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25 August 2015
EMA/CHMP/ICH/83812/2013
Committee for Human Medicinal Products
ICH guideline M7(R1) on assessment and control of DNA
reactive (mutagenic) impurities in pharmaceuticals to
limit potential carcinogenic risk
Step 5
Transmission to CHMP
July 2017
Adoption by CHMP for release for consultation
July 2017
End of consultation (deadline for comments)
January 2018
Final adoption by CHMP
February 2018
Date for coming into effect
February 2018
ICH guideline M7(R1) on assessment and control of DNA reactive (mutagenic) impurities in
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M7(R1)
Document History
Code
History
Date
M7
Approval by the Steering Committee under Step 2 and release
for public consultation.
6 February
2013
M7
Approval by the Steering Committee under Step 4 and
recommendation for adoption to the three ICH regulatory bodies.
5 June 2014
M7
Corrigendum to fix typographical errors and replace word
“degradants” with “degradation products”.
23 June
2014
M7(R1)
Addendum
Endorsement by the Members of the ICH Assembly under Step 2
and release for public consultation.
11 June
2015
Current Step 4 version
M7(R1)
Addendum
Adoption by the Regulatory Members of the ICH Assembly under
Step 4 and recommendation for adoption to the ICH regulatory
bodies
TBC
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ICH guideline M7(R1) on assessment and control of DNA
reactive (mutagenic) impurities in pharmaceuticals to
limit potential carcinogenic risk
Table of contents
1. Introduction ............................................................................................ 5
2. Scope of guideline ................................................................................... 5
3. General principles .................................................................................... 6
4. Considerations for marketed products ..................................................... 7
4.1. Post approval changes to the drug substance chemistry, manufacturing, and controls ... 7
4.2. Post approval changes to the drug product chemistry, manufacturing, and controls ...... 7
4.3. Changes to the clinical use of marketed products ..................................................... 8
4.4. Other considerations for marketed products............................................................. 8
5. Drug substance and drug product impurity assessment .......................... 8
5.1. Synthetic impurities .............................................................................................. 8
5.2. Degradation products............................................................................................ 9
5.3. Considerations for clinical development ................................................................... 9
6. Hazard assessment elements ................................................................ 10
7. Risk characterization ............................................................................. 11
7.1. TTC-based acceptable intakes .............................................................................. 11
7.2. Acceptable intakes based on compound-specific risk assessments ............................ 11
7.2.1. Mutagenic impurities with positive carcinogenicity data (class 1 in table 1) ............. 11
7.2.2. Mutagenic impurities with evidence for a practical threshold ................................. 12
7.3. Acceptable intakes in relation to less-than-lifetime (LTL) exposure ........................... 12
7.3.1. Clinical development ........................................................................................ 12
7.3.2. Marketed products ........................................................................................... 13
7.4. Acceptable intakes for multiple mutagenic impurities .............................................. 13
7.5. Exceptions and flexibility in approaches ................................................................. 13
8. Control .................................................................................................. 14
8.1. Control of process related impurities ..................................................................... 14
8.2. Considerations for control approaches ................................................................... 16
8.3. Considerations for periodic testing ........................................................................ 16
8.4. Control of degradation products ........................................................................... 16
8.5. Lifecycle management ........................................................................................ 17
8.6. Considerations for clinical development ................................................................. 18
9. Documentation ...................................................................................... 18
9.1. Clinical trial applications ...................................................................................... 18
9.2. Common technical document (marketing application) ............................................. 18
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Glossary .................................................................................................... 24
References ................................................................................................ 26
Appendices ................................................................................................ 27
Appendix 1: Scope scenarios for application of the ICH M7 guideline .............................. 27
Appendix 2: Case examples to illustrate potential control approaches ............................. 28
Appendix 3: Addendum to ICH M7 .............................................................................. 32
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1. Introduction
The synthesis of drug substances involves the use of reactive chemicals, reagents, solvents, catalysts,
and other processing aids. As a result of chemical synthesis or subsequent degradation, impurities
reside in all drug substances and associated drug products. While ICH Q3A(R2): Impurities in New
Drug Substances and Q3B(R2): Impurities in New Drug Products (Ref. 1, 2) provides guidance for
qualification and control for the majority of the impurities, limited guidance is provided for those
impurities that are DNA reactive. The purpose of this guideline is to provide a practical framework that
is applicable to the identification, categorization, qualification, and control of these mutagenic
impurities to limit potential carcinogenic risk. This guideline is intended to complement ICH Q3A(R2),
Q3B(R2) (Note 1), and ICH M3(R2): Nonclinical Safety Studies for the Conduct of Human Clinical Trials
and Marketing Authorizations for Pharmaceuticals (Ref. 3).
This guideline emphasizes considerations of both safety and quality risk management in establishing
levels of mutagenic impurities that are expected to pose negligible carcinogenic risk. It outlines
recommendations for assessment and control of mutagenic impurities that reside or are reasonably
expected to reside in final drug substance or product, taking into consideration the intended conditions
of human use.
2. Scope of guideline
This document is intended to provide guidance for new drug substances and new drug products during
their clinical development and subsequent applications for marketing. It also applies to post-approval
submissions of marketed products, and to new marketing applications for products with a drug
substance that is present in a previously approved product, in both cases only where:
Changes to the drug substance synthesis result in new impurities or increased acceptance criteria
for existing impurities;
Changes in the formulation, composition or manufacturing process result in new degradation
products or increased acceptance criteria for existing degradation products;
Changes in indication or dosing regimen are made which significantly affect the acceptable cancer
risk level.
Assessment of the mutagenic potential of impurities as described in this guideline is not intended for
the following types of drug substances and drug products: biological/biotechnological, peptide,
oligonucleotide, radiopharmaceutical, fermentation products, herbal products, and crude products of
animal or plant origin.
This guideline does not apply to drug substances and drug products intended for advanced cancer
indications as defined in the scope of ICH S9 (Ref. 4). Additionally, there may be some cases where a
drug substance intended for other indications is itself genotoxic at therapeutic concentrations and may
be expected to be associated with an increased cancer risk. Exposure to a mutagenic impurity in these
cases would not significantly add to the cancer risk of the drug substance. Therefore, impurities could
be controlled at acceptable levels for non-mutagenic impurities.
