box 12233

Research Triangle Park, NC 27709

May 2004

NTP TR 518

NIH Publication No. 04-4452

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

National Institutes of Health

CONTRIBUTORS

National Toxicology Program

Evaluated and interpreted results and reported findings

F.A. Suarez, M.D., M.S.,

Study Scientist

R.A. Herbert, D.V.M., Ph.D.,

Study Pathologist

D.W. Bristol, Ph.D.

J.R. Bucher, Ph.D.

J.R. Hailey, D.V.M.

J.K. Haseman, Ph.D.

R.R. Maronpot, D.V.M.

D.P. Orzech, M.S.

G. Pearse, B.V.M. & S.

S.D. Peddada, Ph.D.

J.H. Roycroft, Ph.D.

C.S. Smith, Ph.D.

G.S. Travlos, D.V.M.

K.L. Witt, M.S.,

ILS, Inc.

Battelle Columbus Operations

Conducted studies and evaluated pathology findings

M.R. Hejtmancik, Ph.D.,

Principal Investigator

M.J. Ryan, D.V.M., Ph.D.

Experimental Pathology Laboratories, Inc.

Provided pathology quality assurance

J.F. Hardisty, D.V.M., Principal Investigator

C.C. Shackelford, D.V.M., M.S., Ph.D.

G. Willson, B.V.M. & S.

Dynamac Corporation

Prepared quality assurance audits

S. Brecher, Ph.D., Principal Investigator

NTP Pathology Working Group

Evaluated slides and prepared pathology report

(April 1, 2002)

G.A. Parker, D.V.M., Ph.D., Chairperson

ILS, Inc.

R. Bahnemann, D.V.M., Ph.D.,

Observer

BASF Corporation

W.R. Brown, D.V.M., Ph.D.

Research Pathology Services, Inc.

G.P. Flake, M.D.

National Toxicology Program

J.R. Hailey, D.V.M.

National Toxicology Program

A. Nyska, D.V.M.

National Toxicology Program

G. Pearse, B.V.M. & S.

National Toxicology Program

C.C. Shackelford, D.V.M., M.S., Ph.D.

Experimental Pathology Laboratories, Inc.

G. Willson, B.V.M. & S.

Experimental Pathology Laboratories, Inc.

Analytical Sciences, Inc.

Provided statistical analyses

P.W. Crockett, Ph.D.,

Principal Investigator

L.J. Betz, M.S.

K.P. McGowan, M.B.A.

J.T. Scott, M.S.

Biotechnical Services, Inc.

Prepared Technical Report

S.R. Gunnels, M.A., Principal Investigator

B.F. Hall, M.S.

L.M. Harper, B.S.

D.C. Serbus, Ph.D.

R.A. Willis, B.A., B.S.

CONTENTS

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY . . . . . . . . . . . . . . . 8

TECHNICAL REPORTS REVIEW SUBCOMMITTEE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS . . . . . . . . . . . . 10

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

DISCUSSION AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

PPENDIX A Summary of Lesions in Male Mice in the 2-Year Dermal Study

of Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

PPENDIX B Summary of Lesions in Female Mice in the 2-Year Dermal Study

of Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

PPENDIX C Genetic Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

PPENDIX D Chemical Characterization and Dose Formulation Studies . . . . . . . . . . . . . . . . . . . . . . 135

APPENDIX E Ingredients, Nutrient Composition, and Contaminant Levels

in NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

PPENDIX F Sentinel Animal Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

PPENDIX G Absorption, Distribution, Metabolism, and Excretion Studies . . . . . . . . . . . . . . . . . . . . 151

4 Triethanolamine, NTP TR 518

SUMMARY

Background

Triethanolamine is used to make many products, including household detergents and polishes, herbicides,

vegetable and mineral oils, paraffin and waxes, plastics, resins, adhesives, and ointments. We studied the effects of

triethanolamine on male and female mice to identify potential toxic or cancer-related hazards to humans.

Methods

We placed solutions of triethanolamine dissolved in acetone onto the shaved skin on the backs of male and female

mice 5 days per week for 2 years. Male mice received concentrations of 200, 630, or 2,000 milligrams of

triethanolamine per kilogram of body weight, and female mice received half those concentrations. Control groups

received only acetone.

Results

The dosed animals did not have higher death rates than the control animals. The male mice receiving 2,000 mg

triethanolamine per kg body weight weighed less than the male control animals. There was a slight increase in

hemangiosarcomas (tumors of the vascular system) in the livers of males exposed to triethanolamine. In female

mice exposed to triethanolamine, there was a significant increase in liver tumors.

Conclusions

We conclude that triethanolamine caused liver tumors in female mice and may have caused a slight increase in

hemangiosarcomas of the liver in male mice.

ABSTRACT

TRIETHANOLAMINE

CAS No. 102-71-6

Chemical Formula: C

Molecular Weight: 149.19

Synonyms: Nitrilo-2,2N,2NN-triethanol; 2,2N,2NN-nitrilotriethanol; 2,2N,2NN-nitrilotrisethanol; TEA; triaethanolamin-NG; triethanolamin;

triethylolamine; tri(hydroxyethyl)amine; 2,2N,2NN-trihydroxytriethylamine; trihydroxytriethylamine; tris(hydroxyethyl)amine;

tris(2-hydroxyethyl)amine; trolamine

Trade names: Daltogen, Sterolamide, Thiofaco T-35

Triethanolamine is widely used in the manufacturing of marginal increase in the incidence of renal tubule adeno-

household detergents and polishes, textiles, agricultural ma (NTP, 1991). Interpretation of the results from the

herbicides, mineral and vegetable oils, paraffin and 2-year study in mice was complicated by Helicobacter

waxes, pharmaceutical ointments, petroleum demulsi- hepaticus infection, prompting a repeat 2-year study in

fiers, synthetic resins, plasticizers, adhesives, and mice. Male and female B6C3F

mice received tri-

sealants. It is used as a chemical intermediate for anion- ethanolamine (greater than 99% pure) by dermal appli-

ic and nonionic surfactants, a vulcanization accelerator, cation for 2 years; a study of absorption, distribution,

a humectant and softening agent and in many other metabolism, and excretion was performed in additional

industrial applications. The National Cancer Institute mice. Genetic toxicology studies were conducted in

nominated triethanolamine for study because of its wide- Salmonella typhimurium, cultured Chinese hamster

spread use in cosmetics and other consumer products, its ovary cells, Drosophila melanogaster, and mouse

high potential for worker exposure due to its many peripheral blood erythrocytes.

industrial uses, and its potential for conversion to the

carcinogen N-nitrosodiethanolamine. Previous 3-month

and 2-year studies of triethanolamine were conducted by

2-YEAR STUDY

the National Toxicology Program in F344/N rats and

Groups of 50 male and 50 female mice received dermal

B6C3F

mice; results from the 2-year rat study indicated

applications of 0, 200, 630, or 2,000 mg/kg (males) and

equivocal evidence of carcinogenic activity based on a

0, 100, 300, or 1,000 mg/kg (females) triethanolamine in

__________

6 Triethanolamine, NTP TR 518

acetone, 5 days per week, for 104 (males) or 104 to

105 (females) weeks.

Survival of all dosed groups was similar to that of the

vehicle control groups.

Body weights of 2,000 mg/kg

males were less than those of the vehicle controls from

weeks 17 to 37 and at the end of the study; body weights

of dosed groups of females were similar to those of the

vehicle controls throughout the study. Treatment-related

clinical findings included skin irritation at the site of

application, which increased with increasing dose and

was more severe in males than in females.

Gross lesions observed at necropsy included nodules and

masses of the liver in dosed females.

The incidences of

hepatocellular adenoma and hepatocellular adenoma or

carcinoma (combined) were significantly increased in all

dosed groups of females. The incidence of heman-

giosarcoma of the liver in 630 mg/kg males was margin-

ally increased. The incidences of eosinophilic focus in

all dosed groups of mice were greater than those in the

vehicle controls.

Gross lesions observed at necropsy included visible

crusts at the site of application in all dosed groups of

mice.

Treatment-related epidermal hyperplasia, suppu-

rative inflammation, ulceration, and dermal chronic

inflammation occurred at the site of application in most

dosed groups of mice, and the incidences and severities

of these lesions generally increased with increasing

dose.

GENETIC TOXICOLOGY

Triethanolamine was not mutagenic in any of the in vitro

or in vivo tests. It did not induce mutations in

Salmonella typhimurium, and no induction of sister

chromatid exchanges or chromosomal aberrations was

noted in cultured Chinese hamster ovary cells exposed to

triethanolamine. These in vitro tests were all conducted

with and without S9 metabolic activation.

Triethanolamine did not induce sex-linked recessive

lethal mutations in germ cells of adult male Drosophila

melanogaster exposed by feeding or injection. No

increase in the frequency of micronucleated erythrocytes

was observed in peripheral blood samples of male or

female mice that received dermal applications of tri-

ethanolamine for 13 weeks.

CONCLUSIONS

Under the conditions of this 2-year dermal study, there

was equivocal evidence of carcinogenic activity* of

triethanolamine in male B6C3F

mice based on the

occurrence of liver hemangiosarcoma. There was some

evidence of carcinogenic activity in female B6C3F

mice

based on increased incidences of hepatocellular

adenoma.

Exposure to triethanolamine by dermal application

resulted in increased incidences of eosinophilic focus of

the liver in males and females. Dosed mice developed

treatment-related nonneoplastic lesions at the site of

application.

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 8. A summary of the Technical Reports Review Subcommittee

comments and public discussion on this Technical Report appears on page 10.

7 Triethanolamine, NTP TR 518

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Triethanolamine

Male B6C3F

Mice Female B6C3F

Mice

Doses in acetone by dermal application

Survival rates

Body weights

Nonneoplastic effects

Neoplastic effects

Equivocal findings

Level of evidence of carcinogenic activity

Genetic toxicology

Salmonella typhimurium gene mutations:

Sister chromatid exchanges

Cultured Chinese hamster ovary cells in vitro:

Chromosomal aberrations

Cultured Chinese hamster ovary cells in vitro:

Sex-linked recessive lethal mutations

Drosophila melanogaster:

Micronucleated erythrocytes

Mouse peripheral blood in vivo:

Vehicle control, 200, 630, or 2,000 mg/kg

37/50, 43/50, 34/50, 40/50

2,000 mg/kg group less than the vehicle

control group

Liver: eosinophilic focus (9/50, 20/50,

31/50, 30/50)

Skin (site of application)

: epidermis,

hyperplasia (5/50, 44/50, 45/50, 49/50);

epidermis, inflammation, suppurative (1/50,

11/50, 33/50, 42/50); epidermis, ulcer (0/50,

3/50, 20/50, 47/50); dermis, inflammation,

chronic (1/50, 15/50, 40/50, 49/50)

None

Liver: hemangiosarcoma (1/50, 0/50, 6/50,

1/50)

Equivocal evidence

Vehicle control, 100, 300, or 1,000 mg/kg

35/50, 34/50, 41/50, 32/50

Dosed groups similar to the vehicle control

group

Liver: eosinophilic focus (16/50, 22/50,

28/50, 32/50)

Skin (site of application): epidermis,

hyperplasia (14/50, 50/50, 46/50, 50/50);

epidermis, inflammation, suppurative (1/50,

2/50, 20/50, 32/50); epidermis, ulcer (1/50,

1/50, 6/50, 17/50); dermis, inflammation,

chronic (4/50, 27/50, 31/50, 44/50)

Liver

: hepatocellular adenoma (9/50, 18/50,

20/50, 33/50); hepatocellular adenoma or

carcinoma (12/50, 23/50, 24/50, 34/50)

Some evidence

Negative in strains TA98, TA100, TA1535, and TA1537 with and without S9

Negative with and without S9

Negative when administered in feed or by injection

Negative

8 Triethanolamine, NTP TR 518

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY

The National Toxicology Program describes the results of individual experiments on a chemical agent and notes the strength of the evidence for

conclusions regarding each study. Negative results, in which the study animals do not have a greater incidence of neoplasia than control

animals, do not necessarily mean that a chemical is not a carcinogen, inasmuch as the experiments are conducted under a limited set of

conditions. Positive results demonstrate that a chemical is carcinogenic for laboratory animals under the conditions of the study and indicate

that exposure to the chemical has the potential for hazard to humans. Other organizations, such as the International Agency for Research on

Cancer, assign a strength of evidence for conclusions based on an examination of all available evidence, including animal studies such as those

conducted by the NTP, epidemiologic studies, and estimates of exposure. Thus, the actual determination of risk to humans from chemicals

found to be carcinogenic in laboratory animals requires a wider analysis that extends beyond the purview of these studies.

Five categories of evidence of carcinogenic activity are used in the Technical Report series to summarize the strength of the evidence observed

in each experiment: two categories for positive results (clear evidence and some evidence); one category for uncertain findings (equivocal

evidence); one category for no observable effects (no evidence); and one category for experiments that cannot be evaluated because of major

flaws (inadequate study). These categories of interpretative conclusions were first adopted in June 1983 and then revised in March 1986 for

use in the Technical Report series to incorporate more specifically the concept of actual weight of evidence of carcinogenic activity. For each

separate experiment (male rats, female rats, male mice, female mice), one of the following five categories is selected to describe the findings.

These categories refer to the strength of the experimental evidence and not to potency or mechanism.

• Clear evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a dose-related

(i) increase of malignant neoplasms, (ii) increase of a combination of malignant and benign neoplasms, or (iii) marked increase of

benign neoplasms if there is an indication from this or other studies of the ability of such tumors to progress to malignancy.

• Some evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a chemical-related increased

incidence of neoplasms (malignant, benign, or combined) in which the strength of the response is less than that required for clear

evidence.

• Equivocal evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a marginal increase of

neoplasms that may be chemical related.

• No evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing no chemical-related increases in

malignant or benign neoplasms.

• Inadequate study of carcinogenic activity is demonstrated by studies that, because of major qualitative or quantitative limitations,

cannot be interpreted as valid for showing either the presence or absence of carcinogenic activity.

For studies showing multiple chemical-related neoplastic effects that if considered individually would be assigned to different levels of evidence

categories, the following convention has been adopted to convey completely the study results. In a study with clear evidence of carcinogenic

activity at some tissue sites, other responses that alone might be deemed some evidence are indicated as “were also related” to chemical

exposure. In studies with clear or some evidence of carcinogenic activity, other responses that alone might be termed equivocal evidence are

indicated as “may have been” related to chemical exposure.

When a conclusion statement for a particular experiment is selected, consideration must be given to key factors that would extend the actual

boundary of an individual category of evidence. Such consideration should allow for incorporation of scientific experience and current

understanding of long-term carcinogenesis studies in laboratory animals, especially for those evaluations that may be on the borderline between

two adjacent levels. These considerations should include:

• adequacy of the experimental design and conduct;

• occurrence of common versus uncommon neoplasia;

• progression (or lack thereof) from benign to malignant neoplasia as well as from preneoplastic to neoplastic lesions;

• some benign neoplasms have the capacity to regress but others (of the same morphologic type) progress. At present, it is impossible to

identify the difference. Therefore, where progression is known to be a possibility, the most prudent course is to assume that benign

neoplasms of those types have the potential to become malignant;

• combining benign and malignant tumor incidence known or thought to represent stages of progression in the same organ or tissue;

• latency in tumor induction;

• multiplicity in site-specific neoplasia;

• metastases;

• supporting information from proliferative lesions (hyperplasia) in the same site of neoplasia or in other experiments (same lesion in

another sex or species);

• presence or absence of dose relationships;

• statistical significance of the observed tumor increase;

• concurrent control tumor incidence as well as the historical control rate and variability for a specific neoplasm;

• survival-adjusted analyses and false positive or false negative concerns;

• structure-activity correlations; and

• in some cases, genetic toxicology.

__________

9 Triethanolamine, NTP TR 518

NATIONAL TOXICOLOGY PROGRAM BOARD OF SCIENTIFIC COUNSELORS

TECHNICAL REPORTS REVIEW SUBCOMMITTEE

The members of the Technical Reports Review Subcommittee who evaluated the draft NTP Technical Report on triethanolamine on

May 22, 2003, are listed below. Subcommittee members serve as independent scientists, not as representatives of any institution, company, or

governmental agency. In this capacity, subcommittee members have five major responsibilities in reviewing the NTP studies:

• to ascertain that all relevant literature data have been adequately cited and interpreted,

• to determine if the design and conditions of the NTP studies were appropriate,

• to ensure that the Technical Report presents the experimental results and conclusions fully and clearly,

• to judge the significance of the experimental results by scientific criteria, and

• to assess the evaluation of the evidence of carcinogenic activity and other observed toxic responses.

Mary Anna Thrall, D.V.M., Chairperson

Department of Pathology

College of Veterinary Medicine and Biomedical Sciences

Colorado State University

Fort Collins, CO

Kim Boekelheide, M.D., Ph.D.

Division of Biology and Medicine

Department of Pathology and Laboratory Medicine

Brown University

Providence, RI

Hillary M. Carpenter, III, Ph.D.

Minnesota Department of Health

St. Paul, MN

Samuel M. Cohen, M.D., Ph.D.*

Department of Pathology and Microbiology

University of Nebraska Medical Center

Omaha, NE

Michael R. Elwell, D.V.M., Ph.D.

Pfizer, Inc.

Groton, CT

* Did not attend

Walter W. Piegorsch, Ph.D.

Department of Statistics

University of South Carolina

Columbia, SC

Stephen M. Roberts, Ph.D.

Department of Physiological Sciences

College of Veterinary Medicine

University of Florida

Gainesville, FL

Richard D. Storer, M.P.H., Ph.D.

Department of Genetic and Cellular Toxicology

Merck Research Laboratories

West Point, PA

Mary Vore, Ph.D.

Graduate Center for Toxicology

University of Kentucky

Lexington, KY

Cheryl Lyn Walker, Ph.D.

M.D. Anderson Cancer Center

The University of Texas

Smithville, TX

10 Triethanolamine, NTP TR 518

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS

On May 22, 2003, the draft Technical Report on the tox-

icology and carcinogenesis studies of triethanolamine

received public review by the National Toxicology

Program’s Board of Scientific Counselors’ Technical

Reports Review Subcommittee. The review meeting

was held at the National Institute of Environmental

Health Sciences, Research Triangle Park, NC.

Dr. A. Suarez, NIEHS, introduced the toxicology and

carcinogenesis studies of triethanolamine by describing

the uses of the chemical. Dr. Suarez discussed the

results of the previously published NTP Technical

Report on the Toxicology and Carcinogenesis Studies of

Triethanolamine (TR 449) in Rats and Mice. The previ-

ous mouse studies were considered inadequate due to

infection of the mice with Helicobacter hepaticus and an

associated hepatitis in male mice that confounded inter-

pretation of a possible liver neoplasm response.

Therefore, the current mouse studies were repeated

using the same protocol. The proposed conclusions

were equivocal evidence of carcinogenic activity of tri-

ethanolamine in male B6C3F

mice and some evidence

of carcinogenic activity in female B6C3F

mice.

Exposure to triethanolamine by dermal application

resulted in increased incidences of eosinophilic focus of

the liver in males and females. Dosed mice developed

treatment-related nonneoplastic lesions at the site of

application.

Drs. Walker, Roberts, and Carpenter, the three principal

reviewers, all concurred with the proposed conclusions.

Dr. Carpenter asked for an expansion in the discussion

on the relative certainty regarding the hemangiosarco-

mas. Dr. J.R. Hailey, NIEHS, agreed to add more detail

(see pages 33 and 36).

Dr. W.T. Allaben, NCTR, inquired about the extent of

ulceration of the skin at the site of application.

Dr. Hailey explained that even the most severe were

characterized as occasional pinpoint focal crusts, with

most of the skin relatively unaffected.

Dr. Walker moved, and Dr. Carpenter seconded, that the

conclusions be accepted as written. The motion was

accepted unamimously with eight votes.

TRIETHANOLAMINE

INTRODUCTION

CAS No. 102-71-6

Chemical Formula: C

Molecular Weight: 149.19

Synonyms: Nitrilo-2,2N,2NN-triethanol; 2,2N,2NN-nitrilotriethanol; 2,2N,2NN-nitrilotrisethanol; TEA; triaethanolamin-NG; triethanolamin;

triethylolamine; tri(hydroxyethyl)amine; 2,2N,2NN-trihydroxytriethylamine; trihydroxytriethylamine; tris(hydroxyethyl)amine;

tris(2-hydroxyethyl)amine; trolamine

Trade names: Daltogen, Sterolamide, Thiofaco T-35

CHEMICAL AND PHYSICAL PROPERTIES

Triethanolamine is a colorless to pale yellow, viscous,

hygroscopic liquid with a slight ammonia-like odor. It

has a melting point of 21.6° C, boiling point of 335.4° C

at 760 mm, a specific gravity of 1.124 (20/4° C), and

vapor pressure less than 0.01 mm at 20° C.

Triethanolamine is miscible in water, methanol, or ace-

tone; slightly soluble in ether or benzene; and turns

brown when exposed to air and light (Merck Index,

1996). It is combustible when heated, and it may

decompose to oxides of nitrogen (Lewis, 1997).

PRODUCTION, USE,

AND HUMAN EXPOSURE

Ethanolamines are produced on an industrial scale by

aminating ethylene oxide with excess ammonia.

Triethanolamine is separated from the mixture of amines

generated by this reaction (mono- and diethanolamine)

by distillation; in the United States, production of tri-

ethanolamine grew steadily from 13,000 tons in 1960 to

98,000

tons in 1990 (Kirk-Othmer, 1992). Worldwide

production of triethanolamine is estimated to be

500,000 tons per year (United Nations Environment

Program, cited by IARC, 2000).

Triethanolamine is used in manufacturing a variety of

household products and in several industrial processes.

It is a chemical intermediate for anionic and nonionic

surfactants.

Triethanolamine is also widely used in man-

ufacturing emulsifiers and dispersing agents for house-

hold detergents and polishes, textiles (lubricants, dyes,

and antistatic agents), agricultural herbicides, mineral

and vegetable oils, paraffin and waxes, pharmaceutical

ointments, and petroleum demulsifiers. Additional

industrial uses of triethanolamine include as a vulcaniza-

tion accelerator in the rubber industry; as a humectant

and softening agent in hide tanning; and in manufactur-

ing synthetic resins, plasticizers, adhesives, and sealants.

It is a solvent for casein, shellac, and dyes. It also

increases the penetration of organic liquids into wood

12 Triethanolamine, NTP TR 518

and paper. The metal chelating properties of tri-

ethanolamine are used in industrial applications such as

corrosion inhibition, electroplating, metal cleaning and

rust removal, and the preparation of photographic chem-

icals and soldering fluxes. The fatty acid salts of tri-

ethanolamine are used in lubricating and metalworking

fluids (cutting oils). Addition of small amounts of tri-

ethanolamine or its salts reduces particle agglomeration

during the grinding of cement and reduces set time and

increases the early strength of concrete (Kirk-Othmer,

1978; Hawley’s, 1987; Merck Index, 1996; Melnick and

Tomaszewski, 1990). Articles intended for use in the

production, processing, and packaging of food may con-

tain triethanolamine (21 CFR, Parts 175, 176, 177, and

178).

Direct contact of the skin or eyes with undiluted tri-

ethanolamine represents the most significant risk for

acute exposure in industrial settings. Substantial expo-

sure to triethanolamine via inhalation appears unlikely

due to its low vapor pressure (Patty’s, 1981). From 1981

to 1983, The National Occupational Exposure Survey

estimated that more than 1.7 million workers were

potentially exposed to triethanolamine in the United

States (NIOSH, 1990). The threshold limit value for tri-

ethanolamine is 5 mg/m

based primarily on skin and

eye irritation (ACGIH, 2002).

Chronic exposure to triethanolamine may result from

skin contact with household detergents, pharmaceutical

ointments, cutting fluids, adhesives and sealants.

However, dermal exposure to triethanolamine-contain-

ing consumer products (principally personal care prod-

ucts) is considered to be the primary route of general

population exposure. Triethanolamine, in combination

with fatty acids, is used extensively in cosmetic formu-

lations including emulsifiers, thickeners, wetting agents,

detergents, and alkalizing agents. The Cosmetic

Ingredient Review (CIR) Expert Panel (1983) reported

that, in 1981, triethanolamine was present in 2,757 cos-

metic products at concentrations up to 5%; these prod-

ucts included creams, lotions, skin cleansers, shampoos,

hair care and coloring agents, permanent wave lotions,

deodorants, fragrances, makeup, nail polish and polish

remover, and cuticle softeners and removers.

The CIR Expert Panel (1983) concluded that the use of

mono-, di-, and triethanolamine is safe in cosmetic for-

mulations designed for brief, discontinuous use followed

by thorough rinsing of the skin. The CIR Expert Panel

further concluded that the concentration of ethanolamine

should not exceed 5% in cosmetic products intended for

prolonged contact with the skin. In the presence of

nitrite, oxides of nitrogen, or 2-bromo-2-nitropropane-

1,3-diol, an antimicrobial agent used in cosmetics, di-

and triethanolamine may be readily nitrosated to

N-nitrosodiethanolamine, which is a known liver, kid-

ney, and nasal carcinogen in laboratory animals

(Hoffmann et al., 1982; Preussmann et al., 1982;

Lijinsky and Kovatch, 1985). N-Nitrosodiethanolamine

has been identified in a variety of cosmetic products at

concentrations up to 48,000 ppb (Fan et al., 1977); the

CIR Expert Panel (1983) concluded, therefore, that di-

and triethanolamines should not be used in cosmetic

products that contain N-nitrosating agents as intentional

ingredients or potential contaminants.

ENVIRONMENTAL IMPACT

The environmental fate and aquatic toxicology of the

alkanolamines have been reviewed (Davis and

Carpenter, 1997). Triethanolamine may be released into

the environment in emissions or effluents from manu-

facturing or industrial sites, from the disposal of con-

sumer products containing triethanolamine, from the

application of agricultural chemicals in which tri-

ethanolamine is used as a dispersing agent, or during use

of an aquatic herbicide containing a copper-

triethanolamine complex (Hawley’s, 1987). Residual

triethanolamine in soil may also leach into the ground-

water. The half-life of triethanolamine in soil and water

ranges from days to weeks; it biodegrades fairly rapidly

following acclimation (HSDB, 2002). Ethanolamines

have been shown to be selectively toxic to green algae

(Scenedesmus quadricauda) (Bringmann and Kühn,

1980). In the atmosphere, triethanolamine primarily

exists in the vapor phase; the vapor, which has a half-life

of 4 hours, is expected to react with photochemically

generated hydroxyl radicals in the atmosphere. The

complete solubility of triethanolamine in water suggests

that this compound may also be removed from the

atmosphere by precipitation. Volatilization of tri-

ethanolamine from water and moist soil surfaces has

been estimated to be negligible (Eisenreich et al., 1981;

Atkinson, 1987; HSDB, 2002).

ABSORPTION, DISTRIBUTION,

ETABOLISM, AND EXCRETION

Experimental Animals

Triethanolamine is absorbed through the skin and in the

gastrointestinal tract. In oral studies, 63% of

13 Triethanolamine, NTP TR 518

triethanolamine administered to Wistar rats was

absorbed from the gastrointestinal tract within one hour

after administration (IARC, 2000). In the skin, differ-

ences in the rate of absorption between F344 rats and

C3H/HeJ mice have been described. In mice, most of

the topically applied [

C]-triethanolamine is absorbed,

and only 6% to 11% is detected at the site of application

after 48 hours (Stott et al., 2000). In rats, absorption

occurs more slowly and is less extensive (Melnick and

Tomaszewski, 1990; IARC, 2000). Stott et al. (2000)

reported similar levels of dermal absorption between

C3H/HeJ mice and F344 rats 24 to 48 hours after dosing.

In contrast, an absorption, distribution, metabolism, and

excretion study by the National Toxicology Program

(Appendix G) found that after 72 hours of exposure,

only 20% to 30% of the applied dose of triethanolamine

(68 or 276 mg/kg) was absorbed in rats and 80% was

absorbed in mice (79 or 1,120 mg/kg).

These differences in absorption have been attributed

either to the different doses used in comparative studies

or to species-specific factors. In fact, an analysis of

absorption data in Appendix G reveals that a 14-fold

increase in concentration gives a 23-fold increase in

absorption in mice; whereas in rats, a fourfold increase

gives a sixfold increase in absorption. No differences in

tissue distribution were noted after oral or dermal expo-

sure (Appendix G).

The elimination of [

C]-triethanolamine-derived

radioactivity from the blood of mice after a 1 mg/kg

intravenous injection displays two-phase elimination

kinetics with an initial rapid distribution phase (0.3-hour

half-life) followed by a slower elimination phase

(10-hour half-life). Kinetics following an unoccluded

dermal application of neat triethanolamine (200 mL/kg)

was described as an initial rapid absorption phase

(0.7-hour half-life) and two elimination phases with

half-lives of 1.9 and 31 hours (Stott et al., 2000). The

dermal absorption study was unoccluded, so it may be

expected that some triethanolamine was orally absorbed

through grooming.

Excretion of [

C]-triethanolamine is similar among rats

and mice. Following a dermal dose of 1,000 mg/kg,

mice excreted approximately 60% of the radioactivity in

the urine and 20% in the feces 48 hours after dosing; rats

excreted 54% and 9% in the urine and feces, respective-

ly (Stott et al., 2000). Kohri et al. (1982) reported that

urinary excretion of [

C]-triethanolamine in rats

occurred primarily as the parent compound with a small

amount of glucoronide metabolite.

Humans

No information on the absorption, distribution, metabo-

lism, or excretion of triethanolamine in humans was

found in the literature.

TOXICITY

Experimental Animals

The toxicity of triethanolamine was the subject of a

review by Knaak et al. (1997). The acute and chronic

toxicity of triethanolamine is generally considered to be

low (Kindsvatter, 1940; Melnick and Tomaszewski,

1990). Triethanolamine toxicity has been demonstrated

in animals following administration by injection and oral

routes (gavage, feed, and drinking water) and by dermal

application. In a study comparing the relative toxicities

of the ethanolamines (Patty’s, 2000), symptoms in dogs

following intravenous injection included increased

blood pressure, diuresis, salivation, and pupillary dila-

tion; larger doses caused sedation, coma, and death

following a decrease in blood pressure and cardiac

collapse. Symptoms were most severe with mono-

ethanolamine and less severe with diethanolamine and

equivalent doses of triethanolamine.

The acute oral LD

of triethanolamine was reported to

be 8 to 9 g/kg in albino rats and 8 g/kg in guinea pigs

(Kindsvatter, 1940; Smyth et al., 1951). Gross lesions in

the animals that died were confined to the gastrointesti-

nal tract and included gastric dilatation, congestion, and

focal hemorrhage in the stomach and dilatation of intes-

tinal blood vessels. Kindsvatter (1940) suggested that

the acute toxicity of triethanolamine was related to its

alkalinity, based on the survival of one guinea pig fed

10 g/kg neutralized with hydrochloric acid; this dose

exceeds the oral LD

(8 g/kg) of triethanolamine when

administered as the free base. The acute oral LD

val-

ues for triethanolamine ranged from 4.2 to 11.3 g/kg in

rats and 5.4 to 7.8 g/kg in mice (BIBRA, 1990).

In a 90-day study in Carworth-Wistar rats administered

triethanolamine in feed at daily doses of 5 to

2,610 mg/kg, the no-observed-effect level (NOEL) was

80 mg/kg. Decreased body weight gains were observed

in rats administered 1,270 mg/kg or above. Microscopic

lesions of the kidney, liver, lung, or small intestine and a

low incidence of mortality occurred at a concentration of

730 mg/kg or above. Liver and kidney weight effects

occurred at concentrations as low as 170 mg/kg (Smyth

et al., 1951). In other studies (Kindsvatter, 1940), doses

ranging from 200 to 1,600 mg/kg were administered to

14 Triethanolamine, NTP TR 518

albino rats daily in feed for up to 17 weeks or to guinea

pigs 5 days per week by gavage for up to 120 doses.

Slight, reversible changes were present in the kidney

(cloudy swelling of the convoluted tubules and Henle’s

loop) at all doses and in the liver (hepatocellular cloudy

swelling and fatty change) at doses of 400 mg/kg and

higher. Although liver toxicity was not apparent in a

2-year study with Fischer 344/DuCrj rats administered

1% or 2% triethanolamine in drinking water ad libitum,

the occurrence of dose-related kidney toxicity (accelera-

tion of chronic nephropathy, mineralization of the renal

papilla, nodular hyperplasia of the pelvic mucosa, and

pyelonephritis with or without papillary necrosis) indi-

cated that even the 1% dose level was not well tolerated

by rats (Maekawa et al., 1986); effects were more severe

in females than in males. Due to increased mortality and

decreased body weight gains in females that received 2%

triethanolamine, doses administered to females were

halved from week 69 to the end of the study. In contrast,

B6C3F

mice tolerated the same concentrations of tri-

ethanolamine in drinking water for 82 weeks without

adverse effects on survival or organ weights and with no

increased incidences of histopathologic lesions (Konishi

et al., 1992); however, body weights of mice that

received 2% were slightly less than those of the controls.

The dermal toxicity of triethanolamine has been evaluat-

ed in mice, rats, rabbits, and guinea pigs under various

experimental conditions. Guinea pigs dosed with 8 g/kg

of undiluted triethanolamine daily by dermal applica-

tion, 5 days per week, died after 2 to 17 applications

(Kindsvatter, 1940). Histopathologic changes included

generalized congestion of the lungs, kidneys, liver, adre-

nal glands, and peritoneum; extravasation of fibrin into

the alveoli of the lungs; cloudy swelling of the kidneys

and liver; fatty change in the liver; and cellular infiltra-

tion and inflammation at the site of application, indicat-

ing that dermal absorption of triethanolamine is capable

of producing systemic toxicity (Kindsvatter, 1940;

Melnick and Tomaszewski, 1990).

The acute dermal toxicity of a single 2 g/kg application

of undiluted triethanolamine was evaluated over a

24-hour period with a closed-patch test in rabbits (CIR,

1983). Triethanolamine was applied to six rabbits with

intact skin and six with abraded skin. Mild to moderate

erythema without edema occurred on both intact and

abraded skin and resolved within 10 days. In another

dermal study, a single application of 560 mg/kg to rab-

bits resulted in erythema and slight edema at the appli-

cation site. Dermal applications of 2 mL/kg per day of a

2.5% aqueous solution of triethanolamine for 28 days

produced only mild dermatitis in New Zealand rabbits

(CIR, 1983). Triethanolamine (1% to 100%), applied

dermally to male C3H mice 5 days per week for 2 weeks

in 50 µL acetone, caused mild epidermal hyperplasia at

the site of application with dosage solutions as low as

25% (cited by Melnick and Tomaszewski, 1990). In a

follow-up study in which male and female C3H mice

received dermal applications of 0%, 10%, 33%, or 100%

triethanolamine in 50 µL acetone three times per week

for 13 weeks, mild hyperplasia at the application site

was observed in all dosed groups (Melnick and

Tomaszewski, 1990; DePass et al., 1995).

In a combined dermal and drinking water study,

CBA × C57Bl6 mice received dermal applications of a

6.5% or 13% aqueous solution of triethanolamine,

1 hour per day, 5 days per week for 6 months, with or

without additional oral administration of 1.4 mg/L in the

drinking water (Kostrodymova et al., 1976; CIR, 1983).

No toxic effects were present in mice that received the

6.5% solution. However, functional changes in the liver

and central nervous system occurred 1 month after treat-

ment began with the 13% solution, with or without addi-

tional triethanolamine in the drinking water, indicating

that systemic toxicity had resulted from percutaneous

absorption. Clinical pathology changes at 3 months

included elevated lymphocyte and segmented neutrophil

counts.

The eye irritation potential of triethanolamine or cos-

metics containing the chemical has been evaluated in

rabbits and rhesus monkeys; these studies have been

reviewed by the CIR Expert Panel (1983). In a study

with albino rabbits, moderate irritation occurred when

0.1 mL triethanolamine was instilled (Griffith et al.,

1980); application of 0.01 mL caused only negligible

damage. Application of 0.02 mL undiluted tri-

ethanolamine to the cornea of the rabbit eye with the lids

retracted caused necrosis of 63% to 87% of the cornea;

this reaction was graded as 5 on a scale of 1 to 10

(Carpenter and Smyth, 1946). Application of a 0.023 M

aqueous solution of triethanolamine to rabbit eyes, fol-

lowing removal of the corneal epithelium to facilitate

penetration, caused essentially no injuries when the solu-

tion was adjusted to pH 10; application of the same

solution adjusted to pH 11 caused moderate corneal

swelling and hyperemia of the iris and conjunctiva that

reversed within 1 week (Grant, 1974).

15 Triethanolamine, NTP TR 518

Skin and eye irritation in rabbits were evaluated in a

study comparing the effects of ethanolamines (mono-,

di-, and triethanolamine) and three mixtures containing

69%, 74%, or 87% triethanolamine plus varying propor-

tions of other ethanolamines (Dutertre-Catella et al.,

1982). Eye irritation was rated maximum for

monoethanolamine, severe for diethanolamine, mild for

the 69% and 74% mixtures, and minimum for tri-

ethanolamine and the 87% mixture. Skin irritation was

rated severe for monoethanolamine, moderate for

diethanolamine and the 69% mixture, and slight for tri-

ethanolamine and the 74% and 87% mixtures. Although

skin sensitization occurs in humans, no skin sensitizing

responses or delayed hypersensitivity reactions occurred

in studies of guinea pigs treated dermally with 5% to

100% triethanolamine (one application per week for

3 weeks; up to 6 hours per application) and subsequent-

ly challenged with 25% to 100% triethanolamine after 1

to 3 weeks (CIR, 1983).

No clinical evidence of systemic toxicity or histopatho-

logic lesions was observed when hair dye preparations

containing 0.1% to 1.5% triethanolamine were applied to

the clipped backs and sides of New Zealand white rab-

bits at doses of 1 mg/kg twice weekly for 13 weeks, with

or without prior abrasion of the skin (Burnett et al.,

1976). However, in a similar 13-week rabbit study in

which cosmetic formulations containing 14% tri-

ethanolamine stearate were applied to the clipped back

five times per week in doses of 1 or 3 mg/kg, mild to

moderate skin irritation occurred; the irritation, which

cleared within 72 hours, was followed by moderate to

heavy scaling (CIR, 1983). Signs of systemic toxicity

included lower body weight and significantly greater

kidney weights at the 3 mg/kg dose.

NTP (1999) examined both the local and systemic tox-

icity of triethanolamine in prechronic studies. Fischer

344/N rats received dermal applications of tri-

ethanolamine either undiluted (2,000 mg/kg) or in ace-

tone (125 to 1,000 mg/kg) once per day, 5 days per

week. Rats receiving 2,000 mg/kg gained less weight

than controls and had hypertrophy of the pituitary gland.

Additional lesions seen in rats receiving 2,000 mg/kg

and lower doses included increased kidney weights and

acanthosis and chronic inflammation at the skin site of

application. B6C3F

mice were treated topically with

4,000 mg/kg (neat) or 250 to 2,000 mg/kg (diluted) tri-

ethanolamine. Weight gains were marginally less at the

top concentration, and these animals also exhibited

increased liver and kidney weights and chronic active

inflammatiion at the skin site of application. Skin

lesions in mice receiving lower doses of triethanolamine

were generally limited to minimal acanthosis.

In subsequent studies, doses of 32 to 125 mg/kg in male

rats and 63 to 250 mg/kg in female rats were used to

investigate the effects of triethanolamine applied topi-

cally after 103 weeks of treatment (NTP, 1999).

Inflammation (males and females) and ulceration

(females) at the site of application were common find-

ings at the 15-month interim evaluation, and a spectrum

of lesions at the site of application was observed at the

end of the 2-year study. These included acanthosis,

chronic active inflammation, erosions, and ulcers. These

occurred primarily in females and in 125 mg/kg males.

B6C3F

mice were also treated chronically with tri-

ethanolamine at doses of 200 to 2,000 mg/kg in males

and 100 to 1,000 mg/kg in females. Skin lesions at the

site of application included acanthosis and chronic

inflammation. These were generally minimal to mild at

15 months and 2 years and again showed that mice were

less sensitive to triethanolamine than were rats.

More recently, triethanolamine was evaluated in a genet-

ically modified mouse skin papilloma model (Spalding

et al., 2000). Doses up to 30 mg of triethanolamine were

administered topically to groups of 15 to 20 female

Tg.AC mice five times per week for 20 weeks. The

experimental design also included positive and negative

controls. In contrast to the positive controls, which

developed multiple papillomas, there were no increases

in the incidences of skin tumors in mice receiving

triethanolamine.

Humans

There is no appreciable hazard to workers from normal

industrial use of ethanolamines (Kirk-Othmer, 1999).

However, these compounds may cause serious toxic

effects when ingested, as well as local injury to the

mouth, throat, and digestive tract. At ordinary tempera-

tures, triethanolamine presents no hazard from vapor

inhalation, but excessive vapor concentrations may

occur when triethanolamine is heated. These vapors are

irritating to the eyes and nose. Triethanolamine is a skin

and eye irritant; however, it is less irritating to the skin

and mucous membranes than most amines.

In a series of patch tests on healthy volunteers, the high-

est nonirritant concentration of triethanolamine, applied

in petrolatum for 48 hours, was determined to be 50%

16 Triethanolamine, NTP TR 518

(Meneghini et al., 1971). In a different test, the irritan-

cy potential of triethanolamine was designated as slight

(5% concentration) or marked (10% concentration)

when applied topically in ethanol within a chamber to

the scarified forearm skin of volunteers once daily for

3 days; the threshold concentration for skin irritation

was 100% on intact skin and 5% (in ethanol) on scarified

skin (Frosch and Kligman, 1976).

There have been no reports of industrial injuries from tri-

ethanolamine (Patty’s, 2000). However, Shrank (1985)

reported that a lathe operator became sensitized to tri-

ethanolamine, which was an ingredient in a cutting oil,

and 47 positive reactions to triethanolamine (10% in an

aqueous solution) occurred in patch tests of 230 metal

workers with occupational dermatitis (Alomar et al.,

1985). In this study, 43% of all positive responses were

to triethanolamine. Triethanolamine was also the most

frequent sensitizer in a study in which patients with sus-

pected cosmetic- or medicine-related contact dermatoses

were patch tested with common emulsifying agents

(Tosti et al., 1990). It has been identified as a causative

agent in patients with eczema or allergic contact der-

matitis (Venediktova and Gudina, 1976; Angelini et al.,

1985; Jones and Kennedy, 1988) and in a curious case of

intractable sneezing caused by exposure to a laundry

detergent (Herman, 1983).