Assessment of the mutagenic potential of impurities as described in this guideline is not intended for
excipients used in existing marketed products, flavoring agents, colorants, and perfumes. Application
of this guideline to leachables associated with drug product packaging is not intended, but the safety
risk assessment principles outlined in this guideline for limiting potential carcinogenic risk can be used
if warranted. The safety risk assessment principles of this guideline can be used if warranted for
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impurities in excipients that are used for the first time in a drug product and are chemically
synthesized.
3. General principles
The focus of this guideline is on DNA reactive substances that have a potential to directly cause DNA
damage when present at low levels leading to mutations and therefore, potentially causing cancer.
This type of mutagenic carcinogen is usually detected in a bacterial reverse mutation (mutagenicity)
assay. Other types of genotoxicants that are non-mutagenic typically have threshold mechanisms and
usually do not pose carcinogenic risk in humans at the level ordinarily present as impurities. Therefore
to limit a possible human cancer risk associated with the exposure to potentially mutagenic impurities,
the bacterial mutagenicity assay is used to assess the mutagenic potential and the need for controls.
Structure-based assessments are useful for predicting bacterial mutagenicity outcomes based upon the
established knowledge. There are a variety of approaches to conduct this evaluation including a review
of the available literature, and/or computational toxicology assessment.
A Threshold of Toxicological Concern (TTC) concept was developed to define an acceptable intake for
any unstudied chemical that poses a negligible risk of carcinogenicity or other toxic effects. The
methods upon which the TTC is based are generally considered to be very conservative since they
involve a simple linear extrapolation from the dose giving a 50% tumor incidence (TD
50
) to a 1 in 10
6
incidence, using TD
50
data for the most sensitive species and most sensitive site of tumor induction.
For application of a TTC in the assessment of acceptable limits of mutagenic impurities in drug
substances and drug products, a value of 1.5 μg/day corresponding to a theoretical 10
-5
excess lifetime
risk of cancer, can be justified. Some structural groups were identified to be of such high potency that
intakes even below the TTC would theoretically be associated with a potential for a significant
carcinogenic risk. This group of high potency mutagenic carcinogens referred to as the “cohort of
concern”, comprises aflatoxin-like-, N-nitroso-, and alkyl-azoxy compounds.
During clinical development, it is expected that control strategies and approaches will be less
developed in earlier phases where overall development experience is limited. This guideline bases
acceptable intakes for mutagenic impurities on established risk assessment strategies. Acceptable risk
during the early development phase is set at a theoretically calculated level of approximately one
additional cancer per million. For later stages in development and for marketed products, acceptable
increased cancer risk is set at a theoretically calculated level of approximately one in one hundred
thousand. These risk levels represent a small theoretical increase in risk when compared to human
overall lifetime incidence of developing any type of cancer, which is greater than 1 in 3. It is noted
that established cancer risk assessments are based on lifetime exposures. Less-Than-Lifetime (LTL)
exposures both during development and marketing can have higher acceptable intakes of impurities
and still maintain comparable risk levels. The use of a numerical cancer risk value (1 in 100,000) and
its translation into risk-based doses (TTC) is a highly hypothetical concept that should not be regarded
as a realistic indication of the actual risk. Nevertheless, the TTC concept provides an estimate of safe
exposures for any mutagenic compound. However, exceeding the TTC is not necessarily associated
with an increased cancer risk given the conservative assumptions employed in the derivation of the
TTC value. The most likely increase in cancer incidence is actually much less than 1 in 100,000. In
addition, in cases where a mutagenic compound is a non-carcinogen in a rodent bioassay, there would
be no predicted increase in cancer risk. Based on all the above considerations, any exposure to an
impurity that is later identified as a mutagen is not necessarily associated with an increased cancer risk
for patients already exposed to the impurity. A risk assessment would determine whether any further
actions would be taken.
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Where a potential risk has been identified for an impurity, an appropriate control strategy leveraging
process understanding and/or analytical controls should be developed to ensure that the mutagenic
impurity is at or below the acceptable cancer risk level.
There may be cases when an impurity is also a metabolite of the drug substance. In such cases the
risk assessment that addresses mutagenicity of the metabolite can qualify the impurity.
4. Considerations for marketed products
This guideline is not intended to be applied retrospectively (i.e., to products marketed prior to adoption
of this guideline). However, some types of post-approval changes warrant a reassessment of safety
relative to mutagenic impurities. This section applies to these post approval changes for products
marketed prior to, or after, the adoption of this guideline. Section 8.5 (Lifecycle Management)
contains additional recommendations for products marketed after adoption of this guideline.
4.1. Post approval changes to the drug substance chemistry,
manufacturing, and controls
Post approval submissions involving the drug substance chemistry, manufacturing, and controls should
include an evaluation of the potential risk impact associated with mutagenic impurities from changes to
the route of synthesis, reagents, solvents, or process conditions after the starting material.
Specifically, changes should be evaluated to determine if the changes result in any new mutagenic
impurities or higher acceptance criteria for existing mutagenic impurities. Reevaluation of impurities
not impacted by changes is not recommended. For example, when only a portion of the manufacturing
process is changed, the assessment of risk from mutagenic impurities should be limited to whether any
new mutagenic impurities result from the change, whether any mutagenic impurities formed during the
affected step are increased, and whether any known mutagenic impurities from up-stream steps are
increased. Regulatory submissions associated with such changes should describe the assessment as
outlined in Section 9.2. Changing the site of manufacture of drug substance, intermediates, or starting
materials or changing raw materials supplier will not require a reassessment of mutagenic impurity
risk.
When a new drug substance supplier is proposed, evidence that the drug substance produced by this
supplier using the same route of synthesis as an existing drug product marketed in the assessor’s
region is considered to be sufficient evidence of acceptable risk/benefit regarding mutagenic impurities
and an assessment per this guideline is not required. If this is not the case, then an assessment per
this guideline is expected.
4.2. Post approval changes to the drug product chemistry, manufacturing,
and controls
Post approval submissions involving the drug product (e.g., change in composition, manufacturing
process, dosage form) should include an evaluation of the potential risk associated with any new
mutagenic degradation products or higher acceptance criteria for existing mutagenic degradation
products. If appropriate, the regulatory submission would include an updated control strategy.
Reevaluation of the drug substance associated with drug products is not recommended or expected
provided there are no changes to the drug substance. Changing the site of manufacture of drug
product will not require a reassessment of mutagenic impurity risk.