In a study designed to evaluate allergic contact dermati-

tis to substances commonly found in pharmaceutical

ointments, a 24-hour patch test with a 1% aqueous solu-

tion of triethanolamine in 773 patients gave positive

reactions in four (0.5%) of these patients (Iden and

Schroeter, 1977). Positive reactions also occurred in 2%

of 100 subjects in another study with a 48-hour patch

test using 5% triethanolamine in petrolatum (Fisher

et al., 1971; Iden and Schroeter, 1977). In clinical tests

with triethanolamine and cosmetic products containing

triethanolamine, mild skin irritation occurred at concen-

trations above 5%, but there was little skin sensitization.

In addition, there was no evidence of phototoxicity or

photosensitization reactions with products that contained

up to 20% triethanolamine (CIR, 1983). However,

based on positive skin sensitivity reactions (patch-test

results) in 1.6% of a patient population with eczematous

dermatitis who were tested with 5% triethanolamine in

petrolatum, Meneghini et al. (1971) recommended

restricting the use of triethanolamine in cosmetics and

pharmaceutical preparations.

Triethanolamine has been given a toxic hazard rating of

slightly toxic, with a probable oral lethal dose in humans

of 5 to 15 g/kg, which corresponds to between 1 pint and

1 quart for a 70 kg (150 lb) person (Gosselin et al.,

1984). Dreisbach (1980) estimated the lethal dose in

humans to be 50 g. Assuming complete dermal absorp-

tion, a human weighing 50 kg would receive an approx-

imate dose of 10 mg/kg through the use of 10 g of cos-

metics containing 5% triethanolamine.

REPRODUCTIVE

AND DEVELOPMENTAL TOXICITY

Experimental Animals

Triethanolamine has been shown to stimulate neuritoge-

nesis in cultured chick embryo ganglia (Sisken et al.,

1985). However, it was embryotoxic when injected into

3-day chick embryos; the LD

value (the dose causing

early death in 50% of the embryos) was 3 µmol (447 µg)

per egg. Eleven days after the triethanolamine injection,

there was no significant increase in the incidence of

chick embryo malformations (Korhonen et al., 1983;

cited by Melnick and Tomaszewski, 1990).

In an in vivo assay for potential adverse reproductive

effects in mice, di- and triethanolamine, administered

daily by gavage on gestation days 6 through 15 in doses

of 1,125 mg/kg per day, had no effect on maternal mor-

tality, the number of viable litters, litter size, or survival

and body weight of the pups. However, similar admin-

istration of 850 mg/kg per day of monoethanolamine

resulted in 16% mortality in dams and fewer viable lit-

ters (NIOSH, 1987). A preliminary developmental tox-

icity test in mice, used in conjunction with a scoring sys-

tem of indices of developmental toxicity, resulted in a

classification of low priority for further study of

monoethanolamine, intermediate priority or “no deci-

sion” for triethanolamine, and high priority for

diethanolamine (York et al., 1988).

In mating trial studies in male and female Fisher 344

rats, 0.5 g/kg of triethanolamine in acetone, applied der-

mally to the interscapular area of the clipped back in an

approximate volume of 1.8 mL/kg daily for 10 weeks

prior to mating, during breeding, and through gestation

and lactation for females, had no effect on mating, fertil-

ity, or offspring growth and survival. In similar studies

with Swiss (CD-1

) mice administered daily

17 Triethanolamine, NTP TR 518

applications of 2 g/kg in an approximate volume of

3.6 mL/kg, no chemical-related effects occurred other

than ruffled fur in females and irritation at the applica-

tion site in males and females (Battelle, 1988a,b).

No embryotoxic or teratogenic effects were produced by

topical administration of semipermanent hair dye prepa-

rations (2 mL/kg) containing 0.1% to 1.5% tri-

ethanolamine to the shaved backs of pregnant Charles

River CD rats on gestation days 1, 4, 7, 10, 13, 16, and

19 (Burnett et al., 1976).

Humans

No information related to the reproductive or develop-

mental toxicity of triethanolamine in humans was found

in the literature.

CARCINOGENECITY

Experimental Animals

Triethanolamine was not carcinogenic in Fischer

F344/DuCrj rats when administered ad libitum in drink-

ing water at dose levels of 1% or 2%, but was nephro-

toxic, especially in females (Maekawa et al., 1986). Due

to increased mortality associated with nephrotoxicity in

females and decreased body weight gain in females in

the 2% group, the doses of triethanolamine administered

to the female groups were reduced by half after week 68

of the study. There were no statistically significant

increased incidences of primary neoplasms in exposed

groups compared to the controls when analyzed by the

chi-square test. Because of nephrotoxicity, which

appeared to have an adverse effect on the life expectan-

cy of exposed animals, an age-adjusted statistical analy-

sis was performed. The results showed a positive trend

(P<0.05) in the incidence of hepatic neoplasms in males

and uterine endometrial sarcomas and renal tubule ade-

nomas in females. However, the incidences of these

neoplasms in the control groups were lower than those in

historical controls. In a similar study, triethanolamine

had no carcinogenic activity in B6C3F

mice when

administered in drinking water at concentrations of 1%

or 2% for 82 weeks (Konishi et al., 1992). Tri-

ethanolamine was not carcinogenic in male

CBA × C57Bl6 mice when applied dermally for 14 to

18 months (Kostrodymova et al., 1976; CIR, 1983).

Incidences of malignant lymphoma, particularly thymic

lymphoma, were increased in female, but not in male,

ICR-JCL mice fed diets containing 0.03% or 0.3% tri-

ethanolamine throughout their life spans (Hoshino and

Tanooka, 1978). The incidences of total malignant neo-

plasms in treated female mice were significantly greater

than the control incidence (P<0.01); the incidences were

1/36 in controls, 10/37 in the 0.03% group, and 13/36 in

the 0.3% group. In another long-term study with ICR

mice (Inai et al., 1979), the combined incidence of

thymic lymphoma and nonthymic leukemia in control

females at 109 weeks was 5/15. This rate is 10 times

greater than the rate observed in the female control

group of the Hoshino and Tanooka study, and is similar

to that reported for triethanolamine-treated females.

Rust-proofing cutting fluid (containing low levels of tri-

ethanolamine, sodium nitrite, and polyethylene glycol)

was carcinogenic in male Wistar rats (Wang et al.,

1988). Groups of 40 rats were administered either undi-

luted cutting fluid or a threefold dilution of the fluid in

water ad libitum for 2 years. Treated rats had increased

incidences of neoplasms, particularly pancreatic carcino-

ma. The total incidences of malignant neoplasms were

0% in the control group, 10% in the group given diluted

cutting fluid, and 27.5% in the group that received the

undiluted fluid. In another group of rats exposed to

undiluted cutting fluid supplemented with ascorbic acid,

the total incidence of malignant neoplasms was 1/40

(2.5%). Based on the protective action of ascorbic acid

in this study, Wang et al. (1988) concluded that the car-

cinogenic agent was a nitrosamine formed in vivo; the

inhibitory action of ascorbic acid on the in vivo forma-

tion of nitroso compounds has been documented

(Mirvish et al., 1975). Because the cutting fluid con-

tained triethanolamine and sodium nitrite, the most prob-

able nitrosamine formed during this study was

N-nitrosodiethanolamine. However, the neoplasms

induced in rats in previous experiments with

N-nitrosodiethanolamine were primarily hepatocellular

carcinomas, not pancreatic carcinomas (Lijinsky and

Kovatch, 1985).

No neoplastic effects occurred in female Fischer 344/N

rats that received dermal applications of 63, 125, or

250 mg triethanolamine/kg body weight for 2 years

(NTP, 1999); body weights of the highest dose group

were 7% lower than those of the vehicle controls at the

end of the study. Male rats treated with 32, 63, or

125 mg/kg had marginal increases in the incidences of

renal tubule adenoma. Male B6C3F

mice received 200,

630, or 2,000 mg/kg triethanolamine by dermal applica-

tion and female mice received 100, 300, or 1,000 mg/kg;

18 Triethanolamine, NTP TR 518

skin inflammation and liver neoplasms occurred in

males and females. However, the 2-year study in mice

was considered inadequate due to infection with

Helicobacter hepaticus.

Humans

In an epidemiology study conducted by Järvholm et al.

(1986), the mortality and cancer morbidity in 219 men

exposed to cutting fluids for at least 5 years, including at

least 1 year of exposure to a cutting fluid containing both

amines and nitrites (primarily ethanolamines and sodium

nitrite), were not significantly different from those of the

general population. Although the results indicate that

the use of cutting fluids in this industry does not lead to

an increased risk of cancer, the authors were unable to

exclude the possibility of an increased risk for cancer of

a specific site because of the small sample population.

Although an association between exposure to metal-

working fluids and cancer has been reported (Kazerouni

et al., 2000), no conclusions can be drawn regarding the

specific exposure to triethanolamine.

ENETIC TOXICITY

The limited information on the mutagenicity of tri-

ethanolamine indicates that the chemical is not genotox-

ic. A review of the toxicity of triethanolamine, includ-

ing genetic toxicity, was published by IARC (2000).

Triethanolamine did not induce DNA damage in

Escherichia coli (Inoue et al., 1982), mutations in

Salmonella typhimurium (Inoue et al., 1982; Dean et al.,

1985; Mortelmans et al., 1986), or gene conversion in

Saccharomyces cerevisiae (Dean et al., 1985). No

induction of sister chromatid exchanges occurred in cul-

tured Chinese hamster ovary cells treated with tri-

ethanolamine (Galloway et al., 1987), and results of tests

for induction of chromosomal aberrations in cultured rat

liver cells (Dean et al., 1985) and cultured Chinese ham-

ster cells (Inoue et al., 1982; Galloway et al., 1987) were

also negative. The S. typhimurium tests and the rodent

cell cytogenetic tests were conducted with and without

S9 metabolic activation enzymes. No increase in the fre-

quency of sex-linked recessive lethal mutations was

observed in germ cells of male Drosophila melanogaster

administered triethanolamine by feeding or injection

(Yoon et al., 1985).

STUDY RATIONALE

The National Cancer Institute nominated tri-

ethanolamine for study because of its widespread use in

cosmetics and other consumer products, its high poten-

tial for worker exposure due to its many industrial uses,

and its potential for conversion to the carcinogen

N-nitrosodiethanolamine. Concern was also prompted

by the increased incidences of lymphoma and total

malignant neoplasms in female ICR-JCL mice that

received 0.03% or 0.3% triethanolamine in the diet

(Hoshino and Tanooka, 1978). Based on these consider-

ations, the NTP conducted dermal studies in order to

evaluate the toxicity and potential carcinogenic activity

of triethanolamine in mice and rats. Dermal application

was chosen as the route of exposure to mimic the pri-

mary human exposure to triethanolamine and because

considerable systemic exposure is achieved with this

route.

NTP (1999) reported equivocal evidence of carcinogenic

activity of triethanolamine in male F344/N rats.

However, the study in B6C3F

mice was considered

inadequate due to the presence of H. hepaticus infection,

which complicated interpretation of the relationship

between triethanolamine administration and liver neo-

plasms. Evaluating the role of triethanolamine in the

development of liver neoplasms in uninfected mice is

necessary to complete the characterization of the car-

cinogenic hazard of triethanolamine.

MATERIALS AND METHODS

ROCUREMENT

AND

CHARACTERIZATION

Triethanolamine

Triethanolamine was obtained from Texaco Chemical

Company (Division of Texaco, Inc., Bellaire, TX) in one

lot (7G-60). Identity, purity, and moisture content analy-

ses were conducted by the analytical chemistry laborato-

ry (Research Triangle Institute, Research Triangle Park,

NC); identity, purity, and stability analyses were con-

ducted by the study laboratory (Appendix D). Special

analyses of diethanolamine and nitrosamine impurities

in the bulk chemical were conducted by the analytical

chemistry laboratory and Covance Laboratories, Inc.

(Madison, WI). Reports on analyses performed in sup-

port of the triethanolamine study are on file at the

National Institute of Environmental Health Sciences.

The chemical, a clear, colorless liquid, was identified as

triethanolamine using ultraviolet/visible, infrared, and

proton nuclear magnetic resonance spectroscopy and

high- and low-resolution mass spectrometry. Karl

Fischer titration indicated 0.18% water. The purity of

lot 7G-60 was determined using gas chromatography

and high-performance liquid chromotography (HPLC).

Gas chromatography indicated one major peak and no

impurities by two systems and one major peak and one

impurity with an area of 0.88% relative to the major peak

area by a third system; the impurity was determined to

be diethanolamine. Gas chromatography indicated that

the compound contained no impurity other than

diethanolamine at a concentration greater than 0.1% by

a fourth system. HPLC indicated no measurable impuri-

ties with chromophores. The overall purity of lot 7G-60

was determined to be greater than 99%.

A special HPLC/mass spectrometric study was conduct-

ed to determine the relative concentration of

diethanolamine as an impurity in the test chemical and to

conduct an accelerated stability study. Analysis showed

that diethanolamine constituted 0.491% (weight percent

relative to the test chemical weight) of the test chemical.

Solutions of triethanolamine were prepared in acetone or

95% ethanol and stored in sealed glass containers at

32° to 36° C, protected from light, for 1.6 hours or

11 days. After 11 days of storage, both solutions showed

slight increases in diethanolamine concentrations com-

pared to those measured at time 0; no differences were

noted following 1.6 hours of storage.

The concentrations of nonpolar nitrosamines (N-nitroso-

dimethylamine, N-nitrosomethylethylamine, N-nitroso-

diethylamine, N-nitrosodi-n-propylamine, N-nitrosodi-

n-butylamine, N-nitrosopiperidine, N-nitrosopyrrolidine,

and N-nitrosomorpholine) and the polar nitrosamine N-

nitrosodiethanolamine were determined in lot 7G-60 by

gas chromatography and HPLC. No nonpolar or polar

nitrosamines were present at concentrations greater than

the limits of detection (0.1 and 1.0 ppm, respectively).

Stability studies of the bulk chemical were performed on

lot 03601CN (not used in the current study) using gas

chromatography. These studies indicated that tri-

ethanolamine was stable as a bulk chemical for 14 days

when stored in amber glass vials at 5°, 25°, and 60° C,

when compared to a sample stored at –20° C. To ensure

stability, triethanolamine was stored at room temperature

in amber glass containers with Teflon

-lined lids. No

degradation of triethanolamine was observed.

Acetone

Acetone was obtained in four lots (NE0173, NV0163,

OG0513, and OX0312) from Spectrum Chemical

Manufacturing Corporation (Gardena, CA). Identity and

purity analyses of each lot were conducted by the study

laboratory.

The chemical, a clear liquid, was identified as acetone

using infrared spectroscopy. The purity of each lot was

determined using gas chromatography. No significant

impurities were detected in any lot. The overall purity of

each lot was determined to be greater than 99%.

To ensure stability, the bulk chemical was stored at room

temperature in amber glass bottles. No degradation of

the acetone was detected.

20 Triethanolamine, NTP TR 518

PREPARATION AND ANALYSIS

DOSE FORMULATIONS

The dose formulations were prepared approximately

every 2 weeks by mixing triethanolamine and acetone to

give the required concentration (Table D3). The dose

formulations were stored for up to 21 days at 5° C in

amber glass bottles with Teflon

-lined lids.

Stability studies of 10 mg/mL dose formulations in ace-

tone were performed by Midwest Research Institute

(Kansas City, MO) using gas chromatograpy. The dose

formulations were stored in amber glass bottles with

Teflon

-lined lids under nitrogen headspace for up to

21 days at 5° C, and for at least 3 hours under animal

room conditions (open to air and light).

Periodic analyses of the dose formulations of tri-

ethanolamine were conducted by the study laboratory

with gas chromatography. During the 2-year study, the

dose formulations were analyzed approximately every 8

or 12 weeks; animal room samples were also analyzed

periodically (Table D4). Of the dose formulations ana-

lyzed, all 66 were within 10% of the target concentra-

tions; 18 of 24 animal room samples were within 10% of

the target concentrations. The other six dose formula-

tions were higher than the target concentrations and,

with one exception, occurred with the first set of dose

formulations used in the study. The change in concen-

trations was due to evaporation of the acetone during

administration and subsequently, steps were taken to

minimize this effect. The maximum was 15% higher

than the prepared concentration.

2-YEAR STUDY

Study Design

Groups of 50 male and 50 female mice received dermal

applications of 0, 200, 630, or 2,000 mg/kg (males) and

0, 100, 300, or 1,000 mg/kg (females) triethanolamine in

acetone, 5 days per week, for 104 (males) or 104 to

105 (females) weeks; the dosing volume was 2 mL/kg.

Doses were applied to an area extending from the ani-

mal’s mid-back to the intrascapular region; the site of

application was clipped approximately once per week

during the study.

Source and Specification of Animals

Male and female B6C3F

mice were obtained from

Taconic Laboratory Animals and Services

(Germantown, NY) for use in the 2-year study. Mice

were quarantined for 14 (males) or 13 (females) days

before the beginning of the study. Five male and five

female mice were randomly selected for parasite evalua-

tion and gross observation of disease. Mice were

approximately 6 weeks old at the beginning of the study.

The health of the animals was monitored during the stud-

ies according to the protocols of the NTP Sentinel

Animal Program (Appendix F).

Animal Maintenance

Mice were housed individually. Feed and water were

available ad libitum. Cages and racks were rotated every

2 weeks. Further details of animal maintenance are

given in Table 1. Information on feed composition and

contaminants is provided in Appendix E.

Clinical Examinations and Pathology

All mice were observed twice daily and were weighed

initially, weekly for 13 weeks, monthly thereafter, and at

the end of the study. Clinical findings were recorded on

day 29, monthly thereafter, and at the end of the study.

Complete necropsies and microscopic examinations

were performed on all mice. At necropsy, all organs and

tissues were examined for grossly visible lesions, and all

major tissues were fixed and preserved in 10% neutral

buffered formalin, processed and trimmed, embedded in

paraffin, sectioned to a thickness of 4 to 6 µm, and

stained with hematoxylin and eosin for microscopic

examination. For all paired organs (e.g., adrenal gland,

kidney, ovary), samples from each organ were examined.

Tissues examined microscopically are listed in Table 1.

Microscopic evaluations were completed by the study

laboratory pathologist, and the pathology data were

entered into the Toxicology Data Management System.

The slides, paraffin blocks, and residual wet tissues were

sent to the NTP Archives for inventory, slide/block

match, and wet tissue audit. The slides, individual ani-

mal data records, and pathology tables were evaluated

by an independent quality assessment laboratory. The

individual animal records and tables were compared for

21 Triethanolamine, NTP TR 518

accuracy, the slide and tissue counts were verified, and

the histotechnique was evaluated. For the 2-year study,

a quality assessment pathologist evaluated slides from

all tumors and all potential target organs, which includ-

ed the liver and skin.

The quality assessment report and the reviewed slides

were submitted to the NTP Pathology Working Group

(PWG) chairperson, who reviewed the selected tissues

and addressed any inconsistencies in the diagnoses made

by the laboratory and quality assessment pathologists.

Representative histopathology slides containing exam-

ples of lesions related to chemical administration, exam-

ples of disagreements in diagnoses between the labora-

tory and quality assessment pathologists, or lesions of

general interest were presented by the chairperson to the

PWG for review. The PWG consisted of the quality

assessment pathologist and other pathologists experi-

enced in rodent toxicologic pathology. This group

examined the tissues without any knowledge of dose

groups or previously rendered diagnoses. When the

PWG consensus differed from the opinion of the labora-

tory pathologist, the diagnosis was changed. Final diag-

noses for reviewed lesions represent a consensus

between the laboratory pathologist, reviewing patholo-

gist(s), and the PWG. Details of these review proce-

dures have been described, in part, by Maronpot and

Boorman (1982) and Boorman et al. (1985). For subse-

quent analyses of the pathology data, the decision of

whether to evaluate the diagnosed lesions for each tissue

type separately or combined was generally based on the

guidelines of McConnell et al. (1986).

22 Triethanolamine, NTP TR 518

ABLE 1

Experimental Design and Materials and Methods in the 2-Year Dermal Study of Triethanolamine

Study Laboratory

Battelle Columbus Operations (Columbus, OH)

Strain and Species

B6C3F mice

Animal Source

Taconic Laboratory Animals and Services (Germantown, NY)

Time Held Before Studies

14 (males) or 13 (females) days

Average Age When Studies Began

6 weeks

Date of First Dose

September 24 (males) or 23 (females), 1998

Duration of Dosing

104 (males) or 104 to 105 (females) weeks

Date of Last Dose

September 17-19 (males) or 19-21 (females), 2000

Necropsy Dates

September 18-20 (males) or 20-22 (females), 2000

Average Age at Necropsy

110 weeks

Size of Study Groups

50 males and 50 females

Method of Distribution

Animals were distributed randomly into groups of approximately equal initial mean body weights.

Animals per Cage

Method of Animal Identification

Tail tattoo

Diet

Irradiated NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly

Water

Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum

Cages

Polycarbonate (Lab Products, Inc., Montville, NJ), changed weekly

Bedding

Irradiated Sani-Chips

(P.J. Murphy Forest Products Corp., Montville, NJ), changed weekly

23 Triethanolamine, NTP TR 518

ABLE 1

Experimental Design and Materials and Methods in the 2-Year Dermal Study of Triethanolamine

Cage Filters

Spun-bonded polyester (Snow Filtration Co., Cincinnati, OH), changed every 2 weeks

Racks

Stainless steel (Lab Products, Inc., Montville, NJ), changed and rotated every 2 weeks

Animal Room Environment

Temperature: 72° ± 3° F

Relative humidity: 50% ± 15%

Room fluorescent light: 12 hours/day

Room air changes: 10/hour

Doses

0, 200, 630, or 2,000 mg/kg (males) and 0, 100, 300, or 1,000 mg/kg (females) in acetone by dermal application (dosing volume 2 mL/kg)

Type and Frequency of Observation

Observed twice daily; animals were weighed initially, weekly for 13 weeks, monthly thereafter, and at the end of the study; clinical findings

were recorded on day 29, monthly thereafter, and at the end of the study.

Method of Sacrifice

Carbon dioxide asphyxiation

Necropsy

Necropsy was performed on all mice.

Histopathology

Complete histopathology was performed on all mice. In addition to gross lesions and tissue masses, the following tissues were examined:

adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eye, gallbladder, harderian gland, heart with aorta, large intestine (cecum,

colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung with bronchi, lymph nodes (mandibular and mesenteric),

mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin (site of

application), spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary

bladder, and uterus.

STATISTICAL METHODS

Survival Analyses

The probability of survival was estimated by the prod-

uct-limit procedure of Kaplan and Meier (1958) and is

presented in the form of graphs. Animals found dead of

other than natural causes were censored from the sur-

vival analyses; animals dying from natural causes were

not censored. Statistical analyses for possible dose-

related effects on survival used Cox’s (1972) method for

testing two groups for equality and Tarone’s (1975) life

table test to identify dose-related trends. All reported

P values for the survival analyses are two sided.

Calculation of Incidence

The incidences of neoplasms or nonneoplastic lesions

are presented in Tables A1, A5, B1, and B5 as the num-

bers of animals bearing such lesions at a specific

anatomic site and the numbers of animals with that site

examined microscopically. For calculation of statistical

significance, the incidences of most neoplasms

(Tables A3 and B3) and all nonneoplastic lesions are

given as the numbers of animals affected at each site

examined microscopically. However, when macroscop-

ic examination was required to detect neoplasms in cer-

tain tissues (e.g., harderian gland, intestine, and mam-

mary gland) before microscopic evaluation, or when

neoplasms had multiple potential sites of occurrence

(e.g., leukemia or lymphoma), the denominators consist

of the number of animals on which a necropsy was per-

formed. Tables A3 and B3 also give the survival-adjust-

ed neoplasm rate for each group and each site-specific

neoplasm. This survival-adjusted rate (based on the

Poly-3 method described below) accounts for differen-

tial mortality by assigning a reduced risk of neoplasm,

24 Triethanolamine, NTP TR 518

proportional to the third power of the fraction of time on

study, to animals that do not reach terminal sacrifice.

Analysis of Neoplasm

and Nonneoplastic Lesion Incidences

The Poly-k test (Bailer and Portier, 1988; Portier and

Bailer, 1989; Piegorsch and Bailer, 1997) was used to

assess neoplasm and nonneoplastic lesion prevalence.

This test is a survival-adjusted quantal-response proce-

dure that modifies the Cochran-Armitage linear trend

test to take survival differences into account. More

specifically, this method modifies the denominator in the

quantal estimate of lesion incidence to approximate

more closely the total number of animal years at risk.

For analysis of a given site, each animal is assigned a

risk weight. This value is one if the animal had a lesion

at that site or if it survived until terminal sacrifice; if the

animal died prior to terminal sacrifice and did not have a

lesion at that site, its risk weight is the fraction of the

entire study time that it survived, raised to the kth power.

This method yields a lesion prevalence rate that depends

only upon the choice of a shape parameter for a Weibull

hazard function describing cumulative lesion incidence

over time (Bailer and Portier, 1988). Unless otherwise

specified, a value of k=3 was used in the analysis of site-

specific lesions. This value was recommended by Bailer

and Portier (1988) following an evaluation of neoplasm

onset time distributions for a variety of site-specific neo-

plasms in control F344 rats and B6C3F

mice (Portier

et al., 1986). Bailer and Portier (1988) showed that the

Poly-3 test gave valid results if the true value of k was

anywhere in the range from 1 to 5. A further advantage

of the Poly-3 method is that it does not require lesion

lethality assumptions. Variation introduced by the use of

risk weights, which reflect differential mortality, was

accommodated by adjusting the variance of the Poly-3

statistic as recommended by Bieler and Williams (1993).

Tests of significance included pairwise comparisons of

each dosed group with controls and a test for an overall

dose-related trend. Continuity-corrected Poly-3 tests

were used in the analysis of lesion incidence, and report-

ed P values are one sided. The significance of lower

incidences or decreasing trends in lesions is represented

as 1– P with the letter N added (e.g., P=0.99 is present-

ed as P=0.01N).

Analysis of Continuous Variables

Average severity values were analyzed for significance

with the Mann-Whitney U test (Hollander and Wolfe,

1973).

Historical Control Data

The concurrent control group represents the most valid

comparison to the treated groups and is the only control

group analyzed statistically in NTP bioassays. However,

historical control data are often helpful in interpreting

potential treatment-related effects, particularly for

uncommon or rare neoplasm types. For meaningful

comparisons, the conditions for studies in the historical

database must be generally similar. One significant fac-

tor affecting the background incidence of neoplasms at a

variety of sites is diet. In 1995, the NTP incorporated a

new diet (NTP-2000) that contains less protein and more

fiber than the NIH-07 diet used previously in toxicity

and carcinogenicity studies (Rao, 1996, 1997). The cur-

rent NTP historical database contains all 21 studies that

use the NTP-2000 diet with histopathology findings

completed up to the present. A second potential source

of variability is route of administration. In general the

historical database for a given study will include studies

using the same route of administration, and the overall

incidences of neoplasms for all routes of administration

are included for comparison. The triethanolamine study

is the only dermal study in the database; therefore, over-

all incidences for all routes have been used in this

Technical Report.

QUALITY ASSURANCE METHODS

The 2-year study was conducted in compliance with

Food and Drug Administration Good Laboratory

Practice Regulations (21 CFR, Part 58). In addition, as

records from the 2-year study were submitted to the NTP

Archives, this study was audited retrospectively by an

independent quality assurance contractor. Separate

audits covered completeness and accuracy of the pathol-

ogy data, pathology specimens, final pathology tables,

and a draft of this NTP Technical Report. Audit proce-

dures and findings are presented in the reports and are on

file at NIEHS. The audit findings were reviewed and

assessed by NTP staff, and all comments were resolved

or otherwise addressed during the preparation of this

Technical Report.

25 Triethanolamine, NTP TR 518

GENETIC TOXICOLOGY

The genetic toxicity of triethanolamine was assessed by

testing the ability of the chemical to induce mutations in

various strains of Salmonella typhimurium, sister chro-

matid exchanges and chromosomal aberrations in cul-

tured Chinese hamster ovary cells, sex-linked recessive

lethal mutations in Drosophila melanogaster, and

increases in the frequency of micronucleated erythro-

cytes in mouse peripheral blood. The protocols for these

studies and the results are given in Appendix C.

The genetic toxicity studies have evolved from an earli-

er effort by the NTP to develop a comprehensive data-

base permitting a critical anticipation of a chemical’s

carcinogenicity in experimental animals based on

numerous considerations, including the molecular

structure of the chemical and its observed effects in

short-term in vitro and in vivo genetic toxicity tests

(structure-activity relationships). The short-term tests

were originally developed to clarify proposed mecha-

nisms of chemical-induced DNA damage based on the

relationship between electrophilicity and mutagenicity

(Miller and Miller, 1977) and the somatic mutation the-

ory of cancer (Straus, 1981; Crawford, 1985). However,

it should be noted that not all cancers arise through geno-

toxic mechanisms.

DNA reactivity combined with Salmonella mutagenicity

is highly correlated with induction of carcinogenicity in

multiple species/sexes of rodents and at multiple tissue

sites (Ashby and Tennant, 1991). A positive response in

the Salmonella test was shown to be the most predictive

in vitro indicator for rodent carcinogenicity (89% of the

Salmonella mutagens are rodent carcinogens) (Tennant

et al., 1987; Zeiger et al., 1990). Additionally, no bat-

tery of tests that included the Salmonella test improved

the predictivity of the Salmonella test alone. However,

these other tests can provide useful information on the

types of DNA and chromosomal damage induced by the

chemical under investigation.

The predictivity for carcinogenicity of a positive

response in acute in vivo bone marrow chromosome

aberration or micronucleus tests appears to be less than

that in the Salmonella test (Shelby et al., 1993; Shelby

and Witt, 1995). However, clearly positive results in

long-term peripheral blood micronucleus tests have high

predictivity for rodent carcinogenicity (Witt et al.,

2000); negative results in this assay do not correlate well

with either negative or positive results in rodent carcino-

genicity studies. Because of the theoretical and

observed associations between induced genetic damage

and adverse effects in somatic and germ cells, the deter-

mination of in vivo genetic effects is important to the

overall understanding of the risks associated with expo-

sure to a particular chemical. Most organic chemicals

that are identified by the International Agency for

Research on Cancer as human carcinogens, other than

hormones, are genotoxic. The vast majority of these are

detected by both the Salmonella assay and rodent bone

marrow cytogenetics tests (Shelby, 1988; Shelby and

Zeiger, 1990).

26 Triethanolamine, NTP TR 518

RESULTS

2-Y

EAR STUDY

Survival

Estimates of 2-year survival probabilities for male and

Meier survival curves (Figure 1). Survival of all dosed

female mice are shown in Table 2 and in the Kaplan-

groups was similar to that of the vehicle control groups.

TABLE 2

Survival of Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 200 mg/kg 630 mg/kg 2,000 mg/kg

Male

Animals initially in study 50 50 50 50

Moribund 5 2 6 8

Natural deaths 8 5 10 2

Animals surviving to study termination 37 43 34 40

Percent probability of survival at end of study 74 86 68 80

Mean survival (days) 710 705 683 701

Survival analysis P=1.000N P=0.254N P=0.492 P=0.743N

Vehicle Control 100 mg/kg 300 mg/kg 1,000 mg/kg

Female

Animals initially in study 50 50 50 50

Accidental death 0 0 0 1

Moribund 8 10 5 10

Natural deaths 7 6 4 7

Animals surviving to study termination 35 34 41 32

Percent probability of survival at end of study 70 68 82 65

Mean survival (days) 692 682 686 687

Survival analysis P=0.759 P=1.000 P=0.293N P=0.865

Kaplan-Meier determinations

Mean of all deaths (uncensored, censored, and terminal sacrifice).

The result of the life table trend test (Tarone, 1975) is in the vehicle control column, and the results of the life table pairwise comparisons

(Cox, 1972) with the vehicle controls are in the dosed group columns. A negative trend or lower mortality in a dosed group is indicated

by N.

Censored from survival analyses

Includes one animal that died during the last week of the study

...

;l:

:;>

...

1::

::;

iii

...

;l:

:;>

...

::;

iii

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

1.0

0.8

........

0.7

0.8

0.5

0.4

0.2

MALE MICE

• Omgl\g

200

mg/kg

630

mg/kg

u 2,000mgltg

15 30

...

, .

..,

FEMALE MICE

•

Omglkg

0 100tnglkg

A 300rnglkg

U 1 ,000

ml)fk

60 75

105 1

WEEKS

STUDY

...

0.0

-----;----.------,---.-------T----j------T-----l

0 15 45 80 75 90 105 120

WEEKS

STUDY

28 Triethanolamine, NTP TR 518

FIGURE 1

Kaplan-Meier Survival Curves for Male and Female Mice

Administered Triethanolamine Dermally for 2 Years

29 Triethanolamine, NTP TR 518

Body Weights and Clinical Findings

Body weights of 2,000 mg/kg males were less than those

of the vehicle controls from weeks 17 to 37 and at the

end of the study, and body weights of dosed groups of

females were similar to those of the vehicle controls

throughout the study (Tables 3 and 4; Figure 2).

Treatment-related clinical findings included irritation of

the skin at the site of application in dosed males and 300

and 1,000 mg/kg females consisting of small, brown,

crusty, scab-like patches. Skin irritation increased with

increasing dose and was more severe in males than in

females; these lesions were not considered sufficiently

severe to require early termination of any of the mice in

the study.

30 Triethanolamine, NTP TR 518

ABLE 3

Mean Body Weights and Survival of Male Mice in the 2-Year Dermal Study of Triethanolamine

Weeks Vehicle Control 200 mg/kg 630 mg/kg 2,000 mg/kg

on Av. Wt. No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of

Study (g) Survivors (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors

1 22.8 50 22.9 100 50 22.9 100 50 22.8 100 50

2 23.8 50 23.8 100 50 23.7 100 50 24.0 101 50

3 24.9 50 25.2 101 50 25.1 101 50 25.2 101 50

4 26.3 50 26.6 101 50 26.5 101 50 26.6 101 50

5 27.5 50 27.7 101 50 27.6 100 50 27.8 101 50

6 28.5 50 28.0 98 50 28.7 101 50 28.3 99 50

7 29.3 50 29.4 100 50 29.4 100 50 28.9 99 50

8 30.3 50 29.9 99 50 30.4 100 50 29.8 98 50

9 31.4 50 30.8 98 50 31.2 99 50 30.4 97 50

10 32.6 50 31.8 98 50 32.4 99 50 31.3 96 50

11 33.7 50 32.6 97 50 33.2 99 50 32.1 95 50

12 34.8 50 33.6 97 50 34.1 98 50 32.8 94 50

13 35.1 50 34.1 97 50 34.4 98 50 33.3 95 50

17 39.4 50 38.1 97 50 38.6 98 50 36.2 92 50

21 42.5 50 40.6 96 50 41.6 98 50 38.1 90 50

25 45.2 50 43.8 97 50 43.5 96 50 40.9 91 50

29 47.5 50 46.5 98 50 46.9 99 50 43.3 91 50

33 49.0 50 48.0 98 50 48.6 99 50 45.4 93 50

37 50.2 50 49.6 99 50 49.9 99 50 47.3 94 50

41 50.1 50 49.7 99 50 50.9 102 50 48.8 97 50

45 51.4 50 50.4 98 50 51.6 100 50 50.2 98 50

49 51.5 50 50.8 99 49 52.1 101 50 51.0 99 50

53 50.6 50 50.0 99 49 51.3 101 50 50.8 100 50

57 51.0 50 50.5 99 49 51.4 101 50 50.2 98 50

61 51.6 50 51.1 99 49 52.0 101 50 50.7 98 49

65 51.3 50 51.6 101 49 52.4 102 48 51.1 100 49

69 52.0 50 52.6 101 49 52.2 100 48 50.8 98 49

73 52.3 50 53.1 102 48 54.3 104 45 51.7 99 49

77 53.4 49 54.0 101 48 54.2 102 44 52.3 98 49

81 52.8 49 53.1 101 47 53.8 102 44 51.6 98 48

85 51.8 48 52.9 102 47 53.8 104 43 51.7 100 46

89 51.9 48 52.7 102 46 53.0 102 41 50.2 97 46

93 51.9 46 53.4 103 45 53.1 102 41 50.1 97 43

97 50.4 45 51.0 101 45 50.4 100 39 47.1 94 43

101 51.0 41 50.9 100 44 51.2 100 36 46.5 91 40

Mean for weeks

1-13 29.3 29.0 99 29.2 100 28.7 98

14-52 47.4 46.4 98 47.1 99 44.6 94

53-101 51.7 52.1 101 52.5 102 50.4 97

31 Triethanolamine, NTP TR 518

TABLE 4

Mean Body Weights and Survival of Female Mice in the 2-Year Dermal Study of Triethanolamine

Weeks Vehicle Control 100 mg/kg 300 mg/kg 1,000 mg/kg

on Av. Wt. No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of

Study (g) Survivors (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors

1 18.8 50 19.0 101 50 19.0 101 50 19.0 101 50

2 19.4 50 19.7 102 50 19.5 101 50 20.1 104 50

3 21.0 50 21.3 101 50 21.4 102 50 21.8 104 50

4 22.5 50 22.8 101 50 23.1 103 50 23.5 104 50

5 24.0 50 24.4 102 50 24.2 101 50 24.6 103 50

6 24.4 50 24.9 102 50 24.9 102 50 25.5 105 50

7 25.1 50 25.6 102 50 25.6 102 50 26.2 104 50

8 26.2 50 26.4 101 50 26.5 101 50 27.1 103 50

9 26.6 50 27.1 102 50 27.0 102 50 27.8 105 50

10 27.5 50 28.3 103 50 28.0 102 49 28.7 104 50

11 28.3 50 29.0 103 50 28.9 102 49 29.3 104 49

12 28.8 50 29.7 103 50 29.6 103 49 30.0 104 49

13 29.6 50 30.7 104 50 30.6 103 49 31.2 105 49

17 33.3 50 35.0 105 50 34.4 103 49 35.5 107 49

21 36.2 50 37.7 104 50 36.9 102 49 38.3 106 49

25 39.0 50 40.6 104 50 40.7 104 49 41.0 105 49

29 43.0 50 44.3 103 50 43.9 102 49 44.7 104 49

33 46.3 50 47.5 103 49 47.7 103 49 47.9 104 49

37 48.8 50 49.5 101 48 49.5 101 49 50.5 104 49

41 50.8 50 51.2 101 48 51.7 102 49 52.6 104 49

45 53.9 50 54.4 101 48 54.0 100 49 54.9 102 49

49 56.0 49 55.9 100 48 55.3 99 49 56.3 101 49

53 57.3 49 56.1 98 48 56.5 99 48 57.1 100 49

57 57.4 49 56.7 99 48 56.2 98 48 57.5 100 49

61 58.3 49 58.3 100 48 57.9 99 48 58.7 101 48

65 58.6 49 58.8 100 47 59.3 101 46 60.2 103 48

69 60.2 48 59.5 99 47 60.7 101 46 60.3 100 48

73 60.5 48 60.2 100 47 60.9 101 46 61.3 101 48

77 61.3 47 60.9 99 46 61.3 100 46 61.4 100 47

81 62.7 47 59.9 96 46 60.2 96 46 60.9 97 47

85 62.1 47 58.8 95 46 60.7 98 43 60.8 98 46

89 61.8 44 59.7 97 43 60.2 97 43 60.2 97 43

93 60.3 41 58.1 96 43 58.9 98 43 58.4 97 42

97 58.2 37 54.8 94 40 56.8 98 43 55.2 95 41

101 57.1 37 56.8 100 35 55.8 98 41 54.1 95 37

Mean for weeks

1-13 24.8 25.3 102 25.3 102 25.8 104

14-52 45.3 46.2 102 46.0 102 46.9 104

53-101 59.7 58.4 98 58.9 99 58.9 99

••

::c

;;:;

••

::!;

::c

•••

•

45 60

WEEKS

STUDY

WEEKS

STUDY

MALE MICE

•

Om91ki

200~!1

630o¢g

a.ooo

m91\i

FEMALE MICE

• o ..

safki

0 100"'91kg

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300

Mglkg

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0 0

105

32 Triethanolamine, NTP TR 518

FIGURE 2

Growth Curves for Male and Female Mice Administered Triethanolamine Dermally for 2 Years

33 Triethanolamine, NTP TR 518

Pathology and Statistical Analyses

This section describes the statistically significant or bio-

logically noteworthy changes in the incidences of neo-

plasms and/or nonneoplastic lesions of the liver and

skin. Summaries of the incidences of neoplasms and

nonneoplastic lesions, individual animal tumor diag-

noses, statistical analyses of primary neoplasms that

occurred with an incidence of at least 5% in at least one

animal group, and historical incidences for the neo-

plasms mentioned in this section are presented in

Appendix A for male mice and Appendix B for female

mice.

Liver: Gross lesions observed at necropsy included nod-

ules and masses of the liver in dosed females. The inci-

dences of hepatocellular adenoma and hepatocellular

adenoma or carcinoma (combined) occurred with posi-

tive trends in females, and the incidences of these neo-

plasms in all dosed groups of females were significantly

increased. The incidence of hepatocellular adenoma in

300 mg/kg females was at the upper end of the historical

control range, and the incidences of hepatocelluar ade-

noma in 1,000 mg/kg females and of hepatocellular ade-

noma or carcinoma (combined) in all dosed groups of

females exceeded the historical ranges in controls (all

routes) given NTP-2000 diet (Tables 5, B3, and B4).

The incidences of multiple hepatocellular adenoma were

significantly increased in the 300 and 1,000 mg/kg

females. Historically, approximately 18% (31/170) of

female control mice that developed hepatocellular ade-

nomas had multiple adenomas. In the current study,

multiple adenomas did not occur in the vehicle controls;

multiple adenomas occurred in 3 (17%), 7 (35%), and 17

(52%) of the females in the 100, 300, and 1,000 mg/kg

groups, respectively, that developed hepatocellular ade-

nomas.

The incidences of hepatocellular neoplasms in males

were similar to those in the vehicle controls (Tables 5

and A3). The incidences of hepatoblastoma were slight-

ly increased in 630 and 2,000 mg/kg males, but the inci-

dences were within the historical control range and were

not considered treatment-related (Tables 5, A3, and

A4a). The incidence of hemangioma in 2,000 mg/kg

males was greater than that in the vehicle controls, and

the incidence of hemangiosarcoma in 630 mg/kg males

was significantly increased; the incidences of these

lesions in these groups exceeded the historical control

ranges. Two 630 mg/kg males had multiple heman-

giosarcomas of the liver.