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4.3. Changes to the clinical use of marketed products
Changes to the clinical use of marketed products that can warrant a reevaluation of the mutagenic
impurity limits include a significant increase in clinical dose, an increase in duration of use (in particular
when a mutagenic impurity was controlled above the lifetime acceptable intake for a previous
indication that may no longer be appropriate for the longer treatment duration associated with the new
indication), or for a change in indication from a serious or life threatening condition where higher
acceptable intakes were justified (Section 7.5) to an indication for a less serious condition where the
existing impurity acceptable intakes may no longer be appropriate. Changes to the clinical use of
marketed products associated with new routes of administration or expansion into patient populations
that include pregnant women and/or pediatrics will not warrant a reevaluation, assuming no increases
in daily dose or duration of treatment.
4.4. Other considerations for marketed products
Application of this guideline may be warranted to marketed products if there is specific cause for
concern. The existence of impurity structural alerts alone is considered insufficient to trigger follow-up
measures, unless it is a structure in the cohort of concern (Section 3). However a specific cause for
concern would be new relevant impurity hazard data (classified as Class 1 or 2, Section 6) generated
after the overall control strategy and specifications for market authorization were established. This
new relevant impurity hazard data should be derived from high-quality scientific studies consistent with
relevant regulatory testing guidelines, with data records or reports readily available. Similarly, a newly
discovered impurity that is a known Class 1 or Class 2 mutagen that is present in a marketed product
could also be a cause for concern. In both of these cases when the applicant becomes aware of this
new information, an evaluation per this guideline should be conducted.
5. Drug substance and drug product impurity assessment
Actual and potential impurities that are likely to arise during the synthesis and storage of a new drug
substance, and during manufacturing and storage of a new drug product should be assessed.
The impurity assessment is a two-stage process:
Actual impurities that have been identified should be considered for their mutagenic potential.
An assessment of potential impurities likely to be present in the final drug substance is carried out
to determine if further evaluation of their mutagenic potential is required.
The steps as applied to synthetic impurities and degradation products are described in Sections 5.1
and 5.2, respectively.
5.1. Synthetic impurities
Actual impurities include those observed in the drug substance above the ICH Q3A reporting
thresholds. Identification of actual impurities is expected when the levels exceed the identification
thresholds outlined by ICH Q3A. It is acknowledged that some impurities below the identification
threshold may also have been identified.
Potential impurities in the drug substance can include starting materials, reagents and intermediates in
the route of synthesis from the starting material to the drug substance.
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The risk of carryover into the drug substance should be assessed for identified impurities that are
present in starting materials and intermediates, and impurities that are reasonably expected by-
products in the route of synthesis from the starting material to the drug substance. As the risk of
carryover may be negligible for some impurities (e.g., those impurities in early synthetic steps of long
routes of synthesis), a risk-based justification could be provided for the point in the synthesis after
which these types of impurities should be evaluated for mutagenic potential.
For starting materials that are introduced late in the synthesis of the drug substance (and where the
synthetic route of the starting material is known) the final steps of the starting material synthesis
should be evaluated for potential mutagenic impurities.
Actual impurities where the structures are known and potential impurities as defined above should be
evaluated for mutagenic potential as described in Section 6.
5.2. Degradation products
Actual drug substance degradation products include those observed above the ICH Q3A reporting
threshold during storage of the drug substance in the proposed long-term storage conditions and
primary and secondary packaging. Actual degradation products in the drug product include those
observed above the ICH Q3B reporting threshold during storage of the drug product in the proposed
long-term storage conditions and primary and secondary packaging, and also include those impurities
that arise during the manufacture of the drug product. Identification of actual degradation products is
expected when the levels exceed the identification thresholds outlined by ICH Q3A/Q3B. It is
acknowledged that some degradation products below the identification threshold may also have been
identified.
Potential degradation products in the drug substance and drug product are those that may be
reasonably expected to form during long term storage conditions. Potential degradation products
include those that form above the ICHQ 3A/B identification threshold during accelerated stability
studies (e.g., 40
°
C/75% relative humidity for 6 months) and confirmatory photo-stability studies as
described in ICH Q1B (Ref. 5), but are yet to be confirmed in the drug substance or drug product
under long-term storage conditions in the primary packaging.
Knowledge of relevant degradation pathways can be used to help guide decisions on the selection of
potential degradation products to be evaluated for mutagenicity e.g., from degradation chemistry
principles, relevant stress testing studies, and development stability studies.
Actual and potential degradation products likely to be present in the final drug substance or drug
product and where the structure is known should be evaluated for mutagenic potential as described in
Section 6.
5.3. Considerations for clinical development
It is expected that the impurity assessment described in Sections 5.1 and 5.2 applies to products in
clinical development. However, it is acknowledged that the available information is limited. For
example, information from long term stability studies and photo-stability studies may not be available
during clinical development and thus information on potential degradation products may be limited.
Additionally, the thresholds outlined in ICHQ 3A/B do not apply to products in clinical development and
consequently fewer impurities will be identified.
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6. Hazard assessment elements
Hazard assessment involves an initial analysis of actual and potential impurities by conducting
database and literature searches for carcinogenicity and bacterial mutagenicity data in order to classify
them as Class 1, 2, or 5 according to Table 1. If data for such a classification are not available, an
assessment of Structure-Activity Relationships (SAR) that focuses on bacterial mutagenicity predictions
should be performed. This could lead to a classification into Class 3, 4, or 5.
Table 1. Impurities Classification with Respect to Mutagenic and Carcinogenic Potential and Resulting
Control Actions
Class
Definition
Proposed action for control
(details in Section 7 and 8)
1
Known mutagenic carcinogens
Control at or below compound-specific
acceptable limit
2
Known mutagens with
unknown carcinogenic potential
(bacterial mutagenicity positive*, no rodent
carcinogenicity data)
Control at or below acceptable limits
(appropriate TTC)
3
Alerting structure, unrelated to the
structure of the drug substance;
no mutagenicity data
Control at or below acceptable limits
(appropriate TTC) or conduct bacterial
mutagenicity assay;
If non-mutagenic = Class 5
If mutagenic = Class 2
4
Alerting structure, same alert in drug substance
or compounds related to the drug substance
(e.g., process intermediates) which have been
tested and are non-mutagenic
Treat as non-mutagenic impurity
5
No structural alerts, or alerting structure with
sufficient data to demonstrate lack of
mutagenicity or carcinogenicity
Treat as non-mutagenic impurity
*Or other relevant positive mutagenicity data indicative of DNA-reactivity related induction of gene
mutations (e.g., positive findings in in vivo gene mutation studies)
A computational toxicology assessment should be performed using (Q)SAR methodologies that predict
the outcome of a bacterial mutagenicity assay (Ref. 6). Two (Q)SAR prediction methodologies that
complement each other should be applied. One methodology should be expert rule-based and the
second methodology should be statistical-based. (Q)SAR models utilizing these prediction
methodologies should follow the general validation principles set forth by the Organisation for
Economic Co-operation and Development (OECD).
The absence of structural alerts from two complementary (Q)SAR methodologies (expert rule-based
and statistical) is sufficient to conclude that the impurity is of no mutagenic concern, and no further
testing is recommended (Class 5 in Table 1).
If warranted, the outcome of any computer system-based analysis can be reviewed with the use of
expert knowledge in order to provide additional supportive evidence on relevance of any positive,
negative, conflicting or inconclusive prediction and provide a rationale to support the final conclusion.
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To follow up on a relevant structural alert (Class 3 in Table 1), either adequate control measures could
be applied or a bacterial mutagenicity assay with the impurity alone can be conducted. An
appropriately conducted negative bacterial mutagenicity assay (Note 2) would overrule any structure-
based concern, and no further genotoxicity assessments would be recommended (Note 1). These
impurities should be considered non-mutagenic (Class 5 in Table 1). A positive bacterial mutagenicity
result would warrant further hazard assessment and/or control measures (Class 2 in Table 1). For
instance, when levels of the impurity cannot be controlled at an appropriate acceptable limit, it is
recommended that the impurity be tested in an in vivo gene mutation assay in order to understand the
relevance of the bacterial mutagenicity assay result under in vivo conditions. The selection of other in
vivo genotoxicity assays should be scientifically justified based on knowledge of the mechanism of
action of the impurity and expected target tissue exposure (Note 3). In vivo studies should be
designed taking into consideration existing ICH genotoxicity Guidelines. Results in the appropriate in
vivo assay may support setting compound specific impurity limits.
An impurity with a structural alert that is shared (e.g., same structural alert in the same position and
chemical environment) with the drug substance or related compounds can be considered as non-
mutagenic (Class 4 in Table 1) if the testing of such material in the bacterial mutagenicity assay was
negative.
7. Risk characterization
As a result of hazard assessment described in Section 6, each impurity will be assigned to one of the
five classes in Table 1. For impurities belonging in Classes 1, 2, and 3 the principles of risk
characterization used to derive acceptable intakes are described in this section.
7.1. TTC-based acceptable intakes
A TTC-based acceptable intake of a mutagenic impurity of 1.5 µg per person per day is considered to
be associated with a negligible risk (theoretical excess cancer risk of <1 in 100,000 over a lifetime of
exposure) and can in general be used for most pharmaceuticals as a default to derive an acceptable
limit for control. This approach would usually be used for mutagenic impurities present in
pharmaceuticals for long-term treatment (> 10 years) and where no carcinogenicity data are available
(Classes 2 and 3).
7.2. Acceptable intakes based on compound-specific risk assessments
7.2.1. Mutagenic impurities with positive carcinogenicity data (class 1 in
table 1)
Compound-specific risk assessments to derive acceptable intakes should be applied instead of the TTC-
based acceptable intakes where sufficient carcinogenicity data exist. For a known mutagenic
carcinogen, a compound-specific acceptable intake can be calculated based on carcinogenic potency
and linear extrapolation as a default approach. Alternatively, other established risk assessment
practices such as those used by international regulatory bodies may be applied either to calculate
acceptable intakes or to use already existing values published by regulatory authorities (Note 4).
Compound-specific calculations for acceptable intakes can be applied case-by-case for impurities which
are chemically similar to a known carcinogen compound class (class-specific acceptable intakes)
provided that a rationale for chemical similarity and supporting data can be demonstrated (Note 5).
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7.2.2. Mutagenic impurities with evidence for a practical threshold
The existence of mechanisms leading to a dose response that is non-linear or has a practical threshold
is increasingly recognized, not only for compounds that interact with non-DNA targets but also for
DNA-reactive compounds, whose effects may be modulated by, for example, rapid detoxification before
coming into contact with DNA, or by effective repair of induced damage. The regulatory approach to
such compounds can be based on the identification of a No-Observed Effect Level (NOEL) and use of
uncertainty factors (see ICH Q3C(R5), Ref. 7) to calculate a permissible daily exposure (PDE) when
data are available.
The acceptable intakes derived from compound-specific risk assessments (Section 7.2) can be adjusted
for shorter duration of use in the same proportions as defined in the following sections (Section 7.3.1
and 7.3.2) or should be limited to not more than 0.5%, whichever is lower. For example, if the
compound specific acceptable intake is 15 µg/day for lifetime exposure, the less than lifetime limits
(Table 2) can be increased to a daily intake of 100 µg (> 1-10 years treatment duration), 200 µg (> 1-
12 months) or 1200 µg (< 1 month). However, for a drug with a maximum daily dose of, for instance,
100 mg the acceptable daily intake for the < 1 month duration would be limited to 0.5% (500 µg)
rather than 1200 µg.
7.3. Acceptable intakes in relation to less-than-lifetime (LTL) exposure
Standard risk assessments of known carcinogens assume that cancer risk increases as a function of
cumulative dose. Thus, cancer risk of a continuous low dose over a lifetime would be equivalent to the
cancer risk associated with an identical cumulative exposure averaged over a shorter duration.
The TTC-based acceptable intake of 1.5 µg/day is considered to be protective for a lifetime of daily
exposure. To address LTL exposures to mutagenic impurities in pharmaceuticals, an approach is
applied in which the acceptable cumulative lifetime dose (1.5 µg/day x 25,550 days = 38.3 mg) is
uniformly distributed over the total number of exposure days during LTL exposure. This would allow
higher daily intake of mutagenic impurities than would be the case for lifetime exposure and still
maintain comparable risk levels for daily and non-daily treatment regimens. Table 2 is derived from
the above concepts and illustrates the acceptable intakes for LTL to lifetime exposures for clinical
development and marketing. In the case of intermittent dosing, the acceptable daily intake should be
based on the total number of dosing days instead of the time interval over which the doses were
administered and that number of dosing days should be related to the relevant duration category in
Table 2. For example, a drug administered once per week for 2 years (i.e., 104 dosing days) would
have an acceptable intake per dose of 20µg.