Hepatocellular adenomas were nodular, expansile

lesions that occupied an area greater than one liver

lobule. They were well demarcated from surrounding

parenchyma by a zone of compression or lack of conti-

nuity between the hepatic cords within the nodule and

those of the surrounding parenchyma. There was loss of

normal lobular architecture, with a lack of portal triads

and haphazardly arranged hepatic cords, often with areas

of atypia. Neoplastic cells were generally large, with

abundant eosinophilic and variably vacuolated cyto-

plasm, increased nuclear to cytoplasmic ratio, and

nuclear atypia and with an increased mitotic index.

Hepatoblastomas are uncommon neoplasms in mice and

may occur spontaneously or be chemically induced.

Histologically, they have a characteristic appearance of

small, dark, ovoid- to spindle-shaped cells with round to

oval nuclei and scant amounts of eosinophilic cytoplasm

arranged in compact sheets, islands, or trabeculae.

Hepatoblastomas almost always occur within an existing

proliferative lesion, most often a hepatocellular carcino-

ma. In NTP studies, the diagnosis of hepatoblastoma is

made whenever this distinctive lesion is observed. To

avoid duplicate diagnoses, no separate diagnosis is made

for the lesion within which the hepatoblastoma occurs.

Hemangio-sarcomas are malignant neoplasms of the

vasculature, and those in the current study were charac-

terized by pleomorphic, proliferative endothelial cells

forming irregular vascular spaces, occasionally with

areas of thrombosis. In contrast, hemangiomas are

benign neoplasms, and those in the current study were

characterized by irregular vascular spaces lined by a sin-

gle layer of flattened, mature appearing endothelial cells.

Hemangiosarcoma also occurred in the spleen in two

males from each of the vehicle control, 200, and

630 mg/kg groups, and the aorta of one male in the

2,000 mg/kg group (Tables 6 and A1). In one male in

each of the vehicle control and dosed groups, heman-

giosarcoma metastasized from the spleen or aorta to the

liver, and hemangiosarcoma metastasized from the

spleen to the bone marrow in one vehicle control and one

630 mg/kg male. The incidence of hemangiosarcoma in

all organs was significantly increased in the 630 mg/kg

group and exceeded the historical control range; this

increase was attributed to the high incidence of heman-

giosarcoma in the liver of mice in this group (Tables 6,

A3, and A4b). Hemangiosarcoma is a malignant neo-

plasm of the vascular endothelium. Spontaneous

hemangiosarcomas occur in 3.0% of male B6C3F

mice

and 3.6% of females. While hemangiosarcomas may

occur at a variety of sites, the liver and spleen are the

34 Triethanolamine, NTP TR 518

ABLE 5

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Mice

in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 200 mg/kg 630 mg/kg 2,000 mg/kg

Male

Number Examined Microscopically

Eosinophilic Focus

20*

31**

30**

Hemangioma

0 1 0 2

Hemangiosarcoma, Multiple 0 0 2 0

Hemangiosarcoma (includes multiple)

Overall rate

Adjusted rate

Terminal rate

First incidence (days)

Poly-3 test

1/50 (2%)

2.1%

1/37 (3%)

726 (T)

P=0.587

0/50 (0%)

0.0%

0/43 (0%)

—

P=0.501N

6/50 (12%)

13.5%

3/34 (9%)

517

P=0.047

1/50 (2%)

2.2%

1/40 (3%)

726 (T)

P=0.755

Hepatocellular Adenoma 19 18 23 20

Hepatocellular Carcinoma 17 14 14 11

Hepatocellular Adenoma or Carcinoma 33 27 33 25

Hepatoblastoma

1 1 2 3

Vehicle Control 100 mg/kg 300 mg/kg 1,000 mg/kg

Female

Number Examined Microscopically 50 50 50 50

Eosinophilic Focus 16 22 28* 32**

Mixed Cell Focus 5 8 14* 11

Hepatocellular Adenoma, Multiple 0 3 7* 17**

Hepatocellular Adenoma (includes multiple)

Overall rate 9/50 (18%) 18/50 (36%) 20/50 (40%) 33/50 (66%)

Adjusted rate 19.9% 41.0% 43.5% 72.4%

Terminal rate 6/35 (17%) 16/34 (47%) 18/41 (44%) 25/32 (78%)

First incidence (days) 617 665 444 604

Poly-3 test P<0.001 P=0.024 P=0.012 P<0.001

Hepatocellular Carcinoma 6 8 4 5

Hepatocellular Adenoma or Carcinoma

Overall rate 12/50 (24%) 23/50 (46%) 24/50 (48%) 34/50 (68%)

Adjusted rate 26.3% 51.0% 51.7% 74.6%

Terminal rate 7/35 (20%) 17/34 (50%) 21/41 (51%) 26/32 (81%)

First incidence (days) 595 601 444 604

Poly-3 test P<0.001 P=0.011 P=0.009 P<0.001

* Significantly different (P#0.05) from the vehicle control group by the Poly-3 test

** P#0.01

(T)Terminal sacrifice

Number of animals with lesion

Historical incidence for 2-year studies with controls given NTP-2000 diet (mean ± standard deviation): 2/1,159 (0.2% ± 0.6%),

range 0%-2%

Historical incidence: 28/1,159 (2.5% ± 1.4%), range 0%-4%

Number of animals with neoplasm per number of animals with liver examined microscopically

Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality

Observed incidence at terminal kill

Beneath the vehicle control incidence is the P value associated with the trend test. Beneath the dosed group incidence are the P values

corresponding to pairwise comparisons between the vehicle controls and that dosed group. The Poly-3 test accounts for differential

mortality in animals that do not reach terminal sacrifice. A lower incidence in a dosed group is indicated by N.

Not applicable; no neoplasms in animal group

Historical incidence: 16/1,159 (1.5% ± 2.6%), range 0%-10%

Historical incidence: 179/1,152 (16.3% ± 6.6%), range 6%-28%

Historical incidence: 250/1,152 (22.8% ± 9.4%), range 8%-40%

Triethanolamine, NTP TR 518

ABLE 6

Incidences of Hemangioma and Hemangiosarcoma in Male Mice in the 2-Year Dermal Study

of Triethanolamine

Vehicle Control 200 mg/kg 630 mg/kg 2,000 mg/kg

Number Necropsied 50 50 50 50

Hemangioma, Liver

0 1 0 2

Hemangiosarcoma, Liver 1 0 6* 1

Hemangiosarcoma, Liver, Metastatic, Aorta 0 0 0 1

Hemangiosarcoma, Liver, Metastatic, Spleen 1 1 1 0

Hemangiosarcoma, Aorta 0 0 0 1

Hemangiosarcoma, Bone Marrow, Metastatic, Spleen 1 0 1 0

Hemangioma, Spleen 1 0 0 0

Hemangiosarcoma, Spleen 2 2 2 0

Hemangiosarcoma (All Organs)

Overall rate

Adjusted rate

3/50 (6%)

6.3%

2/50 (4%)

4.3%

9/50 (18%)

20.1%

2/50 (4%)

4.3%

Terminal rate

1/37 (3%) 2/43 (5%) 5/34 (15%) 1/40 (3%)

First incidence (days)

Poly-3 test

624

P=0.442N

726 (T)

P=0.508N

517

P=0.046

618

P=0.513N

Hemangioma or Hemangiosarcoma (All Organs)

Overall rate 4/50 (8%) 3/50 (6%) 9/50 (18%) 4/50 (8%)

Adjusted rate 8.4% 6.4% 20.1% 8.6%

Terminal rate 2/37 (5%) 3/43 (7%) 5/34 (15%) 3/40 (8%)

First incidence (days) 624 726 (T) 517 618

Poly-3 test P=0.550 P=0.509N P=0.092 P=0.628

* Significantly different (P#0.05) from the vehicle control group by the Poly-3 test

(T)Terminal sacrifice

Number of animals with lesion

Historical incidence for 2-year studies with controls given NTP-2000 diet (mean ± standard deviation): 59/1,159 (5.3% ± 3.4%),

range 0%-14%

Number of animals with neoplasm per number of animals necropsied

Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality

Observed incidence at terminal kill

Beneath the vehicle control incidence is the P value associated with the trend test. Beneath the dosed group incidence are the P values

corresponding to pairwise comparisons between the vehicle controls and that dosed group. The Poly-3 test accounts for differential

mortality in animals that do not reach terminal sacrifice. A lower incidence in a dosed group is indicated by N.

Historical incidence: 73/1,159 (6.4% ± 3.3%), range 2%-14%

36 Triethanolamine, NTP TR 518

most common sites in male B6C3F

mice, and the spleen

and subcutis are the most common sites in females

(Chandra, S.A., Hardisty, J.F., Seely, J.C., Haseman,

J.K., and Maronpot, R.R., unpublished). In 20 chemical

studies for which there was a chemical-related increased

incidence in vascular neoplasms, the increased inci-

dences occurred most commonly at a specific site, and

less commonly at two or more specific sites. In general,

the vasculature as a whole is not affected; rather the vas-

culature within a specific organ/tissue is affected. The

most common site of chemically induced vascular neo-

plasms in NTP studies is the liver.

In the current study, the incidences at individual sites and

all sites combined are presented in the results. Only the

incidence in the liver stands out as being statistically sig-

nificant and/or outside of historical control ranges, and

only in the 630 mg/kg group. The incidences of heman-

gioma of the liver were low in each group and not sig-

nificantly different between groups, although they did

exceed historical control ranges. When interpreting

potential treatment-related incidences of many neoplasm

types, the combined incidence of the benign and malig-

nant forms is an important consideration. However,

unlike the liver and kidney, for example, where there is

evidence of a morphological and biological continuum

between benign and malignant neoplasms, the link

between hemangioma and hemangiosarcoma is not as

strong. Also, the majority of NTP studies with chemi-

cal-related increases in the incidences of vasculature

neoplasms have involved hemangiosarcomas without an

increase in hemangiomas. However, there are

exceptions.

The incidences of eosinophilic focus in all dosed groups

of males and in 300 and 1,000 mg/kg females were sig-

nificantly increased (Tables 5, A5, and B5). The inci-

dences of mixed cell focus in dosed groups of females

were greater than that in the vehicle controls, and the

incidence in the 300 mg/kg group was significantly

increased (Tables 5 and B5). Foci of cellular alteration

were sharply demarcated clusters of cells with altered

cytoplasmic tinctoral properties. Eosinophilic and

mixed foci were variably sized and ranged from approx-

imately one hepatic lobule to several hepatic lobules,

generally causing little compression of the adjacent

parenchyma. There was little or no alteration of normal

hepatic architecture within the focus, and cellular atypia

was generally absent. Component cells of the eosin-

ophilic foci were large with abundant eosinophilic cyto-

plasm. In the mixed foci, the eosinophilic cells were

admixed with a second population of cells with promi-

nent fine to coarse cytoplasmic vacuolation.

Skin: Gross lesions observed at necropsy included visi-

ble crusts at the site of application in all dosed groups of

mice, and more lesions occurred in 630 and 2,000 mg/kg

males. Treatment-related epidermal hyperplasia, suppu-

rative inflammation, and ulceration and dermal chronic

inflammation occurred at the site of application in most

dosed groups of mice, and the incidences and severities

of these lesions generally increased with increasing dose

(Tables 7, A5, and B5). Epidermal hyperplasia varied

from minimal to moderate in males and minimal to mild

in females. Moderate hyperplasia in males was charac-

terized by rete peg formation and an epidermal thickness

approximately six times the normal thickness.

Epidermal suppurative inflammation generally occurred

in conjunction with the epidermal hyperplasia and con-

sisted of heavy, focal aggregations of viable and degen-

erate neutrophils, predominantly within or superficial to

the stratum corneum (superficial intracorneal pustule

formation). Aggregates superficial to the stratum

corneum also occurred and were admixed with keratin

and proteinaceous material (serocellular crusts). Ulcers

were less common and were characterized by complete

necrosis of the epidermis, variable subjacent dermal ero-

sion and associated chronic inflammatory cell infiltra-

tion and fibroplasia. Ulcers were covered with serocel-

lular crusts comprising cellular debris, keratin, fibrin,

and inflammatory cells. Chronic inflammation of the

dermis occurred in association with areas of epidermal

hyperplasia and/or ulceration. It consisted of focal and

more diffuse, loose aggregates of mixed inflammatory

cells (neutrophils, eosinophils, mast cells, lymphoctes

and plasma cells), often admixed with areas of active

fibroblasts aligned parallel to the surface of the skin.

ABSORPTION

ISTRIBUTION, METABOLISM,

AND EXCRETION STUDIES

Data for the disposition of a 3 mg/kg intravenous dose of

[

C]-triethanolamine female rats are presented in

Table G1. The radioactivity was rapidly excreted in the

urine, and 90% of the dosed radioactivity was recovered

in the urine within 24 hours. An average of 98% of the

dose was recovered in the urine within 72 hours after

dosing, and approximately 0.6% of the radioactivity was

recovered in the feces during this time. Less than 0.5%

of the dose was recovered in carbon dioxide traps, and

less than 0.1% was recovered in volatiles traps.

37 Triethanolamine, NTP TR 518

TABLE

Incidences of Nonneoplastic Lesions of the Skin (Site of Application) in Mice

in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 200 mg/kg 630 mg/kg 2,000 mg/kg

Male

Number Examined Microscopically

Epidermis, Hyperplasia

(1.2)

44** (1.5)

45** (2.0)

49** (2.7)

Epidermis, Inflammation, Suppurative 1 (1.0) 11** (1.5) 33** (1.7) 42** (2.8)

Epidermis, Ulcer 0 3 (1.0) 20** (1.4) 47** (2.6)

Dermis, Inflammation, Chronic 1 (1.0) 15** (1.1) 40** (1.6) 49** (2.5)

Vehicle Control 100 mg/kg 300 mg/kg 1,000 mg/kg

Female

Number Examined Microscopically 50 50 50 50

Epidermis, Hyperplasia 14 (1.3) 50** (1.9) 46** (2.0) 50** (2.0)

Epidermis, Inflammation, Suppurative 1 (2.0) 2 (2.0) 20** (1.5) 32** (2.2)

Epidermis, Ulcer 1 (3.0) 1 (3.0) 6 (1.5) 17** (1.6)

Dermis, Inflammation, Chronic 4 (1.8) 27** (1.1) 31** (1.6) 44** (1.8)

** Significantly different (P#0.01) from the vehicle control group by the Poly-3 test

Number of animals with lesion

Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked

38 Triethanolamine, NTP TR 518

The distribution of radioactivity from female rats

following intravenous administration of [

C]-tri-

ethanolamine is presented in Table G2. Only 0.9% of

the dose remained in the tissues 72 hours after dosing.

In contrast to diethanolamine, which was only slowly

excreted (30% of dose within 48 hours) and accumulat-

ed in the brain, heart, kidney, spleen (5% of dose at

48 hours), and liver (27% of dose at 48 hours) in a

process thought to involve biochemical mimicry with the

natural alkanolamine ethanolamine (Mathews et al.,

1995), comparatively little triethanol-amine bioaccumu-

lated in the tissues.

Data for the excretion of radioactivity following dermal

administration of 68 and 276 mg/kg [

C]-triethanol-

amine in female rats is presented in Table G3, and the

distribution of radioactivity present in tissues 72 hours

after dosing is presented in Table G4. Approximately

20% to 30% of the dose was absorbed within 72 hours

following dermal exposure. The mean percentage of

dose absorbed increased with increasing dose, but the

increase was not statistically significant (Table G5).

The disposition of a 3 mg/kg intravenous dose of

[

C]-triethanolamine in female mice is presented in

Table G6. With only 40% of the dose excreted within

24 hours, mice appeared to excrete intravenously admin-

istered triethanolamine more slowly than did rats.

Considerably more of the dose was recovered in the

feces of mice (28%) than in rats (0.6%). However, it is

common for mice to shred and powder their food pellets,

and the feces collections are often contaminated with

urine-soaked solids. Less than 0.5% of the dose was

recovered in carbon dioxide traps, and less than 0.1%

was recovered in volatiles traps.

The distribution of radioactivity in tissue samples from

female mice is presented in Table G7. As was the case

for rats, the heart, kidney, liver, lung, and spleen con-

tained higher concentrations of triethanolamine equiva-

lents relative to blood.

Mice absorbed dermally applied triethanolamine more

readily than did rats (Tables G8 and G9) and excreted the

absorbed radioactivity in the urine and feces (which

were probably contaminated with the urine as noted

above), with 28% to 48% of the dose recovered in ex-

creta within 24 hours. The percentage of the dose

absorbed increased with increasing dose.

Urine collected 6 to 24 hours after intravenous dosing

and 48 to 72 hours after dermal application of tri-

ethanolamine in female rats was analyzed by HPLC

(Figure G1). The chromatogram contains a peak that

coelutes with triethanolamine and two other peaks that

comprise about 5% of the radioactivity in the sample.

These peaks, however, were also present in the chro-

matogram of the radiolabeled test article and may reflect

the presence of impurities rather than metabolites. The

metabolite fractions were collected and incubated with

purified $-glucuronidase, as was an aliquot of the whole

urine. Analysis of these samples showed no change in

the metabolite profile. Similarly, urine collected 6 to

24 hours after intravenous or dermal dosing contained

more than 95% radiolabeled components that coeluted

with unchanged triethanolamine, with minor compo-

nents eluting the same fractions in mice as those in rats.

GENETIC TOXICOLOGY

Triethanolamine (33 to 3,333 µg/plate) was negative for

induction of mutations in Salmonella typhimurium

strains TA98, TA100, TA1535, and TA1537 when tested

with or without S9 metabolic activation (Table C1;

Mortelmans et al., 1986). In cytogenetic tests with cul-

tured Chinese hamster ovary cells, no induction of sister

chromatid exchanges (SCEs) (Table C2) or chromoso-

mal aberrations (Abs) (Table C3) was observed with or

without S9 (Galloway et al., 1987). In the SCE test

without S9, the first of two trials was negative. In the

second trial, a significant increase in SCEs was observed

at the highest dose tested (2,520 µg/mL), but the trend

test was negative (P$0.025), and the trial was concluded

to be equivocal. Severe cytotoxicity limited the number

of cells that could be scored at this dose. Overall, the

SCE test was considered to be negative. Cytotoxicity

was also noted at the highest dose tested (4,030 µg/mL)

in the Abs test without S9.

Triethanolamine administered by feeding or injection at

doses up to 30,000 ppm did not induce sex-linked reces-

sive lethal mutations in germ cells of male Drosophila

melanogaster (Table C4; Yoon et al., 1985). Results of

an in vivo peripheral blood micronucleus test in mice

were also negative (Table C5; Witt et al., 2000). In this

test, blood samples were obtained from male and female

mice after 13 weeks of dermal applications of 1,000 to

4,000 mg/kg triethanolamine. No significant increases

in the frequencies of micronucleated normochromatic

erythrocytes were observed at any dose level, and the

percentages of polychromatic erythrocytes in the dosed

groups were similar to those in the vehicle control

groups, indicating an absence of bone marrow toxicity.

DISCUSSION AND CONCLUSIONS

The National Cancer Institute nominated tri-

ethanolamine for evaluation of its safety and potential

carcinogenicity due to its widespread use in cosmetics

and other consumer products. In 1999, the National

Toxicology Program completed and published the results

of 3-month and 2-year studies in F344/N rats and

B6C3F

mice administered topical applications of tri-

ethanolamine (NTP, 1999). The 2-year F344/N rat study

indicated equivocal evidence of carcinogenic activity in

males based on a marginal increase in the incidence of

renal tubule cell adenoma. No evidence of carcinogenic

activity was found in female rats. Treated male and

female B6C3F

mice developed liver neoplasms; how-

ever, male mice had a pattern of nonneoplastic liver

lesions (hepatitis) along with microscopic silver-staining

helical organisms within the liver, which suggested an

infection with Helicobacter hepaticus. Polymerase

chain reaction (PCR) based assays and culture confirmed

infection in male and female mice even though females

had no evidence of H. hepaticus-associated hepatitis.

Increases in the incidences of hepatocellular neoplasms

in male mice have been shown to be associated with H.

hepaticus infection when hepatitis is also present (Ward

et al., 1994; Fox et al., 1996; Hailey et al., 1998).

Therefore, the NTP proposed that the male mouse study

be considered inadequate. Because there was no evi-

dence that females had H. hepaticus-associated hepatitis,

the female mouse study was interpreted as showing

some evidence of carcinogenic activity based on an

increase in hepatocellular neoplasms. However, the

NTP Board of Scientific Counselors Technical Reports

Review Subcommittee considered the entire mouse

study to be inadequate because of uncertainty about the

potential for H. hepaticus infection to influence the liver

tumor response to triethanolamine.

Due to the uncertainty about the potential of H. hepati-

cus to influence the incidence of liver neoplasms in

B6C3F

mice, the NTP decided to repeat the 2-year

study. With the exception of the diet, the design of the

preceding study including route of administration, dose

selection, and treatment duration was preserved in the

present study with the purpose of reproducing as closely

as possible the exposure conditions used previously.

The NTP-2000 diet was used in the current study, replac-

ing the NIH-07 diet used in the previous study. This new

diet has been used in all NTP studies since 1994, and

composition and performance data have been published

(Rao, et al., 1997).

Following detection of the H. hepaticus infection in

12 NTP studies (Hailey et al., 1998), the breeding

colony was rederived and was determined to be free of

infection. Also, in order to assure that animals of the

repeat triethanolamine study were not infected with

H. hepaticus, sentinel animals, as well as four male and

five female mice from the study groups were analyzed

for H. hepaticus infection at 18 months. PCR of fecal

pellets, serology, and bacteriological cultures found no

evidence of H. hepaticus in any of the animals evaluated

(Appendix F).

In general, results reported here are consistent with those

reported in NTP Technical Report 449 on triethanol-

amine (NTP, 1999). Treatment did not affect survival of

animals of either sex after 104 weeks of exposure.

Similar to the previous study, treatment-related effects

on body weights of dosed mice were observed only in

males in the highest dosed group by the end of the study.

However, body weights of the animals used in this study

were higher than those of the mice in the previous study

(approximately 20% in females and 5% in males).

Consistent with the first study (NTP, 1999), female mice

in the current study again showed an increase in the inci-

dences of hepatocellular neoplasms. In the current

study, increased incidences of hepatocellular adenomas

and multiple hepatocelluar adenomas were observed in

all dosed groups of females (in contrast to the first study

where this was a high-dose effect only), and the inci-

dences in the dosed groups exceeded the upper historical

control incidence. The primary difference in the liver

neoplasm incidences between the two triethanolamine

studies was in the control groups, with the first study

showing a control rate greater than twice the control

incidence seen in the second study. This may be associ-

ated with the change from the use of the NIH-07 diet to

the NTP-2000 diet in the intervening period. In that

40 Triethanolamine, NTP TR 518

respect, Rao and Crockett (2003) demonstrated that

female B6C3F

mice fed the NTP-2000 diet have

markedly (P<0.01) reduced liver neoplasm rates com-

pared to female mice fed the NIH-07 diet (14.3% versus

34.2% for dosed feed studies). This was in part attrib-

uted to a reduction in average body weight of mice fed

the NTP-2000 diet. However, a reduction in liver neo-

plasms in control mice in inhalation studies was also

noted using the NTP-2000 diet, and this occurred with-

out a concurrent reduction in body weight.

Application of a prediction model derived by Haseman

et al. (1997) indicates that the control liver tumor inci-

dence of 46% in the first study (NTP, 1999) was very

similar to what would be expected for individually

housed control B6C3F

mice of equivalent age and body

weight fed the NIH-07 diet (42%). Using this same

approach and the historical control data for the 21 NTP

studies that have used the NTP-2000 diet, a regression

analysis was carried out to determine the best fitting

model relating liver tumor occurrence to 2-year survival

and 1-year body weight. This model was then applied to

the data in the current study to determine if the observed

control tumor rates were consistent with this model. The

observed and predicted liver tumor rates were similar for

both liver adenoma (19% predicted, 18% observed) and

liver adenoma or carcinoma (combined) (27% predicted,

24% observed). Thus, the control liver tumor incidences

in this study were essentially those expected in control

animals of this size and longevity. Based on this, it

appears likely that diet was the primary factor responsi-

ble for the difference in the liver tumor rates between the

control groups of the two studies.

A significant increase in the incidences of multiple hepa-

tocellular adenomas were also observed in treated

female mice with adenomas in the current study (35%

and 52% in the 300 and 1,000 mg/kg groups, respective-

ly). Historically, approximately 18% of female control

mice fed the NTP-2000 diet that developed hepatocellu-

lar adenomas had multiple adenomas. None of the nine

adenomas observed in the control group were multiple,

and the proportion of hepatocellular adenomas that were

multiple showed a clear dose-response trend.

Treated animals of both sexes also developed significant

increases in eosinophilic foci in the liver in all dosed

groups. Compared to the previous study, the overall

incidence of this lesion in females was about two times

the incidence reported in the 1999 study. However,

taken together, incidences of hepatocellular adenomas

and eosinophilic foci result in a rate of proliferative he-

patic lesions that is reasonably similar in the two studies.

Male mice receiving 630 mg/kg triethanolamine devel-

oped an increased incidence of hemangiosarcoma that

exceeded the historical control range. Similar to other

chemical-related vascular tumors observed in previous

NTP studies, the liver was the organ with the highest

incidence of hemangiosarcoma. However, there was no

increase in the next dose group (2,000 mg/kg), or in any

of the dosed groups of females, nor was there an increase

in the incidence of these tumors in the previous study.

Therefore, the association between triethanolamine and

hemangiosarcoma of the liver was considered an uncer-

tain finding.

A major difference between the results of previous and

current NTP triethanolamine studies was the unexpected

disparity in skin response to the application of tri-

ethanolamine. Because of this discrepancy, records from

both studies were evaluated to determine if any differ-

ences in the chemical (dose formulation or preparation),

administration of the chemical, animal preparation (clip-

ping, etc.) and/or handling of the animals could explain

the different response. The formalin-fixed skin from the

site of application of several animals from the original

study was reevaluated to determine if lesions had been

missed. None of these evaluations revealed any infor-

mation that could explain the differences in the local

effect. Although lesions were qualitatively similar,

males were more severely affected in the current study.

Based on the microscopic diagnoses, the changes in the

skin at the site of application appear fairly severe, par-

ticularly in males. However, the gross evaluation indi-

cated that the ulcerative lesions were small, focal to mul-

tifocal, affecting a relatively small portion of the skin at

the site of application. In this study, as in all skin paint

studies, a routine section of skin was taken from a spe-

cific area of the site of application. Additional sections

were taken of grossly observed lesions, which in this

study generally consisted of small focal crusty areas.

Microscopically, the ulcers were most commonly identi-

fied within the crusty areas from the additional sections,

and not from the routine section. Also, in general, the

most severe suppurative inflammation and hyperplasia

were associated with existing and/or healing ulcers,

particularly in females. The lesions in the skin were not

considered sufficiently severe to require early termina-

tion of affected animals.

__________

41 Triethanolamine, NTP TR 518

Males appeared to be more sensitive to the local effects

of triethanolamine, although the major treatment-related

response outside the skin at the site of application was

the increased incidence of hepatocellular adenomas seen

in females. However, in females, both incidence and

severity of the skin lesions, and the incidence of hepato-

cellular adenomas appeared to increase with increasing

dose. Therefore, there was a concern that enhanced

absorption across lesioned skin at the site of application

might have played a role in some female mice,

predisposing those animals to develop hepatocellular

adenomas.

In order to address this concern, logistic regression pro-

cedures were used to determine the relative importance

of these two factors (incidence and severity of skin

lesions) in the prediction of liver tumor occurrence. The

severities of the following skin lesions were considered:

epidermal hyperplasia, suppurative inflammation, and

ulceration and dermal chronic inflammation. The

summed severities of these four lesions were also evalu-

ated. Logistic regression analysis revealed that while the

occurrence of chronic dermal inflammation was signifi-

cantly (P<0.05) correlated with the occurrence of liver

neoplasms, the increasing trend in liver neoplasm inci-

dence due to triethanolamine remained significant

(P<0.05) even after adjusting for the possible influence

of dermal chronic inflammation. Thus, the increased

incidences of liver neoplasms observed in female mice

receiving triethanolamine cannot be attributed solely to

the concurrently observed increase in nonneoplastic skin

lesions at the site of application.

Finally, increased incidences of malignant lymphoma

reported previously in female ICL-JCR mice treated

with 0.03% or 0.3% of triethanolamine in the diet

(Hoshino and Tanooka, 1978) raised concerns regarding

the carcinogenic potential of triethanolamine. However,

a treatment-related increase in the incidence of malig-

nant lymphoma in B6C3F

mice was not identified in the

current or the previous NTP (1999) study.

Results from the current study demonstrated that chron-

ic administration of triethanolamine does not significant-

ly affect the hepatic carcinoma rates in B6C3F

mice.

Although hepatocellular adenoma is a common benign

tumor that occurs with variable incidence in female

B6C3F

mice, the dose-related increases in the inci-

dences of hepatocellular adenomas and multiple hepato-

cellular adenomas described in this report constitute

some evidence of carcinogenic activity of triethanol-

amine in female B6C3F

mice. Potential mechanisms

accounting for this fact will require further study.

CONCLUSIONS

Under the conditions of this 2-year dermal study, there

was equivocal evidence of carcinogenic activity* of

triethanolamine in male B6C3F

mice based on the

occurrence of liver hemangiosarcoma. There was some

evidence of carcinogenic activity in female B6C3F

mice

based on increased incidences of hepatocellular

adenoma.

Exposure to triethanolamine by dermal application

resulted in increased incidences of eosinophilic focus of

the liver in males and females. Dosed mice developed

treatment-related nonneoplastic lesions at the site of

application.

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 8. A summary of the Technical Reports Review Subcommittee

comments and public discussion on this Technical Report appears on page 10.

42 Triethanolamine, NTP TR 518

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APPENDIX A

SUMMARY OF LESIONS IN MALE MICE

IN THE 2-YEAR DERMAL STUDY

OF TRIETHANOLAMINE

TABLE A1

ABLE A2

ABLE A3

ABLE A4a

ABLE A4b

ABLE A5

Summary of the Incidence of Neoplasms in Male Mice

in the 2-Year Dermal Study of Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Individual Animal Tumor Pathology of Male Mice

in the 2-Year Dermal Study of Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Statistical Analysis of Primary Neoplasms in Male Mice

in the 2-Year Dermal Study of Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Historical Incidence of Liver Neoplasms in Control Male B6C3F

Mice . . . . . . . . . . . . . . .

Historical Incidence of Hemangioma or Hemangiosarcoma (All Organs)

in Control Male B6C3F

Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Summary of the Incidence of Nonneoplastic Lesions in Male Mice

in the 2-Year Dermal Study of Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

50 Triethanolamine, NTP TR 518

TABLE A1

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 200 mg/kg 630 mg/kg 2,000 mg/kg

Disposition Summary

Animals initially in study 50 50 50 50

Early deaths

Moribund 5 2 6 8

Natural deaths 8 5 10 2

Survivors

Terminal sacrifice 37 43 34 40

Animals examined microscopically 50 50 50 50

Alimentary System

Intestine small, jejunum (50) (50) (50) (50)

Adenoma 1 (2%)

Carcinoma 2 (4%) 1 (2%) 1 (2%)

Carcinoma, multiple 1 (2%)

Sarcoma, metastatic, adrenal medulla 1 (2%)

Liver (50) (50) (50) (50)

Hemangioma 1 (2%) 2 (4%)

Hemangiosarcoma 1 (2%) 4 (8%) 1 (2%)

Hemangiosarcoma, multiple 2 (4%)

Hemangiosarcoma, metastatic, blood vessel 1 (2%)

Hemangiosarcoma, metastatic, spleen 1 (2%) 1 (2%) 1 (2%)

Hepatoblastoma 1 (2%) 1 (2%) 2 (4%) 3 (6%)

Hepatocellular carcinoma 11 (22%) 10 (20%) 13 (26%) 10 (20%)

Hepatocellular carcinoma, multiple 6 (12%) 4 (8%) 1 (2%) 1 (2%)

Hepatocellular adenoma 13 (26%) 14 (28%) 12 (24%) 15 (30%)

Hepatocellular adenoma, multiple 6 (12%) 4 (8%) 11 (22%) 5 (10%)

Hepatocholangiocarcinoma 1 (2%)

Histiocytic sarcoma 1 (2%) 2 (4%)

Sarcoma, metastatic, adrenal medulla 1 (2%)

Mesentery (3) (3) (5) (7)

Fibrous histiocytoma 1 (14%)

Stomach, forestomach (50) (50) (50) (50)

Squamous cell papilloma 1 (2%) 2 (4%) 1 (2%)

Tongue (1)

Squamous cell carcinoma 1 (100%)

Tooth (19) (18) (14) (13)

Odontoma 1 (5%)

Cardiovascular System

Blood vessel (50) (50) (50) (50)

Aorta, hemangiosarcoma 1 (2%)

Heart (50) (50) (50) (50)

Carcinoma, metastatic, kidney 1 (2%)

Fibrous histiocytoma, metastatic, mesentery 1 (2%)

Histiocytic sarcoma 1 (2%)

Triethanolamine, NTP TR 518

TABLE A1

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 200 mg/kg 630 mg/kg 2,000 mg/kg

Endocrine System

Adrenal cortex (50) (50) (50) (50)

Alveolar/bronchiolar carcinoma, metastatic, lung 1 (2%)

Subcapsular, adenoma 3 (6%) 3 (6%) 6 (12%) 5 (10%)

Adrenal medulla (50) (50) (50) (50)

Pheochromocytoma benign 1 (2%) 1 (2%)

Sarcoma 1 (2%)

Pituitary gland (50) (49) (49) (50)

Pars distalis, adenoma 1 (2%)

Pars intermedia, adenoma 1 (2%) 1 (2%)

Thyroid gland (50) (50) (49) (50)

Follicle, adenoma 1 (2%) 1 (2%)

General Body System

None

Genital System

Epididymis (50) (50) (50) (50)

Fibrous histiocytoma, metastatic, mesentery 1 (2%)

Testes (50) (50) (50) (50)

Interstitial cell, adenoma 1 (2%)

Hematopoietic System

Bone marrow (50) (50) (50) (50)

Hemangiosarcoma, metastatic, spleen 1 (2%) 1 (2%)

Histiocytic sarcoma 1 (2%)

Lymph node (1) (1) (1)

Mediastinal, alveolar/bronchiolar carcinoma,

metastatic, lung 1 (100%)

Mediastinal, fibrous histiocytoma, metastatic, mesentery 1 (100%)

Mediastinal, hepatocellular carcinoma, metastatic, liver 1 (100%)

Pancreatic, fibrous histiocytoma, metastatic, mesentery 1 (100%)

Renal, fibrous histiocytoma 1 (100%)

Lymph node, mesenteric (50) (48) (48) (50)

Histiocytic sarcoma 1 (2%)

Spleen (49) (50) (50) (50)

Hemangioma 1 (2%)

Hemangiosarcoma 2 (4%) 2 (4%) 2 (4%)

Histiocytic sarcoma 1 (2%) 1 (2%)

Thymus (48) (47) (48) (48)

Alveolar/bronchiolar carcinoma, metastatic, lung 1 (2%)

Carcinoma, metastatic, kidney 1 (2%)

Fibrous histiocytoma, metastatic, mesentery 1 (2%)

52 Triethanolamine, NTP TR 518

ABLE A1

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 200 mg/kg 630 mg/kg 2,000 mg/kg

Integumentary System

Skin (50) (50) (50) (50)

Squamous cell carcinoma 1 (2%)

Subcutaneous tissue, hibernoma 1 (2%)

Subcutaneous tissue, melanoma malignant 1 (2%)

Subcutaneous tissue, skin, site of application,

hemangiosarcoma 1 (2%)

Subcutaneous tissue, site of application, dermis,

mast cell tumor benign 1 (2%)

Musculoskeletal System

Bone (50) (50) (50) (50)

Osteosarcoma 1 (2%)

Nervous System

None

Respiratory System

Lung (50) (50) (50) (50)

Alveolar/bronchiolar adenoma 6 (12%) 6 (12%) 11 (22%) 7 (14%)

Alveolar/bronchiolar adenoma, multiple 1 (2%) 1 (2%) 2 (4%)

Alveolar/bronchiolar carcinoma 6 (12%) 6 (12%) 8 (16%) 3 (6%)

Alveolar/bronchiolar carcinoma, multiple 1 (2%) 1 (2%)

Carcinoma, metastatic, harderian gland 1 (2%)

Carcinoma, metastatic, kidney 1 (2%)

Fibrous histiocytoma, metastatic, mesentery 1 (2%)

Hepatoblastoma, metastatic, liver 1 (2%) 1 (2%)

Hepatocellular carcinoma, metastatic, liver 8 (16%) 6 (12%) 6 (12%) 2 (4%)

Hepatocholangiocarcinoma, metastatic, liver 1 (2%)

Histiocytic sarcoma 1 (2%) 2 (4%)

Special Senses System

Harderian gland (50) (50) (49) (50)

Adenoma 6 (12%) 2 (4%) 2 (4%) 5 (10%)

Carcinoma 1 (2%) 1 (2%)

Bilateral, adenoma 1 (2%) 1 (2%)

Urinary System

Kidney (50) (50) (50) (50)

Histiocytic sarcoma 1 (2%) 1 (2%)

Renal tubule, adenoma 1 (2%) 1 (2%) 1 (2%)

Renal tubule, carcinoma, multiple 1 (2%)

Triethanolamine, NTP TR 518

ABLE A1

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 200 mg/kg 630 mg/kg 2,000 mg/kg

Systemic Lesions

Multiple organs

Histiocytic sarcoma

Lymphoma malignant

(50) (50)

1 (2%)

(50)

1 (2%)

(50)

(4%)

(2%)

Neoplasm Summary

Total animals with primary neoplasms

Total primary neoplasms

Total animals with benign neoplasms

Total benign neoplasms

Total animals with malignant neoplasms

Total malignant neoplasms

Total animals with metastatic neoplasms

Total metastatic neoplasms

Number of animals examined microscopically at the site and the number of animals with neoplasm

Number of animals with any tissue examined microscopically

Primary neoplasms: all neoplasms except metastatic neoplasms

54 Triethanolamine, NTP TR 518

TABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: Vehicle Control

5566666667777777777777777

Number of Days on Study 1822688891122222222222222

1048444844655666666666666

0000000000000000000000000

Carcass ID Number 2053022130124011111233444

3604329109017725689815026

Alimentary System

Esophagus +++++++++++++++++++++++++

Gallbladder + M + + + M +++++++++++++++++++

Intestine large, colon +++++++++++++++++++++++++

Intestine large, rectum +++++++++++++++++++++++++

Intestine large, cecum +++++++++++++++++++++++++

Intestine small, duodenum +++++++++++++++++++++++++

Intestine small, jejunum +++++++++++++++++++++++++

Carcinoma

Carcinoma, multiple

Intestine small, ileum +++++++++++++++++++++++++

Liver +++++++++++++++++++++++++

Hemangiosarcoma

Hemangiosarcoma, metastatic, spleen X

Hepatoblastoma

Hepatocellular carcinoma X X X X X X X X X

Hepatocellular carcinoma, multiple X X X X X

Hepatocellular adenoma XXX

Hepatocellular adenoma, multiple X X X

Mesentery + + +

Oral mucosa + + + + + + + + + + + + + + +

Pancreas +++++++++++++++++++++++++

Salivary glands +++++++++++++++++++++++++

Stomach, forestomach +++++++++++++++++++++++++

Squamous cell papilloma

Stomach, glandular +++++++++++++++++++++++++

Tooth + + +++++ ++

Odontoma X

Cardiovascular System

Blood vessel +++++++++++++++++++++++++

Heart +++++++++++++++++++++++++

Endocrine System

Adrenal cortex +++++++++++++++++++++++++

Subcapsular, adenoma X

Adrenal medulla +++++++++++++++++++++++++

Pheochromocytoma benign

Islets, pancreatic +++++++++++++++++++++++++

Parathyroid gland +++++MM ++++++++MM +++++++M

Pituitary gland +++++++++++++++++++++++++

Pars intermedia, adenoma

Thyroid gland +++++++++++++++++++++++++

Follicle, adenoma X

+: Tissue examined microscopically M: Missing tissue X: Lesion present

A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined

Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: Vehicle Control

7777777777777777777777777

Number of Days on Study 2222222222222222222222222

7777777777777778888888888

0000000000000000000000000 Total

Carcass ID Number 0000122223333440112334444 Tissues/

1458405672368452374791389 Tumors

Alimentary System

Esophagus +++++++++++++++++++++++++ 50

Gallbladder +++++++++++++++++++++++++ 48

Intestine large, colon +++++++++++++++++++++++++ 50

Intestine large, rectum +++++++++++++++++++++++++ 50

Intestine large, cecum +++++++++++++++++++++++++ 50

Intestine small, duodenum +++++++++++++++++++++++++ 50

Intestine small, jejunum +++++++++++++++++++++++++ 50

Carcinoma X X 2

Carcinoma, multiple X 1

Intestine small, ileum +++++++++++++++++++++++++ 50

Liver +++++++++++++++++++++++++ 50

Hemangiosarcoma X 1

Hemangiosarcoma, metastatic, spleen 1

Hepatoblastoma X 1

Hepatocellular carcinoma X X 11

Hepatocellular carcinoma, multiple X 6

Hepatocellular adenoma X X X X X X X X X X 13

Hepatocellular adenoma, multiple X X X 6

Mesentery 3

Oral mucosa + +++++ +++ + + ++ +++ 31

Pancreas +++++++++++++++++++++++++ 50

Salivary glands +++++++++++++++++++++++++ 50

Stomach, forestomach +++++++++++++++++++++++++ 50

Squamous cell papilloma X 1

Stomach, glandular +++++++++++++++++++++++++ 50

Tooth ++++++++++ 19

Odontoma 1

Cardiovascular System

Blood vessel +++++++++++++++++++++++++ 50

Heart +++++++++++++++++++++++++ 50

Endocrine System

Adrenal cortex +++++++++++++++++++++++++ 50

Subcapsular, adenoma X X 3

Adrenal medulla +++++++++++++++++++++++++ 50

Pheochromocytoma benign X 1

Islets, pancreatic +++++++++++++++++++++++++ 50

Parathyroid gland + + M M + M + + + M ++++++M+++M+M+ + 38

Pituitary gland +++++++++++++++++++++++++ 50

Pars intermedia, adenoma X 1

Thyroid gland +++++++++++++++++++++++++ 50

Follicle, adenoma 1

56 Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: Vehicle Control

5566666667777777777777777

Number of Days on Study 1822688891122222222222222

1048444844655666666666666

Carcass ID Number

0000000000000000000000000

2053022130124011111233444

3604329109017725689815026

General Body System

None

Genital System

Epididymis

Preputial gland

Prostate

Seminal vesicle

Testes

+++++++++++++++++++++++++

Hematopoietic System

Bone marrow

Hemangiosarcoma, metastatic, spleen

Lymph node

Mediastinal, alveolar/bronchiolar carcinoma,

metastatic, lung

Lymph node, mandibular

Lymph node, mesenteric

Spleen

Hemangioma

Hemangiosarcoma

Thymus

+++++++++++++++++++++++++

++++++++++M++++++++++++++

+++++++++++++++++++++++++

++++++M++++++++++++++++++

X X

++++++M++++++++++++++++++

Integumentary System

Mammary gland

Skin

Subcutaneous tissue, melanoma malignant

MMMMMMMMMMMMMM + M + MMM + MMMM

+++++++++++++++++++++++++

Musculoskeletal System

Bone

Osteosarcoma

+++++++++++++++++++++++++

Nervous System

Brain

Peripheral nerve

Spinal cord

+++++++++++++++++++++++++

Respiratory System

Lung

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma

Alveolar/bronchiolar carcinoma, multiple

Hepatocellular carcinoma, metastatic, liver

Nose

Trachea

+++++++++++++++++++++++++

X X X

X X X X X X X

+++++++++++++++++++++++++

Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: Vehicle Control

7777777777777777777777777

Number of Days on Study 2222222222222222222222222

7777777777777778888888888

Carcass ID Number

0000000000000000000000000

0000122223333440112334444

1458405672368452374791389

Total

Tissues/

Tumors

General Body System

None

Genital System

Epididymis

Preputial gland

Prostate

Seminal vesicle

Testes

+++++++++++++++++++++++++

Hematopoietic System

Bone marrow

Hemangiosarcoma, metastatic, spleen

Lymph node

Mediastinal, alveolar/bronchiolar carcinoma,

metastatic, lung

Lymph node, mandibular

Lymph node, mesenteric

Spleen

Hemangioma

Hemangiosarcoma

Thymus

+++++++++++++++++++++++++

++++++++++++++++++++M++++

Integumentary System

Mammary gland

Skin

Subcutaneous tissue, melanoma malignant

MMMMMMMMMMMMMMMMMMMMMMMMM

+++++++++++++++++++++++++

Musculoskeletal System

Bone

Osteosarcoma

+++++++++++++++++++++++++ 50

Nervous System

Brain

Peripheral nerve

Spinal cord

+++++++++++++++++++++++++ 50

Respiratory System

Lung

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma

Alveolar/bronchiolar carcinoma, multiple

Hepatocellular carcinoma, metastatic, liver

Nose

Trachea

+++++++++++++++++++++++++

X X X X X

X X X

+++++++++++++++++++++++++

58 Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: Vehicle Control

5566666667777777777777777

Number of Days on Study 1822688891122222222222222

1048444844655666666666666

0000000000000000000000000

Carcass ID Number 2053022130124011111233444

3604329109017725689815026

Special Senses System

Eye +++++++++++++++++++++++++

Harderian gland +++++++++++++++++++++++++

Adenoma X X

Bilateral, adenoma X

Urinary System

Kidney +++++++++++++++++++++++++

Renal tubule, adenoma X

Urinary bladder +++++++++++++++++++++++++

Systemic Lesions

Multiple organs +++++++++++++++++++++++++

Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: Vehicle Control

7777777777777777777777777

Number of Days on Study 2222222222222222222222222

7777777777777778888888888

0000000000000000000000000 Total

Carcass ID Number 0000122223333440112334444 Tissues/

1458405672368452374791389 Tumors

Special Senses System

Eye +++++++++++++++++++++++++ 50

Harderian gland +++++++++++++++++++++++++ 50

Adenoma XX X X 6

Bilateral, adenoma 1

Urinary System

Kidney +++++++++++++++++++++++++ 50

Renal tubule, adenoma 1

Urinary bladder +++++++++++++++++++++++++ 50

Systemic Lesions

Multiple organs +++++++++++++++++++++++++ 50

60 Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 200 mg/kg

3556667777777777777777777

Number of Days on Study 3051371222222222222222222

4245760666666666666666666

0000000000000000000000000

Carcass ID Number 5857678556677778888899999

7384215493837890124756789

Alimentary System

Esophagus +++++++++++++++++++++++++

Gallbladder +++++++++++++++++++++++++

Intestine large, colon +++++++++++++++++++++++++

Intestine large, rectum +++++++++++++++++++++++++

Intestine large, cecum +++++++++++++++++++++++++

Intestine small, duodenum +++++++++++++++++++++++++

Intestine small, jejunum +++++++++++++++++++++++++

Carcinoma X

Sarcoma, metastatic, adrenal medulla X

Intestine small, ileum +++++++++++++++++++++++++

Liver +++++++++++++++++++++++++

Hemangioma X

Hemangiosarcoma, metastatic, spleen

Hepatoblastoma

Hepatocellular carcinoma X X X

Hepatocellular carcinoma, multiple X X X

Hepatocellular adenoma X X X X

Hepatocellular adenoma, multiple X X

Histiocytic sarcoma X

Sarcoma, metastatic, adrenal medulla X

Mesentery +

Oral mucosa + + + + + + +

Pancreas +++++++++++++++++++++++++

Salivary glands +++++++++++++++++++++++++

Stomach, forestomach +++++++++++++++++++++++++

Squamous cell papilloma X X

Stomach, glandular +++++++++++++++++++++++++

Tooth + + + + + + + + +

Cardiovascular System

Blood vessel +++++++++++++++++++++++++

Heart +++++++++++++++++++++++++

Endocrine System

Adrenal cortex +++++++++++++++++++++++++

Subcapsular, adenoma X

Adrenal medulla +++++++++++++++++++++++++

Sarcoma X

Islets, pancreatic +++++++++++++++++++++++++

Parathyroid gland + M + + + M +++++++++++++M++++M

Pituitary gland ++++M++++++++++++++++++++

Thyroid gland +++++++++++++++++++++++++

General Body System

None

Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 200 mg/kg

7777777777777777777777777

Number of Days on Study 2222222222222222222222222

6777777777777777788888888

1000000000000000000000000 Total

Carcass ID Number 0555566666778899956677899 Tissues/

0123504579566803461602912 Tumors

Alimentary System

Esophagus +++++++++++++++++++++++++ 50

Gallbladder ++++++++++++++++++++++M+ + 49

Intestine large, colon +++++++++++++++++++++++++ 50

Intestine large, rectum +++++++++++++++++++++++++ 50

Intestine large, cecum +++++++++++++++++++++++++ 50

Intestine small, duodenum +++++++++++++++++++++++++ 50

Intestine small, jejunum +++++++++++++++++++++++++ 50

Carcinoma 1

Sarcoma, metastatic, adrenal medulla 1

Intestine small, ileum +++++++++++++++++++++++++ 50

Liver +++++++++++++++++++++++++ 50

Hemangioma 1

Hemangiosarcoma, metastatic, spleen X 1

Hepatoblastoma X 1

Hepatocellular carcinoma X X X X X X X 10

Hepatocellular carcinoma, multiple X 4

Hepatocellular adenoma XXXX XXXX X X 14

Hepatocellular adenoma, multiple X X 4

Histiocytic sarcoma 1

Sarcoma, metastatic, adrenal medulla 1

Mesentery + + 3

Oral mucosa ++++++ +++++++ + +++++ 26

Pancreas +++++++++++++++++++++++++ 50

Salivary glands +++++++++++++++++++++++++ 50

Stomach, forestomach +++++++++++++++++++++++++ 50

Squamous cell papilloma 2

Stomach, glandular +++++++++++++++++++++++++ 50

Tooth + + + + + + + + + 18

Cardiovascular System

Blood vessel +++++++++++++++++++++++++ 50

Heart +++++++++++++++++++++++++ 50

Endocrine System

Adrenal cortex +++++++++++++++++++++++++ 50

Subcapsular, adenoma X X 3

Adrenal medulla +++++++++++++++++++++++++ 50

Sarcoma 1

Islets, pancreatic +++++++++++++++++++++++++ 50

Parathyroid gland +++++++M+++++++++MM++++++ 43

Pituitary gland +++++++++++++++++++++++++ 49

Thyroid gland +++++++++++++++++++++++++ 50

General Body System

None

62 Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 200 mg/kg

3556667777777777777777777

Number of Days on Study 3051371222222222222222222

4245760666666666666666666

Carcass ID Number

0000000000000000000000000

5857678556677778888899999

7384215493837890124756789

Genital System

Epididymis

Preputial gland

Prostate

Seminal vesicle

Testes

Interstitial cell, adenoma

+++++++++++++++++++++++++

Hematopoietic System

Bone marrow

Lymph node, mandibular

Lymph node, mesenteric

Spleen

Hemangiosarcoma

Histiocytic sarcoma

Thymus

+++++++++++++++++++++++++

+ M +++++++++++++++++++++++

+++++++++++++++++++++++++

++++M++++++++++++++++++++

Integumentary System

Mammary gland

Skin

Subcutaneous tissue, site of application, dermis,

mast cell tumor benign

MMMMMMMMMMMMMMM + MMMMMMMMM

+++++++++++++++++++++++++

Musculoskeletal System

Bone +++++++++++++++++++++++++

Nervous System

Brain +++++++++++++++++++++++++

Respiratory System

Lung

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar adenoma, multiple

Alveolar/bronchiolar carcinoma

Carcinoma, metastatic, harderian gland

Hepatocellular carcinoma, metastatic, liver

Histiocytic sarcoma

Nose

Trachea

+++++++++++++++++++++++++

X X X

X X X X X

+++++++++++++++++++++++++

Special Senses System

Eye

Harderian gland

Adenoma

Carcinoma

+++++++++++++++++++++++++

Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 200 mg/kg

7777777777777777777777777

Number of Days on Study 2222222222222222222222222

6777777777777777788888888

Carcass ID Number

1000000000000000000000000

0555566666778899956677899

0123504579566803461602912

Total

Tissues/

Tumors

Genital System

Epididymis

Preputial gland

Prostate

Seminal vesicle

Testes

Interstitial cell, adenoma

+++++++++++++++++++++++++

Hematopoietic System

Bone marrow

Lymph node, mandibular

Lymph node, mesenteric

Spleen

Hemangiosarcoma

Histiocytic sarcoma

Thymus

+++++++++++++++++++++++++

+++++M+++++++++++++++++++

+++++++++++++++++++++++++

X X

++++++++++++++++++++++MM+

Integumentary System

Mammary gland

Skin

Subcutaneous tissue, site of application, dermis,

mast cell tumor benign

MMMMMMMMMMMMMMMMMMMMM + M + M

+++++++++++++++++++++++++

Musculoskeletal System

Bone +++++++++++++++++++++++++ 50

Nervous System

Brain +++++++++++++++++++++++++ 50

Respiratory System

Lung

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar adenoma, multiple

Alveolar/bronchiolar carcinoma

Carcinoma, metastatic, harderian gland

Hepatocellular carcinoma, metastatic, liver

Histiocytic sarcoma

Nose

Trachea

+++++++++++++++++++++++++

X X X

X X X X X

+++++++++++++++++++++++++

Special Senses System

Eye

Harderian gland

Adenoma

Carcinoma

+++++++++++++++++++++++++

X X

64 Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 200 mg/kg

3556667777777777777777777

Number of Days on Study 3051371222222222222222222

4245760666666666666666666

0000000000000000000000000

Carcass ID Number 5857678556677778888899999

7384215493837890124756789

Urinary System

Kidney +++++++++++++++++++++++++

Histiocytic sarcoma X

Urinary bladder +++++++++++++++++++++++++

Systemic Lesions

Multiple organs +++++++++++++++++++++++++

Histiocytic sarcoma X

Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 200 mg/kg

7777777777777777777777777

Number of Days on Study 2222222222222222222222222

6777777777777777788888888

1000000000000000000000000 Total

Carcass ID Number 0555566666778899956677899 Tissues/

0123504579566803461602912 Tumors

Urinary System

Kidney +++++++++++++++++++++++++ 50

Histiocytic sarcoma 1

Urinary bladder +++++++++++++++++++++++++ 50

Systemic Lesions

Multiple organs +++++++++++++++++++++++++ 50

Histiocytic sarcoma 1

66 Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 630 mg/kg

4444555666666677777777777

Number of Days on Study 3488016015779900222222222

9367376349361958666666666

1111111111111111111111111

Carcass ID Number 2110112421331342000022233

4874020655289418357813703

Alimentary System

Esophagus +++++++++++++++++++++++++

Gallbladder +++++++++++++++++++++++++

Intestine large, colon +++++++++++++++++++++++++

Intestine large, rectum +++++++++++++++++++++++++

Intestine large, cecum +++++++++++++++++++++++++

Intestine small, duodenum +++++++++++++++++++++++++

Intestine small, jejunum +++++++++++++++++++++++++

Intestine small, ileum +++++++++++++++++++++++++

Liver +++++++++++++++++++++++++

Hemangiosarcoma X X

Hemangiosarcoma, multiple X X

Hemangiosarcoma, metastatic, spleen X

Hepatoblastoma X

Hepatocellular carcinoma X X X X XXXXXX X X

Hepatocellular carcinoma, multiple X

Hepatocellular adenoma X X XXXX

Hepatocellular adenoma, multiple X X X X

Mesentery + +

Oral mucosa + + + + + + + +++++++++

Pancreas +++++++++++++++++++++++++

Salivary glands +++++++++++++++++++++++++

Stomach, forestomach +++++++++++++++++++++++++

Stomach, glandular +++++++++++++++++++++++++

Tooth + + + + + +

Cardiovascular System

Blood vessel +++++++++++++++++++++++++

Heart +++++++++++++++++++++++++

Endocrine System

Adrenal cortex +++++++++++++++++++++++++

Alveolar/bronchiolar carcinoma, metastatic, lung X

Subcapsular, adenoma X X X

Adrenal medulla +++++++++++++++++++++++++

Islets, pancreatic +++++++++++++++++++++++++

Parathyroid gland ++++++++++M+++MMM+M++++M+

Pituitary gland +++++++++++++++++++++++++

Pars distalis, adenoma X

Thyroid gland ++++++++++M++++++++++++++

Follicle, adenoma X

General Body System

None

Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 630 mg/kg

7777777777777777777777777

Number of Days on Study 2222222222222222222222222

6677777777777777788888888

1111111111111111111111111 Total

Carcass ID Number 3400011333444444501122234 Tissues/

6512646579234789091326910 Tumors

Alimentary System

Esophagus +++++++++++++++++++++++++ 50

Gallbladder +++++++++++++++++++++++++ 50

Intestine large, colon +++++++++++++++++++++++++ 50

Intestine large, rectum +++++++++++++++++++++++++ 50

Intestine large, cecum +++++++++++++++++++++++++ 50

Intestine small, duodenum +++++++++++++++++++++++++ 50

Intestine small, jejunum +++++++++++++++++++++++++ 50

Intestine small, ileum +++++++++++++++++++++++++ 50

Liver +++++++++++++++++++++++++ 50

Hemangiosarcoma X X 4

Hemangiosarcoma, multiple 2

Hemangiosarcoma, metastatic, spleen 1

Hepatoblastoma X 2

Hepatocellular carcinoma X 13

Hepatocellular carcinoma, multiple 1

Hepatocellular adenoma X X X X X X 12

Hepatocellular adenoma, multiple X X X X X X X 11

Mesentery + + + 5

Oral mucosa + + +++++++++++ + ++ +++ 35

Pancreas +++++++++++++++++++++++++ 50

Salivary glands +++++++++++++++++++++++++ 50

Stomach, forestomach +++++++++++++++++++++++++ 50

Stomach, glandular +++++++++++++++++++++++++ 50

Tooth + + ++++ + + 14

Cardiovascular System

Blood vessel +++++++++++++++++++++++++ 50

Heart +++++++++++++++++++++++++ 50

Endocrine System

Adrenal cortex +++++++++++++++++++++++++ 50

Alveolar/bronchiolar carcinoma, metastatic, lung 1

Subcapsular, adenoma X X X 6

Adrenal medulla +++++++++++++++++++++++++ 50

Islets, pancreatic +++++++++++++++++++++++++ 50

Parathyroid gland M + + + M + + + M +++++++++M+M++++ 39

Pituitary gland M ++++++++++++++++++++++++ 49

Pars distalis, adenoma 1

Thyroid gland +++++++++++++++++++++++++ 49

Follicle, adenoma 1

General Body System

None

68 Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 630 mg/kg

4444555666666677777777777

Number of Days on Study 3488016015779900222222222

9367376349361958666666666

Carcass ID Number

1111111111111111111111111

2110112421331342000022233

4874020655289418357813703

Genital System

Coagulating gland

Epididymis

Preputial gland

Prostate

Seminal vesicle

Testes

+++++++++++++++++++++++++

Hematopoietic System

Bone marrow

Hemangiosarcoma, metastatic, spleen

Lymph node

Mediastinal, hepatocellular carcinoma,

metastatic, liver

Lymph node, mandibular

Lymph node, mesenteric

Spleen

Hemangiosarcoma

Thymus

Alveolar/bronchiolar carcinoma, metastatic, lung

+++++++++++++++++++++++++

++++++++++++++++++++++++ M

++++++++++ M ++++++++++++++

+++++++++++++++++++++++++

++++++++++++++++++++++++ M

Integumentary System

Mammary gland

Skin

Subcutaneous tissue, skin, site of application,

hemangiosarcoma

MMMMMMMMMMMMMMMMMMMMMMMMM

+++++++++++++++++++++++++

Musculoskeletal System

Bone +++++++++++++++++++++++++

Nervous System

Brain +++++++++++++++++++++++++

Respiratory System

Lung

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar adenoma, multiple

Alveolar/bronchiolar carcinoma

Hepatoblastoma, metastatic, liver

Hepatocellular carcinoma, metastatic, liver

Nose

Trachea

+++++++++++++++++++++++++

X X X X X X

X X X

X X X X X X

+++++++++++++++++++++++++

Special Senses System

Eye

Harderian gland

Adenoma

+++++++++++++++++++++++++

+++++ M +++++++++++++++++++

Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 630 mg/kg

7777777777777777777777777

Number of Days on Study 2222222222222222222222222

6677777777777777788888888

Carcass ID Number

1111111111111111111111111

3400011333444444501122234

6512646579234789091326910

Total

Tissues/

Tumors

Genital System

Coagulating gland

Epididymis

Preputial gland

Prostate

Seminal vesicle

Testes

+++++++++++++++++++++++++

Hematopoietic System

Bone marrow

Hemangiosarcoma, metastatic, spleen

Lymph node

Mediastinal, hepatocellular carcinoma,

metastatic, liver

Lymph node, mandibular

Lymph node, mesenteric

Spleen

Hemangiosarcoma

Thymus

Alveolar/bronchiolar carcinoma, metastatic, lung

+++++++++++++++++++++++++

+++++++ M +++++++++++++++++

+++++++++++++++++++++++++

++++++++++++++++++++++++ M

Integumentary System

Mammary gland

Skin

Subcutaneous tissue, skin, site of application,

hemangiosarcoma

MMMMMMMMMMMMMMMMMMMMMMMMM

+++++++++++++++++++++++++ 50

Musculoskeletal System

Bone +++++++++++++++++++++++++ 50

Nervous System

Brain +++++++++++++++++++++++++ 50

Respiratory System

Lung

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar adenoma, multiple

Alveolar/bronchiolar carcinoma

Hepatoblastoma, metastatic, liver

Hepatocellular carcinoma, metastatic, liver

Nose

Trachea

+++++++++++++++++++++++++

X X X X X

+++++++++++++++++++++++++

Special Senses System

Eye

Harderian gland

Adenoma

+++++++++++++++++++++++++

70 Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 630 mg/kg

4444555666666677777777777

Number of Days on Study 3488016015779900222222222

9367376349361958666666666

1111111111111111111111111

Carcass ID Number 2110112421331342000022233

4874020655289418357813703

Urinary System

Kidney +++++++++++++++++++++++++

Renal tubule, adenoma

Urinary bladder +++++++++++++++++++++++++

Systemic Lesions

Multiple organs +++++++++++++++++++++++++

Lymphoma malignant X

Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 630 mg/kg

7777777777777777777777777

Number of Days on Study 2222222222222222222222222

6677777777777777788888888

1111111111111111111111111 Total

Carcass ID Number 3400011333444444501122234 Tissues/

6512646579234789091326910 Tumors

Urinary System

Kidney +++++++++++++++++++++++++ 50

Renal tubule, adenoma X 1

Urinary bladder +++++++++++++++++++++++++ 50

Systemic Lesions

Multiple organs +++++++++++++++++++++++++ 50

Lymphoma malignant 1

72 Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 2,000 mg/kg

3555666666777777777777777

Number of Days on Study 9567124888222222222222222

9462884344666666666666666

1111111111111111111111111

Carcass ID Number 6887976857555566677777888

8531661622356734534589018

Alimentary System

Esophagus +++++++++++++++++++++++++

Gallbladder + + + M +++++++++++++++++++++

Intestine large, colon +++++++++++++++++++++++++

Intestine large, rectum +++++++++++++++++++++++++

Intestine large, cecum +++++++++++++++++++++++++

Intestine small, duodenum +++++++++++++++++++++++++

Intestine small, jejunum +++++++++++++++++++++++++

Adenoma X

Carcinoma X

Intestine small, ileum +++++++++++++++++++++++++

Liver +++++++++++++++++++++++++

Hemangioma X

Hemangiosarcoma X

Hemangiosarcoma, metastatic, blood vessel X

Hepatoblastoma X X

Hepatocellular carcinoma X X X X X X

Hepatocellular carcinoma, multiple

Hepatocellular adenoma X X X X X X X

Hepatocellular adenoma, multiple X X X

Hepatocholangiocarcinoma X

Histiocytic sarcoma X X

Mesentery + + + +

Fibrous histiocytoma X

Oral mucosa + + + + + +++++

Pancreas +++++++++++++++++++++++++

Salivary glands +++++++++++++++++++++++++

Stomach, forestomach +++++++++++++++++++++++++

Squamous cell papilloma X

Stomach, glandular +++++++++++++++++++++++++

Tongue

Squamous cell carcinoma

Tooth + + + + + + +

Cardiovascular System

Blood vessel +++++++++++++++++++++++++

Aorta, hemangiosarcoma X

Heart +++++++++++++++++++++++++

Carcinoma, metastatic, kidney X

Fibrous histiocytoma, metastatic, mesentery X

Histiocytic sarcoma X

Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 2,000 mg/kg

7777777777777777777777777

Number of Days on Study 2222222222222222222222222

6667777777777777888888888

1111111111111111111111112 Total

Carcass ID Number 9995566678889999556678990 Tissues/

1391902902474578486779020 Tumors

Alimentary System

Esophagus +++++++++++++++++++++++++ 50

Gallbladder +++++++++++M+++++++M+++++ 47

Intestine large, colon +++++++++++++++++++++++++ 50

Intestine large, rectum +++++++++++++++++++++++++ 50

Intestine large, cecum +++++++++++++++++++++++++ 50

Intestine small, duodenum +++++++++++++++++++++++++ 50

Intestine small, jejunum +++++++++++++++++++++++++ 50

Adenoma 1

Carcinoma 1

Intestine small, ileum +++++++++++++++++++++++++ 50

Liver +++++++++++++++++++++++++ 50

Hemangioma X 2

Hemangiosarcoma 1

Hemangiosarcoma, metastatic, blood vessel 1

Hepatoblastoma X 3

Hepatocellular carcinoma X X X X 10

Hepatocellular carcinoma, multiple X 1

Hepatocellular adenoma X X X X X X X X 15

Hepatocellular adenoma, multiple X X 5

Hepatocholangiocarcinoma 1

Histiocytic sarcoma 2

Mesentery + + + 7

Fibrous histiocytoma 1

Oral mucosa +++++ ++++ +++ ++++++ ++ 30

Pancreas +++++++++++++++++++++++++ 50

Salivary glands +++++++++++++++++++++++++ 50

Stomach, forestomach +++++++++++++++++++++++++ 50

Squamous cell papilloma 1

Stomach, glandular +++++++++++++++++++++++++ 50

Tongue + 1

Squamous cell carcinoma X 1

Tooth + + + + + + 13

Cardiovascular System

Blood vessel +++++++++++++++++++++++++ 50

Aorta, hemangiosarcoma 1

Heart +++++++++++++++++++++++++ 50

Carcinoma, metastatic, kidney 1

Fibrous histiocytoma, metastatic, mesentery 1

Histiocytic sarcoma 1

74 Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 2,000 mg/kg

3555666666777777777777777

Number of Days on Study 9567124888222222222222222

9462884344666666666666666

1111111111111111111111111

Carcass ID Number 6887976857555566677777888

8531661622356734534589018

Endocrine System

Adrenal cortex +++++++++++++++++++++++++

Subcapsular, adenoma X X

Adrenal medulla +++++++++++++++++++++++++

Pheochromocytoma benign X

Islets, pancreatic +++++++++++++++++++++++++

Parathyroid gland +++++++++++++++++++++++++

Pituitary gland +++++++++++++++++++++++++

Pars intermedia, adenoma X

Thyroid gland +++++++++++++++++++++++++

General Body System

None

Genital System

Coagulating gland

Epididymis +++++++++++++++++++++++++

Fibrous histiocytoma, metastatic, mesentery X

Preputial gland +++++++++++++++++++++++++

Prostate +++++++++++++++++++++++++

Seminal vesicle +++++++++++++++++++++++++

Testes +++++++++++++++++++++++++

Hematopoietic System

Bone marrow +++++++++++++++++++++++++

Histiocytic sarcoma X

Lymph node +

Mediastinal, fibrous histiocytoma,

metastatic, mesentery X

Pancreatic, fibrous histiocytoma,

metastatic, mesentery X

Renal, fibrous histiocytoma X

Lymph node, mandibular +++++++++++++M+++++++++++

Lymph node, mesenteric +++++++++++++++++++++++++

Histiocytic sarcoma X

Spleen +++++++++++++++++++++++++

Histiocytic sarcoma X

Thymus +++++++++++++++++++++++++

Carcinoma, metastatic, kidney X

Fibrous histiocytoma, metastatic, mesentery X

Integumentary System

Mammary gland MMMMMMMMMMMMMMMMMMMMM + MMM

Skin +++++++++++++++++++++++++

Squamous cell carcinoma X

Subcutaneous tissue, hibernoma X

Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 2,000 mg/kg

7777777777777777777777777

Number of Days on Study 2222222222222222222222222

6667777777777777888888888

1111111111111111111111112 Total

Carcass ID Number 9995566678889999556678990 Tissues/

1391902902474578486779020 Tumors

Endocrine System

Adrenal cortex +++++++++++++++++++++++++ 50

Subcapsular, adenoma X X X 5

Adrenal medulla +++++++++++++++++++++++++ 50

Pheochromocytoma benign 1

Islets, pancreatic +++++++++++++++++++++++++ 50

Parathyroid gland + M + + M ++++++++++++++++++++ 48

Pituitary gland +++++++++++++++++++++++++ 50

Pars intermedia, adenoma 1

Thyroid gland +++++++++++++++++++++++++ 50

General Body System

None

Genital System

Coagulating gland + 1

Epididymis +++++++++++++++++++++++++ 50

Fibrous histiocytoma, metastatic, mesentery 1

Preputial gland +++++++++++++++++++++++++ 50

Prostate +++++++++++++++++++++++++ 50

Seminal vesicle +++++++++++++++++++++++++ 50

Testes +++++++++++++++++++++++++ 50

Hematopoietic System

Bone marrow +++++++++++++++++++++++++ 50

Histiocytic sarcoma 1

Lymph node 1

Mediastinal, fibrous histiocytoma,

metastatic, mesentery 1

Pancreatic, fibrous histiocytoma,

metastatic, mesentery 1

Renal, fibrous histiocytoma 1

Lymph node, mandibular ++++++++++++++++++++M++++ 48

Lymph node, mesenteric +++++++++++++++++++++++++ 50

Histiocytic sarcoma 1

Spleen +++++++++++++++++++++++++ 50

Histiocytic sarcoma 1

Thymus ++++M++M+++++++++++++++++ 48

Carcinoma, metastatic, kidney 1

Fibrous histiocytoma, metastatic, mesentery 1

Integumentary System

Mammary gland MMMMMMMMMMMMMMMMMMMMMMMMM 1

Skin +++++++++++++++++++++++++ 50

Squamous cell carcinoma 1

Subcutaneous tissue, hibernoma 1

76 Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 2,000 mg/kg

3555666666777777777777777

Number of Days on Study 9567124888222222222222222

9462884344666666666666666

1111111111111111111111111

Carcass ID Number 6887976857555566677777888

8531661622356734534589018

Musculoskeletal System

Bone +++++++++++++++++++++++++

Nervous System

Brain +++++++++++++++++++++++++

Respiratory System

Lung +++++++++++++++++++++++++

Alveolar/bronchiolar adenoma X X X

Alveolar/bronchiolar adenoma, multiple

Alveolar/bronchiolar carcinoma

Alveolar/bronchiolar carcinoma, multiple

Carcinoma, metastatic, kidney X

Fibrous histiocytoma, metastatic, mesentery X

Hepatoblastoma, metastatic, liver X

Hepatocellular carcinoma, metastatic, liver X X

Hepatocholangiocarcinoma, metastatic, liver X

Histiocytic sarcoma X X

Nose +++++++++++++++++++++++++

Trachea +++++++++++++++++++++++++

Special Senses System

Eye +++++++++++++++++++++++++

Harderian gland +++++++++++++++++++++++++

Adenoma X X X

Carcinoma X

Bilateral, adenoma X

Urinary System

Kidney +++++++++++++++++++++++++

Histiocytic sarcoma X

Renal tubule, adenoma

Renal tubule, carcinoma, multiple X

Urinary bladder +++++++++++++++++++++++++

Systemic Lesions

Multiple organs +++++++++++++++++++++++++

Histiocytic sarcoma X X

Lymphoma malignant

Triethanolamine, NTP TR 518

ABLE A2

Individual Animal Tumor Pathology of Male Mice in the 2-Year Dermal Study of Triethanolamine: 2,000 mg/kg

7777777777777777777777777

Number of Days on Study 2222222222222222222222222

6667777777777777888888888

1111111111111111111111112 Total

Carcass ID Number 9995566678889999556678990 Tissues/

1391902902474578486779020 Tumors

Musculoskeletal System

Bone +++++++++++++++++++++++++ 50

Nervous System

Brain +++++++++++++++++++++++++ 50

Respiratory System

Lung +++++++++++++++++++++++++ 50

Alveolar/bronchiolar adenoma X X X X 7

Alveolar/bronchiolar adenoma, multiple X X 2

Alveolar/bronchiolar carcinoma X X X 3

Alveolar/bronchiolar carcinoma, multiple X 1

Carcinoma, metastatic, kidney 1

Fibrous histiocytoma, metastatic, mesentery 1

Hepatoblastoma, metastatic, liver 1

Hepatocellular carcinoma, metastatic, liver 2

Hepatocholangiocarcinoma, metastatic, liver 1

Histiocytic sarcoma 2

Nose +++++++++++++++++++++++++ 50

Trachea +++++++++++++++++++++++++ 50

Special Senses System

Eye +++++++++++++++++++++++++ 50

Harderian gland +++++++++++++++++++++++++ 50

Adenoma X X 5

Carcinoma 1

Bilateral, adenoma 1

Urinary System

Kidney +++++++++++++++++++++++++ 50

Histiocytic sarcoma 1

Renal tubule, adenoma X 1

Renal tubule, carcinoma, multiple 1

Urinary bladder +++++++++++++++++++++++++ 50

Systemic Lesions

Multiple organs +++++++++++++++++++++++++ 50

Histiocytic sarcoma 2

Lymphoma malignant X 1

78 Triethanolamine, NTP TR 518

TABLE A3

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 200 mg/kg 630 mg/kg 2,000 mg/kg

Adrenal Cortex: Adenoma

Overall rate

Adjusted rate

Terminal rate

3/50 (6%)

6.4%

3/37 (8%)

3/50 (6%)

6.4%

3/43 (7%)

6/50 (12%)

13.8%

4/34 (12%)

5/50 (10%)

10.8%

4/40 (10%)

First incidence (days)

Poly-3 test

726 (T)

P=0.285

726 (T)

P=0.659

676

P=0.204

618

P=0.347

Harderian Gland: Adenoma

Overall rate 7/50 (14%) 2/50 (4%) 2/50 (4%) 6/50 (12%)

Adjusted rate 14.6% 4.3% 4.6% 13.0%

Terminal rate 6/37 (16%) 2/43 (5%) 2/34 (6%) 6/40 (15%)

First incidence (days) 511 726 (T) 726 (T) 726 (T)

Poly-3 test P=0.369 P=0.084N P=0.105N P=0.530N

Harderian Gland: Adenoma or Carcinoma

Overall rate 7/50 (14%) 3/50 (6%) 2/50 (4%) 7/50 (14%)

Adjusted rate 14.6% 6.4% 4.6% 15.1%

Terminal rate 6/37 (16%) 3/43 (7%) 2/34 (6%) 6/40 (15%)

First incidence (days) 511 726 (T) 726 (T) 628

Poly-3 test P=0.289 P=0.166N P=0.105N P=0.589

Intestine (Small): Carcinoma

Overall rate 3/50 (6%) 1/50 (2%) 0/50 (0%) 1/50 (2%)

Adjusted rate 6.4% 2.1% 0.0% 2.2%

Terminal rate

First incidence (days)

3/37 (8%)

726 (T)

1/43 (2%)

726 (T)

0/34 (0%)

—

1/40 (3%)

726 (T)

Poly-3 test P=0.352N P=0.308N P=0.135N P=0.314N

Intestine (Small): Adenoma or Carcinoma

Overall rate 3/50 (6%) 1/50 (2%) 0/50 (0%) 2/50 (4%)

Adjusted rate 6.4% 2.1% 0.0% 4.3%

Terminal rate 3/37 (8%) 1/43 (2%) 0/34 (0%) 1/40 (3%)

First incidence (days) 726 (T) 726 (T) — 399

Poly-3 test P=0.629N P=0.308N P=0.135N P=0.503N

Liver: Hemangiosarcoma

Overall rate 1/50 (2%) 0/50 (0%) 6/50 (12%) 1/50 (2%)

Adjusted rate 2.1% 0.0% 13.5% 2.2%

Terminal rate 1/37 (3%) 0/43 (0%) 3/34 (9%) 1/40 (3%)

First incidence (days) 726 (T) — 517 726 (T)

Poly-3 test P=0.587 P=0.501N P=0.047 P=0.755

Liver: Hepatocellular Adenoma

Overall rate 19/50 (38%) 18/50 (36%) 23/50 (46%) 20/50 (40%)

Adjusted rate 40.0% 38.4% 52.4% 42.8%

Terminal rate 17/37 (46%) 18/43 (42%) 20/34 (59%) 18/40 (45%)

First incidence (days) 688 726 (T) 659 618

Poly-3 test P=0.425 P=0.519N P=0.162 P=0.476

Liver: Hepatocellular Carcinoma

Overall rate 17/50 (34%) 14/50 (28%) 14/50 (28%) 11/50 (22%)

Adjusted rate 34.4% 29.1% 29.6% 23.6%

Terminal rate 8/37 (22%) 11/43 (26%) 5/34 (15%) 9/40 (23%)

First incidence (days) 511 554 439 572

Poly-3 test P=0.177N P=0.365N P=0.390N P=0.172N

Triethanolamine, NTP TR 518

ABLE A3

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 200 mg/kg 630 mg/kg 2,000 mg/kg

Liver: Hepatocellular Adenoma or Carcinoma

Overall rate 33/50 (66%) 27/50 (54%) 33/50 (66%) 25/50 (50%)

Adjusted rate 66.6% 56.1% 69.2% 52.7%

Terminal rate 23/37 (62%) 24/43 (56%) 22/34 (65%) 21/40 (53%)

First incidence (days) 511 554 439 572

Poly-3 test P=0.148N P=0.195N P=0.475 P=0.116N

Liver: Hepatoblastoma

Overall rate 1/50 (2%) 1/50 (2%) 2/50 (4%) 3/50 (6%)

Adjusted rate 2.1% 2.1% 4.6% 6.5%

Terminal rate 1/37 (3%) 1/43 (2%) 2/34 (6%) 2/40 (5%)

First incidence (days) 726 (T) 726 (T) 726 (T) 618

Poly-3 test P=0.181 P=0.759 P=0.470 P=0.299

Liver: Hepatocellular Carcinoma or Hepatoblastoma

Overall rate 18/50 (36%) 15/50 (30%) 15/50 (30%) 13/50 (26%)

Adjusted rate 36.4% 31.1% 31.8% 27.6%

Terminal rate 9/37 (24%) 12/43 (28%) 6/34 (18%) 10/40 (25%)

First incidence (days) 511 554 439 572

Poly-3 test P=0.253N P=0.369N P=0.395N P=0.240N

Liver: Hepatocellular Adenoma, Hepatocellular Carcinoma, or Hepatoblastoma

Overall rate 33/50 (66%) 27/50 (54%) 34/50 (68%) 26/50 (52%)

Adjusted rate 66.6% 56.1% 71.3% 54.8%

Terminal rate 23/37 (62%) 24/43 (56%) 23/34 (68%) 22/40 (55%)

First incidence (days) 511 554 439 572

Poly-3 test P=0.208N P=0.195N P=0.386 P=0.162N

Lung: Alveolar/bronchiolar Adenoma

Overall rate 6/50 (12%) 7/50 (14%) 12/50 (24%) 9/50 (18%)

Adjusted rate 12.7% 14.9% 26.8% 19.6%

Terminal rate 6/37 (16%) 7/43 (16%) 7/34 (21%) 9/40 (23%)

First incidence (days) 726 (T) 726 (T) 487 726 (T)

Poly-3 test P=0.272 P=0.495 P=0.074 P=0.269

Lung: Alveolar/bronchiolar Carcinoma

Overall rate 7/50 (14%) 6/50 (12%) 8/50 (16%) 4/50 (8%)

Adjusted rate 14.6% 12.8% 17.8% 8.7%

Terminal rate 5/37 (14%) 6/43 (14%) 5/34 (15%) 4/40 (10%)

First incidence (days) 511 726 (T) 503 726 (T)

Poly-3 test P=0.246N P=0.519N P=0.444 P=0.287N

Lung: Alveolar/bronchiolar Adenoma or Carcinoma

Overall rate 12/50 (24%) 13/50 (26%) 19/50 (38%) 13/50 (26%)

Adjusted rate 25.0% 27.7% 40.9% 28.2%

Terminal rate 10/37 (27%) 13/43 (30%) 11/34 (32%) 13/40 (33%)

First incidence (days) 511 726 (T) 487 726 (T)

Poly-3 test P=0.481 P=0.471 P=0.074 P=0.450

All Organs: Hemangiosarcoma

Overall rate 3/50 (6%) 2/50 (4%) 9/50 (18%) 2/50 (4%)

Adjusted rate 6.3% 4.3% 20.1% 4.3%

Terminal rate 1/37 (3%) 2/43 (5%) 5/34 (15%) 1/40 (3%)

First incidence (days) 624 726 (T) 517 618

Poly-3 test P=0.442N P=0.508N P=0.046 P=0.513N

Triethanolamine, NTP TR 518

ABLE A3

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 200 mg/kg 630 mg/kg 2,000 mg/kg

All Organs: Hemangioma or Hemangiosarcoma

Overall rate 4/50 (8%) 3/50 (6%) 9/50 (18%) 4/50 (8%)

Adjusted rate 8.4% 6.4% 20.1% 8.6%

Terminal rate 2/37 (5%) 3/43 (7%) 5/34 (15%) 3/40 (8%)

First incidence (days) 624 726 (T) 517 618

Poly-3 test P=0.550 P=0.509N P=0.092 P=0.628

All Organs: Benign Neoplasms

Overall rate 30/50 (60%) 25/50 (50%) 32/50 (64%) 32/50 (64%)

Adjusted rate 62.3% 53.3% 70.4% 66.2%

Terminal rate 27/37 (73%) 25/43 (58%) 23/34 (68%) 27/40 (68%)

First incidence (days) 511 726 (T) 487 399

Poly-3 test P=0.241 P=0.245N P=0.269 P=0.426

All Organs: Malignant Neoplasms

Overall rate 28/50 (56%) 24/50 (48%) 27/50 (54%) 21/50 (42%)

Adjusted rate 56.0% 49.8% 55.3% 43.5%

Terminal rate 16/37 (43%) 20/43 (47%) 14/34 (41%) 14/40 (35%)

First incidence (days) 511 554 439 566

Poly-3 test P=0.150N P=0.339N P=0.552N P=0.149N

All Organs: Benign or Malignant Neoplasms

Overall rate 45/50 (90%) 35/50 (70%) 43/50 (86%) 41/50 (82%)

Adjusted rate 90.0% 72.6% 87.4% 82.5%

Terminal rate 33/37 (89%) 31/43 (72%) 28/34 (82%) 32/40 (80%)

First incidence (days) 511 554 439 399

Poly-3 test P=0.518N P=0.022N P=0.460N P=0.212N

(T)Terminal sacrifice

Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland,

liver, and lung; for other tissues, denominator is number of animals necropsied.

Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality

Observed incidence at terminal kill

Beneath the vehicle control incidence is the P value associated with the trend test. Beneath the dosed group incidence are the P values corresponding to

pairwise comparisons between the vehicle controls and that dosed group. The Poly-3 test accounts for the differential mortality in animals that do not reach

terminal sacrifice. A negative trend or a lower incidence in a dosed group is indicated by N.