Table 2. Acceptable Intakes for an Individual Impurity
Duration of
treatment
< 1 month
>1 - 12 months
>1 - 10 years
>10 years to
lifetime
Daily intake
[µg/day]
120
20
10
1.5
7.3.1. Clinical development
Using this LTL concept, acceptable intakes of mutagenic impurities are recommended for limited
treatment periods during clinical development of up to 1 month, 1 to 12 months and more than one
year up to completion of Phase 3 clinical trials (Table 2). These adjusted acceptable intake values
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maintain a 10
-6
risk level in early clinical development when benefit has not yet been established and a
10
-5
risk level for later stages in development (Note 6).
An alternative approach to the strict use of an adjusted acceptable intake for any mutagenic impurity
could be applied for Phase 1 clinical trials for dosing up to 14 days. For this approach, only impurities
that are known mutagenic carcinogens (Class 1) and known mutagens of unknown carcinogenic
potential (Class 2), as well as impurities in the cohort of concern chemical class, should be controlled
(see Section 8) to acceptable limits as described in Section 7. All other impurities would be treated as
non-mutagenic impurities. This includes impurities which contain structural alerts (Class 3), which
alone would not trigger action for an assessment for this limited Phase 1 duration.
7.3.2. Marketed products
The treatment duration categories with acceptable intakes in Table 2 for marketed products are
intended to be applied to anticipated exposure durations for the great majority of patients. The
proposed intakes along with various scenarios for applying those intakes are described in Table 4, Note
7. In some cases, a subset of the population of patients may extend treatment beyond the marketed
drugs categorical upper limit (e.g., treatment exceeding 10 years for an acceptable intake of 10
µg/day, perhaps receiving 15 years of treatment). This would result in a negligible increase (in the
example given, a fractional increase to 1.5/100,000) compared to the overall calculated risk for the
majority of patients treated for 10 years.
7.4. Acceptable intakes for multiple mutagenic impurities
The TTC-based acceptable intakes should be applied to each individual impurity. When there are two
Class 2 or Class 3 impurities, individual limits apply. When there are three or more Class 2 or Class 3
impurities specified on the drug substance specification, total mutagenic impurities should be limited as
described in Table 3 for clinical development and marketed products.
For combination products each active ingredient should be regulated separately.
Table 3. Acceptable Total Daily Intakes for Multiple Impurities
Duration of
treatment
< 1 month
>1 - 12 months
>1 - 10 years
>10 years to
lifetime
Total Daily
intake
[µg/day]
120
60
30
5
Only specified Class 2 and 3 impurities on the drug substance specification are included in the
calculation of the total limit. However, impurities with compound-specific or class-related acceptable
intake limits (Class 1) should not be included in the total limits of Class 2 and Class 3 impurities. Also,
degradation products which form in the drug product would be controlled individually and a total limit
would not be applied.
7.5. Exceptions and flexibility in approaches
Higher acceptable intakes may be justified when human exposure to the impurity will be much
greater from other sources e.g., food, or endogenous metabolism (e.g., formaldehyde).
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Case-by-case exceptions to the use of the appropriate acceptable intake can be justified in cases of
severe disease, reduced life expectancy, late onset but chronic disease, or with limited therapeutic
alternatives.
Compounds from some structural classes of mutagens can display extremely high carcinogenic
potency (cohort of concern), i.e., aflatoxin-like-, N-nitroso-, and alkyl-azoxy structures. If these
compounds are found as impurities in pharmaceuticals, acceptable intakes for these high-potency
carcinogens would likely be significantly lower than the acceptable intakes defined in this guideline.
Although the principles of this guideline can be used, a case-by-case approach using e.g.,
carcinogenicity data from closely related structures, if available, should usually be developed to
justify acceptable intakes for pharmaceutical development and marketed products.
The above risk approaches described in Section 7 are applicable to all routes of administration and no
corrections to acceptable intakes are generally warranted. Exceptions to consider may include
situations where data justify route-specific concerns that should be evaluated case-by-case. These
approaches are also applicable to all patient populations based upon the conservative nature of the risk
approaches being applied.
8. Control
A control strategy is a planned set of controls, derived from current product and process understanding
that assures process performance and product quality (ICH Q10, Ref. 8). A control strategy can
include, but is not limited to, the following:
Controls on material attributes (including raw materials, starting materials, intermediates,
reagents, solvents, primary packaging materials);
Facility and equipment operating conditions;
Controls implicit in the design of the manufacturing process;
In-process controls (including in-process tests and process parameters);
Controls on drug substance and drug product (e.g., release testing).
When an impurity has been characterized as Classes 1, 2, or 3 in Table 1, it is important to develop a
control strategy that assures that the level of this impurity in the drug substance and drug product is
below the acceptable limit. A thorough knowledge of the chemistry associated with the drug substance
manufacturing process, and of the drug product manufacturing process, along with an understanding
of the overall stability of the drug substance and drug product is fundamental to developing the
appropriate controls. Developing a strategy to control mutagenic impurities in the drug product is
consistent with risk management processes identified in ICH Q9 (Ref. 9). A control strategy that is
based on product and process understanding and utilisation of risk management principles will lead to
a combination of process design and control and appropriate analytical testing, which can also provide
an opportunity to shift controls upstream and minimize the need for end-product testing.
8.1. Control of process related impurities
There are 4 potential approaches to development of a control strategy for drug substance:
Option 1
Include a test for the impurity in the drug substance specification with an acceptance criterion at or
below the acceptable limit using an appropriate analytical procedure.
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For an Option 1 control approach, it is possible to apply periodic verification testing per ICH Q6A (Ref
10). Periodic verification testing is justified when it can be shown that levels of the mutagenic impurity
in the drug substance are less than 30% of the acceptable limit for at least 6 consecutive pilot scale or
3 consecutive production scale batches. If this condition is not fulfilled, a routine test in the drug
substance specification is recommended. See Section 8.3 for additional considerations.
Option 2
Include a test for the impurity in the specification for a raw material, starting material or intermediate,
or as an in-process control, with an acceptance criterion at or below the acceptable limit using an
appropriate analytical procedure.
Option 3
Include a test for the impurity in the specification for a raw material, starting material or intermediate,
or as an in-process control, with an acceptance criterion above the acceptable limit of the impurity in
the drug substance, using an appropriate analytical procedure coupled with demonstrated
understanding of fate and purge and associated process controls that assure the level in the drug
substance is below the acceptable limit without the need for any additional testing later in the process.
This option can be justified when the level of the impurity in the drug substance will be less than 30%
of the acceptable limit by review of data from laboratory scale experiments (spiking experiments are
encouraged) and where necessary supported by data from pilot scale or commercial scale batches. See
Case Examples 1 and 2. Alternative approaches can be used to justify Option 3.