Not applicable; no neoplasms in animal group

Triethanolamine, NTP TR 518

TABLE

A4a

Historical Incidence of Liver Neoplasms in Control Male B6C3F

Mice

Incidence in Controls

Study Hemangioma Hemangiosarcoma Hepatoblastoma

Historical Incidence in Controls Given NTP-2000 Diet

Acrylonitrile (gavage) 0/50 2/50 0/50

trans-Cinnamaldehyde (feed) 0/100 0/100 0/100

Citral (feed) 0/100 2/100 0/100

Decalin (inhalation) 0/50 1/50 0/50

p,pN-Dichlorodiphenyl sulfone (feed) 0/50 2/50 0/50

Dipropylene glycol (drinking water) 1/50 0/50 0/50

Elmiron

(gavage) 0/50 2/50 1/50

2,4-Hexadienal (gavage) 0/50 1/50 2/50

Indium phosphide (inhalation) 0/50 2/50 0/50

60-Hz Magnetic fields (whole body exposure) 0/100 4/100 2/100

Methacrylonitrile (gavage) 0/49 1/49 1/49

2-Methylimidazole (feed) 0/50 1/50 0/50

o-Nitrotoluene (feed) 0/60 1/60 1/60

p-Nitrotoluene (feed) 0/50 1/50 0/50

Propylene glycol mono-t-butyl ether (inhalation) 0/50 2/50 0/50

Riddelliine (gavage) 0/50 2/50 3/50

Sodium nitrite (drinking water) 0/50 2/50 5/50

Stoddard solvent (Type IIC) (inhalation) 1/50 0/50 0/50

Triethanolamine (dermal) 0/50 1/50 1/50

Vanadium pentoxide (inhalation) 0/50 1/50 0/50

Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 2/1,159 (0.2%) 28/1,159 (2.4%) 16/1,159 (1.4%)

Mean ± standard deviation 0.2% ± 0.6% 2.5% ± 1.4% 1.5% ± 2.6%

Range 0%-2% 0%-4% 0%-10%

Data as of March 3, 2003

82 Triethanolamine, NTP TR 518

ABLE A4b

Historical Incidence of Hemangioma or Hemangiosarcoma (All Organs) in Control Male B6C3F

Mice

Incidence in Controls

Study Hemangioma Hemangiosarcoma Hemangioma

or Hemangiosarcoma

Historical Incidence in Controls Given NTP-2000 Diet

Acrylonitrile (gavage) 2/50 3/50 5/50

trans-Cinnamaldehyde (feed) 3/100 2/100 5/100

Citral (feed) 2/100 3/100 5/100

Decalin (inhalation) 0/50 1/50 1/50

p,pN-Dichlorodiphenyl sulfone (feed) 0/50 3/50 3/50

Dipropylene glycol (drinking water) 2/50 0/50 2/50

Elmiron

(gavage) 0/50 6/50 6/50

2,4-Hexadienal (gavage) 1/50 4/50 5/50

Indium phosphide (inhalation) 0/50 3/50 3/50

60-Hz Magnetic fields (whole body exposure) 0/100 6/100 6/100

Methacrylonitrile (gavage) 0/49 3/49 3/49

2-Methylimidazole (feed) 0/50 2/50 2/50

o-Nitrotoluene (feed) 1/60 4/60 5/60

p-Nitrotoluene (feed) 0/50 1/50 1/50

Propylene glycol mono-t-butyl ether (inhalation) 1/50 3/50 4/50

Riddelliine (gavage) 0/50 3/50 3/50

Sodium nitrite (drinking water) 0/50 7/50 7/50

Stoddard solvent (Type IIC) (inhalation) 1/50 1/50 2/50

Triethanolamine (dermal) 1/50 3/50 4/50

Vanadium pentoxide (inhalation) 0/50 1/50 1/50

Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 14/1,159 (1.2%) 59/1,159 (5.1%) 73/1,159 (6.3%)

Mean ± standard deviation 1.1% ± 1.4% 5.3% ± 3.4% 6.4% ± 3.3%

Range 0%-4% 0%-14% 2%-14%

Data as of March 3, 2003

Triethanolamine, NTP TR 518

ABLE A5

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 200 mg/kg 630 mg/kg 2,000 mg/kg

Disposition Summary

Animals initially in study 50 50 50 50

Early deaths

Moribund 5 2 6 8

Natural deaths 8 5 10 2

Survivors

Terminal sacrifice 37 43 34 40

Animals examined microscopically 50 50 50 50

Alimentary System

Gallbladder (48) (49) (50) (47)

Necrosis 1 (2%)

Intestine large, colon (50) (50) (50) (50)

Inflammation, chronic 1 (2%)

Goblet cell, hyperplasia 1 (2%)

Intestine large, cecum (50) (50) (50) (50)

Hemorrhage 1 (2%)

Intestine small, duodenum (50) (50) (50) (50)

Epithelium, hyperplasia 1 (2%)

Intestine small, jejunum (50) (50) (50) (50)

Diverticulum 1 (2%)

Epithelium, hyperplasia 1 (2%)

Peyer’s patch, hyperplasia, lymphoid 1 (2%) 1 (2%)

Serosa, inflammation, granulomatous 1 (2%)

Intestine small, ileum (50) (50) (50) (50)

Muscularis, infiltration cellular, mononuclear cell 1 (2%)

Liver (50) (50) (50) (50)

Basophilic focus 2 (4%) 6 (12%) 3 (6%) 2 (4%)

Clear cell focus 19 (38%) 23 (46%) 14 (28%) 11 (22%)

Eosinophilic focus 9 (18%) 20 (40%) 31 (62%) 30 (60%)

Fibrosis 1 (2%)

Hematopoietic cell proliferation 3 (6%) 4 (8%) 1 (2%) 2 (4%)

Hepatodiaphragmatic nodule 1 (2%) 1 (2%)

Infiltration cellular, lymphoid 3 (6%) 2 (4%) 2 (4%) 3 (6%)

Inflammation, acute 1 (2%)

Inflammation, chronic 9 (18%) 14 (28%) 10 (20%) 11 (22%)

Mixed cell focus 13 (26%) 11 (22%) 15 (30%) 8 (16%)

Necrosis, focal 4 (8%) 3 (6%) 7 (14%) 6 (12%)

Vacuolization cytoplasmic, focal 2 (4%) 1 (2%) 3 (6%) 5 (10%)

Centrilobular, necrosis 1 (2%) 1 (2%)

Mesentery (3) (3) (5) (7)

Inflammation, granulomatous 1 (20%)

Artery, inflammation, chronic 1 (14%)

Fat, necrosis 3 (100%) 3 (100%) 4 (80%) 5 (71%)

Oral mucosa (31) (26) (35) (30)

Inflammation, chronic 31 (100%) 26 (100%) 35 (100%) 30 (100%)

Pancreas (50) (50) (50) (50)

Acinus, atrophy 1 (2%)

Duct, cyst 1 (2%)

Number of animals examined microscopically at the site and the number of animals with lesion

84 Triethanolamine, NTP TR 518

TABLE

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 200 mg/kg 630 mg/kg 2,000 mg/kg

Alimentary System (continued)

Stomach, forestomach (50) (50) (50) (50)

Inflammation, suppurative 1 (2%)

Ulcer 1 (2%) 1 (2%)

Epithelium, cyst 1 (2%)

Epithelium, hyperplasia, focal 3 (6%) 3 (6%) 2 (4%)

Stomach, glandular (50) (50) (50) (50)

Hyperplasia, lymphoid 1 (2%)

Ulcer 2 (4%)

Epithelium, hyperplasia, focal 1 (2%)

Glands, ectasia 1 (2%)

Tooth (19) (18) (14) (13)

Malformation 19 (100%) 18 (100%) 14 (100%) 13 (100%)

Cardiovascular System

Blood vessel (50) (50) (50) (50)

Aorta, inflammation, chronic 1 (2%)

Aorta, mineralization 1 (2%)

Pulmonary vein, thrombosis 1 (2%)

Heart (50) (50) (50) (50)

Infiltration cellular, mononuclear cell 3 (6%) 1 (2%)

Inflammation, acute 1 (2%)

Inflammation, chronic 2 (4%)

Artery, inflammation, chronic 1 (2%) 3 (6%)

Atrium, thrombosis 1 (2%) 1 (2%)

Coronary artery, inflammation, chronic 1 (2%)

Myocardium, degeneration 1 (2%)

Myocardium, mineralization 1 (2%) 1 (2%) 2 (4%)

Valve, inflammation, chronic 1 (2%)

Ventricle, thrombosis 1 (2%)

Endocrine System

Adrenal cortex (50) (50) (50) (50)

Hyperplasia, focal 8 (16%) 4 (8%) 3 (6%) 1 (2%)

Hypertrophy, focal 16 (32%) 21 (42%) 10 (20%) 10 (20%)

Necrosis 1 (2%)

Subcapsular, hyperplasia 39 (78%) 45 (90%) 40 (80%) 40 (80%)

Zona glomerulosa, hyperplasia 2 (4%) 3 (6%) 1 (2%)

Adrenal medulla (50) (50) (50) (50)

Hyperplasia, focal 2 (4%)

Necrosis 1 (2%)

Islets, pancreatic (50) (50) (50) (50)

Hyperplasia 45 (90%) 43 (86%) 43 (86%) 32 (64%)

Pituitary gland (50) (49) (49) (50)

Pars distalis, hyperplasia 1 (2%) 2 (4%) 1 (2%)

Pars intermedia, hyperplasia 1 (2%) 3 (6%)

Thyroid gland (50) (50) (49) (50)

Follicle, cyst 1 (2%) 1 (2%) 1 (2%)

General Body System

None

Triethanolamine, NTP TR 518

ABLE A5

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 200 mg/kg 630 mg/kg 2,000 mg/kg

Genital System

Coagulating gland (1) (1)

Cyst 1 (100%)

Epididymis (50) (50) (50) (50)

Granuloma sperm 1 (2%) 1 (2%) 2 (4%)

Inflammation, chronic 1 (2%)

Preputial gland (50) (50) (50) (50)

Atrophy 1 (2%)

Cyst 10 (20%) 14 (28%) 11 (22%) 15 (30%)

Inflammation, chronic 1 (2%) 2 (4%)

Inflammation, suppurative 1 (2%) 3 (6%)

Prostate (50) (50) (50) (50)

Hyperplasia 1 (2%)

Seminal vesicle (50) (50) (50) (50)

Atrophy 1 (2%)

Hyperplasia 1 (2%) 2 (4%) 2 (4%)

Testes (50) (50) (50) (50)

Inflammation, granulomatous 1 (2%)

Germinal epithelium, atrophy 4 (8%) 2 (4%) 1 (2%) 2 (4%)

Germinal epithelium, mineralization 2 (4%) 1 (2%)

Hematopoietic System

Lymph node, mesenteric (50) (48) (48) (50)

Congestion 1 (2%)

Hyperplasia, lymphoid 1 (2%)

Spleen (49) (50) (50) (50)

Atrophy 4 (8%) 1 (2%) 1 (2%)

Hematopoietic cell proliferation 14 (29%) 20 (40%) 17 (34%) 19 (38%)

Lymphoid follicle, hematopoietic cell proliferation 1 (2%) 1 (2%)

Red pulp, infiltration cellular, mononuclear cell 1 (2%)

Thymus (48) (47) (48) (48)

Atrophy 38 (79%) 37 (79%) 37 (77%) 38 (79%)

Integumentary System

Skin (50) (50) (50) (50)

Dermis, edema 1 (2%)

Epidermis, hyperkeratosis 1 (2%)

Epidermis, ulcer 1 (2%)

Site of application, epidermis, hyperkeratosis 3 (6%)

Site of application, epidermis, hyperplasia 5 (10%) 44 (88%) 45 (90%) 49 (98%)

Site of application, epidermis, inflammation, suppurative 1 (2%) 11 (22%) 33 (66%) 42 (84%)

Site of application, epidermis, ulcer 3 (6%) 20 (40%) 47 (94%)

Subcutaneous tissue, site of application, dermis,

inflammation, chronic 1 (2%) 15 (30%) 40 (80%) 49 (98%)

Musculoskeletal System

Bone (50) (50) (50) (50)

Fibrosis 1 (2%)

86 Triethanolamine, NTP TR 518

ABLE A5

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 200 mg/kg 630 mg/kg 2,000 mg/kg

Nervous System

Brain (50) (50) (50) (50)

Compression 1 (2%)

Spinal cord (1)

Necrosis 1 (100%)

Respiratory System

Lung (50) (50) (50) (50)

Infiltration cellular, lymphoid 1 (2%)

Inflammation, acute 1 (2%)

Inflammation, chronic 1 (2%)

Inflammation, granulomatous 2 (4%)

Alveolar epithelium, hyperplasia, focal 2 (4%) 2 (4%) 1 (2%) 2 (4%)

Bronchus, hyperplasia, focal 1 (2%)

Perivascular, infiltration cellular, lymphoid 1 (2%)

Vein, thrombosis 1 (2%)

Nose (50) (50) (50) (50)

Inflammation, suppurative 2 (4%) 1 (2%)

Polyp, inflammatory 1 (2%)

Respiratory epithelium, mineralization 1 (2%)

Special Senses System

Eye (50) (50) (50) (50)

Degeneration 1 (2%)

Cornea, inflammation, acute 1 (2%)

Retrobulbar, inflammation, suppurative 1 (2%)

Harderian gland (50) (50) (49) (50)

Hyperplasia 3 (6%) 5 (10%) 6 (12%) 7 (14%)

Urinary System

Kidney (50) (50) (50) (50)

Hydronephrosis 1 (2%)

Infarct 2 (4%) 3 (6%) 2 (4%) 4 (8%)

Inflammation, acute 2 (4%)

Metaplasia, osseous 1 (2%) 4 (8%) 2 (4%) 2 (4%)

Nephropathy 39 (78%) 42 (84%) 37 (74%) 40 (80%)

Artery, thrombosis 1 (2%)

Capsule, inflammation, chronic 1 (2%)

Glomerulus, cyst 2 (4%) 2 (4%)

Papilla, necrosis 1 (2%)

Renal tubule, cyst 4 (8%) 2 (4%) 4 (8%)

Renal tubule, hyperplasia 1 (2%) 1 (2%) 2 (4%)

Urinary bladder (50) (50) (50) (50)

Cyst 1 (2%)

Inflammation, acute 1 (2%)

APPENDIX B

SUMMARY OF LESIONS IN FEMALE MICE

IN THE 2-YEAR DERMAL STUDY

OF TRIETHANOLAMINE

TABLE

B1 Summary of the Incidence of Neoplasms in Female Mice

in the 2-Year Dermal Study of Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

ABLE B2 Individual Animal Tumor Pathology of Female Mice

in the 2-Year Dermal Study of Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

ABLE B3 Statistical Analysis of Primary Neoplasms in Female Mice

in the 2-Year Dermal Study of Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

TABLE B4 Historical Incidence of Liver Neoplasms in Control Female B6C3F

Mice . . . . . . . . . . . . . 119

ABLE B5 Summary of the Incidence of Nonneoplastic Lesions in Female Mice

in the 2-Year Dermal Study of Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

88 Triethanolamine, NTP TR 518

TABLE B1

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 100 mg/kg 300 mg/kg 1,000 mg/kg

Disposition Summary

Animals initially in study 50 50 50 50

Early deaths

Accidental death 1

Moribund 8 10 5 10

Natural deaths 7 6 4 7

Survivors

Died last week of study 1

Terminal sacrifice 34 34 41 32

Animals examined microscopically 50 50 50 50

Alimentary System

Intestine large, colon (50) (50) (50) (50)

Leiomyoma 1 (2%)

Intestine large, rectum (50) (50) (50) (50)

Leiomyosarcoma, metastatic, uterus 1 (2%)

Intestine large, cecum (50) (50) (50) (50)

Intestine small, jejunum (50) (50) (50) (50)

Carcinoma 1 (2%)

Polyp adenomatous 1 (2%)

Liver (50) (50) (50) (50)

Hemangioma 1 (2%)

Hemangiosarcoma 1 (2%)

Hepatocellular carcinoma 5 (10%) 8 (16%) 4 (8%) 5 (10%)

Hepatocellular carcinoma, multiple 1 (2%)

Hepatocellular adenoma 9 (18%) 15 (30%) 13 (26%) 16 (32%)

Hepatocellular adenoma, multiple 3 (6%) 7 (14%) 17 (34%)

Histiocytic sarcoma 4 (8%) 5 (10%) 3 (6%)

Mesentery (11) (6) (4) (9)

Liposarcoma 1 (25%)

Oral mucosa (26) (26) (31) (31)

Histiocytic sarcoma 1 (4%)

Pancreas (50) (50) (50) (50)

Histiocytic sarcoma 1 (2%)

Salivary glands (50) (50) (50) (50)

Carcinoma 1 (2%)

Stomach, forestomach (50) (50) (50) (50)

Squamous cell papilloma 2 (4%) 2 (4%) 4 (8%)

Squamous cell papilloma, multiple 1 (2%)

Stomach, glandular (50) (50) (50) (50)

Cardiovascular System

Heart (50) (50) (50) (50)

Hemangiosarcoma 1 (2%)

Histiocytic sarcoma 1 (2%) 2 (4%) 1 (2%)

Triethanolamine, NTP TR 518

TABLE B1

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 100 mg/kg 300 mg/kg 1,000 mg/kg

Endocrine System

Adrenal cortex (50) (50) (50) (50)

Carcinoma 1 (2%)

Histiocytic sarcoma 1 (2%)

Subcapsular, adenoma 1 (2%)

Adrenal medulla (49) (50) (50) (50)

Pheochromocytoma benign 1 (2%) 1 (2%)

Islets, pancreatic (50) (50) (50) (50)

Adenoma 1 (2%) 1 (2%) 1 (2%)

Pituitary gland (50) (50) (50) (50)

Pars distalis, adenoma 8 (16%) 5 (10%) 5 (10%) 5 (10%)

Pars distalis, carcinoma 1 (2%)

Pars intermedia, adenoma 3 (6%) 1 (2%)

Thyroid gland (50) (50) (50) (50)

Follicle, adenoma 1 (2%) 3 (6%)

Follicle, carcinoma 1 (2%)

General Body System

None

Genital System

Ovary (50) (50) (50) (50)

Cystadenoma 1 (2%) 1 (2%) 1 (2%)

Granulosa cell tumor malignant 1 (2%)

Histiocytic sarcoma 2 (4%) 2 (4%) 1 (2%)

Luteoma 1 (2%) 1 (2%)

Teratoma benign 1 (2%)

Tubulostromal adenoma 1 (2%)

Uterus (50) (50) (50) (50)

Histiocytic sarcoma 3 (6%) 3 (6%) 1 (2%)

Leiomyosarcoma 1 (2%) 2 (4%)

Polyp stromal 1 (2%) 3 (6%) 1 (2%)

Vagina (1)

Leiomyosarcoma, metastatic, uterus 1 (100%)

Hematopoietic System

Bone marrow (50) (50) (50) (50)

Histiocytic sarcoma 1 (2%) 1 (2%) 1 (2%)

Lymph node (6) (7) (4) (6)

Iliac, histiocytic sarcoma 1 (14%)

Lumbar, histiocytic sarcoma 1 (17%)

Mediastinal, liposarcoma, metastatic, mesentery 1 (25%)

Renal, histiocytic sarcoma 1 (14%)

Lymph node, mandibular (49) (50) (50) (50)

Histiocytic sarcoma 1 (2%)

Lymph node, mesenteric (50) (49) (48) (48)

Histiocytic sarcoma 3 (6%)

Spleen (50) (50) (49) (50)

Hemangiosarcoma 1 (2%) 1 (2%) 1 (2%)

Histiocytic sarcoma 2 (4%) 3 (6%)

Thymus (49) (49) (49) (49)

Histiocytic sarcoma 1 (2%)

90 Triethanolamine, NTP TR 518

ABLE B1

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 100 mg/kg 300 mg/kg 1,000 mg/kg

Integumentary System

Skin

Sebaceous gland, adenoma

Subcutaneous tissue, fibrosarcoma

Subcutaneous tissue, hemangiosarcoma

Subcutaneous tissue, hemangiosarcoma, multiple

Subcutaneous tissue, histiocytic sarcoma

Subcutaneous tissue, melanoma malignant multiple

Subcutaneous tissue, osteosarcoma

(50)

(2%)

(50)

1 (2%)

(50)

2 (4%)

(50)

(2%)

Musculoskeletal System

Bone

Osteosarcoma

Skeletal muscle

Hemangiosarcoma, metastatic, spleen

Rhabdomyosarcoma

(50)

(1)

1 (100%)

(50)

(2)

(2%)

(100%)

(50)

(1)

1 (100%)

(50)

(3)

2 (67%)

Nervous System

Brain

Histiocytic sarcoma

(50)

1 (2%)

(50) (49) (50)

1 (2%)

Respiratory System

Lung

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma

Carcinoma, metastatic, harderian gland

Hemangiosarcoma, metastatic, heart

Hepatocellular carcinoma, metastatic, liver

Histiocytic sarcoma

Liposarcoma, metastatic, mesentery

Osteosarcoma, metastatic, bone

Osteosarcoma, metastatic, skin

Nose

(50)

(6%)

(4%)

(2%)

(6%)

(8%)

(2%)

(50)

(2%)

(4%)

(6%)

(8%)

(2%)

(50)

(4%)

(6%)

(2%)

(50)

(2%)

(4%)

(2%)

Special Senses System

Eye

Harderian gland

Adenoma

Carcinoma

(49)

(10%)

(4%)

(50)

5 (10%)

(50)

3 (6%)

(50)

3 (6%)

Urinary System

Kidney

Histiocytic sarcoma

Renal tubule, adenoma

Urinary bladder

(50)

(4%)

(50)

(6%)

(50)

(49)

(50)

(49)

(2%)

Triethanolamine, NTP TR 518

ABLE B1

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 100 mg/kg 300 mg/kg 1,000 mg/kg

Systemic Lesions

Multiple organs

Histiocytic sarcoma

Lymphoma malignant

(50)

(8%)

(16%)

(50)

(10%)

(18%)

(50)

6 (12%)

(50)

(6%)

(16%)

Neoplasm Summary

Total animals with primary neoplasms

Total primary neoplasms

Total animals with benign neoplasms

Total benign neoplasms

Total animals with malignant neoplasms

Total malignant neoplasms

Total animals with metastatic neoplasms

Total metastatic neoplasms

Number of animals examined microscopically at the site and the number of animals with neoplasm