Option 4
Understand process parameters and impact on residual impurity levels (including fate and purge
knowledge) with sufficient confidence that the level of the impurity in the drug substance will be below
the acceptable limit such that no analytical testing is recommended for this impurity. (i.e., the impurity
does not need to be listed on any specification).
A control strategy that relies on process controls in lieu of analytical testing can be appropriate if the
process chemistry and process parameters that impact levels of mutagenic impurities are understood
and the risk of an impurity residing in the final drug substance above the acceptable limit is
determined to be negligible. In many cases justification of this control approach based on scientific
principles alone is sufficient. Elements of a scientific risk assessment can be used to justify an option 4
approach. The risk assessment can be based on physicochemical properties and process factors that
influence the fate and purge of an impurity including chemical reactivity, solubility, volatility,
ionizability and any physical process steps designed to remove impurities. The result of this risk
assessment might be shown as an estimated purge factor for clearance of the impurity by the process
(Ref. 11).
Option 4 is especially useful for those impurities that are inherently unstable (e.g., thionyl chloride that
reacts rapidly and completely with water) or for those impurities that are introduced early in the
synthesis and are effectively purged.
In some cases an Option 4 approach can be appropriate when the impurity is known to form, or is
introduced late in the synthesis, however process-specific data should then be provided to justify this
approach.
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8.2. Considerations for control approaches
For Option 4 approaches where justification based on scientific principles alone is not considered
sufficient, as well as for Option 3 approaches, analytical data to support the control approach is
expected. This could include as appropriate information on the structural changes to the impurity
caused by downstream chemistry (“fate”), analytical data on pilot scale batches, and in some cases,
laboratory scale studies with intentional addition of the impurity (“spiking studies”). In these cases, it
is important to demonstrate that the fate/purge argument for the impurity is robust and will
consistently assure a negligible probability of an impurity residing in the final drug substance above the
acceptable limit. Where the purge factor is based on developmental data, it is important to address
the expected scale-dependence or independence. In the case that the small scale model used in the
development stage is considered to not represent the commercial scale, confirmation of suitable
control in pilot scale and/or initial commercial batches is generally appropriate. The need for data from
pilot/commercial batches is influenced by the magnitude of the purge factor calculated from laboratory
or pilot scale data, point of entry of the impurity, and knowledge of downstream process purge points.
If Options 3 and 4 cannot be justified, then a test for the impurity on the specification for a raw
material, starting material or intermediate, or as an in-process control (Option 2) or drug substance
(Option 1) at the acceptable limit should be included. For impurities introduced in the last synthetic
step, an Option 1 control approach would be expected unless otherwise justified.
The application of “As Low As Reasonably Practicable” (ALARP) is not necessary if the level of the
mutagenic impurity is below acceptable limits. Similarly, it is not necessary to demonstrate that
alternate routes of synthesis have been explored.
In cases where control efforts cannot reduce the level of the mutagenic impurity to below the
acceptable limit and levels are as low as reasonably practical, a higher limit may be justified based on
a risk/benefit analysis.
8.3. Considerations for periodic testing
The above options include situations where a test is recommended to be included in the specification,
but where routine measurement for release of every batch may not be necessary. This approach,
referred to as periodic or skip testing in ICH Q6A could also be called “Periodic Verification Testing.”
This approach may be appropriate when it can be demonstrated that processing subsequent to
impurity formation/introduction clears the impurity. It should be noted that allowance of Periodic
Verification Testing is contingent upon use of a process that is under a state of control (i.e., produces a
quality product that consistently meets specifications and conforms to an appropriately established
facility, equipment, processing, and operational control regimen). If upon testing, the level of the
mutagenic impurity fails to meet the acceptance criteria established for the periodic test, the drug
producer should immediately commence full testing (i.e., testing of every batch for the attribute
specified) until the cause of the failure has been conclusively determined, corrective action has been
implemented, and the process is again documented to be in a state of control. As noted in ICH Q6A,
regulatory authorities should be notified of a periodic verification test failure to evaluate the
risk/benefit of previously released batches that were not tested.
8.4. Control of degradation products
For a potential degradation product that has been characterized as mutagenic, it is important to
understand if the degradation pathway is relevant to the drug substance and drug product
manufacturing processes and/or their proposed packaging and storage conditions. A well-designed
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accelerated stability study (e.g., 40
°
C/75% relative humidity, 6 months) in the proposed packaging,
with appropriate analytical procedures is recommended to determine the relevance of the potential
degradation product. Alternatively, well designed kinetically equivalent shorter term stability studies
at higher temperatures in the proposed commercial package may be used to determine the relevance
of the degradation pathway prior to initiating longer term stability studies. This type of study would be
especially useful to understand the relevance of those potential degradation products that are based on
knowledge of potential degradation pathways but not yet observed in the product.
Based on the result of these accelerated studies, if it is anticipated that the degradation product will
form at levels approaching the acceptable limit under the proposed packaging and storage conditions,
then efforts to control formation of the degradation product is expected. In these cases, monitoring for
the drug substance or drug product degradation product in long term primary stability studies at the
proposed storage conditions (in the proposed commercial pack) is expected unless otherwise justified.
Whether or not a specification limit for the mutagenic degradation product is appropriate will generally
depend on the results from these stability studies.
If it is anticipated that formulation development and packaging design options are unable to control
mutagenic degradation product levels to less than the acceptable limit and levels are as low as
reasonably practicable, a higher limit can be justified based on a risk/benefit analysis.
8.5. Lifecycle management
This section is intended to apply to those products approved after the issuance of this guideline.
The quality system elements and management responsibilities described in ICH Q10 are intended to
encourage the use of science-based and risk-based approaches at each lifecycle stage, thereby
promoting continual improvement across the entire product lifecycle. Product and process knowledge
should be managed from development through the commercial life of the product up to and including
product discontinuation.
The development and improvement of a drug substance or drug product manufacturing process usually
continues over its lifecycle. Manufacturing process performance, including the effectiveness of the
control strategy, should be periodically evaluated. Knowledge gained from commercial manufacturing
can be used to further improve process understanding and process performance and to adjust the
control strategy.
Any proposed change to the manufacturing process should be evaluated for the impact on the quality
of drug substance and drug product. This evaluation should be based on understanding of the
manufacturing process and should determine if appropriate testing to analyze the impact of the
proposed changes is required. Additionally, improvements in analytical procedures may lead to
structural identification of an impurity. In those cases the new structure would be assessed for
mutagenicity as described in this guideline.