Number of animals with any tissue examined microscopically

Primary neoplasms: all neoplasms except metastatic neoplasms

92 Triethanolamine, NTP TR 518

TABLE

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: Vehicle Control

3455666666666777777777777

Number of Days on Study 2639111145556112222223333

9925779902376179999990000

2222222222222222222222222

Carcass ID Number 3104230330211320012440011

0462379314710823780341226

Alimentary System

Esophagus +++++++++++++++++++++++++

Gallbladder ++++++++++++++M++++M+++++

Intestine large, colon +++++++++++++++++++++++++

Intestine large, rectum +++++++++++++++++++++++++

Intestine large, cecum +++++++++++++++++++++++++

Intestine small, duodenum +++++++++++++++++++++++++

Intestine small, jejunum +++++++++++++++++++++++++

Intestine small, ileum +++++++++++++++++++++++++

Liver +++++++++++++++++++++++++

Hemangioma

Hepatocellular carcinoma X X X X

Hepatocellular carcinoma, multiple X

Hepatocellular adenoma X X X X

Histiocytic sarcoma X X X X

Mesentery + + + + +

Oral mucosa + + + + + + + +

Histiocytic sarcoma X

Pancreas +++++++++++++++++++++++++

Salivary glands +++++++++++++++++++++++++

Stomach, forestomach +++++++++++++++++++++++++

Squamous cell papilloma X

Stomach, glandular +++++++++++++++++++++++++

Cardiovascular System

Blood vessel +++++++++++++++++++++++++

Heart +++++++++++++++++++++++++

Hemangiosarcoma X

Histiocytic sarcoma X

Endocrine System

Adrenal cortex +++++++++++++++++++++++++

Adrenal medulla +++++++++++++M+++++++++++

Pheochromocytoma benign

Islets, pancreatic +++++++++++++++++++++++++

Parathyroid gland ++++++++++M+++M+M++++++M+

Pituitary gland +++++++++++++++++++++++++

Pars distalis, adenoma X X X X

Thyroid gland +++++++++++++++++++++++++

Follicle, carcinoma

General Body System

None

+: Tissue examined microscopically M: Missing tissue X: Lesion present

A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined

Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: Vehicle Control

7777777777777777777777777

Number of Days on Study 3333333333333333333333333

0000000000111111111111111

2222222222222222222222222 Total

Carcass ID Number 2223334445001111222334444 Tissues/

1592491580583579468560679 Tumors

Alimentary System

Esophagus +++++++++++++++++++++++++ 50

Gallbladder +++++++++++++++++++++++++ 48

Intestine large, colon +++++++++++++++++++++++++ 50

Intestine large, rectum +++++++++++++++++++++++++ 50

Intestine large, cecum +++++++++++++++++++++++++ 50

Intestine small, duodenum +++++++++++++++++++++++++ 50

Intestine small, jejunum +++++++++++++++++++++++++ 50

Intestine small, ileum +++++++++++++++++++++++++ 50

Liver +++++++++++++++++++++++++ 50

Hemangioma X 1

Hepatocellular carcinoma X 5

Hepatocellular carcinoma, multiple 1

Hepatocellular adenoma X X X X X 9

Histiocytic sarcoma 4

Mesentery + + + + + + 11

Oral mucosa + + + + + + ++++ +++ ++ +++ 26

Histiocytic sarcoma 1

Pancreas +++++++++++++++++++++++++ 50

Salivary glands +++++++++++++++++++++++++ 50

Stomach, forestomach +++++++++++++++++++++++++ 50

Squamous cell papilloma X 2

Stomach, glandular +++++++++++++++++++++++++ 50

Cardiovascular System

Blood vessel +++++++++++++++++++++++++ 50

Heart +++++++++++++++++++++++++ 50

Hemangiosarcoma 1

Histiocytic sarcoma 1

Endocrine System

Adrenal cortex +++++++++++++++++++++++++ 50

Adrenal medulla +++++++++++++++++++++++++ 49

Pheochromocytoma benign X 1

Islets, pancreatic +++++++++++++++++++++++++ 50

Parathyroid gland M ++++++M++++MM+M+++M++M++ 39

Pituitary gland +++++++++++++++++++++++++ 50

Pars distalis, adenoma X X X X 8

Thyroid gland +++++++++++++++++++++++++ 50

Follicle, carcinoma X 1

General Body System

None

94 Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: Vehicle Control

3455666666666777777777777

Number of Days on Study 2639111145556112222223333

9925779902376179999990000

2222222222222222222222222

Carcass ID Number 3104230330211320012440011

0462379314710823780341226

Genital System

Clitoral gland +++++++++++++++++++++++++

Ovary +++++++++++++++++++++++++

Cystadenoma X

Histiocytic sarcoma X X

Luteoma

Uterus +++++++++++++++++++++++++

Histiocytic sarcoma X X X

Polyp stromal X

Hematopoietic System

Bone marrow +++++++++++++++++++++++++

Histiocytic sarcoma X

Lymph node + +

Lumbar, histiocytic sarcoma X

Lymph node, mandibular +++++++++++++++++++++++++

Lymph node, mesenteric +++++++++++++++++++++++++

Spleen +++++++++++++++++++++++++

Hemangiosarcoma

Histiocytic sarcoma X X

Thymus +++++++++++++++++++++++++

Integumentary System

Mammary gland +++++++++++++++++++++++++

Skin +++++++++++++++++++++++++

Sebaceous gland, adenoma

Subcutaneous tissue, fibrosarcoma X

Subcutaneous tissue, hemangiosarcoma, multiple

Subcutaneous tissue, histiocytic sarcoma X

Subcutaneous tissue, melanoma malignant,

multiple X

Subcutaneous tissue, osteosarcoma X

Musculoskeletal System

Bone +++++++++++++++++++++++++

Skeletal muscle +

Rhabdomyosarcoma X

Nervous System

Brain +++++++++++++++++++++++++

Histiocytic sarcoma X

Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: Vehicle Control

7777777777777777777777777

Number of Days on Study 3333333333333333333333333

0000000000111111111111111

2222222222222222222222222 Total

Carcass ID Number 2223334445001111222334444 Tissues/

1592491580583579468560679 Tumors

Genital System

Clitoral gland +++++++++++++++++++++++++ 50

Ovary +++++++++++++++++++++++++ 50

Cystadenoma 1

Histiocytic sarcoma 2

Luteoma X 1

Uterus +++++++++++++++++++++++++ 50

Histiocytic sarcoma 3

Polyp stromal 1

Hematopoietic System

Bone marrow +++++++++++++++++++++++++ 50

Histiocytic sarcoma 1

Lymph node + + + + 6

Lumbar, histiocytic sarcoma 1

Lymph node, mandibular +++++++++++++++++++++M+++ 49

Lymph node, mesenteric +++++++++++++++++++++++++ 50

Spleen +++++++++++++++++++++++++ 50

Hemangiosarcoma X 1

Histiocytic sarcoma 2

Thymus +++++++++++++++++++++M+++ 49

Integumentary System

Mammary gland +++++++++++++++++++++++++ 50

Skin +++++++++++++++++++++++++ 50

Sebaceous gland, adenoma X 1

Subcutaneous tissue, fibrosarcoma 1

Subcutaneous tissue, hemangiosarcoma, multiple X 1

Subcutaneous tissue, histiocytic sarcoma 1

Subcutaneous tissue, melanoma malignant,

multiple 1

Subcutaneous tissue, osteosarcoma 1

Musculoskeletal System

Bone +++++++++++++++++++++++++ 50

Skeletal muscle 1

Rhabdomyosarcoma 1

Nervous System

Brain +++++++++++++++++++++++++ 50

Histiocytic sarcoma 1

96 Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: Vehicle Control

3455666666666777777777777

Number of Days on Study 2639111145556112222223333

9925779902376179999990000

2222222222222222222222222

Carcass ID Number 3104230330211320012440011

0462379314710823780341226

Respiratory System

Lung +++++++++++++++++++++++++

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma X

Carcinoma, metastatic, harderian gland X

Hemangiosarcoma, metastatic, heart X

Hepatocellular carcinoma, metastatic, liver X X X

Histiocytic sarcoma X X X X

Osteosarcoma, metastatic, skin X

Nose +++++++++++++++++++++++++

Trachea +++++++++++++++++++++++++

Special Senses System

Eye +++M+++++++++++++++++++++

Harderian gland + + + M +++++++++++++++++++++

Adenoma X

Carcinoma X X

Urinary System

Kidney +++++++++++++++++++++++++

Histiocytic sarcoma X X

Urinary bladder +++++++++++++++++++++++++

Systemic Lesions

Multiple organs +++++++++++++++++++++++++

Histiocytic sarcoma X X X X

Lymphoma malignant X X X

Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: Vehicle Control

7777777777777777777777777

Number of Days on Study 3333333333333333333333333

0000000000111111111111111

Carcass ID Number

2222222222222222222222222

2223334445001111222334444

1592491580583579468560679

Total

Tissues/

Tumors

Respiratory System

Lung

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma

Carcinoma, metastatic, harderian gland

Hemangiosarcoma, metastatic, heart

Hepatocellular carcinoma, metastatic, liver

Histiocytic sarcoma

Osteosarcoma, metastatic, skin

Nose

Trachea

+++++++++++++++++++++++++

X X X

+++++++++++++++++++++++++

Special Senses System

Eye

Harderian gland

Adenoma

Carcinoma

+++++++++++++++++++++++++

X X X X

Urinary System

Kidney

Histiocytic sarcoma

Urinary bladder

+++++++++++++++++++++++++

Systemic Lesions

Multiple organs

Histiocytic sarcoma

Lymphoma malignant

+++++++++++++++++++++++++

X X X X X

98 Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 100 mg/kg

2245566666666667777777777

Number of Days on Study 2341901666777890222222233

1940410555444688999999900

2222222222222232222222222

Carcass ID Number 9685785677899708556667755

7526469735726108344690225

Alimentary System

Esophagus +++++++++++++++++++++++++

Gallbladder +++++++++++++++++++++++++

Intestine large, colon +++++++++++++++++++++++++

Intestine large, rectum +++++++++++++++++++++++++

Intestine large, cecum +++++++++++++++++++++++++

Intestine small, duodenum +++++++++++++++++++++++++

Intestine small, jejunum +++++++++++++++++++++++++

Carcinoma X

Intestine small, ileum +++++++++++++++++++++++++

Liver +++++++++++++++++++++++++

Hemangiosarcoma X

Hepatocellular carcinoma X X X X X

Hepatocellular adenoma X X XXXX X

Hepatocellular adenoma, multiple X

Histiocytic sarcoma X X X

Mesentery + + + +

Oral mucosa + + + + + ++++ ++

Pancreas +++++++++++++++++++++++++

Histiocytic sarcoma X

Salivary glands +++++++++++++++++++++++++

Stomach, forestomach +++++++++++++++++++++++++

Stomach, glandular +++++++++++++++++++++++++

Cardiovascular System

Blood vessel +++++++++++++++++++++++++

Heart +++++++++++++++++++++++++

Histiocytic sarcoma X X

Endocrine System

Adrenal cortex +++++++++++++++++++++++++

Carcinoma

Histiocytic sarcoma X

Adrenal medulla +++++++++++++++++++++++++

Islets, pancreatic +++++++++++++++++++++++++

Adenoma

Parathyroid gland + M M ++++++M+++M++++MM+M+++

Pituitary gland +++++++++++++++++++++++++

Pars distalis, adenoma

Pars intermedia, adenoma X

Thyroid gland +++++++++++++++++++++++++

General Body System

None

Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 100 mg/kg

7777777777777777777777777

Number of Days on Study 3333333333333333333333333

0000000000111111111111111

2222222222222222222222222 Total

Carcass ID Number 5667888999556667778889999 Tissues/

7018049034182386791351589 Tumors

Alimentary System

Esophagus +++++++++++++++++++++++++ 50

Gallbladder +++++++++M+++++++++++++++ 49

Intestine large, colon +++++++++++++++++++++++++ 50

Intestine large, rectum +++++++++++++++++++++++++ 50

Intestine large, cecum +++++++++++++++++++++++++ 50

Intestine small, duodenum +++++++++++++++++++++++++ 50

Intestine small, jejunum +++++++++++++++++++++++++ 50

Carcinoma 1

Intestine small, ileum +++++++++++++++++++++++++ 50

Liver +++++++++++++++++++++++++ 50

Hemangiosarcoma 1

Hepatocellular carcinoma X X X 8

Hepatocellular adenoma X X X X X X X X 15

Hepatocellular adenoma, multiple X X 3

Histiocytic sarcoma X X 5

Mesentery ++ 6

Oral mucosa + + + ++++++ ++ +++ + 26

Pancreas +++++++++++++++++++++++++ 50

Histiocytic sarcoma 1

Salivary glands +++++++++++++++++++++++++ 50

Stomach, forestomach +++++++++++++++++++++++++ 50

Stomach, glandular +++++++++++++++++++++++++ 50

Cardiovascular System

Blood vessel +++++++++++++++++++++++++ 50

Heart +++++++++++++++++++++++++ 50

Histiocytic sarcoma 2

Endocrine System

Adrenal cortex +++++++++++++++++++++++++ 50

Carcinoma X 1

Histiocytic sarcoma 1

Adrenal medulla +++++++++++++++++++++++++ 50

Islets, pancreatic +++++++++++++++++++++++++ 50

Adenoma X 1

Parathyroid gland +++++++M++++++++++++++M++ 41

Pituitary gland +++++++++++++++++++++++++ 50

Pars distalis, adenoma X X X X X 5

Pars intermedia, adenoma X X 3

Thyroid gland +++++++++++++++++++++++++ 50

General Body System

None

100 Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 100 mg/kg

2245566666666667777777777

Number of Days on Study 2341901666777890222222233

1940410555444688999999900

2222222222222232222222222

Carcass ID Number 9685785677899708556667755

7526469735726108344690225

Genital System

Clitoral gland + + + M +++++++++++++++++++++

Ovary +++++++++++++++++++++++++

Cystadenoma X

Histiocytic sarcoma X X

Luteoma X

Uterus +++++++++++++++++++++++++

Histiocytic sarcoma X X X

Leiomyosarcoma

Hematopoietic System

Bone marrow +++++++++++++++++++++++++

Histiocytic sarcoma X

Lymph node + + + + + + M

Iliac, histiocytic sarcoma X

Renal, histiocytic sarcoma X

Lymph node, mandibular +++++++++++++++++++++++++

Histiocytic sarcoma X

Lymph node, mesenteric +++++++++++++++++++++++++

Histiocytic sarcoma X X

Spleen +++++++++++++++++++++++++

Histiocytic sarcoma X X

Thymus +++++++M+++++++++++++++++

Histiocytic sarcoma X

Integumentary System

Mammary gland +++++++++++++++++++++++++

Skin +++++++++++++++++++++++++

Subcutaneous tissue, fibrosarcoma X

Musculoskeletal System

Bone +++++++++++++++++++++++++

Osteosarcoma X

Skeletal muscle + +

Rhabdomyosarcoma X X

Nervous System

Brain +++++++++++++++++++++++++

Respiratory System

Lung +++++++++++++++++++++++++

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma

Hepatocellular carcinoma, metastatic, liver X

Histiocytic sarcoma X X X

Osteosarcoma, metastatic, bone X

Nose +++++++++++++++++++++++++

Trachea +++++++++++++++++++++++++

101

Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 100 mg/kg

7777777777777777777777777

Number of Days on Study 3333333333333333333333333

0000000000111111111111111

2222222222222222222222222 Total

Carcass ID Number 5667888999556667778889999 Tissues/

7018049034182386791351589 Tumors

Genital System

Clitoral gland +++++++++++++++++++++++++ 49

Ovary +++++++++++++++++++++++++ 50

Cystadenoma 1

Histiocytic sarcoma 2

Luteoma 1

Uterus +++++++++++++++++++++++++ 50

Histiocytic sarcoma 3

Leiomyosarcoma X 1

Hematopoietic System

Bone marrow +++++++++++++++++++++++++ 50

Histiocytic sarcoma 1

Lymph node + 7

Iliac, histiocytic sarcoma 1

Renal, histiocytic sarcoma 1

Lymph node, mandibular +++++++++++++++++++++++++ 50

Histiocytic sarcoma 1

Lymph node, mesenteric ++++++++++ M ++++++++++++++ 49

Histiocytic sarcoma X 3

Spleen +++++++++++++++++++++++++ 50

Histiocytic sarcoma X 3

Thymus +++++++++++++++++++++++++ 49

Histiocytic sarcoma 1

Integumentary System

Mammary gland +++++++++++++++++++++++++ 50

Skin +++++++++++++++++++++++++ 50

Subcutaneous tissue, fibrosarcoma 1

Musculoskeletal System

Bone +++++++++++++++++++++++++ 50

Osteosarcoma 1

Skeletal muscle 2

Rhabdomyosarcoma 2

Nervous System

Brain +++++++++++++++++++++++++ 50

Respiratory System

Lung +++++++++++++++++++++++++ 50

Alveolar/bronchiolar adenoma X 1

Alveolar/bronchiolar carcinoma X X 2

Hepatocellular carcinoma, metastatic, liver X X 3

Histiocytic sarcoma X 4

Osteosarcoma, metastatic, bone 1

Nose +++++++++++++++++++++++++ 50

Trachea +++++++++++++++++++++++++ 50

102 Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 100 mg/kg

2245566666666667777777777

Number of Days on Study 2341901666777890222222233

1940410555444688999999900

2222222222222232222222222

Carcass ID Number 9685785677899708556667755

7526469735726108344690225

Special Senses System

Eye +++++++++++++++++++++++++

Harderian gland +++++++++++++++++++++++++

Adenoma X X X X

Urinary System

Kidney +++++++++++++++++++++++++

Histiocytic sarcoma X X X

Urinary bladder +++++++++++++++++++++++++

Systemic Lesions

Multiple organs +++++++++++++++++++++++++

Histiocytic sarcoma X X X

Lymphoma malignant X X X X X X

103

Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 100 mg/kg

7777777777777777777777777

Number of Days on Study 3333333333333333333333333

0000000000111111111111111

2222222222222222222222222 Total

Carcass ID Number 5667888999556667778889999 Tissues/

7018049034182386791351589 Tumors

Special Senses System

Eye +++++++++++++++++++++++++ 50

Harderian gland +++++++++++++++++++++++++ 50

Adenoma X 5

Urinary System

Kidney +++++++++++++++++++++++++ 50

Histiocytic sarcoma 3

Urinary bladder +++++++++++++++++++++++++ 50

Systemic Lesions

Multiple organs +++++++++++++++++++++++++ 50

Histiocytic sarcoma X X 5

Lymphoma malignant X X X 9

104 Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 300 mg/kg

0344555667777777777777777

Number of Days on Study 6344788792222222333333333

2847316429999999000000000

3333333333333333333333333

Carcass ID Number 0412214030000344000111122

5916723771348447269457905

Alimentary System

Esophagus +++++++++++++++++++++++++

Gallbladder +++++++++++++++++++++++++

Intestine large, colon +++++++++++++++++++++++++

Intestine large, rectum +++++++++++++++++++++++++

Intestine large, cecum +++++++++++++++++++++++++

Intestine small, duodenum +++++++++++++++++++++++++

Intestine small, jejunum +++++++++++++++++++++++++

Polyp adenomatous

Intestine small, ileum +++++++++++++++++++++++++

Liver +++++++++++++++++++++++++

Hepatocellular carcinoma X

Hepatocellular adenoma X X X X X X X

Hepatocellular adenoma, multiple X X X

Mesentery +

Liposarcoma X

Oral mucosa + +++++++ ++++ +++

Pancreas +++++++++++++++++++++++++

Salivary glands +++++++++++++++++++++++++

Stomach, forestomach +++++++++++++++++++++++++

Squamous cell papilloma X X

Squamous cell papilloma, multiple

Stomach, glandular +++++++++++++++++++++++++

Tooth

Cardiovascular System

Blood vessel +++++++++++++++++++++++++

Heart +++++++++++++++++++++++++

Endocrine System

Adrenal cortex +++++++++++++++++++++++++

Adrenal medulla +++++++++++++++++++++++++

Pheochromocytoma benign

Islets, pancreatic +++++++++++++++++++++++++

Adenoma X

Parathyroid gland M +++++++++++M+++++M++++++

Pituitary gland +++++++++++++++++++++++++

Pars distalis, adenoma X X

Pars intermedia, adenoma

Thyroid gland +++++++++++++++++++++++++

Follicle, adenoma X

General Body System

None

105

Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 300 mg/kg

7777777777777777777777777

Number of Days on Study 3333333333333333333333333

0000000011111111111111111

3333333333333333333333333 Total

Carcass ID Number 2233334411112222333344445 Tissues/

8901282603681234356901580 Tumors

Alimentary System

Esophagus +++++++++++++++++++++++++ 50

Gallbladder + + M +++++++++++++M++++++++ 48

Intestine large, colon +++++++++++++++++++++++++ 50

Intestine large, rectum +++++++++++++++++++++++++ 50

Intestine large, cecum +++++++++++++++++++++++++ 50

Intestine small, duodenum +++++++++++++++++++++++++ 50

Intestine small, jejunum +++++++++++++++++++++++++ 50

Polyp adenomatous X 1

Intestine small, ileum +++++++++++++++++++++++++ 50

Liver +++++++++++++++++++++++++ 50

Hepatocellular carcinoma X X X 4

Hepatocellular adenoma X X X X X X 13

Hepatocellular adenoma, multiple X X X X 7

Mesentery + + + 4

Liposarcoma 1

Oral mucosa +++++ ++ + ++ + +++++ 31

Pancreas +++++++++++++++++++++++++ 50

Salivary glands +++++++++++++++++++++++++ 50

Stomach, forestomach +++++++++++++++++++++++++ 50

Squamous cell papilloma 2

Squamous cell papilloma, multiple X 1

Stomach, glandular +++++++++++++++++++++++++ 50

Tooth + 1

Cardiovascular System

Blood vessel +++++++++++++++++++++++++ 50

Heart +++++++++++++++++++++++++ 50

Endocrine System

Adrenal cortex +++++++++++++++++++++++++ 50

Adrenal medulla +++++++++++++++++++++++++ 50

Pheochromocytoma benign X 1

Islets, pancreatic +++++++++++++++++++++++++ 50

Adenoma 1

Parathyroid gland ++++++++++++M+++++++++++M 45

Pituitary gland +++++++++++++++++++++++++ 50

Pars distalis, adenoma X X X 5

Pars intermedia, adenoma X 1

Thyroid gland +++++++++++++++++++++++++ 50

Follicle, adenoma 1

General Body System

None

106 Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 300 mg/kg

0344555667777777777777777

Number of Days on Study 6344788792222222333333333

2847316429999999000000000

Carcass ID Number

3333333333333333333333333

0412214030000344000111122

5916723771348447269457905

Genital System

Clitoral gland

Ovary

Uterus

Polyp stromal

+ + M ++++++++++++++++++++++

+++++++++++++++++++++++++

Hematopoietic System

Bone marrow

Lymph node

Mediastinal, liposarcoma, metastatic, mesentery

Lymph node, mandibular

Lymph node, mesenteric

Spleen

Hemangiosarcoma

Thymus

+++++++++++++++++++++++++

+ +

+++++++++++++++++++++++++

M ++++++++++++++++++++++++

++++++++++++M++++++++++++

Integumentary System

Mammary gland

Skin

Subcutaneous tissue, fibrosarcoma

+++++++++++++++++++++++++

X X

Musculoskeletal System

Bone

Skeletal muscle

Hemangiosarcoma, metastatic, spleen

+++++++++++++++++++++++++

Nervous System

Brain

Peripheral nerve

Spinal cord

+++++++++++++++++++++++++

Respiratory System

Lung

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma

Liposarcoma, metastatic, mesentery

Nose

Trachea

+++++++++++++++++++++++++

X X

+++++++++++++++++++++++++

Special Senses System

Eye

Harderian gland

Adenoma

+++++++++++++++++++++++++

107

Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 300 mg/kg

7777777777777777777777777

Number of Days on Study 3333333333333333333333333

0000000011111111111111111

Carcass ID Number

3333333333333333333333333

2233334411112222333344445

8901282603681234356901580

Total

Tissues/

Tumors

Genital System

Clitoral gland

Ovary

Uterus

Polyp stromal

+++++++++++++++++++++++++

X X X

Hematopoietic System

Bone marrow

Lymph node

Mediastinal, liposarcoma, metastatic, mesentery

Lymph node, mandibular

Lymph node, mesenteric

Spleen

Hemangiosarcoma

Thymus

+++++++++++++++++++++++++

+ +

+++++++++++++++++++++++++

++++++M++++++++++++++++++

+++++++++++++++++++++++++

Integumentary System

Mammary gland

Skin

Subcutaneous tissue, fibrosarcoma

+++++++++++++++++++++++++

Musculoskeletal System

Bone

Skeletal muscle

Hemangiosarcoma, metastatic, spleen

+++++++++++++++++++++++++ 50

Nervous System

Brain

Peripheral nerve

Spinal cord

+++++++++++++++++++++M+++ 49

Respiratory System

Lung

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma

Liposarcoma, metastatic, mesentery

Nose

Trachea

+++++++++++++++++++++++++

X X

+++++++++++++++++++++++++

Special Senses System

Eye

Harderian gland

Adenoma

+++++++++++++++++++++++++

X X

108 Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 300 mg/kg

0344555667777777777777777

Number of Days on Study 6344788792222222333333333

2847316429999999000000000

3333333333333333333333333

Carcass ID Number 0412214030000344000111122

5916723771348447269457905

Urinary System

Kidney +++++++++++++++++++++++++

Urinary bladder +++++++++++++++++++++++++

Systemic Lesions

Multiple organs +++++++++++++++++++++++++

Lymphoma malignant X X X X

109

Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 300 mg/kg

7777777777777777777777777

Number of Days on Study 3333333333333333333333333

0000000011111111111111111

3333333333333333333333333 Total

Carcass ID Number 2233334411112222333344445 Tissues/

8901282603681234356901580 Tumors

Urinary System

Kidney +++++++++++++++++++++++++ 50

Urinary bladder + + + M +++++++++++++++++++++ 49

Systemic Lesions

Multiple organs +++++++++++++++++++++++++ 50

Lymphoma malignant X X 6

110 Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 1,000 mg/kg

0455566666677777777777777

Number of Days on Study 7126901158800011222222333

0077547935511927669999000

3333333333333333333333333

Carcass ID Number 6755965857888896795679556

5743182078537884006763180

Alimentary System

Esophagus +++++++++++++++++++++++++

Gallbladder +++++++++++++++++++++++++

Intestine large, colon +++++++++++++++++++++++++

Leiomyoma

Intestine large, rectum +++++++++++++++++++++++++

Leiomyosarcoma, metastatic, uterus X

Intestine large, cecum +++++++++++++++++++++++++

Intestine small, duodenum +++++++++++++++++++++++++

Intestine small, jejunum +++++++++++++++++++++++++

Intestine small, ileum +++++++++++++++++++++++++

Liver +++++++++++++++++++++++++

Hepatocellular carcinoma X X

Hepatocellular adenoma X X X X X X X X X

Hepatocellular adenoma, multiple X X X X X X

Histiocytic sarcoma X X

Mesentery + +

Oral mucosa + + + + + + +++++++

Pancreas +++++++++++++++++++++++++

Salivary glands +++++++++++++++++++++++++

Carcinoma X

Stomach, forestomach +++++++++++++++++++++++++

Squamous cell papilloma X X

Stomach, glandular +++++++++++++++++++++++++

Cardiovascular System

Blood vessel +++++++++++++++++++++++++

Heart +++++++++++++++++++++++++

Histiocytic sarcoma X

Endocrine System

Adrenal cortex +++++++++++++++++++++++++

Subcapsular, adenoma

Adrenal medulla +++++++++++++++++++++++++

Islets, pancreatic +++++++++++++++++++++++++

Adenoma X

Parathyroid gland +++++++++++++++++++++++++

Pituitary gland +++++++++++++++++++++++++

Pars distalis, adenoma X X

Pars distalis, carcinoma

Thyroid gland +++++++++++++++++++++++++

Follicle, adenoma X X

General Body System

None

111

Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 1,000 mg/kg

7777777777777777777777777

Number of Days on Study 3333333333333333333333333

0000000000000111111111111

3333333333333333333333334 Total

Carcass ID Number 6666777788999556778889990 Tissues/

1369235949249592141265670 Tumors

Alimentary System

Esophagus +++++++++++++++++++++++++ 50

Gallbladder +++++++++++++++++++++++++ 50

Intestine large, colon +++++++++++++++++++++++++ 50

Leiomyoma X 1

Intestine large, rectum +++++++++++++++++++++++++ 50

Leiomyosarcoma, metastatic, uterus 1

Intestine large, cecum +++++++++++++++++++++++++ 50

Intestine small, duodenum +++++++++++++++++++++++++ 50

Intestine small, jejunum +++++++++++++++++++++++++ 50

Intestine small, ileum +++++++++++++++++++++++++ 50

Liver +++++++++++++++++++++++++ 50

Hepatocellular carcinoma X X X 5

Hepatocellular adenoma X X X X X X X 16

Hepatocellular adenoma, multiple X X X X X X X X X X X 17

Histiocytic sarcoma X 3

Mesentery + + + + + + + 9

Oral mucosa + + + + + + + +++++++++ ++ 31

Pancreas +++++++++++++++++++++++++ 50

Salivary glands +++++++++++++++++++++++++ 50

Carcinoma 1

Stomach, forestomach +++++++++++++++++++++++++ 50

Squamous cell papilloma X X 4

Stomach, glandular +++++++++++++++++++++++++ 50

Cardiovascular System

Blood vessel +++++++++++++++++++++++++ 50

Heart +++++++++++++++++++++++++ 50

Histiocytic sarcoma 1

Endocrine System

Adrenal cortex +++++++++++++++++++++++++ 50

Subcapsular, adenoma X 1

Adrenal medulla +++++++++++++++++++++++++ 50

Islets, pancreatic +++++++++++++++++++++++++ 50

Adenoma 1

Parathyroid gland + + M ++++++++++++M+++++MM++ 46

Pituitary gland +++++++++++++++++++++++++ 50

Pars distalis, adenoma X X X 5

Pars distalis, carcinoma X 1

Thyroid gland +++++++++++++++++++++++++ 50

Follicle, adenoma X 3

General Body System

None

112 Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 1,000 mg/kg

0455566666677777777777777

Number of Days on Study 7126901158800011222222333

0077547935511927669999000

3333333333333333333333333

Carcass ID Number 6755965857888896795679556

5743182078537884006763180

Genital System

Clitoral gland ++++M++++++++++++++++++++

Ovary +++++++++++++++++++++++++

Cystadenoma X

Granulosa cell tumor malignant X

Histiocytic sarcoma X

Teratoma benign X

Tubulostromal adenoma

Uterus +++++++++++++++++++++++++

Histiocytic sarcoma X

Leiomyosarcoma X X

Polyp stromal X

Vagina +

Leiomyosarcoma, metastatic, uterus X

Hematopoietic System

Bone marrow +++++++++++++++++++++++++

Histiocytic sarcoma X

Lymph node + + +

Lymph node, mandibular +++++++++++++++++++++++++

Lymph node, mesenteric +++++++++M+++++M+++++++++

Spleen +++++++++++++++++++++++++

Hemangiosarcoma

Thymus +++++++++++++++++++++++++

Integumentary System

Mammary gland +++++++++++++++++++++++++

Skin +++++++++++++++++++++++++

Subcutaneous tissue, fibrosarcoma X

Subcutaneous tissue, hemangiosarcoma X

Subcutaneous tissue, histiocytic sarcoma X

Musculoskeletal System

Bone +++++++++++++++++++++++++

Skeletal muscle + +

Rhabdomyosarcoma X

Nervous System

Brain +++++++++++++++++++++++++

Histiocytic sarcoma X

Respiratory System

Lung +++++++++++++++++++++++++

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma

Hepatocellular carcinoma, metastatic, liver X X

Histiocytic sarcoma X

Nose +++++++++++++++++++++++++

Trachea +++++++++++++++++++++++++

113

Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 1,000 mg/kg

7777777777777777777777777

Number of Days on Study 3333333333333333333333333

0000000000000111111111111

3333333333333333333333334 Total

Carcass ID Number 6666777788999556778889990 Tissues/

1369235949249592141265670 Tumors

Genital System

Clitoral gland +++++++++++++++++++++++++ 49

Ovary +++++++++++++++++++++++++ 50

Cystadenoma 1

Granulosa cell tumor malignant 1

Histiocytic sarcoma 1

Teratoma benign 1

Tubulostromal adenoma X 1

Uterus +++++++++++++++++++++++++ 50

Histiocytic sarcoma 1

Leiomyosarcoma 2

Polyp stromal 1

Vagina 1

Leiomyosarcoma, metastatic, uterus 1

Hematopoietic System

Bone marrow +++++++++++++++++++++++++ 50

Histiocytic sarcoma 1

Lymph node + + + 6

Lymph node, mandibular +++++++++++++++++++++++++ 50

Lymph node, mesenteric +++++++++++++++++++++++++ 48

Spleen +++++++++++++++++++++++++ 50

Hemangiosarcoma X 1

Thymus ++++++++++++M++++++++++++ 49

Integumentary System

Mammary gland +++++++++++++++++++++++++ 50

Skin +++++++++++++++++++++++++ 50

Subcutaneous tissue, fibrosarcoma 1

Subcutaneous tissue, hemangiosarcoma 1

Subcutaneous tissue, histiocytic sarcoma 1

Musculoskeletal System

Bone +++++++++++++++++++++++++ 50

Skeletal muscle + 3

Rhabdomyosarcoma X 2

Nervous System

Brain +++++++++++++++++++++++++ 50

Histiocytic sarcoma 1

Respiratory System

Lung +++++++++++++++++++++++++ 50

Alveolar/bronchiolar adenoma X 1

Alveolar/bronchiolar carcinoma X 1

Hepatocellular carcinoma, metastatic, liver 2

Histiocytic sarcoma 1

Nose +++++++++++++++++++++++++ 50

Trachea +++++++++++++++++++++++++ 50

114 Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 1,000 mg/kg

0455566666677777777777777

Number of Days on Study 7126901158800011222222333

0077547935511927669999000

3333333333333333333333333

Carcass ID Number 6755965857888896795679556

5743182078537884006763180

Special Senses System

Eye +++++++++++++++++++++++++

Harderian gland +++++++++++++++++++++++++

Adenoma X

Urinary System

Kidney +++++++++++++++++++++++++

Renal tubule, adenoma X

Urinary bladder ++++M++++++++++++++++++++

Systemic Lesions

Multiple organs +++++++++++++++++++++++++

Histiocytic sarcoma X X

Lymphoma malignant X X X X

115

Triethanolamine, NTP TR 518

ABLE B2

Individual Animal Tumor Pathology of Female Mice in the 2-Year Dermal Study of Triethanolamine: 1,000 mg/kg

7777777777777777777777777

Number of Days on Study 3333333333333333333333333

0000000000000111111111111

3333333333333333333333334 Total

Carcass ID Number 6666777788999556778889990 Tissues/

1369235949249592141265670 Tumors

Special Senses System

Eye +++++++++++++++++++++++++ 50

Harderian gland +++++++++++++++++++++++++ 50

Adenoma XX 3

Urinary System

Kidney +++++++++++++++++++++++++ 50

Renal tubule, adenoma 1

Urinary bladder +++++++++++++++++++++++++ 49

Systemic Lesions

Multiple organs +++++++++++++++++++++++++ 50

Histiocytic sarcoma X 3

Lymphoma malignant X X X X 8

116 Triethanolamine, NTP TR 518

ABLE B3

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 100 mg/kg 300 mg/kg 1,000 mg/kg

Harderian Gland: Adenoma

Overall rate

Adjusted rate

Terminal rate

5/50 (10%)

11.3%

4/35 (11%)

5/50 (10%)

11.3%

3/34 (9%)

3/50 (6%)

6.7%

3/41 (7%)

3/50 (6%)

6.7%

2/32 (6%)

First incidence (days)

Poly-3 test

666

P=0.279N

594

P=0.628

729 (T)

P=0.353N

527

P=0.350N

Harderian Gland: Adenoma or Carcinoma

Overall rate 7/50 (14%) 5/50 (10%) 3/50 (6%) 3/50 (6%)

Adjusted rate 15.4% 11.3% 6.7% 6.7%

Terminal rate 4/35 (11%) 3/34 (9%) 3/41 (7%) 2/32 (6%)

First incidence (days) 532 594 729 (T) 527

Poly-3 test P=0.161N P=0.397N P=0.164N P=0.162N

Liver: Hepatocellular Adenoma

Overall rate 9/50 (18%) 18/50 (36%) 20/50 (40%) 33/50 (66%)

Adjusted rate 19.9% 41.0% 43.5% 72.4%

Terminal rate 6/35 (17%) 16/34 (47%) 18/41 (44%) 25/32 (78%)

First incidence (days) 617 665 444 604

Poly-3 test P<0.001 P=0.024 P=0.012 P<0.001

Liver: Hepatocellular Carcinoma

Overall rate 6/50 (12%) 8/50 (16%) 4/50 (8%) 5/50 (10%)

Adjusted rate 13.3% 17.9% 8.9% 11.3%

Terminal rate 3/35 (9%) 4/34 (12%) 3/41 (7%) 4/32 (13%)

First incidence (days) 595 601 586 701

Poly-3 test P=0.354N P=0.382 P=0.367N P=0.509N

Liver: Hepatocellular Adenoma or Carcinoma

Overall rate 12/50 (24%) 23/50 (46%) 24/50 (48%) 34/50 (68%)

Adjusted rate 26.3% 51.0% 51.7% 74.6%

Terminal rate 7/35 (20%) 17/34 (50%) 21/41 (51%) 26/32 (81%)

First incidence (days) 595 601 444 604

Poly-3 test P<0.001 P=0.011 P=0.009 P<0.001

Lung: Alveolar/bronchiolar Adenoma

Overall rate 3/50 (6%) 1/50 (2%) 2/50 (4%) 1/50 (2%)

Adjusted rate 6.8% 2.3% 4.5% 2.3%

Terminal rate 3/35 (9%) 1/34 (3%) 2/41 (5%) 1/32 (3%)

First incidence (days) 729 (T) 729 (T) 729 (T) 729 (T)

Poly-3 test P=0.344N P=0.310N P=0.495N P=0.304N

Lung: Alveolar/bronchiolar Carcinoma

Overall rate 2/50 (4%) 2/50 (4%) 3/50 (6%) 1/50 (2%)

Adjusted rate 4.5% 4.6% 6.7% 2.3%

Terminal rate 2/35 (6%) 2/34 (6%) 3/41 (7%) 1/32 (3%)

First incidence (days) 729 (T) 729 (T) 729 (T) 729 (T)

Poly-3 test P=0.370N P=0.689 P=0.505 P=0.499N

Lung: Alveolar/bronchiolar Adenoma or Carcinoma

Overall rate 5/50 (10%) 3/50 (6%) 4/50 (8%) 2/50 (4%)

Adjusted rate 11.3% 6.9% 9.0% 4.5%

Terminal rate 5/35 (14%) 3/34 (9%) 4/41 (10%) 2/32 (6%)

First incidence (days) 729 (T) 729 (T) 729 (T) 729 (T)

Poly-3 test P=0.224N P=0.364N P=0.493N P=0.215N

117

Triethanolamine, NTP TR 518

ABLE B3

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 100 mg/kg 300 mg/kg 1,000 mg/kg

Pituitary Gland (Pars Distalis): Adenoma

Overall rate 8/50 (16%) 5/50 (10%) 5/50 (10%) 5/50 (10%)

Adjusted rate 17.8% 11.5% 11.2% 11.3%

Terminal rate 6/35 (17%) 5/34 (15%) 5/41 (12%) 5/32 (16%)

First incidence (days) 619 729 (T) 729 (T) 729 (T)

Poly-3 test P=0.344N P=0.294N P=0.277N P=0.283N

Pituitary Gland (Pars Distalis): Adenoma or Carcinoma

Overall rate 8/50 (16%) 5/50 (10%) 5/50 (10%) 6/50 (12%)

Adjusted rate 17.8% 11.5% 11.2% 13.6%

Terminal rate 6/35 (17%) 5/34 (15%) 5/41 (12%) 6/32 (19%)

First incidence (days) 619 729 (T) 729 (T) 729 (T)

Poly-3 test P=0.488N P=0.294N P=0.277N P=0.396N

Pituitary Gland (Pars Intermedia): Adenoma

Overall rate 0/50 (0%) 3/50 (6%) 1/50 (2%) 0/50 (0%)

Adjusted rate 0.0% 6.9% 2.2% 0.0%

Terminal rate

First incidence (days)

Poly-3 test

0/35 (0%)

—

P=0.280N

3/34 (9%)

729 (T)

P=0.116

1/41 (2%)

729 (T)

P=0.502

0/32 (0%)

—

Stomach (Forestomach): Squamous Cell Papilloma

Overall rate 2/50 (4%) 0/50 (0%) 3/50 (6%) 4/50 (8%)

Adjusted rate 4.5% 0.0% 6.7% 9.0%

Terminal rate 2/35 (6%) 0/34 (0%) 3/41 (7%) 3/32 (9%)

First incidence (days) 729 (T) — 729 (T) 712

Poly-3 test P=0.113 P=0.240N P=0.505 P=0.338

Thyroid Gland (Follicular Cell): Adenoma

Overall rate 0/50 (0%) 0/50 (0%) 1/50 (2%) 3/50 (6%)

Adjusted rate 0.0% 0.0% 2.2% 6.7%

Terminal rate 0/35 (0%) 0/34 (0%) 0/41 (0%) 1/32 (3%)

First incidence (days) — — 692 653

Poly-3 test P=0.023 — P=0.503 P=0.120

Thyroid Gland (Follicular Cell): Adenoma or Carcinoma

Overall rate 1/50 (2%) 0/50 (0%) 1/50 (2%) 3/50 (6%)

Adjusted rate 2.3% 0.0% 2.2% 6.7%

Terminal rate 1/35 (3%) 0/34 (0%) 0/41 (0%) 1/32 (3%)

First incidence (days) 729 (T) — 692 653

Poly-3 test P=0.084 P=0.503N P=0.757N P=0.309

Uterus: Stromal Polyp

Overall rate 1/50 (2%) 0/50 (0%) 3/50 (6%) 1/50 (2%)

Adjusted rate 2.3% 0.0% 6.7% 2.2%

Terminal rate 1/35 (3%) 0/34 (0%) 3/41 (7%) 0/32 (0%)

First incidence (days) 729 (T) — 729 (T) 619

Poly-3 test P=0.602 P=0.503N P=0.309 P=0.758N

All Organs: Hemangiosarcoma

Overall rate 3/50 (6%) 1/50 (2%) 1/50 (2%) 2/50 (4%)

Adjusted rate 6.7% 2.3% 2.2% 4.5%

Terminal rate 2/35 (6%) 1/34 (3%) 1/41 (2%) 1/32 (3%)

First incidence (days) 619 729 (T) 729 (T) 717

Poly-3 test P=0.613N P=0.313N P=0.304N P=0.503N

118 Triethanolamine, NTP TR 518

ABLE B3

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 100 mg/kg 300 mg/kg 1,000 mg/kg

All Organs: Hemangioma or Hemangiosarcoma

Overall rate 4/50 (8%) 1/50 (2%) 1/50 (2%) 2/50 (4%)

Adjusted rate 9.0% 2.3% 2.2% 4.5%

Terminal rate 3/35 (9%) 1/34 (3%) 1/41 (2%) 1/32 (3%)

First incidence (days) 619 729 (T) 729 (T) 717

Poly-3 test P=0.477N P=0.185N P=0.178N P=0.340N

All Organs: Histiocytic Sarcoma

Overall rate 4/50 (8%) 5/50 (10%) 0/50 (0%) 3/50 (6%)

Adjusted rate 8.8% 11.1% 0.0% 6.7%

Terminal rate 0/35 (0%) 2/34 (6%) 0/41 (0%) 1/32 (3%)

First incidence (days) 617 444 — 617

Poly-3 test P=0.396N P=0.495 P=0.063N P=0.506N

All Organs: Malignant Lymphoma

Overall rate 8/50 (16%) 9/50 (18%) 6/50 (12%) 8/50 (16%)

Adjusted rate 18.1% 20.3% 13.2% 17.8%

Terminal rate 8/35 (23%) 6/34 (18%) 3/41 (7%) 6/32 (19%)

First incidence (days) 729 (T) 610 586 567

Poly-3 test P=0.530N P=0.504 P=0.363N P=0.595N

All Organs: Benign Neoplasms

Overall rate 24/50 (48%) 28/50 (56%) 27/50 (54%) 38/50 (76%)

Adjusted rate 52.7% 62.6% 58.5% 79.8%

Terminal rate 20/35 (57%) 23/34 (68%) 24/41 (59%) 27/32 (84%)

First incidence (days) 617 594 444 70

Poly-3 test P=0.003 P=0.226 P=0.362 P=0.003

All Organs: Malignant Neoplasms

Overall rate 27/50 (54%) 26/50 (52%) 16/50 (32%) 21/50 (42%)

Adjusted rate 56.2% 54.5% 34.7% 45.1%

Terminal rate 16/35 (46%) 14/34 (41%) 12/41 (29%) 12/32 (38%)

First incidence (days) 532 444 573 567

Poly-3 test P=0.179N P=0.514N P=0.027N P=0.188N

All Organs: Benign or Malignant

Overall rate 38/50 (76%) 41/50 (82%) 35/50 (70%) 44/50 (88%)

Adjusted rate 79.2% 85.3% 74.0% 89.7%

Terminal rate 27/35 (77%) 27/34 (79%) 29/41 (71%) 29/32 (91%)

First incidence (days) 532 444 444 70

Poly-3 test P=0.122 P=0.300 P=0.359N P=0.119

(T)Terminal sacrifice

Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for liver, lung,

pituitary gland, and thyroid gland; for other tissues, denominator is number of animals necropsied.

Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality

Observed incidence at terminal kill

Beneath the vehicle control incidence is the P value associated with the trend test. Beneath the dosed group incidence are the P values corresponding to

pairwise comparisons between the vehicle controls and that dosed group. The Poly-3 test accounts for the differential mortality in animals that do not reach

terminal sacrifice. A negative trend or a lower incidence in a dosed group is indicated by N.

Not applicable; no neoplasms in animal group

Value of statistic cannot be computed

119

Triethanolamine, NTP TR 518

TABLE B4

Historical Incidence of Liver Neoplasms in Control Female B6C3F

Mice

Incidence in Controls

Hepatocellular Hepatocellular Hepatocellular

Adenoma Carcinoma Adenoma

Study or Carcinoma

Historical Incidence in Controls Given NTP-2000 Diet

Acrylonitrile (gavage) 14/50 7/50 20/50

trans-Cinnamaldehyde (feed) 7/99 3/99 9/99

Citral (feed) 8/99 4/99 12/99

Decalin (inhalation) 7/49 4/49 11/49

p,pN-Dichlorodiphenyl sulfone (feed) 4/50 3/50 6/50

Dipropylene glycol (drinking water) 11/50 7/50 17/50

Elmiron

(gavage) 7/50 3/50 10/50

2,4-Hexadienal (gavage) 11/50 3/50 13/50

Indium phosphide (inhalation) 12/50 6/50 18/50

60-Hz Magnetic fields (whole body exposure) 17/98 6/98 22/98

Methacrylonitrile (gavage) 9/50 2/50 10/50

2-Methylimidazole (feed) 3/50 2/50 4/50

o-Nitrotoluene (feed) 7/60 2/60 9/60

p-Nitrotoluene (feed) 6/49 3/49 8/49

Propylene glycol mono-t-butyl ether (inhalation) 14/49 4/49 18/49

Riddelliine (gavage) 9/49 8/49 16/49

Sodium nitrite (drinking water) 9/50 2/50 10/50

Stoddard solvent (Type IIC) (inhalation) 9/50 6/50 13/50

Triethanolamine (dermal 9/50 6/50 12/50

Vanadium pentoxide (inhalation) 6/50 6/50 12/50

Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 179/1,152 (15.5%) 87/1,152 (7.6%) 250/1,152 (21.7%)

Mean ± standard deviation 16.3% ± 6.6% 8.1% ± 4.2% 22.8% ± 9.4%

Range 6%-29% 3%-14% 8%-40%

Data as of March 3, 2003

120 Triethanolamine, NTP TR 518

ABLE B5

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 100 mg/kg 300 mg/kg 1,000 mg/kg

Disposition Summary

Animals initially in study 50 50 50 50

Early deaths

Accidental death 1

Moribund 8 10 5 10

Natural deaths 7 6 4 7

Survivors

Died last week of study 1

Terminal sacrifice 34 34 41 32

Animals examined microscopically 50 50 50 50

Alimentary System

Intestine large, cecum (50) (50) (50) (50)

Serosa, inflammation, chronic 1 (2%)

Intestine small, duodenum (50) (50) (50) (50)

Epithelium, necrosis 1 (2%)

Intestine small, jejunum (50) (50) (50) (50)

Diverticulum 1 (2%)

Ulcer 1 (2%)

Peyer’s patch, hyperplasia 1 (2%)

Peyer’s patch, inflammation, suppurative 1 (2%)

Serosa, inflammation, granulomatous 1 (2%)

Liver (50) (50) (50) (50)

Angiectasis, focal 1 (2%)

Basophilic focus 4 (8%) 3 (6%) 3 (6%)

Clear cell focus 4 (8%) 3 (6%) 3 (6%) 5 (10%)

Eosinophilic focus 16 (32%) 22 (44%) 28 (56%) 32 (64%)

Fibrosis, focal 1 (2%)

Hematopoietic cell proliferation 4 (8%) 3 (6%) 3 (6%) 4 (8%)

Hepatodiaphragmatic nodule 2 (4%)

Infiltration cellular, lymphoid 26 (52%) 30 (60%) 36 (72%) 20 (40%)

Inflammation, chronic 14 (28%) 27 (54%) 22 (44%) 15 (30%)

Mixed cell focus 5 (10%) 8 (16%) 14 (28%) 11 (22%)

Necrosis, focal 5 (10%) 1 (2%) 3 (6%)

Vacuolization cytoplasmic, focal 8 (16%) 1 (2%) 6 (12%) 6 (12%)

Centrilobular, cytomegaly, diffuse 1 (2%)

Centrilobular, necrosis 1 (2%) 1 (2%)

Mesentery (11) (6) (4) (9)

Inflammation, chronic 1 (9%)

Fat, necrosis 10 (91%) 6 (100%) 3 (75%) 9 (100%)

Lymphatic, angiectasis 1 (9%)

Oral mucosa (26) (26) (31) (31)

Inflammation, chronic 26 (100%) 26 (100%) 31 (100%) 31 (100%)

Pancreas (50) (50) (50) (50)

Acinus, atrophy 1 (2%) 3 (6%) 1 (2%)

Acinus, hyperplasia 1 (2%)

Duct, cyst 1 (2%) 2 (4%) 1 (2%)

Number of animals examined microscopically at the site and the number of animals with lesion

121

Triethanolamine, NTP TR 518

TABLE B5

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 100 mg/kg 300 mg/kg 1,000 mg/kg

Alimentary System (continued)

Stomach, forestomach (50) (50) (50) (50)

Inflammation, chronic 1 (2%) 1 (2%)

Inflammation, suppurative 1 (2%)

Ulcer 1 (2%) 2 (4%)

Epithelium, cyst 1 (2%)

Epithelium, hyperplasia, focal 1 (2%) 1 (2%)

Epithelium, mineralization 1 (2%)

Stomach, glandular (50) (50) (50) (50)

Inflammation, acute 1 (2%)

Ulcer 1 (2%) 1 (2%)

Epithelium, cyst 1 (2%) 1 (2%)

Epithelium, hyperplasia, focal 1 (2%) 1 (2%) 1 (2%)

Serosa, inflammation, chronic 1 (2%)

Tooth (1)

Inflammation, chronic 1 (100%)

Cardiovascular System

Blood vessel (50) (50) (50) (50)

Aorta, inflammation, chronic 2 (4%)

Aorta, mineralization 1 (2%) 2 (4%)

Heart (50) (50) (50) (50)

Artery, inflammation, chronic 3 (6%) 1 (2%) 2 (4%) 1 (2%)

Atrium, thrombosis 2 (4%)

Myocardium, degeneration 4 (8%) 1 (2%)

Myocardium, inflammation, acute 1 (2%)

Myocardium, inflammation, chronic 1 (2%)

Myocardium, mineralization 1 (2%) 2 (4%)

Myocardium, necrosis 1 (2%)

Pericardium, inflammation, chronic 1 (2%)

Valve, inflammation 1 (2%)

Endocrine System

Adrenal cortex (50) (50) (50) (50)

Hematopoietic cell proliferation 1 (2%)

Hyperplasia, focal 1 (2%) 1 (2%)

Hypertrophy, focal 2 (4%) 1 (2%) 1 (2%)

Inflammation, suppurative 1 (2%)

Subcapsular, hyperplasia 49 (98%) 49 (98%) 50 (100%) 50 (100%)

Adrenal medulla (49) (50) (50) (50)

Hyperplasia, focal 1 (2%) 1 (2%)

Infiltration cellular, histiocyte 1 (2%)

Islets, pancreatic (50) (50) (50) (50)

Hyperplasia 25 (50%) 18 (36%) 20 (40%) 18 (36%)

Parathyroid gland (39) (41) (45) (46)

Cyst 1 (2%)

Pituitary gland (50) (50) (50) (50)

Angiectasis 1 (2%)

Pars distalis, hyperplasia 11 (22%) 3 (6%) 9 (18%) 12 (24%)

Pars intermedia, hyperplasia 1 (2%)

Thyroid gland (50) (50) (50) (50)

Inflammation, granulomatous 1 (2%) 1 (2%)

C-cell, hyperplasia 2 (4%)

Follicle, cyst 1 (2%) 1 (2%) 2 (4%) 1 (2%)

Follicular cell, necrosis 1 (2%)

122 Triethanolamine, NTP TR 518

ABLE B5

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 100 mg/kg 300 mg/kg 1,000 mg/kg

General Body System

None

Genital System

Clitoral gland (50) (49) (49) (49)

Cyst 2 (4%) 1 (2%)

Inflammation, suppurative 1 (2%)

Ovary (50) (50) (50) (50)

Angiectasis 2 (4%) 2 (4%)

Cyst 27 (54%) 22 (44%) 21 (42%) 25 (50%)

Mineralization 1 (2%)

Thrombosis 1 (2%) 3 (6%) 1 (2%)

Uterus (50) (50) (50) (50)

Angiectasis 1 (2%) 2 (4%) 2 (4%) 2 (4%)

Thrombosis 1 (2%) 1 (2%)

Endometrium, hyperplasia, cystic 49 (98%) 48 (96%) 47 (94%) 47 (94%)

Hematopoietic System

Bone marrow (50) (50) (50) (50)

Fibrosis 1 (2%)

Myeloid cell, hyperplasia 1 (2%)

Lymph node (6) (7) (4) (6)

Deep cervical, hyperplasia, lymphoid 1 (14%)

Iliac, hyperplasia, lymphoid 1 (14%) 1 (25%)

Lumbar, hyperplasia, lymphoid 1 (17%) 1 (25%)

Lumbar, inflammation, acute 1 (17%)

Mediastinal, hyperplasia, lymphoid 1 (17%) 1 (14%) 2 (33%)

Renal, hyperplasia, lymphoid 1 (17%)

Lymph node, mandibular (49) (50) (50) (50)

Hematopoietic cell proliferation 1 (2%)

Hyperplasia, lymphoid 1 (2%) 2 (4%)

Lymph node, mesenteric (50) (49) (48) (48)

Hyperplasia, histiocytic 1 (2%)

Spleen (50) (50) (49) (50)

Atrophy 1 (2%) 2 (4%) 1 (2%) 1 (2%)

Hematopoietic cell proliferation 32 (64%) 24 (48%) 30 (61%) 33 (66%)

Hyperplasia, lymphoid 1 (2%)

Thymus (49) (49) (49) (49)

Atrophy 13 (27%) 17 (35%) 19 (39%) 16 (33%)

Hyperplasia, focal 1 (2%)

Hyperplasia, histiocytic 1 (2%)

Integumentary System

Mammary gland (50) (50) (50) (50)

Duct, dilatation 1 (2%) 1 (2%)

Skin (50) (50) (50) (50)

Epidermis, hyperkeratosis 1 (2%)

Epidermis, hyperplasia 1 (2%)

Epidermis, inflammation, suppurative 1 (2%)

Epidermis, ulcer 1 (2%)

Site of application, epidermis, hyperkeratosis 3 (6%)

Site of application, epidermis, hyperplasia 14 (28%) 50 (100%) 46 (92%) 50 (100%)

Site of application, epidermis, inflammation, suppurative 1 (2%) 2 (4%) 20 (40%) 32 (64%)

Site of application, epidermis, ulcer 1 (2%) 1 (2%) 6 (12%) 17 (34%)

123

Triethanolamine, NTP TR 518

ABLE B5

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 100 mg/kg 300 mg/kg 1,000 mg/kg

Integumentary System (continued)

Skin (continued) (50) (50) (50) (50)

Subcutaneous tissue, edema 1 (2%)

Subcutaneous tissue, fibrosis 1 (2%)

Subcutaneous tissue, inflammation, chronic 1 (2%)

Subcutaneous tissue, necrosis 1 (2%)

Subcutaneous tissue, site of application, dermis,

inflammation, chronic 4 (8%) 27 (54%) 31 (62%) 44 (88%)

Musculoskeletal System

Bone (50) (50) (50) (50)

Fibrosis 7 (14%) 6 (12%) 13 (26%) 10 (20%)

Fracture 1 (2%)

Hyperostosis 1 (2%)

Cranium, callus 1 (2%)

Femur, hyperostosis 1 (2%)

Nervous System

Brain (50) (50) (49) (50)

Cyst epithelial inclusion 1 (2%)

Hydrocephalus 1 (2%)

Artery, inflammation, chronic 1 (2%)

Artery, meninges, inflammation, chronic 1 (2%)

Hypothalamus, compression 3 (6%) 1 (2%)

Respiratory System

Lung (50) (50) (50) (50)

Inflammation, acute 1 (2%)

Inflammation, chronic 2 (4%) 3 (6%) 1 (2%)

Inflammation, granulomatous 1 (2%)

Mineralization 2 (4%)

Alveolar epithelium, hyperplasia, focal 1 (2%) 1 (2%)

Bronchus, hyperplasia, focal 1 (2%)

Bronchus, hyperplasia, lymphoid 1 (2%)

Mediastinum, inflammation, chronic 1 (2%)

Serosa, inflammation, chronic 1 (2%)

Nose (50) (50) (50) (50)

Inflammation, chronic 1 (2%)

Sinus, inflammation, suppurative 1 (2%)

Special Senses System

Eye (49) (50) (50) (50)

Degeneration 1 (2%)

Inflammation, chronic 1 (2%)

Harderian gland (49) (50) (50) (50)

Hyperplasia 4 (8%) 3 (6%) 4 (8%) 5 (10%)

124 Triethanolamine, NTP TR 518

ABLE B5

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Dermal Study of Triethanolamine

Vehicle Control 100 mg/kg 300 mg/kg 1,000 mg/kg

Urinary System

Kidney (50) (50) (50) (50)

Hydronephrosis 1 (2%)

Infarct 1 (2%) 5 (10%) 3 (6%) 3 (6%)

Metaplasia, osseous 1 (2%) 3 (6%)

Nephropathy 24 (48%) 18 (36%) 23 (46%) 20 (40%)

Glomerulus, amyloid deposition 2 (4%)

Glomerulus, cyst 1 (2%)

Papilla, necrosis 1 (2%) 1 (2%) 1 (2%)

Papilla, renal tubule, atrophy 1 (2%)

Pelvis, inflammation, granulomatous 1 (2%)

Renal tubule, cyst 1 (2%)

Renal tubule, cytoplasmic alteration 1 (2%)

Renal tubule, mineralization 1 (2%)

Urinary bladder (50) (50) (49) (49)

Cyst 1 (2%)

125

APPENDIX C

GENETIC TOXICOLOGY

SALMONELLA TYPHIMURIUM MUTAGENICITY T

EST PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

HINESE HAMSTER

OVARY C

ELL CYTOGENETICS PROTOCOLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

ROSOPHILA MELANOGASTER

EST PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

OUSE P

ERIPHERAL BLOOD MICRONUCLEUS TEST P

ROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

VALUATION PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

ABLE C1 Mutagenicity of Triethanolamine in Salmonella typhimurium . . . . . . . . . . . . . . . . . . . . . . . 130

ABLE C2 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells

by Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

TABLE C3 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells

by Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

ABLE C4 Induction of Sex-Linked Recessive Lethal Mutations in Drosophila melanogaster

by Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

ABLE C5 Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice

Following Treatment with Triethanolamine by Dermal Application for 13 Weeks . . . . . . 133

126 Triethanolamine, NTP TR 518

GENETIC TOXICOLOGY

SALMONELLA TYPHIMURIUM MUTAGENICITY TEST PROTOCOL

Testing was performed as reported by Mortelmans et al. (1986). Triethanolamine was sent to the laboratory as a

coded aliquot from Radian Corporation (Austin, TX). It was incubated with the Salmonella typhimurium tester

strains TA98, TA100, TA1535, and TA1537 either in buffer or S9 mix (metabolic activation enzymes and cofactors

from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver) for 20 minutes at 37° C. Top agar

supplemented with

L-histidine and d-biotin was added, and the contents of the tubes were mixed and poured onto

the surfaces of minimal glucose agar plates. Histidine-independent mutant colonies arising on these plates were

counted following incubation for 2 days at 37° C.

Each trial consisted of triplicate plates of concurrent positive and negative controls and five doses of

triethanolamine. The high dose was limited by toxicity. All trials were repeated.

In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent

(revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in

revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a determination

of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following

chemical treatment. There is no minimum percentage or fold increase required for a chemical to be judged

positive or weakly positive.

CHINESE HAMSTER OVARY CELL CYTOGENETICS PROTOCOLS

Testing was performed as reported by Galloway et al. (1987). Triethanolamine was sent to the laboratory as a

coded aliquot by Radian Corporation. It was tested in cultured Chinese hamster ovary (CHO) cells for induction of

sister chromatid exchanges (SCEs) and chromosomal aberrations (Abs), both in the presence and absence of

Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix. Cultures were handled under gold

lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Each test consisted of concurrent solvent and

positive controls and three doses of triethanolamine. In the trials conducted without S9, the high dose was limited

by toxicity; in the trials with S9, the high dose was limited to 10,100 µg/mL. A single flask per dose was used,

and tests yielding equivocal or positive results were repeated.

Sister Chromatid Exchange Test: In the SCE test without S9, CHO cells were incubated for 25.5 to 28 hours with

triethanolamine in supplemented McCoy’s 5A medium. Bromodeoxyuridine (BrdU) was added 2 hours after

culture initiation. After 25.5 to 28 hours, the medium containing triethanolamine was removed and replaced with

fresh medium plus BrdU and Colcemid, and incubation was continued for up to 2 hours. Cells were then harvested

by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa. In the SCE test with S9, cells were

incubated with triethanolamine, serum-free medium, and S9 for 2 hours. The medium was then removed and

replaced with medium containing serum and BrdU and no triethanolamine. Incubation proceeded for an additional

25.8 hours, with Colcemid present for the final 2 hours. Harvesting and staining were the same as for cells treated

without S9. All slides were scored blind, and those from a single test were read by the same person. Fifty second-

division metaphase cells were scored for frequency of SCEs/cell from each dose level.