Throughout the lifecycle of the product, it will be important to reassess if testing is recommended when
intended or unintended changes occur in the process. This applies when there is no routine monitoring
at the acceptable limit (Option 3 or Option 4 control approaches), or when applying periodic rather
than batch-by-batch testing. This testing should be performed at an appropriate point in the
manufacturing process.
In some cases, the use of statistical process control and trending of process measurements can be
useful for continued suitability and capability of processes to provide adequate control on the impurity.
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Statistical process control can be based on process parameters that influence impurity formation or
clearance, even when that impurity is not routinely monitored (e.g., Option 4).
All changes should be subject to internal change management processes as part of the quality system
(ICH Q10). Changes to information filed and approved in a dossier should be reported to regulatory
authorities in accordance with regional regulations and guidelines.
8.6. Considerations for clinical development
It is recognized that product and process knowledge increases over the course of development and
therefore it is expected that data to support control strategies in the clinical development trial phases
will be less than at the marketing registration phase. A risk-based approach based on process
chemistry fundamentals is encouraged to prioritize analytical efforts on those impurities with the
highest likelihood of being present in the drug substance or drug product. Analytical data may not be
expected to support early clinical development when the likelihood of an impurity being present is low,
but in a similar situation analytical data may be appropriate to support the control approach for the
marketing application. It is also recognized that commercial formulation design occurs later in clinical
development and therefore efforts associated with drug product degradation products will be limited in
the earlier phases.
9. Documentation
Information relevant to the application of this guideline should be provided at the following stages:
9.1. Clinical trial applications
It is expected that the number of structures assessed for mutagenicity, and the collection of
analytical data will both increase throughout the clinical development period.
For Phase 1 studies of 14 days or less a description of efforts to mitigate risks of mutagenic
impurities focused on Class 1, and Class 2 impurities and those in the cohort of concern as outlined
in Section 7 should be included. For Phase 1 clinical trials greater than 14 days and for Phase 2a
clinical trials additionally Class 3 impurities that require analytical controls should be included.
For Phase 2b and Phase 3 clinical development trials, a list of the impurities assessed by (Q)SAR
should be included, and any Class 1, 2 or 3 actual and potential impurities should be described
along with plans for control. The in silico (Q)SAR systems used to perform the assessments should
be described. The results of bacterial mutagenicity tests of actual impurities should be reported.
Chemistry arguments may be appropriate instead of analytical data for potential impurities that
present a low likelihood of being present as described in Section 8.6.
9.2. Common technical document (marketing application)
For actual and potential process related impurities and degradation products where assessments
according to this guideline are conducted, the mutagenic impurity classification and rationale for
this classification should be provided:
This would include the results and description of in silico (Q)SAR systems used, and as
appropriate, supporting information to arrive at the overall conclusion for Class 4 and 5
impurities.
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When bacterial mutagenicity assays were performed on impurities, study reports should be
provided for bacterial mutagenicity assays on impurities.
Justification for the proposed specification and the approach to control should be provided (e.g.,
ICH Q11 example 5b, Ref. 12). For example, this information could include the acceptable intake,
the location and sensitivity of relevant routine monitoring. For Option 3 and Option 4 control
approaches, a summary of knowledge of the purge factor, and identification of factors providing
control (e.g., process steps, solubility in wash solutions, etc.) is important.
Notes
Note 1 The ICH M7 Guideline recommendations provide a state-of-the-art approach for assessing the
potential of impurities to induce point mutations and ensure that such impurities are controlled
to safe levels so that below or above the ICH Q3A/B qualification threshold no further
qualification for mutagenic potential is required. This includes the initial use of (Q)SAR tools to
predict bacterial mutagenicity. In cases where the amount of the impurity exceeds 1 mg daily
dose for chronic administration, evaluation of genotoxic potential as recommended in ICH
Q3A/B could be considered. In cases where the amount of the impurity is less than 1 mg, no
further genotoxicity testing is required regardless of other qualification thresholds.
Note 2 To assess the mutagenic potential of impurities, a single bacterial mutagenicity assay can be
carried out with a fully adequate protocol according to ICH S2(R1) and OECD 471 guidelines
(Ref. 13 and 14). The assays are expected to be performed in compliance with Good
Laboratory Practices (GLP) regulations; however, lack of full GLP compliance does not
necessarily mean that the data cannot be used to support clinical trials and marketing
authorizations. Such deviations should be described in the study report. For example, the test
article may not be prepared or analyzed in compliance with GLP regulations. In some cases,
the selection of bacterial tester strains may be limited to those proven to be sensitive to the
identified alert. For impurities that are not feasible to isolate or synthesize or when compound
quantity is limited, it may not be possible to achieve the highest test concentrations
recommended for an ICH-compliant bacterial mutagenicity assay according to the current
testing guidelines. In this case, bacterial mutagenicity testing could be carried out using a
miniaturized assay format with proven high concordance to the ICH-compliant assay to enable
testing at higher concentrations with justification.
Note 3 Tests to Investigate the in vivo Relevance of in vitro Mutagens (Positive Bacterial Mutagenicity)
In vivo test
Factors to justify choice of test
as fit-for-purpose
Transgenic mutation assays
For any bacterial mutagenicity positive. Justify selection of assay
tissue/organ
Pig-a assay
(blood)
For directly acting mutagens (bacterial mutagenicity positive
without S9)*
Micronucleus test
(blood or bone marrow)
For directly acting mutagens (bacterial mutagenicity positive
without S9) and compounds known to be clastogenic*
Rat liver Unscheduled DNA
Synthesis (UDS) test
In particular for bacterial mutagenicity positive with S9 only
Responsible liver metabolite known
to be generated in test species used
to induce bulky adducts
Comet assay
Justification needed (chemical class specific mode of action to form
alkaline labile sites or single-strand breaks as preceding DNA
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damage that can potentially lead to mutations
Justify selection of assay tissue/organ
Others
With convincing justification
*For indirect acting mutagens (requiring metabolic activation), adequate exposure to metabolite(s)
should be demonstrated.
Note 4 Example of linear extrapolation from the TD
50
It is possible to calculate a compound-specific acceptable intake based on rodent
carcinogenicity potency data such as TD
50
values (doses giving a 50% tumor incidence
equivalent to a cancer risk probability level of 1:2). Linear extrapolation to a probability of 1 in
100,000 (i.e., the accepted lifetime risk level used) is achieved by simply dividing the TD
50
by
50,000. This procedure is similar to that employed for derivation of the TTC.