Statistical analyses were conducted on the slopes of the dose-response curves and the individual dose points

(Galloway et al., 1987). An SCE frequency 20% above the concurrent solvent control value was chosen as a

statistically conservative positive response. The probability of this level of difference occurring by chance at one

dose point is less than 0.01; the probability for such a chance occurrence at two dose points is less than 0.001. An

increase of 20% or greater at any single dose was considered weak evidence of activity; increases at two or more

doses resulted in a determination that the trial was positive. A statistically significant trend (P<0.005) in the

absence of any responses reaching 20% above background led to a call of equivocal.

127 Triethanolamine, NTP TR 518

Chromosomal Aberrations Test: In the Abs test without S9, cells were incubated in McCoy’s 5A medium with

triethanolamine for 8.5 hours; Colcemid was added, and incubation continued for 2 hours. The cells were then

harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with

triethanolamine and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated

for 8.5 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same

manner as for the treatment without S9.

Cells were selected for scoring on the basis of good morphology and completeness of karyotype

(21 ± 2 chromosomes). All slides were scored blind, and those from a single test were read by the same person.

One hundred first-division metaphase cells were scored at each dose level. Classes of aberrations included simple

(breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells,

despiralized chromosomes, and cells containing 10 or more aberrations).

Chromosomal aberration data are presented as percentage of cells with aberrations. To arrive at a statistical call for

a trial, analyses were conducted on both the dose response curve and individual dose points. For a single trial, a

statistically significant (P#0.05) difference for one dose point and a significant trend (P#0.015) were considered

weak evidence for a positive response; significant differences for two or more doses indicated the trial was

positive. A positive trend test in the absence of a statistically significant increase at any one dose resulted in an

equivocal call (Galloway et al., 1987). Ultimately, the trial calls were based on a consideration of the statistical

analyses as well as the biological information available to the reviewers.

ROSOPHILA MELANOGASTER

EST PROTOCOL

The assays for induction of sex-linked recessive lethal (SLRL) mutations were performed with adult flies as

described by Yoon et al. (1985). Triethanolamine was supplied as a coded aliquot by Radian Corporation. It was

assayed in the SLRL test by feeding for 3 days to adult Canton-S wild-type males no more than 24 hours old at the

beginning of treatment. Because no response was obtained, triethanolamine was retested by injection into adult

males. Three dose levels were tested to ensure adequate testing of the chemical.

To administer triethanolamine by injection, a glass Pasteur pipette was drawn out in a flame to a microfine

filament, and the tip was broken off to allow delivery of the test solution. Injection was performed either

manually, by attaching a rubber bulb to the other end of the pipette and forcing through sufficient solution (0.2 to

0.3 µL) to slightly distend the abdomen of the fly, or by attaching the pipette to a microinjector that automatically

delivered a calibrated volume. Flies were anesthetized with ether and immobilized on a strip of tape. Injection

into the thorax, under the wing, was performed with the aid of a dissecting microscope.

Toxicity tests were performed to set concentrations of triethanolamine at a level that would induce 30% mortality

after 72 hours of feeding or 24 hours after injection, while keeping induced sterility at an acceptable level.

Canton-S males were allowed to feed for 72 hours on a solution of triethanolamine in 5% sucrose. In the injection

experiments, 24- to 72-hour old Canton-S males were treated with a solution of triethanolamine dissolved in saline

or peanut oil and allowed to recover for 24 hours. A concurrent saline or peanut oil control group was also

included. In the adult exposures, treated males were mated to three Basc females for 3 days and were given fresh

females at 2-day intervals to produce three matings of 3, 2, and 2 days (in each case, sample sperm from

successive matings was treated at successively earlier postmeiotic stages). F

heterozygous females were mated

with their siblings and then placed in individual vials. F

daughters from the same parental male were kept

together to identify clusters. (A cluster occurs when a number of mutants from a given male result from a single

spontaneous premeiotic mutation event and is identified when the number of mutants from that male exceeds the

number predicted by a Poisson distribution.) If a cluster was identified, all data from the male in question were

discarded. Clusters were identified in four dose groups in this series of experiments (Table C4). Presumptive

lethal mutations were identified as vials containing fewer than 5% of the expected number of wild-type males after

17 days; these were retested to confirm the response.

128 Triethanolamine, NTP TR 518

SLRL data were analyzed by simultaneous comparison with the concurrent and historical controls (Mason et al.,

1992) using a normal approximation to the binomial test (Margolin et al., 1983). A test result was considered

positive if the P value was less than or equal to 0.01 and the mutation frequency in the tested group was greater

than 0.10% or if the P value was less than or equal to 0.05 and the frequency in the treatment group was greater

than 0.15%. A test was considered to be inconclusive if the P value was between 0.05 and 0.01 but the frequency

in the treatment group was between 0.10% and 0.15% or if the P value was between 0.10 and 0.05 but the

frequency in the treatment group was greater than 0.10%. A test was considered negative if the P value was

greater than or equal to 0.10 or if the frequency in the treatment group was less than 0.10%.

MOUSE PERIPHERAL

BLOOD MICRONUCLEUS TEST PROTOCOL

A detailed discussion of this assay is presented by MacGregor et al. (1990). At the end of the 13-week toxicity

study (NTP, 1999), peripheral blood samples were obtained from male and female mice. Smears were immediately

prepared and fixed in absolute methanol. The methanol-fixed slides were stained with a chromatin-specific

fluorescent dye mixture of Hoechst 33258/pyronin Y (MacGregor et al., 1983) and coded. Slides were scanned to

determine the frequency of micronuclei in 10,000 normochromatic erythrocytes (NCEs) in each of 10 animals per

dose group. In addition, the percentage of polychromatic erythrocytes among 10,000 erythrocytes was determined

as a measure of bone marrow toxicity.

The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or

minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a

statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran-Armitage

trend test, followed by pairwise comparisons between each dosed group and the control group (ILS, 1990). In the

presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the

Cochran-Armitage test was adjusted upward in proportion to the excess variation. In the micronucleus test, an

individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any

single dosed group is less than or equal to 0.025 divided by the number of dosed groups. A final call of positive

for micronucleus induction is preferably based on reproducibly positive trials (as noted above). Results of the

13-week study were accepted without repeat tests, because additional test data could not be obtained. Ultimately,

the final call is determined by the scientific staff after considering the results of statistical analyses, the

reproducibility of any effects observed, and the magnitudes of those effects.

EVALUATION PROTOCOL

These are the basic guidelines for arriving at an overall assay result for assays performed by the National

Toxicology Program. Statistical as well as biological factors are considered. For an individual assay, the statistical

procedures for data analysis have been described in the preceding protocols. There have been instances, however,

in which multiple aliquots of a chemical were tested in the same assay, and different results were obtained among

aliquots and/or among laboratories. Results from more than one aliquot or from more than one laboratory are not

simply combined into an overall result. Rather, all the data are critically evaluated, particularly with regard to

pertinent protocol variations, in determining the weight of evidence for an overall conclusion of chemical activity

in an assay. In addition to multiple aliquots, the in vitro assays have another variable that must be considered in

arriving at an overall test result. In vitro assays are conducted with and without exogenous metabolic activation.

Results obtained in the absence of activation are not combined with results obtained in the presence of activation;

each testing condition is evaluated separately. The summary table in the Abstract of this Technical Report presents

a result that represents a scientific judgement of the overall evidence for activity of the chemical in an assay.

129 Triethanolamine, NTP TR 518

RESULTS

Triethanolamine (33 to 3,333 µg/plate) was negative for induction of mutations in S. typhimurium strains TA98,

TA100, TA1535, and TA1537 when tested with or without S9 metabolic activation (Table C1; Mortelmans et al.,

1986). In cytogenetic tests with cultured CHO cells, no induction of SCEs (Table C2) or Abs (Table C3) was

observed with or without S9 (Galloway et al., 1987). In the SCE test without S9, the first of two trials was

negative. In the second trial, a significant increase in SCEs was observed at the highest dose tested (2,520 µg/mL),

but the trend test was negative (P$0.025), and the trial was concluded to be equivocal. Severe cytotoxicity limited

the number of cells that could be scored at this dose. Overall, the SCE test was considered to be negative.

Cytotoxicity was also noted at the highest dose tested (4,030 µg/mL) in the Abs test without S9.

Triethanolamine administered by feeding or injection at doses up to 30,000 ppm did not induce SLRL mutations in

germ cells of male D. melanogaster (Table C4; Yoon et al., 1985). Results of an in vivo peripheral blood

micronucleus test in mice were also negative (Table C5; Witt et al., 2000). In this test, blood samples were

obtained from male and female mice after 13 weeks of dermal applications of 1,000 to 4,000 mg/kg

triethanolamine. No significant increases in the frequencies of micronucleated NCEs were observed at any dose

level, and the percentages of PCEs in the dosed groups were similar to those in the vehicle control groups,

indicating an absence of bone marrow toxicity.

130 Triethanolamine, NTP TR 518

ABLE C1

Mutagenicity of Triethanolamine in Salmonella typhimurium

Revertants/Plate

Strain Dose –S9 +10% hamster S9 +10% rat S9

(µg/plate) Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2

TA100 0 89 ± 2.6 104 ± 11.8 192 ± 13.5 163 ± 14.8 159 ± 15.2 164 ± 8.4

33 96 ± 8.5 112 ± 6.1 180 ± 7.9 145 ± 4.2 162 ± 9.9 143 ± 14.5

100 87 ± 3.1 98 ± 2.2 215 ± 26.7 162 ± 11.0 151 ± 6.1 157 ± 4.4

333 74 ± 12.7 93 ± 5.2 188 ± 10.0 154 ± 4.6 165 ± 3.7 147 ± 12.2

1,000 73 ± 3.2 106 ± 10.6 145 ± 5.2 148 ± 13.6 152 ± 6.1 149 ± 5.4

3,333 74 ± 4.8 96 ± 5.3 155 ± 3.8 137 ± 2.6 153 ± 14.2 122 ± 20.8

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 359 ± 21.0 425 ± 29.6 668 ± 41.6 935 ± 188.8 299 ± 30.2 293 ± 1.2

TA1535 0 7 ± 0.9 4 ± 1.3 9 ± 1.5 6 ± 1.5 11 ± 1.9 7 ± 2.0

33 5 ± 1.7 2 ± 0.3 9 ± 0.9 4 ± 1.2 8 ± 2.2 3 ± 1.2

100 5 ± 2.1 3 ± 0.3 6 ± 0.9 2 ± 1.2 8 ± 2.3 4 ± 1.0

333 10 ± 1.5 3 ± 1.5 6 ± 1.8 4 ± 0.0 9 ± 1.5 7 ± 2.1

1,000 7 ± 0.9 3 ± 0.7 7 ± 1.8 6 ± 0.7 9 ± 1.9 4 ± 1.2

3,333 7 ± 0.6 4 ± 0.6 8 ± 1.8 3 ± 1.5 8 ± 2.1 5 ± 1.5

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 193 ± 19.5 291 ± 39.0 61 ± 11.8 47 ± 7.7 71 ± 4.6 19 ± 2.0

TA1537 0 9 ± 2.4 6 ± 1.5 8 ± 1.5 9 ± 0.3 8 ± 2.0 5 ± 1.8

33 6 ± 2.2 5 ± 0.7 10 ± 1.3 9 ± 2.5 11 ± 1.8 5 ± 1.2

100 7 ± 1.2 6 ± 0.6 10 ± 3.0 10 ± 1.0 8 ± 1.2 4 ± 1.2

333 7 ± 1.2 6 ± 1.3 8 ± 3.5 8 ± 1.3 12 ± 2.5 5 ± 1.7

1,000 9 ± 0.9 6 ± 0.9 7 ± 2.4 7 ± 1.0 7 ± 0.9 6 ± 0.9

3,333 3 ± 0.3 5 ± 2.7 9 ± 1.8 8 ± 1.5 9 ± 1.5 8 ± 1.9

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 300 ± 35.1 198 ± 22.5 37 ± 10.7 44 ± 3.2 35 ± 5.8 19 ± 1.2

TA98 0 18 ± 2.3 15 ± 2.0 23 ± 2.7 18 ± 2.9 23 ± 1.0 19 ± 2.1

33 13 ± 1.7 12 ± 2.6 18 ± 1.0 15 ± 1.3 20 ± 3.7 15 ± 1.5

100 14 ± 2.3 11 ± 0.7 31 ± 9.4 18 ± 3.1 27 ± 1.7 18 ± 3.5

333 15 ± 1.5 12 ± 0.9 23 ± 4.4 17 ± 0.3 17 ± 1.7 13 ± 2.6

1,000 9 ± 1.5 12 ± 2.2 25 ± 2.0 19 ± 0.6 18 ± 3.4 15 ± 0.9

3,333 14 ± 1.9 12 ± 1.2 17 ± 3.5 13 ± 0.9 16 ± 0.7 11 ± 0.9

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 261 ± 25.1 245 ± 30.7 330 ± 15.5 420 ± 7.1 94 ± 2.5 166 ± 1.2

The study was performed at Case Western Reserve University. The detailed protocol and these data are presented

by Mortelmans et al. (1986).

Revertants are presented as mean ± standard error from three plates.

The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 9-aminoacridine (TA1537), and

4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene.

131 Triethanolamine, NTP TR 518

ABLE C2

Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by Triethanolamine

Total Relative

Dose Cells No. of No. of SCEs/ SCEs/ Hrs Change of SCEs/

Compound (µg/mL) Scored Chromosomes SCEs Chromosomes Cell in BrdU Chromosome

(%)

–S9

Trial 1

Summary: Negative

Medium 50 1,047 398 0.38 8.0 25.8

Triethanolamine 100 50 1,050 443 0.42 8.9 25.8 10.99

330 50 1,050 417 0.39 8.3 25.8 4.47

1,010 50 1,048 387 0.36 7.7 25.8 –2.86

P=0.745

Mitomycin-C 0 50 1,049 1,887 1.79 37.7 25.8 373.22

Trial 2

Summary: Equivocal

Medium 50 1,043 435 0.41 8.7 25.5

Triethanolamine 630 50 1,041 447 0.42 8.9 25.5 2.96

1,260 50 1,043 438 0.41 8.8 25.5 0.69

2,520 8 163 84 0.51 10.5 28.0 23.56*

P=0.174

Mitomycin-C 0.005 50 1,036 1,648 1.59 33.0 25.5 281.42

+S9

Summary: Negative

Medium 50 1,034 426 0.41 8.5 25.8

Triethanolamine 330 50 1,031 446 0.43 8.9 25.8 5.00

1,010 50 1,045 453 0.43 9.1 25.8 5.22

10,100 50 1,039 463 0.44 9.3 25.8 8.16

P=0.130

Cyclophosphamide 1.5 50 1,039 1,389 1.33 27.8 25.8 224.49

* Positive response ($20% increase over solvent control)

The study was performed at Litton Bionetics, Inc. The detailed protocol and these data are presented by Galloway et al. (1987).

SCE=sister chromatid exchange; BrdU=bromodeoxyuridine.

SCEs/chromosome in treated cells versus SCEs/chromosome in solvent control cells

Solvent control

Significance of SCEs/chromosome tested by the linear regression trend test versus log of the dose

Positive control

132

Triethanolamine, NTP TR 518

ABLE C3

Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by Triethanolamine

Dose Total Cells Number Aberrations/ Cells with

Compound (µg/mL) Scored of Aberrations Cell Aberrations (%)

–S9

Trial 1

Harvest time: 10.5 hours

Summary: Negative

Dimethylsulfoxide 100 7 0.07 5.0

Triethanolamine 1,510 100 2 0.02 2.0

2,010 100 1 0.01 1.0

4,030 56 0 0.00 1.0

P=0.985

Mitomycin-C 0.5 100 24 0.24 20.0

+S9

Trial 1

Harvest time: 10.5 hours

Summary: Negative

Dimethylsulfoxide 100 2 0.02 2.0

Triethanolamine 6,040 100 3 0.03 3.0

8,060 100 6 0.06 5.0

10,070 100 2 0.02 2.0

P=0.373

Cyclophosphamide 25 100 32 0.32 26.0

Study was performed at Litton Bionetics, Inc. The detailed protocol and these data are presented by Galloway et al. (1987).

Solvent control

Significance of percent cells with aberrations tested by the linear regression trend test versus log of the dose

Positive control

133

Triethanolamine, NTP TR 518

ABLE C4

Induction of Sex-Linked Recessive Lethal Mutations in Drosophila melanogaster by Triethanolamine

Route of

Exposure

Dose

(ppm)

Incidence of

Death (%)

Incidence of

Sterility (%)

No. of Lethals/No. of X Chromosomes Tested

Mating 1 Mating 2 Mating 3 Total

Feeding 20,000

30,000

0/1,011

1/909

0/1,190

2/2,091

1/1,262

2/1,467

1/1,924

2/2,509

1/570

1/735

0/739

2/1,989

2/2,843 (0.07%)

4/3,111 (0.13%)

1/3,853 (0.03%)

6/6,589 (0.09%)

Injection 10,000

20,000

30,000

0/1,016

0/1,024

0/524

0/374

1/247

1/864

0/1,185

0/1,179

0/1,127

0/1,268

0/113

2/1,078

2/1,000

0/981

1/984

2/871

1/108

1/1,091

2/3,201 (0.06%)

0/3,184 (0.00%)

1/2,635 (0.04%)

2/2,513 (0.08%)

2/468 (0.43%)

4/3,033 (0.13%)

The study was performed at Brown University. The detailed protocol and these data are presented by Yoon et al. (1985). The mean mutant

frequency from 518 negative control experiments is 0.074% (Mason et al., 1992). Results were not significant at the 5% level

(Margolin et al., 1983).

Total number of lethal mutations/number of X chromosomes tested for three mating trials

Data were corrected for the occurrence of spontaneous clusters.

TABLE C5

Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice

Following Treatment with Triethanolamine by Dermal Application for 13 Weeks

Dose

(mg/kg)

Number of Mice

with Erythrocytes

Scored

Micronucleated NCEs/

1,000 NCEs

P Value

PCEs (%)

Male

Vehicle control

1,000

2,000

4,000

1.75 ± 0.20

1.88 ± 0.17

1.36 ± 0.10

1.90 ± 0.30

P=0.420

0.3138

0.9350

0.3047

2.2

2.4

2.1

Female

Vehicle control

1,000

2,000

4,000

1.16 ± 0.12

1.20 ± 0.08

1.08 ± 0.12

0.99 ± 0.11

0.3890

0.7393

0.8813

2.1

2.2

2.0

P=0.925

The detailed protocol is presented by MacGregor et al. (1990), and these data are presented by Witt et al. (2000).

NCE=normochromatic erythrocyte; PCE=polychromatic erythrocyte

Mean ± standard error

Pairwise comparison with the vehicle controls, significant at P#0.008 (ILS, 1990)

Significance of micronucleated NCEs/1,000 NCEs tested by the one-tailed trend test, significant at P#0.025 (ILS, 1990)

134 Triethanolamine, NTP TR 518

135

APPENDIX D

CHEMICAL CHARACTERIZATION

AND DOSE FORMULATION STUDIES

PROCUREMENT AND

CHARACTERIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

REPARATION AND

ANALYSIS OF DOSE

FORMULATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

IGURE D1 Infrared Absorption Spectrum of Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

FIGURE D2 Nuclear Magnetic Resonance Spectrum of Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . 139

TABLE D1 Gas Chromatography Systems Used in the 2-Year Dermal Study of Triethanolamine . . . . 140

ABLE D2 High-Performance Liquid Chromatography Systems Used in the 2-Year Dermal Study

of Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

TABLE D3 Preparation and Storage of Dose Formulations in the 2-Year Dermal Study

of Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

ABLE D4 Results of Analyses of Dose Formulations Administered to Mice

in the 2-Year Dermal Study of Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

136 Triethanolamine, NTP TR 518

CHEMICAL CHARACTERIZATION

AND DOSE FORMULATION STUDIES

PROCUREMENT AND

CHARACTERIZATION

Triethanolamine

Triethanolamine was obtained from Texaco Chemical Company (Division of Texaco, Inc., Bellaire, TX) in one lot

(7G-60) for use during the 2-year study. Identity, purity, and moisture content analyses were conducted by the

analytical chemistry laboratory (Research Triangle Institute, Research Triangle Park, NC); identity, purity, and

stability analyses were conducted by the study laboratory. Special analyses of diethanolamine and nitrosamine

impurities in the bulk chemical were conducted by the analytical chemistry laboratory and Covance Laboratories,

Inc. (Madison, WI), respectively. Reports on analyses performed in support of the triethanolamine study are on

file at the National Institute of Environmental Health Sciences.

The chemical, a clear, colorless liquid, was identified as triethanolamine by the analytical chemistry laboratory

using ultraviolet/visible, infrared, and proton nuclear magnetic resonance (NMR) spectroscopy and high- and

low-resolution mass spectrometry and by the study laboratory using infrared spectroscopy. All spectra were

consistent with the structure of triethanolamine and with the literature spectra (Aldrich, 1981, 1983, 1993; NIST

Standard Reference Database). The observed mass of the high-resolution mass spectrometry base peak was within

acceptable limits of the calculated mass. The infrared and NMR spectra are presented in Figures D1 and D2.

The moisture content of lot 7G-60 was determined by Galbraith Laboratories, Inc. (Knoxville, TN) using Karl

Fischer titration. The purity of lot 7G-60 was determined by the analytical chemistry laboratory (systems A, B,

and C) and the study laboratory (system D) using gas chromatography with flame ionization detection (Table D1).

Purity of lot 7G-60 was also determined by the analytical chemistry laboratory using high-performance liquid

chromotography (HPLC) with photodiode array detection (system 1; Table D2).

Karl Fischer titration indicated 0.18% water. Gas chromatography using systems B and D indicated one major

peak and no impurities. Gas chromatography using system A indicated one major peak and one impurity with an

area of 0.88% relative to the major peak area; the impurity was determined to be diethanolamine. Gas

chromatography using system C indicated that the compound contained no impurity other than diethanolamine at a

concentration greater than 0.1%. HPLC using system 1 indicated no measurable impurities with chromophores in

a triethanolamine solution scanned for 30 minutes from 200 to 400 nm. The overall purity of lot 7G-60 was

determined to be greater than 99%.

A special HPLC/mass spectrometric study was conducted by the analytical chemistry laboratory using system 2

(Table D2) to determine the relative concentration of diethanolamine as an impurity in the test chemical

(lot 7G-60), and to conduct an accelerated stability study. A low resolution electrospray ionization detector was

used in the mass spectrometer. Analysis showed that diethanolamine constituted 0.491% (weight percent relative

to the test chemical weight) of the test chemical. Solutions of triethanolamine were prepared in acetone or 95%

ethanol at approximately 500 mg/mL for the accelerated stability study. Aliquots of the solutions were stored in

sealed glass containers at 32° to 36° C, protected from light, for 1.6 hours or 11 days. After 11 days of storage,

both solutions showed slight increases in diethanolamine concentrations compared to those measured at time 0; no

differences were noted following 1.6 hours of storage.

The concentrations of nonpolar nitrosamines (N-nitrosodimethylamine, N-nitrosomethylethylamine,

N-nitrosodiethylamine, N-nitrosodi-n-propylamine, N-nitrosodi-n-butylamine, N-nitrosopiperidine,

N-nitrosopyrrolidine, and N-nitrosomorpholine) and the polar nitrosamine N-nitrosodiethanolamine in

triethanolamine, lot 7G-60, were determined by Covance Laboratories, Inc. Nonpolar nitrosamines were analyzed

137 Triethanolamine, NTP TR 518

by gas chromatography (system E), and the polar nitrosamine N-nitrosodiethanolamine was analyzed by HPLC

(system 3); both systems used a thermal energy analyzer for detection. No nonpolar or polar nitrosamines were

present at concentrations greater than the limits of detection (0.1 and 1.0 ppm, respectively).

Stability studies of the bulk chemical were performed on lot 03601CN (not used in the current study) by the study

laboratory using gas chromatography with flame ionization detection (system F). Triethanolamine was stable for

14 days when stored in amber glass vials at 5°, 25°, and 60° C, when compared to a sample stored at –20°C. To

ensure stability, triethanolamine was stored at room temperature in amber glass containers with Teflon

-lined lids.

No degradation of triethanolamine was observed.

Acetone

Acetone was obtained in four lots (NE0173, NV0163, OG0513, and OX0312) from Spectrum Chemical

Manufacturing Corporation (Gardena, CA) for use during the 2-year study. Identity and purity analyses of each lot

were conducted by the study laboratory.

The chemical, a clear liquid, was identified as acetone using infrared spectroscopy. The purity of each lot was

determined using gas chromatography with flame ionization detection (system G). No significant impurities were

detected in any lot. The overall purity of each lot was determined to be greater than 99%.

To ensure stability, the bulk chemical was stored at room temperature in amber glass bottles. No degradation of

the acetone was detected.

PREPARATION AND ANALYSIS OF DOSE FORMULATIONS

The dose formulations were prepared approximately every 2 weeks by mixing triethanolamine and acetone to give

the required concentration (Table D3). The dose formulations were stored for up to 21 days at 5° C in amber glass

bottles with Teflon

-lined lids.

Stability studies of 10 mg/mL dose formulations in acetone were performed by Midwest Research Institute (Kansas

City, MO) using gas chromatograpy with flame ionization detection (system H). The stability of the dose

formulations was confirmed for at least 3 weeks when stored at room temperature in sealed amber glass vials under

a nitrogen headspace, and for at least 3 hours under animal room conditions (open to air and light).

Periodic analyses of the dose formulations of triethanolamine were conducted by the study laboratory with gas

chromatography using system D. During the 2-year study, the dose formulations were analyzed approximately

every 8 or 12 weeks (Table D4); animal room samples were also analyzed periodically. Of the dose formulations

analyzed, all 66 were within 10% of the target concentrations; 18 of 24 animal room samples were within 10% of

the target concentrations.

It)

..;

.....

138 Triethanolamine, NTP TR 518

FIGURE D1

Infrared Absorption Spectrum of Triethanolamine

...

~('I)

_.1

~It)!

z::

:c:

(/)

iii

~CD

·e

Cl)

()

.....

Solvent-

~::

139 Triethanolamine, NTP TR 518

FIGURE D2

Nuclear Magnetic Resonance Spectrum of Triethanolamine

140 Triethanolamine, NTP TR 518

TABLE D1

Gas Chromatography Systems Used in the 2-Year Dermal Study of Triethanolamine

Oven Temperature

Detection System Column Carrier Gas Program

System A

Flame ionization

System B

Flame ionization

System C

Flame ionization

System D

Flame ionization

System E

Thermal energy analyzer

System F

Flame ionization

System G

Flame ionization

System H

Flame ionization

Capillary, Supelco Carbowax

Amine, 30 m × 0.53 mm, 1.0-µm

film thickness (Supelco,

Bellefonte, PA)

Packed, Supelco Tenax,

1.8 m × 0.25 inches, 60/80 mesh

Capillary, Supelco Carbowax

Amine (30 m × 0.53 mm,

1.0-µm film thickness

Packed, Supelco Tenax TA or

GC 60/80 mesh, 1.8 m × 2 mm

Packed 10% Carbowax 1540

100/120 mesh WHP in 5%

KOH, 6 ft × 4 mm

Packed, Supelco Tenax TA,

60/80 mesh, 6 ft × 2 mm

Packed 20% SP-2401/0.1%

Carbopack 1500 on 100/120

Supelcoport, 2.4 m × 2mm

Capillary, Supelco DB-1,

15 m × 0.51 mm, 1.5-µm film

thickness

Helium at 2.1 mL/minute

Helium at 33.5 mL/minute

Helium at 10 mL/minute

Helium at approximately

15 mL/minute

Argon at 25 mL/minute

Helium at 15 mL/minute

Helium at 30 mL/minute

Helium at 26 mL/minute

35° C for 1 minute, then

10° C/minute to 280° C,

held 13.5 minutes

150° C to 300° C at

10° C/minute, held 25 minutes

60° C for 1 minute, then

20° C/minute to 200° C,

held 32 minutes

220° C to 280° C at

10° C/minute, held 4 minutes

120° C for 4 minutes, then

4° C/minute to 180° C, held for

8 minutes

220° C for 7 minutes, then

10° C/minute to 260° C,

held for 6 minutes

50° C for 4 minutes, then

10° C/minute to 170° C

80° C for 5 minutes, then

15° C/minute to 190° C, held for

3 minutes

Gas chromatographs were manufactured by Hewlett-Packard (Palo Alto, CA) (systems A, B, C, E, and G), Varian (Palo Alto, CA) (system H).

141 Triethanolamine, NTP TR 518

TABLE

High-Performance Liquid Chromatography Systems Used in the 2-Year Dermal Study of Triethanolamine

Detection System Column Solvent System

System 1

Photodiode array (200 to 400 nm scan)

Hamilton PRP-X200, 15 cm × 4.1 mm

(Hamilton Co., Reno, NV)

System 2

Low resolution mass spectrometry with

Agilent/ZORBAX 300-SCX,

electrospray ionization

15 cm × 2.1 mm, 5 µm particle size (Agilent

Technologies, Palo Alto, CA)

System 3

Thermal energy analyzer

Alltech Platinum CN, 25 cm × 2.1 mm, 5 µm

particle size (Alltech Associates, Inc.,

Deerfield, IL)

4.0 mM nitric acid:methanol (70:30),

1.0 mL/minute

Water with trifluoroacetic acid (TFA)

(10 mM) to methanol:water (90:10) with

TFA (10 mM) in 15 minutes, held for

3 minutes, flow rate 0.3 mL/minute

Isooctane:dichloromethane:methanol

(71:18:11), 0.4 mL/minute

The high-performance liquid chromatographs were manufactured by Waters, Inc. (Milford, MA) (system 1), Agilent, Inc. (Palo Alto, CA)

(system 2), and Hewlett-Packard (Palo Alto, CA) (system 3). The mass spectrometer used in system 2 was manufactured by PerkinElmer,

Inc. (Shelton, CT).

TABLE D3

Preparation and Storage of Dose Formulations in the 2-Year Dermal Study of Triethanolamine

Preparation

Dose formulations were prepared by mixing triethanolamine with acetone. Dose formulations were prepared every 2 weeks.

Chemical Lot Number

7G-60

Maximum Storage Time

21 days

Storage Conditions

Stored in amber glass bottles with Teflon

-lined lids at 5° C

Study Laboratory

Battelle Columbus Laboratories (Columbus, OH)

142 Triethanolamine, NTP TR 518

ABLE D4

Results of Analyses of Dose Formulations Administered to Mice in the 2-Year Dermal Study

of Triethanolamine

Target Determined Difference

Date Prepared Date Analyzed Concentration Concentration

from Target

(mg/mL) (mg/mL) (%)

September 14, 1998 September 16, 1998 50 54.80 +10

100 101.7 +2

150 153.5 +2

315 326.8 +4

500 512.2 +2

1,000 1,041 +4

October 7, 1998 50 62.33 +25

100 114.4 +14

150 171.7 +14

315 350.5 +11

500 557.8 +12

1,000 1,096 +10

November 9, 1998 November 10, 1998 50 50.65 +1

100 99.15 –1

150 148.6 –1

315 320.5 +2

500 505.3 +1

1,000 1,029 +3

February 1, 1999 February 1, 1999 50 48.85 –2

100 99.77 0

150 151.5 +1

315 316.5 0

500 509.7 +2

1,000 1,005 +1

February 23-24, 1999 50 54.93 +10

100 107.3 +7

150 160.5 +7

315 337.8 +7

500 534.2 +7

1,000 1,020 +2

March 29, 1999 April 1, 1999 50 48.55 –3

100 99.63 0

150 149.8 0

315 324.5 +3

500 503.2 +1

1,000 1,022 +2

June 21, 1999 June 22, 1999 50 51.85 +4

100 101.7 +2

150 153.7 +2

315 325.2 +3

500 512.0 +2

1,000 967.3 –3

143 Triethanolamine, NTP TR 518

ABLE D4

Results of Analyses of Dose Formulations Administered to Mice in the 2-Year Dermal Study

of Triethanolamine

Date Prepared Date Analyzed

Target

Concentration

(mg/mL)

Determined

Concentration

(mg/mL)

Difference

from Target

(%)

August 16, 1999 August 17, 1999 50

100

150

315

500

1,000

53.18

101.1

153.6

326.1

514.2

1,037

September 7, 1999

100

150

315

500

1,000

54.10

108.1

160.8

342.6

530.1

1,009

November 8, 1999 November 10, 1999 50

100

150

315

500

1,000

52.62

101.2

150.6

322.2

508.8

1,014

January 4, 2000 January 5-6, 2000 50

100

150

315

500

1,000

47.38

95.57

145.2

311.4

488.4

1,023

–5

–4

–3

–1

–2

March 27, 2000 March 31-April 1, 2000 50

100

150

315

500

1,000

51.73

97.52

149.3

316.6

511.2

961.0

–2

–4

April 18, 2000

100

150

315

500

1,000

58.42

105.4

159.2

331.4

525.5

1,054

+17

May 22, 2000 May 23-24, 2000 50

100

150

315

500

1,000

49.96

103.8 ± 4.6

155.7

343.7

524.3

1,033

144

Triethanolamine, NTP TR 518

ABLE D4

Results of Analyses of Dose Formulations Administered to Mice in the 2-Year Dermal Study

of Triethanolamine

Target Determined Difference

Date Prepared Date Analyzed Concentration Concentration from Target

(mg/mL) (mg/mL) (%)

August 14, 2000 August 15, 2000 50 51.63 +3

100 100.6 +1

150 152.4 +2

315 312.8 –1

500 499.8 0

1,000 1,005 +1

Results of duplicate analyses. 50 mg/mL=100 mg/kg; 100 mg/mL=200 mg/kg; 150 mg/mL=300 mg/kg; 315 mg/mL=630 mg/kg;

500 mg/mL=1,000 mg/kg; 1,000 mg/mL=2,000 mg/kg

Animal room samples

Due to poor agreement between the duplicates originally analyzed, two additional replicates were prepared and analyzed; the reported values

are the average ± standard deviation of the four replicates.

145

APPENDIX E

INGREDIENTS, NUTRIENT COMPOSITION,

AND CONTAMINANT LEVELS

IN NTP-2000 RAT AND MOUSE RATION

TABLE

ABLE E2

TABLE E3

TABLE E4

Ingredients of NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Vitamins and Minerals in NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . .

Nutrient Composition of NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . .

Contaminant Levels in NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . .

146

147

148

146 Triethanolamine, NTP TR 518

ABLE E1

Ingredients of NTP-2000 Rat and Mouse Ration

Ingredients Percent by Weight

Ground hard winter wheat 22.26

Ground #2 yellow shelled corn 22.18

Wheat middlings 15.0

Oat hulls 8.5

Alfalfa meal (dehydrated, 17% protein) 7.5

Purified cellulose 5.5

Soybean meal (49% protein) 5.0

Fish meal (60% protein) 4.0

Corn oil (without preservatives) 3.0

Soy oil (without preservatives) 3.0

Dried brewer’s yeast 1.0

Calcium carbonate (USP)

Vitamin premix

Mineral premix

0.9

0.5

Calcium phosphate, dibasic (USP) 0.4

Sodium chloride 0.3

Choline chloride (70% choline) 0.26

Methionine 0.2

Wheat middlings as carrier

Calcium carbonate as carrier

TABLE E2

Vitamins and Minerals in NTP-2000 Rat and Mouse Ration

Amount Source

Vitamins

A 4,000 IU Stabilized vitamin A palmitate or acetate

D 1,000 IU D-activated animal sterol

K 1.0 mg Menadione sodium bisulfite complex

"-Tocopheryl acetate 100 IU

Niacin 23 mg

Folic acid 1.1 mg

d-Pantothenic acid 10 mg d-Calcium pantothenate

Riboflavin 3.3 mg

Thiamine 4 mg Thiamine mononitrate

52 µg

Pyridoxine 6.3 mg Pyridoxine hydrochloride

Biotin 0.2 mg d-Biotin

Minerals

Magnesium 514 mg Magnesium oxide

Iron 35 mg Iron sulfate

Zinc 12 mg Zinc oxide

Manganese 10 mg Manganese oxide

Copper 2.0 mg Copper sulfate

Iodine 0.2 mg Calcium iodate

Chromium 0.2 mg Chromium acetate

Per kg of finished product

147 Triethanolamine, NTP TR 518

ABLE E3

Nutrient Composition of NTP-2000 Rat and Mouse Ration

Mean ± Standard

Nutrient Deviation Range Number of Samples

Protein (% by weight) 13.5 ± 0.44 12.7 – 14.5 24

Crude fat (% by weight) 8.1 ± 0.25 7.6 – 8.6 24

Crude fiber (% by weight) 9.2 ± 0.67 7.9 – 10.5 24

Ash (% by weight) 5.0 ± 0.20 4.7 – 5.4 24

Amino Acids (% of total diet)

Arginine 0.731 ± 0.050 0.670 – 0.800 8

Cystine 0.224 ± 0.012 0.210 – 0.240 8

Glycine 0.684 ± 0.041 0.620 – 0.740 8

Histidine 0.333 ± 0.018 0.310 – 0.350 8

Isoleucine 0.524 ± 0.046 0.430 – 0.590 8

Leucine 1.061 ± 0.061 0.960 – 1.130 8

Lysine 0.708 ± 0.056 0.620 – 0.790 8

Methionine 0.401 ± 0.035 0.350 – 0.460 8

Phenylalanine 0.598 ± 0.036 0.540 – 0.640 8

Threonine 0.501 ± 0.051 0.430 – 0.590 8

Tryptophan 0.126 ± 0.014 0.110 – 0.150 8

Tyrosine 0.390 ± 0.056 0.280 – 0.460 8

Valine 0.640 ± 0.049 0.550 – 0.690 8

Essential Fatty Acids (% of total diet)

Linoleic 3.97 ± 0.284 3.59 – 4.54 8

Linolenic 0.30 ± 0.042 0.21 – 0.35 8

Vitamins

Vitamin A (IU/kg) 5,733 ± 924 4,220 – 7,790 24

Vitamin D (IU/kg) 1,000

"-Tocopherol (ppm) 82.2 ± 14.08 62.2 – 107.0 8

Thiamine (ppm)

7.9 ± 0.73 6.3 – 9.2 24

Riboflavin (ppm) 5.6 ± 1.12 4.20 – 7.70 8

Niacin (ppm) 74.3 ± 5.94 66.4 – 85.8 8

Pantothenic acid (ppm) 22.5 ± 3.96 17.4 – 29.1 8

Pyridoxine (ppm) 9.04

± 2.37 6.4 – 12.4 8

Folic acid (ppm) 1.64 ± 0.38 1.26 – 2.32 8

Biotin (ppm) 0.333 ± 0.15 0.225 – 0.704 8

Vitamin B

(ppb) 68.7 ± 63.0 18.3 – 174.0 8

Choline (ppm) 3,155 ± 325 2,700 – 3,790 8

Minerals

Calcium (%) 0.999 ± 0.045 0.903 – 1.090 24

Phosphorus (%) 0.563 ± 0.029 0.505 – 0.618 24

Potassium (%) 0.659 ± 0.022 0.627 – 0.691 8

Chloride (%) 0.357 ± 0.027 0.300 – 0.392 8

Sodium (%) 0.189 ± 0.019 0.160 – 0.212 8

Magnesium (%) 0.199 ± 0.009 0.185 – 0.213 8

Sulfur (%) 0.178 ± 0.021 0.153 – 0.209 8

Iron (ppm) 160 ± 14.7 135 – 177 8

Manganese (ppm) 50.3 ± 4.82 42.1 – 56.0 8

Zinc (ppm) 50.7 ± 6.59 43.3 – 61.1 8

Copper (ppm) 6.29 ± 0.828 5.08 – 7.59 8

Iodine (ppm) 0.461 ± 0.187 0.233 – 0.843 8

Chromium (ppm) 0.542 ± 0.128 0.330 – 0.707 7

Cobalt (ppm) 0.23 ± 0.049 0.20 – 0.30 7

From formulation

As hydrochloride (thiamine and pyridoxine) or chloride (choline)

148 Triethanolamine, NTP TR 518

ABLE E4

Contaminant Levels in NTP-2000 Rat and Mouse Ration

Mean ± StandardDeviation

Range Number of Samples

Contaminants

Arsenic (ppm) 0.17 ± 0.081 0.10 – 0.37 24

Cadmium (ppm) 0.04 ± 0.007 0.04 – 0.07 24

Lead (ppm) 0.11 ± 0.106 0.05 – 0.54 24

Mercury (ppm) <0.02 24

Selenium (ppm) 0.19 ± 0.035 0.14 – 0.28 24

Aflatoxins (ppb) <5.00 24

Nitrate nitrogen (ppm) 10.8 ± 3.04 9.04 – 21.1 24

Nitrite nitrogen (ppm) <0.61 24

BHA (ppm) <1.0 24

BHT (ppm) <1.0 24

Aerobic plate count (CFU/g) 10.0 ± 2.0 10.0 – 20.0 24

Coliform (MPN/g) 0.30 ± 1.0 0.0 – 3.6 24

Escherichia coli (MPN/g) <10 24

Salmonella (MPN/g) Negative 24

Total nitrosoamines (ppb) 4.5 ± 1.56 2.1 – 8.8 24

N-Nitrosodimethylamine (ppb) 1.8 ± 0.88 1.0 – 5.1 24

N-Nitrosopyrrolidine (ppb) 2.6 ± 1.00 1.0 – 5.6 24

Pesticides (ppm)

"-BHC <0.01 24

$-BHC <0.02 24

(-BHC <0.01 24

*-BHC <0.01 24

Heptachlor <0.01 24

Aldrin <0.01 24

Heptachlor epoxide <0.01 24

DDE <0.01 24

DDD <0.01 24

DDT <0.01 24

HCB <0.01 24

Mirex <0.01 24

Methoxychlor <0.05 24

Dieldrin <0.01 24

Endrin <0.01 24

Telodrin <0.01 24

Chlordane <0.05 24

Toxaphene <0.10 24

Estimated PCBs <0.20 24

Pesticides (ppm)

Ronnel <0.01 24

Ethion <0.02 24

Trithion <0.05 24

Diazinon <0.10 24

Methyl chlorpyrifos 0.145 ± 0.123 0.023 – 0.499 24

Methyl parathion <0.02 24

Ethyl parathion <0.02 24

Malathion 0.207 ± 0.190 0.020 – 0.826 24

Endosulfan I <0.01 24

Endosulfan II <0.01 24

Endosulfan sulfate <0.03 24

All samples were irradiated. CFU=colony-forming units; MPN=most probable number; BHC=hexachlorocyclohexane or benzene

hexachloride

For values less than the limit of detection, the detection limit is given as the mean.

Sources of contamination: Alfalfa, grains, and fish meal

Sources of contamination: Soy oil and fish meal

All values were corrected for percent recovery.

149

APPENDIX F

SENTINEL ANIMAL PROGRAM

METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

150 Triethanolamine, NTP TR 518

SENTINEL ANIMAL PROGRAM

METHODS

Rodents used in the Carcinogenesis Program of the National Toxicology Program are produced in optimally clean

facilities to eliminate potential pathogens that may affect study results. The Sentinel Animal Program is part of the

periodic monitoring of animal health that occurs during the toxicologic evaluation of chemical compounds. Under

this program, the disease state of the rodents is monitored via serology on sera from extra (sentinel) animals in the

study rooms. These animals and the study animals are subject to identical environmental conditions. The sentinel

animals come from the same production source and weanling groups as the animals used for the studies of

chemical compounds.

Serum samples were collected from randomly selected mice during the 2-year study. Blood from each animal was

collected and allowed to clot, and the serum was separated. The samples were processed appropriately and sent to

MABioservices/BioReliance Corporation (Rockville, MD) for determination of antibody titers. The laboratory

serology methods and viral agents for which testing was performed are tabulated below; the times at which blood

was collected during the study are also listed.

Method and Test

Bacterial Assays

Oral

Fecal [including PCR (polymerase chain reaction)

analysis for Helicobacter hepaticus sp.]

ELISA

Ectromelia virus

EDIM (epizootic diarrhea of infant mice)

GDVII (mouse encephalomyelitis virus)

LCM (lymphocytic choriomeningitis virus)

Mouse adenoma virus-FL

MHV (mouse hepatitis virus)

Mycoplasma arthritidis

Mycoplasma pulmonis

PVM (pneumonia virus of mice)

Reovirus 3

Sendai

Immunofluorescence Assay

Ectromelia virus

GDVII

LCM

Mouse adenoma virus-FL

MCMV (mouse cytomegalovirus)

MHV

Parvovirus

RESULTS

All test results were negative.

Time of Analysis

18 months

1, 5, 12, and 18 months, study termination

18 months, study termination

1, 5, 12, and 18 months, study termination

Study termination

18 months

12 and 18 months

18 months, study termination

5 months, study termination

1, 5, 12, and 18 months, study termination

151

APPENDIX G

ABSORPTION, DISTRIBUTION,

METABOLISM, AND EXCRETION STUDIES

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

EFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

TABLE G1 Excretion of Radioactivity by Female Rats Following an Intravenous Dose

of [

C]-Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

ABLE G2 Distribution of Radioactivity in Tissue of Female Rats Following an Intravenous Dose

of [

C]-Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

ABLE G3 Excretion of Radioactivity by Female Rats Following Dermal Administration

of [

C]-Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

TABLE

G4 Distribution of Radioactivity in Tissue of Female Rats Following Dermal Administration

of [

C]-Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

TABLE

G5 Distribution of Absorbed and Unabsorbed Radioactivity in Female Rats

Following Dermal Administration of [

C]-Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . 160

TABLE G6 Excretion of Radioactivity by Female Mice Following an Intravenous Dose

of [

C]-Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

ABLE G7 Distribution of Radioactivity in Tissue of Female Mice Following an Intravenous Dose

of [

C]-Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

TABLE G8 Excretion of Radioactivity by Female Mice Following Dermal Administration

of [

C]-Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

TABLE G9 Distribution of Absorbed and Unabsorbed Radioactivity in Female Mice

Following Dermal Administration of [

C]-Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . 162

IGURE G1 Radiochromatogram of Urine from Female Rats Following Dermal Administration

of [

C]-Triethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

152 Triethanolamine, NTP TR 518

ABSORPTION, DISTRIBUTION, METABOLISM,

AND EXCRETION STUDIES

NTRODUCTION

Triethanolamine is used extensively as an ingredient or chemical intermediate in detergents, cosmetic products,

agrichemicals, and cutting oils and as a vulcanization accelerator in rubber manufacturing. The reported annual

production of triethanolamine in the United States during 1991 was 86 million kilograms (USITC, 1993). In the

presence of nitrosating agents, triethanolamine may be converted to N-nitrosodiethanolamine (CIR, 1983), a known

liver, kidney, and nasal carcinogen in laboratory animals (Hoffman et al., 1982; Preussman et al., 1982; Lijinsky

and Kovatch, 1985).

Triethanolamine is absorbed through the skin and gastrointestinal tract. In mice administered a single 1,000 mg/kg

dermal dose of triethanolamine, approximately 60% of the radioactivity was recovered in the urine and 20% in the

feces 48 hours after dosing (Melnick and Tomaszewski, 1990). Urinary radioactivity was composed mostly of

parent compound. Orally administered triethanolamine was rapidly absorbed and excreted mostly in the urine as

the parent compound along with a small amount of the glucuronide (Kohri et al., 1982).

The purpose of this study was to provide absorption, distribution, metabolism, and excretion data for

[

C]-triethanolamine in the test animals used in the toxicity testing of triethanolamine by the NTP.

MATERIALS AND METHODS

Study Design

Groups of four female rats received a single intravenous dose of 3 mg/kg [

C]-triethanolamine or a single dermal

dose of 68 or 276 mg/kg. Groups of four female mice received a single intravenous dose of 3 mg/kg

[

C]-triethanolamine or a single dermal dose of 79 or 1,120 mg/kg. Expired radioactivity was trapped and

quantitated and urine and feces were collected from all F344/N rats and B6C3F

mice dosed intravenously up to

72 hours after dosing, and urine and feces were collected from all F344/N rats and B6C3F

mice dosed via skin

painting up to 72 hours after dosing. Tissue samples from all intravenous animals and all dermal rats 72 hours

after dosing were also examined, and the skin site of application from all dermal mice was examined.

Procurement and Characterization of Radiolabeled and Nonradiolabeled Triethanolamine

[1,2-

C]-Triethanolamine was obtained from Chemsyn Science Laboratories (Lenexa, KS) in one lot

(CSL-96-663-38-35). The radiochemical preparation (6 mCi) was supplied in a flame-sealed ampoule solution in

ethanol at a concentration of 1.37 mCi/mL. The specific activity of [

C]-triethanolamine was 13.8 mCi/mmol.

The radiochemical purity of [

C]-triethanolamine was determined by high performance liquid chromatography

(HPLC) and was 97% pure. The chromatographic system consisted of a Whatman Partisil 10 SCX column

(250 × 4.6 mm) and a mobile phase of 0.1 M citrate with 5% sodium dihydrogen citrate; the flow rate was

1 mL/minute. Column effluent was monitored with a $-RAM radioactivity detector with a 500 µL solid scintillant

flow cell. Following the injection of [

C]-triethanolamine, the column effluent was collected in fractions, and the

radioactivity eluting in each fraction was measured by liquid scintillation spectrometry.

Nonradiolabeled triethanolamine was obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI) in one lot

(05601PN) and stored within secondary containers with solid sodium hydroxide to minimize uptake of acidic

gases. The identity of the nonradiolabeled triethanolamine was confirmed by mass spectrometry and proton

nuclear magnetic resonance spectroscopy.

153 Triethanolamine, NTP TR 518

Preparation and Administration of Dose Formulations

Single intravenous doses contained approximately 47 µCi radiolabel for rats, approximately 6 µCi radiolabel for

mice, an appropriate amount of nonradiolabeled triethanolamine, and isotonic saline as a vehicle that delivered a

total dosing volume of 1 mL/kg to rats and 2 mL/kg to mice. Intravenous doses were drawn into a syringe

equipped with a Teflon

-tipped plunger (Hamilton) and a 27 (rats) or 30 (mice) gauge hypodermic needle. Excess

dose formulation was wiped off the needle before weighing the filled dosing syringe. Intravenous doses were

injected into one lateral tail vein. After dosing, the needle was wiped clean with a Kimwipe

, and the empty

syringe was reweighed. The Kimwipe

was placed into a vial containing 2 mL ethanol and analyzed by liquid

scintillation spectrometry. Each dose was calculated as the difference between the weights of the filled and empty

dosing apparatus less the amount found in the Kimwipe

. To determine the concentration of [

C]-triethanolamine

in the dose formulation, two weighed aliquots were taken before, two after, and one during dosing.

Dermal doses were formulated in acetone and contained 65 µCi radiolabel for rats and 12 to 15 µCi for mice with

an appropriate amount of nonradiolabeled triethanolamine in a volume of approximately 190 µL per dose. A dose

area of 12 cm

for rats and 1.44 cm

for mice was located on each animal’s back. Approximately 24 hours prior to

dosing, rats were anesthetized with 7:1 ketamine:xylazine (60 mg/kg) by intramuscular injection and mice with

sodium pentobarbitol (60 mg/kg) by intraperitoneal injection. Fur at the dose area was clipped with a No. 40 blade

(Oster Professional Products). The clipped area on each animal was wiped with a gauze soaked with acetone,

dried, and then examined for abrasions. Any animal with abrasions in the clipped area was excluded from the

study. The dose area was outlined on the animal’s back with a permanent marker, and the animal was placed in a

metabolism cage.

Prior to dermal dosing, a protective foam appliance was glued to each rat’s back using Hollister’s Medical

Adhesive. The doses were administered evenly over the dose area using a 500 µL syringe equipped with a

Teflon

-tipped plunger and a gavage needle. A nonocclusive cloth cover was attached to the appliance, and a

protective metal mesh cover was secured over the appliance with Elastoplast adhesive bandage prior to returning

the rat to its cage. The doses for mice were administered over the dose area using a 100 or 250 µL syringe

equipped with a Teflon

-tipped plunger and a gavage needle. A metal tissue capsule was glued in place over the

dose area using Duro Quick Gel™ Super Glue. The doses were measured and quantitated as described above.

Biological Sample Collection in the Disposition Studies

Urine and feces from rats and mice were collected separately into round-bottom flasks cooled with dry ice at

6 (urine only), 24, 48, and 72 hours after dosing. The samples were stored in the dark at –20° C until analysis.

Radiolabeled components in breath were collected by passing air from the metabolism cage (flow rate of 200 to

500 mL/minute) through two cold traps, each containing 100 mL of ethanol, and then through a series of two traps,

each containing 400 mL of 1 N sodium hydroxide. The first cold trap was maintained at 0° C by an ice/water bath,

and the second at –60° C by an isopropanol/dry ice bath. Traps were changed at 6, 24, 32, 48, and 56 hours, and

the total weight of the solutions was measured.

At the end of the intravenous studies, rats were anesthetized with 7:1 ketamine:xylazine (60 mg/kg) by

intramuscular injection, and mice were anesthetized with 60 mg/kg sodium pentobarbital by intraperitoneal

injection. Blood was drawn by cardiac puncture into heparinized syringes, and rats and mice were sacrificed.

Samples of adipose tissue, muscle, skin, and the entire brain, heart, kidney, liver, lung, and spleen were removed

and assayed for [

C] content.

At the end of the dermal studies, rats and mice were anesthetized, blood was drawn, animals were sacrificed, and

tissue samples from rats were taken as described above. With the appliance still attached, the dose site skin from

rats and mice was excised, with care taken not to remove muscle or adipose tissue. The elastoplast was removed

and placed in a bag. For the rat study, the appliance, including the cloth cover, was removed from the dose site

154 Triethanolamine, NTP TR 518

skin. The cloth cover was removed from the appliance and was analyzed separately by liquid scintillation

spectrometry. For the mouse study, the metal appliance was removed from the skin using acetone to dissolve the

adhesive and then placed in a vial that contained water. The dose site was thoroughly rinsed with ethanol then

washed using cotton gauze soaked with soapy water. Next, the skin was rinsed with water-soaked gauze, and all

rinses were collected. The gauze was placed into individual scintillation vials for analysis by liquid scintillation

spectrometry. The dose site skin was dissolved in 2 N ethanolic sodium hydroxide.

Analysis of Biological Samples for Total Radioactivity

Aliquots of urine, trapping solution from the breath traps, skin wash, mouse appliance collection, and all dose site

digests were added directly to vials that contained scintillation cocktail (Ultima Gold, Packard Instrument

Company). Samples of tissue, feces, and blood (0.1 to 0.3 g) were digested in Soluene –350 (2 mL). After

digestion, samples that required bleaching were decolorized with perchloric acid/hydrogen peroxide prior to

addition of the scintillation cocktail. Ultima Gold and either water or ethanol were added to the rat appliance and

all skin gauze samples before analysis by liquid scintillation spectrometry.

Analysis of Biological Samples by HPLC

Urine samples were filtered through an Alltech 0.45 µm PTFE syringe filter unit prior to analysis by HPLC. The

HPLC system consisted of a Whatman Partisil 10 SCX analytical column with a mobile phase of 0.1 M citric

acid/5% sodium dihydrogen citrate buffer that contained 0.1 M sodium chloride and a flow rate of 1 mL/minute.

Analytes eluting from the column were detected using a $-RAM Flow-Through Radioactivity Detector equipped

with a 600 µL solid scintillate flow cell. After the metabolite profile was established, urine and isolated metabolite

peak fractions were incubated overnight at 37° C with purified $-glucoronidase (Sigma, 300 U from Escherichia

coli). The samples were later analyzed with HPLC using the system described above.

RESULTS AND

DISCUSSION

Data for the disposition of a 3 mg/kg intravenous dose in female rats are presented in Table G1. The radioactivity

was rapidly excreted in the urine, and 90% of the dosed radioactivity was recovered in the urine within 24 hours.

An average of 98% of the dose was recovered in the urine within 72 hours after dosing, and approximately 0.6% of

the radioactivity was recovered in the feces during this time. Less than 0.5% of the dose was recovered in carbon

dioxide traps, and less than 0.1% was recovered in volatiles traps.

The distribution of radioactivity present in tissue samples from female rats is presented in Table G2. Only 0.9% of

the dose remained in the tissues 72 hours after dosing. In contrast to diethanolamine, which was only slowly

excreted (30% of dose within 48 hours) and accumulated in the brain, heart, kidney, spleen (5% of dose at

48 hours), and liver (27% of dose at 48 hours) in a process thought to involve biochemical mimicry with the

natural alkanolamine ethanolamine (Mathews et al., 1995), comparatively little triethanolamine bioaccumulated in

the tissues.

Data for the excretion of radioactivity following dermal administration of 68 and 276 mg/kg [

C]-triethanolamine

in female rats is presented in Table G3, and the distribution of equivalents present in tissues 72 hours after dosing

is presented in Table G4. Approximately 20% to 30% of the dose was absorbed within 72 hours following dermal

exposure. The mean percentage of dose absorbed increased with increasing dose, but the increase was not

statistically significant (Table G5).

The disposition of a 3 mg/kg intravenous dose in female mice is presented in Table G6. With only 40% of the dose

excreted within 24 hours, mice appeared to excrete intravenously administered triethanolamine much slower than

did rats (90% in 24 hours; Table G1). Less than 0.5% of the dose was recovered in carbon dioxide traps, and less

than 0.1% was recovered in volatiles traps.

155 Triethanolamine, NTP TR 518

The distribution of radioactivity present in tissue samples from female mice is presented in Table G7. As was the

case for rats, the heart, kidney, liver, lung, and spleen contained higher concentrations of triethanolamine

equivalents relative to blood.

Mice absorbed dermally applied 79 and 1,120 mg/kg triethanolamine more readily than did rats (Tables G8 and

G9). Mice rapidly excreted the absorbed radioactivity in the urine and feces with 28% to 48% of the dose

recovered in excreta within 24 hours. The percent of the dose absorbed increased with increasing dose.

Urine collected 6 to 24 hours after intravenous dosing and 48 to 72 hours after dermal application of

triethanolamine in female rats was analyzed by HPLC (Figure G1). The chromatogram contains a peak that

coelutes with triethanolamine and two other peaks that comprise about 5% of the radioactivity in the sample.

These peaks, however, were also present in the chromatogram of the radiolabeled test article and may reflect the

presence of impurities rather than metabolites. The metabolite fractions were collected and incubated with purified

$-glucuronidase, as was an aliquot of the whole urine. Analysis of these samples showed no change in the

metabolite profile. Similarly, urine collected 6 to 24 hours after intravenous or dermal dosing contained more than

95% radiolabeled components that coeluted with unchanged triethanolamine, with minor components eluting in the

same fractions in mice as those in rats.

SUMMARY

Intravenously administered triethanolamine was rapidly excreted by female rats and mice, primarily in the urine.

Less than 1% of the dose was present in tissues sampled 72 hours after dosing. In both species, the heart, kidney,

liver, lung, and spleen contained higher concentrations of triethanolamine equivalents than did blood.

Only 20% to 30% of dermally applied 68 and 276 mg/kg triethanolamine was absorbed by female rats within

72 hours. Mice absorbed dermally applied 79 and 1,120 mg/kg triethanolamine more extensively (60% to 80%)

than did rats. On the basis of mass of triethanolamine per area of skin, the lowest dermal dose levels for rats and

mice were equal at 1.09 mg/cm

. The skin of mice is thinner than that of rats, and this difference may explain the

higher percentage of dose absorbed by mice. The highest dermal doses were 4 and 15 mg/cm

for rats and mice,

respectively. Triethanolamine enhances its own absorption, and the pronounced difference between the species

was not unexpected. The percent of dose absorbed in each species increased with increasing dose, but in rats, the

increase was not statistically significant. Both species rapidly excreted the absorbed dose, primarily in urine. In

rats, less than 1% of the dose was present in the tissue samples (except the dose site) 72 hours after treatment; the

heart, kidney, liver, lung, and spleen contained elevated concentrations of radiolabel relative to blood.

After intravenous and dermal dosing in female rats and mice, triethanolamine was excreted, for the most part,

unchanged in urine. Two additional polar peaks, each less than or equal to 5% of the total, were present in the

urine. At least one of these polar peaks may have originated from impurities in the [

C]-triethanolamine stock,

which were better resolved from the test article peak during development of HPLC conditions for metabolite

analysis.

REFERENCES

Cosmetic Ingredient Review (CIR) Expert Panel (1983). Final report on the safety assessment of triethanolamine,

diethanolamine, and monoethanolamine. J. Am. Coll. Toxicol. 2, 183-235.

Hoffman, D., Brunnemann, K.D., Rivenson, A., and Hecht, S.S. (1982). N-Nitrosodiethanolamine: Analysis,

formation in tobacco products and carcinogenicity in Syrian golden hamsters. IARC Sci. Publ. 41, 299-308.

Kohri, N., Matsuda, T., Umeniwa, K., Miyazaki, K., and Arita, T. (1982). Development of assay method in

biological fluids and biological fate of triethanolamine [in Japanese, English summary]. Yakuzaigaku 42, 342-348.

156 Triethanolamine, NTP TR 518

Lijinsky, W., and Kovatch, R.M. (1985). Induction of liver tumors in rats by nitrosodiethanolamine at low doses.

Carcinogenesis 6, 1679-1681.

Mathews, J.M., Garner, C.E., and Matthews, H.B. (1995). Metabolism, bioaccumulation, and incorporation of

diethanolamine into phospholipids. Chem. Res. Toxicol. 8, 625-633.

Melnick, R.L., and Tomaszewski, K.E. (1990). Triethanolamine. In Ethel Browning’s Toxicity and Metabolism of

Industrial Solvents, Vol. 2: Nitrogen and Phosphorus Solvents, 2nd ed. (D.R. Buhler and D.J. Reed, Eds.),

pp. 441-450. Elsevier Science Publishers, New York.

Preussmann, R., Habs, M., Habs, H., and Schmähl, D. (1982). Carcinogenicity of N-nitrosodiethanolamine in rats

at five different dose levels. Cancer Res. 42, 5167-5171.

U.S. International Trade Commission (USITC) (1993). Synthetic Organic Chemicals. United States Production

and Sales, 1991, p. 15-4. USITC Publication 2607. U.S. International Trade Commission, Washington, DC.

157 Triethanolamine, NTP TR 518

ABLE G1

Excretion of Radioactivity by Female Rats Following an Intravenous Dose of [

C]-Triethanolamine

Time (hours) Urine Feces Total

6 38.2 ± 26.0 38.2 ± 26.0

24 90.5 ± 1.23 0.373 ± 0.106 90.9 ± 1.33

96.3 ± 1.38

98.2 ± 1.85

0.580 ± 0.120

0.630 ± 0.147

96.9 ± 1.50

98.9 ± 1.99

Samples were collected from four female rats per time point (feces not collected at 6 hours) after a 3 mg/kg intravenous dose; data are

cumulative radioactivity excreted and presented as percent dose recovered (mean ± standard deviation).

Includes urine from the bladder and final cage rinse

TABLE G2

Distribution of Radioactivity in Tissue of Female Rats Following an Intravenous Dose

of [

C]-Triethanolamine

ng-Eq

[

C]-Triethanolamine/ Tissue/Blood % Dose

Tissue g Tissue Ratio in Total Tissue

Adipose 6.89 ± 0.508 0.857 ± 0.141 0.0167 ± 0.00101

Blood 8.13 ± 0.752 Unity 0.0146 ± 0.00145

Brain 21.3 ± 1.43 2.63 ± 0.103 0.00681 ± 0.000588

Heart 41.0 ± 2.97 5.06 ± 0.354 0.00447 ± 0.000231

Kidney 271 ± 16.5 33.6 ± 3.42 0.0597 ± 0.00515

Liver 257 ± 17.3 31.9 ± 5.11 0.272 ± 0.0189

Lung 109 ± 6.21 13.4 ± 0.747 0.0150 ± 0.00102

Muscle 26.9 ± 2.03 3.32 ± 0.215 0.447 ± 0.0332

Skin 15.1 ± 1.61 1.86 ± 0.158 0.0886 ± 0.00939

Spleen 67.8 ± 5.59 8.36 ± 0.710 0.00567 ± 0.0000251

Total in Tissues 0.930 ± 0.0384

Samples were collected from four female rats at 72 hours after a 3 mg/kg intravenous dose; data are presented as mean ± standard deviation.

Eq=Equivalents

158 Triethanolamine, NTP TR 518

ABLE G3

Excretion of Radioactivity by Female Rats Following Dermal Administration of [

C]-Triethanolamine

Time (hours) Urine Feces Total

68 mg/kg Dose Group

6 0.171 ± 0.172 0.171 ± 0.172

24 1.33 ± 0.496 0.0080 ± 0.00446 1.34 ± 0.494

5.95 ± 2.71

12.9 ± 5.84

0.0541 ± 0.0493

0.247 ± 0.167

6.01 ± 2.75

13.1 ± 5.70

276 mg/kg Dose Group

6 0.135 ± 0.0793 0.135 ± 0.0793

24 3.03 ± 1.36 0.0148 ± 0.00738 3.04 ± 1.36

9.90 ± 2.52

23.8 ± 7.82

0.0664 ± 0.0189

0.142 ± 0.0471

9.96 ± 2.53

23.9 ± 7.87

Samples were collected from four (68 mg/kg) or three (276 mg/kg) female rats per time point (feces not collected at 6 hours); data are

cumulative radioactivity excreted and presented as percent dose recovered (mean ± standard deviation).

Includes urine from the bladder and final cage rinse

159 Triethanolamine, NTP TR 518

ABLE G4

Distribution of Radioactivity in Tissue of Female Rats Following Dermal Administration

of [

C]-Triethanolamine

Tissue

ng-Eq

[

C]-Triethanolamine/

g Tissue

Tissue/Blood

Ratio

% Dose

in Total Tissue

68 mg/kg Dose Group

Adipose

Blood

Brain

Heart

Kidney

Liver

Lung

Muscle

Skin

Spleen

136 ± 72.0

174 ± 82.2

214 ± 94.9

682 ± 305

4,890 ± 2,250

2,840 ± 1,280

1,520 ± 695

584 ± 280

341 ± 198

919 ± 389

0.746 ± 0.150

Unity

1.30 ± 0.301

3.98 ± 0.327

28.2 ± 2.05

16.9 ± 3.44

8.83 ± 0.867

3.34 ± 0.440

2.03 ± 0.707

5.47 ± 0.658

0.0131 ± 0.00703

0.0124 ± 0.00599

0.00265 ± 0.00107

0.00293 ± 0.00135

0.0467 ± 0.0226

0.113 ± 0.0471

0.00773 ± 0.00360

0.386 ± 0.187

0.0799 ± 0.0471

0.00290 ± 0.00134

Total in Tissues 0.668 ± 0.308

276 mg/kg Dose Group

Adipose

Blood

Brain

Heart

Kidney

Liver

Lung

Muscle

Skin

Spleen

1,200 ± 539

1,700 ± 758

1,480 ± 685

6,080 ± 2,740

39,300 ± 20,600

19,000 ± 8,270

12,800 ± 5,660

5,060 ± 1,630

2,640 ± 832

7,970 ± 3,680

0.713 ± 0.144

Unity

0.870 ± 0.144

3.57 ± 0.223

22.4 ± 2.67

11.3 ± 1.42

7.54 ± 0.569

3.15 ± 0.566

1.64 ± 0.299

4.66 ± 0.336

0.0282 ± 0.0126

0.0298 ± 0.0142

0.00463 ± 0.00234

0.00682 ± 0.00341

0.0949 ± 0.0543

0.206 ± 0.0998

0.0169 ± 0.00844

0.820 ± 0.289

0.151 ± 0.0520

0.00627 ± 0.00331

Total in Tissues 1.36 ± 0.531

Samples were collected from four (68 mg/kg) or three (276 mg/kg) female rats at 72 hours; data are presented as mean ± standard deviation.

Eq=Equivalents

160 Triethanolamine, NTP TR 518

ABLE G5

Distribution of Absorbed and Unabsorbed Radioactivity in Female Rats

Following Dermal Administration of [

C]-Triethanolamine

68 mg/kg 276 mg/kg

Absorbed

Tissues 0.668 ± 0.308 1.36 ± 0.531

Dose site 5.62 ± 2.32 3.12 ± 0.881

Feces 0.247 ± 0.167 0.142 ± 0.0471

Urine 12.9 ± 5.84 23.8 ± 7.82

Total 19.4 ± 5.43 28.4 ± 9.08

Unabsorbed

Dosing appliance 6.35 ± 4.11 2.99 ± 2.01

Skin gauze 22.9 ± 4.04 10.2 ± 4.39

Skin wash 37.2 ± 9.13 41.4 ± 3.54

Total 66.5 ± 5.26 54.6 ± 6.96

Samples were collected from four (68 mg/kg) or three (276 mg/kg) female rats; data are presented as percent dose recovered

(mean ± standard deviation).

161 Triethanolamine, NTP TR 518

ABLE G6

Excretion of Radioactivity by Female Mice Following an Intravenous Dose of [

C]-Triethanolamine

Time (hours) Urine Feces Total

6 6.76 ± 8.29 6.76 ± 8.29

24 26.0 ± 17.2 14.3 ± 7.56 40.3 ± 10.3

42.6 ± 22.8

61.9 ± 14.6

23.7 ± 13.1

27.6 ± 10.4

66.2 ± 14.6

89.5 ± 4.62

Samples were collected from four female mice per time point (feces not collected at 6 hours) after a 3 mg/kg intravenous dose; data are

cumulative radioactivity excreted and presented as percent dose recovered (mean ± standard deviation).

Includes urine from the bladder and final cage rinse

TABLE G7

Distribution of Radioactivity in Tissue of Female Mice Following an Intravenous Dose

of [

C]-Triethanolamine

ng-Eq

[

C]-Triethanolamine/ Tissue/Blood % Dose

Tissue g Tissue Ratio in Total Tissue

Adipose 17.1 ± 2.7 4.65 ± 1.29 0.0384 ± 0.00623

Blood 3.79 ± 0.578 Unity 0.00631 ± 0.000932

Brain 15.0 ± 1.12 4.02 ± 0.490 0.00835 ± 0.00158

Heart 32.7 ± 1.01 8.77 ± 1.28 0.00587 ± 0.000527

Kidney 106 ± 11.4 28.2 ± 3.75 0.0489 ± 0.00467

Liver 215 ± 33.8 57.7 ± 13.5 0.305 ± 0.0484

Lung 68.4 ± 5.45 18.2 ± 1.94 0.0123 ± 0.00193

Muscle 14.4 ± 1.27 3.87 ± 0.720 0.221 ± 0.0145

Skin 12.1 ± 1.19 3.24 ± 0.627 0.0658 ± 0.00797

Spleen 48.4 ± 2.15 12.9 ± 1.54 0.00425 ± 0.000225

Total in Tissues 0.716 ± 0.0685

Samples were collected from four female mice 72 hours after a 3 mg/kg intravenous dose; data are presented as mean ± standard deviation.

Eq=Equivalents

162 Triethanolamine, NTP TR 518

ABLE G8

Excretion of Radioactivity by Female Mice Following Dermal Administration of [

C]-Triethanolamine

Time (hours) Urine Feces Total

79 mg/kg Dose Group

6 3.04 ± 4.84 3.04 ± 4.84

24 22.5 ± 8.28 5.03 ± 3.09 27.5 ± 6.52

39.7 ± 4.96

48.2 ± 5.09

7.07 ± 4.65

7.8 ± 4.73

46.8 ± 7.28

56.0 ± 8.05

1,120 mg/kg Dose Group

6 0.000105 ± 0.00009 0.000105 ± 0.00009

24 39.7 ± 21.5 8.75 ± 6.02 48.4 ± 20.5

57.1 ± 18.1

67.7 ± 14.9

11.8 ± 7.05

13.0 ± 7.60

68.9 ± 13.1

80.7 ± 7.96

Samples were collected from four (79 mg/kg) or three (1,120 mg/kg) female mice per time point (feces not collected at 6 hours); data are

cumulative radioactivity excreted and presented as percent dose recovered (mean ± standard deviation).

Includes urine from the bladder and final cage rinse

TABLE G9

Distribution of Absorbed and Unabsorbed Radioactivity in Female Mice

Following Dermal Administration of [

C]-Triethanolamine

79 mg/kg 1,120 mg/kg

Absorbed

Blood 0.00590 ± 0.00144 0.00627 ± 0.000412

Dose site 1.35 ± 0.317 0.576 ± 0.118

Feces 7.80 ± 4.73 13.0 ± 7.60

Urine 48.2 ± 5.09 67.7 ± 14.9

Total 57.3 ± 8.34 81.3 ± 8.02

Unabsorbed

Dosing appliance 0.481 ± 0.301 1.68 ± 2.02

Skin gauze 2.30 ± 2.00 0.824 ± 0.737

Skin wash 11.8 ± 4.65 4.24 ± 2.13

Total 14.6 ± 2.84 6.75 ± 3.41

Samples were collected from four (79 mg/kg) or three (1,120 mg/kg) female mice; data are presented as percent dose recovered

(mean ± standard deviation).

A B

Triethanolamine

Time (minutes)

20 25

163 Triethanolamine, NTP TR 518

IGURE G1

Radiochromatogram of Urine from Female Rats Following Dermal Administration

of [

C]-Triethanolamine

(urine collected 48 to 72 hours after dosing)

National Toxicology Program Technical Reports

Printed as of May 2004

Environmental Health Persepctives (EHP) maintains the library of NTP Technical Reports in electronic and print format.

To gain access to these reports, contact EHP online at http://ehp.niehs.nih.gov or call 866-541-3841 or 919-653-2590.

Chemical

Acetaminophen

Acetonitrile

Acrylonitrile

Agar

Allyl Glycidyl Ether

Allyl Isothiocyanate

Allyl Isovalerate

1-Amino-2,4-Dibromoanthraquinone

2-Amino-4-Nitrophenol

2-Amino-5-Nitrophenol

11-Aminoundecanoic Acid

dl-Amphetamine Sulfate

Ampicillin Trihydrate

Asbestos, Amosite (Hamsters)

Asbestos, Amosite (Rats)

Asbestos, Chrysotile (Hamsters)

Asbestos, Chrysotile (Rats)

Asbestos, Crocidolite

Asbestos, Tremolite

L-Ascorbic Acid

AZT and AZT/"-Interferon A/D

Barium Chloride Dihydrate

Benzaldehyde

Benzene

Benzethonium Chloride

Benzofuran

Benzyl Acetate (Gavage)

Benzyl Acetate (Feed)

Benzyl Alcohol

o-Benzyl-p-Chlorophenol (Gavage)

o-Benzyl-p-Chlorophenol (Mouse Skin)

2-Biphenylamine Hydrochloride

2,2-Bis(Bromomethyl)-1,3-Propanediol

Bis(2-Chloro-1-Methylethyl) Ether

Bisphenol A

Boric Acid

Bromodichloromethane

Bromoethane

1,3-Butadiene

t-Butyl Alcohol

Butyl Benzyl Phthalate

n-Butyl Chloride

t-Butylhydroquinone

(-Butyrolactone

Caprolactam

d-Carvone

Chloral Hydrate

Chlorinated and Chloraminated Water

Chlorendic Acid

Chlorinated Paraffins: C

, 43% Chlorine

Chlorinated Paraffins: C

, 60% Chlorine

Chlorinated Trisodium Phosphate

2-Chloroacetophenone

p-Chloroaniline Hydrochloride

Chlorobenzene

Chlorodibromomethane

Chloroethane

2-Chloroethanol

3-Chloro-2-Methylpropene

Chloroprene

1-Chloro-2-Propanol

TR No.

394

447

506

230

376

234

253

383

339

334

216

387

318

249

279

246

295

280

277

247

469

432

378

289

438

370

250

431

343

424

444

233

452

239

215

324

321

363

288

434

436

213

458

312

459

406

214

381

502

503

392

304

305

308

294

379

351

261

282

346

275

300

467

477

Chemical TR No.

Chlorpheniramine Maleate 317

C.I. Acid Orange 3 335

C.I. Acid Orange 10 211

C.I. Acid Red 14 220

C.I. Acid Red 114 405

C.I. Basic Red 9 Monohydrochloride 285

C.I. Direct Blue 15 397

C.I. Direct Blue 218 430

C.I. Disperse Blue 1 299

C.I. Disperse Yellow 3 222

C.I. Pigment Red 3 407

C.I. Pigment Red 23 411

C.I. Solvent Yellow 14 226

trans-Cinnamaldehyde 514

Citral 505

Cobalt Sulfate Heptahydrate 471

Coconut Oil Acid Diethanolamine Condensate 479

Codeine 455

Comparative Initiation/Promotion Studies (Mouse Skin) 441

Corn Oil, Safflower Oil, and Tricaprylin 426

Coumarin 422

CS2 377

Cytembena 207

D&C Red No. 9 225

D&C Yellow No. 11 463

Decabromodiphenyl Oxide 309

Diallyl Phthalate (Mice) 242

Diallyl Phthalate (Rats) 284

4,4´-Diamino-2,2´-Stilbenedisulfonic Acid, Disodium Salt 412

2,4-Diaminophenol Dihydrochloride 401

1,2-Dibromo-3-Chloropropane 206

1,2-Dibromoethane 210

2,3-Dibromo-1-Propanol 400

1,2-Dichlorobenzene (o-Dichlorobenzene) 255

1,4-Dichlorobenzene (p-Dichlorobenzene) 319

p,p´-Dichlorodiphenyl sulfone 501

2,4-Dichlorophenol 353

2,6-Dichloro-p-Phenylenediamine 219

1,2-Dichloropropane 263

1,3-Dichloropropene (Telone II) 269

Dichlorvos 342

Dietary Restriction 460

Diethanolamine 478

Di(2-Ethylhexyl) Adipate 212

Di(2-Ethylhexyl) Phthalate 217

Diethyl Phthalate 429

Diglycidyl Resorcinol Ether 257

3,4-Dihydrocoumarin 423

1,2-Dihydro-2,2,4-Trimethylquinoline (Monomer) 456

Dimethoxane 354

3,3´-Dimethoxybenzidine Dihydrochloride 372

N,N-Dimethylaniline 360

3,3´-Dimethylbenzidine Dihydrochloride 390

Dimethyl Hydrogen Phosphite 287

Dimethyl Methylphosphonate 323

Dimethyl Morpholinophosphoramidate 298

Dimethylvinyl Chloride 316

Diphenhydramine Hydrochloride 355

5,5-Diphenylhydantoin 404

Elmiron

512

Emodin 493

Ephedrine Sulfate 307

Epinephrine Hydrochloride 380

1,2-Epoxybutane 329

Chemical TR No. Chemical TR No.

Erythromycin Stearate

Ethyl Acrylate

Ethylbenzene

Ethylene Glycol

Ethylene Glycol Monobutyl Ether

Ethylene Oxide

Ethylene Thiourea

Eugenol

FD&C Yellow No. 6

Fumonisin B

Furan

Furfural

Furfuryl Alcohol

Furosemide

Gallium Arsenide

Geranyl Acetate

Glutaraldehyde

Glycidol

Guar Gum

Gum Arabic

HC Blue 1

HC Blue 2

HC Red 3

HC Yellow 4

Hexachlorocyclopentadiene

Hexachloroethane

2,4-Hexadienal

4-Hexylresorcinol

Hydrochlorothiazide

Hydroquinone

8-Hydroxyquinoline

Indium Phosphide

Iodinated Glycerol

Isobutene

Isobutyl Nitrite

Isobutyraldehyde

Isophorone

Isoprene

Lauric Acid Diethanolamine Condensate

d-Limonene

Locust Bean Gum

60-Hz Magnetic Fields

Magnetic Field Promotion

Malonaldehyde, Sodium Salt

Manganese Sulfate Monohydrate

D-Mannitol

Marine Diesel Fuel and JP-5 Navy Fuel

Melamine

2-Mercaptobenzothiazole

Mercuric Chloride

Methacrylonitrile

8-Methoxypsoralen

"-Methylbenzyl Alcohol

Methyl Bromide

Methyl Carbamate

Methyldopa Sesquihydrate

Methylene Chloride

4,4´-Methylenedianiline Dihydrochloride

Methyleugenol

Methyl Methacrylate

N-Methylolacrylamide

Methylphenidate Hydrochloride

Mirex

Molybdenum Trioxide

Monochloroacetic Acid

Monuron

Nalidixic Acid

Naphthalene (Mice)

Naphthalene (Rats)

Nickel (II) Oxide

Nickel Sulfate Hexahydrate

338

259

466

413

484

326

388

223

208

496

402

382

482

356

492

252

490

374

229

227

271

293

281

419

437

361

509

330

357

366

276

499

340

487

448

472

291

486

480

347

221

488

489

331

428

236

310

245

332

408

497

359

369

385

328

348

306

248

491

314

352

439

313

462

396

266

368

410

500

451

454

Nickel Subsulfide 453

p-Nitroaniline 418

o-Nitroanisole 416

p-Nitrobenzoic Acid 442

Nitrofurantoin 341

Nitrofurazone 337

Nitromethane 461

p-Nitrophenol 417

o-Nitrotoluene 504

p-Nitrotoluene 498

Ochratoxin A 358

Oleic Acid Diethanolamine Condensate 481

Oxazepam (Mice) 443

Oxazepam (Rats) 468

Oxymetholone 485

Oxytetracycline Hydrochloride 315

Ozone and Ozone/NNK 440

Penicillin VK 336

Pentachloroanisole 414

Pentachloroethane 232

Pentachloronitrobenzene 325

Pentachlorophenol, Purified 483

Pentachlorophenol, Technical Grade 349

Pentaerythritol Tetranitrate 365

Phenolphthalein 465

Phenylbutazone 367

Phenylephrine Hydrochloride 322

N-Phenyl-2-Naphthylamine 333

o-Phenylphenol 301

Polybrominated Biphenyl Mixture (Firemaster FF-1) (Gavage) 244

Polybrominated Biphenyl Mixture (Firemaster FF-1) (Feed) 398

Polysorbate 80 (Glycol) 415

Polyvinyl Alcohol 474

Primidone 476

Probenecid 395

Promethazine Hydrochloride 425

Propylene 272

Propylene Glycol Mono-t-butyl Ether 515

1,2-Propylene Oxide 267

Propyl Gallate 240

Pyridine 470

Quercetin 409

Riddelliine 508

Resorcinol 403

Rhodamine 6G 364

Rotenone 320

Roxarsone 345

Salicylazosulfapyridine 457

Scopolamine Hydrobromide Trihydrate 445

Sodium Azide 389

Sodium Fluoride 393

Sodium Nitrite 495

Sodium Xylenesulfonate 464

Stannous Chloride 231

Succinic Anhydride 373

Talc 421

Tara Gum 224

2,3,7,8-Tetrachlorodibenzo-p-Dioxin (Dermal) 201

2,3,7,8-Tetrachlorodibenzo-p-Dioxin (Gavage) 209

1,1,1,2-Tetrachloroethane 237

Tetrachloroethylene 311

Tetracycline Hydrochloride 344

Tetrafluoroethylene 450

1-Trans-Delta

-Tetrahydrocannabinol 446

Tetrahydrofuran 475

Tetrakis(Hydroxymethyl)Phosphonium Sulfate 296

Tetrakis(Hydroxymethyl)Phosphonium Chloride 296

Tetranitromethane 386

Theophylline 473

4,4-Thiobis(6-t-Butyl-m-Cresol) 435

Titanocene Dichloride 399

Chemical TR No. Chemical TR No.

Toluene 371 Tris(2-Ethylhexyl) Phosphate 274

2,4- & 2,6-Toluene Diisocyanate 251 Turmeric Oleoresin (Curcumin) 427

Triamterene 420 Vanadium Pentoxide 507

Tribromomethane 350 4-Vinylcyclohexene 303

Trichloroethylene 243 4-Vinyl-1-Cyclohexene Diepoxide 362

Trichloroethylene 273 Vinylidene Chloride 228

1,2,3-Trichloropropane 384 Vinyl Toluene 375

Tricresyl Phosphate 433 Xylenes (Mixed) 327

Triethanolamine 449 2,6-Xylidine 278

Triethanolamine 518 Zearalenone 235

Tris(2-Chloroethyl) Phosphate 391 Ziram 238

National Toxicology Program

National Institute of Environmental Health Sciences

National Institutes of Health

P.O. Box 12233, MD K2-05

Durham, NC 27709

Tel: 984-287-3211

[email protected]

https://ntp.niehs.nih.gov

ISSN 2378-8925