Calculation example: Ethylene oxide
TD
50
values for ethylene oxide according to the Carcinogenic Potency Database are 21.3 mg/kg
body weight/day (rat) and 63.7 mg/kg body weight/day (mouse). For the calculation of an
acceptable intake, the lower (i.e., more conservative) value of the rat is used.
To derive a dose to cause tumors in 1 in 100,000 animals, divide by 50,000:
21.3 mg/kg 50,000 = 0.42 µg/kg
To derive a total human daily dose:
0.42 µg/kg/day x 50 kg body weight = 21.3 µg/person/day
Hence, a daily life-long intake of 21.3 µg ethylene oxide would correspond to a theoretical
cancer risk of 10
-5
and therefore be an acceptable intake when present as an impurity in a drug
substance.
Alternative methods and published regulatory limits for cancer risk assessment
As an alternative of using the most conservative TD50 value from rodent carcinogenicity
studies irrespective of its relevance to humans, an in-depth toxicological expert assessment of
the available carcinogenicity data can be done in order to initially identify the findings (species,
organ, etc.) with highest relevance to human risk assessment as a basis for deriving a
reference point for linear extrapolation. Also, in order to better take into account directly the
shape of the dose-response curve, a benchmark dose such as a benchmark dose lower
confidence limit 10% (BMDL10, an estimate of the lowest dose which is 95% certain to cause
no more than a 10% cancer incidence in rodents) may be used instead of TD50 values as a
numerical index for carcinogenic potency. Linear extrapolation to a probability of 1 in 100,000
(i.e., the accepted lifetime risk level used) is then achieved by simply dividing the BMDL10 by
10,000.
Compound-specific acceptable intakes can also be derived from published recommended values
from internationally recognized bodies such as World Health Organization (WHO, International
Program on Chemical Safety [IPCS] Cancer Risk Assessment Programme) and others using the
appropriate 10-5 lifetime risk level. In general, a regulatory limit that is applied should be
based on the most current and scientifically supported data and/or methodology.
Note 5 A compound-specific calculation of acceptable intakes for mutagenic impurities may be applied
for mutagenic impurities (without carcinogenicity data) which are structurally similar to a
chemically-defined class of known carcinogen. For example, factors that are associated with
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the carcinogenic potency of monofunctional alkyl chlorides have been identified (Ref. 15) and
can be used to modify the safe acceptable intake of monofunctional alkyl chlorides, a group of
alkyl chlorides commonly used in drug synthesis. Compared to multifunctional alkyl chlorides
the monofunctional compounds are much less potent carcinogens with TD50 values ranging
from 36 to 1810 mg/kg/day (n=15; epichlorohydrin with two distinctly different functional
groups is excluded). A TD50 value of 36 mg/kg/day can thus be used as a still very
conservative class-specific potency reference point for calculation of acceptable intakes for
monofunctional alkyl chlorides. This potency level is at least ten-fold lower than the TD50 of
1.25 mg/kg/day corresponding to the default lifetime TTC (1.5 µg/day) and therefore justifies
lifetime and less-than-lifetime daily intakes for monofunctional alkyl chlorides ten times the
default ones.
Note 6 Establishing less-than-lifetime acceptable intakes for mutagenic impurities in pharmaceuticals
has precedent in the establishment of the staged TTC limits for clinical development (Ref. 16).
The calculation of less-than-lifetime Acceptable Intakes (AI) is predicated on the principle of
Haber’s rule, a fundamental concept in toxicology where concentration (C) x time (T) = a
constant (k). Therefore, the carcinogenic effect is based on both dose and duration of
exposure.
Figure 1: Illustration of calculated daily dose of a mutagenic impurity corresponding to a
theoretical 1:100,000 cancer risk as a function of duration of treatment in comparison to the
acceptable intake levels as recommended in Section 7.3.
The solid line in Figure 1 represents the linear relationship between the amount of daily
intake of a mutagenic impurity corresponding to a 10
-5
cancer risk and the number of
1
year
1
month
1
day
5
years
1
1
10
100
1000
10000
1 10 100 1000
Number of treatment days
Dose[µg/person/day] given on treatment days
38250 µg
1270 µg
100 µg
10 µg
1 day
1 month
1 year
10 years
70 years
1,5 µg
120 µg
20 µg
10 µg
1,5 µg
SF: 60-5x
SF: 10-1x
SF: 300-10x
SF: 7-1x
30
365
3650
25500
Calculated dose corresp. to 10
-5
cancer risk
Proposed acceptable dose
SF: “Safety Factor” (difference (max./min.) between
calculated and proposed doses
1
year
1
month
1
day
5
years
1
1
10
100
1000
10000
1 10 100 1000
Number of treatment days
Dose[µg/person/day] given on treatment days
38250 µg
1270 µg
100 µg
10 µg
1 day
1 month
1 year
10 years
70 years
1,5 µg
120 µg
20 µg
10 µg
1,5 µg
SF: 60-5x
SF: 10-1x
SF: 300-10x
SF: 7-1x
30
365
3650
25500
Calculated dose corresp. to 10
-5
cancer risk
Proposed acceptable dose
SF: “Safety Factor” (difference (max./min.) between
calculated and proposed doses
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treatment days. The calculation is based on the TTC level as applied in this guideline for life-
long treatment i.e., 1.5 µg per person per day using the formula:
Less-than-lifetime AI = 1.5 µg x (365 days x 70 years lifetime = 25,550)
Total number of treatment days
The calculated daily intake levels would thus be 1.5 µg for treatment duration of 70 years, 10
µg for 10 years, 100 µg for 1 year, 1270 µg for 1 month and approximately 38.3 mg as a
single dose, all resulting in the same cumulative intake and therefore theoretically in the
same cancer risk (1 in 100,000).
The dashed step-shaped curve represents the actual daily intake levels adjusted to less-
than-lifetime exposure as recommended in Section 7 of this guideline for products in clinical
development and marketed products. These proposed levels are in general significantly
lower than the calculated values thus providing safety factors that increase with shorter
treatment durations.
The proposed accepted daily intakes are also in compliance with a 10
-6
cancer risk level if
treatment durations are not longer than 6 months and are therefore applicable in early
clinical trials with volunteers/patients where benefit has not yet been established. In this
case the safety factors as shown in the upper graph would be reduced by a factor of 10.
Note 7
Table 4. Examples of clinical use scenarios with different treatment durations for applying acceptable
intakes
Scenario
1
Acceptable Intake
(µg/day)
Treatment duration of < 1 month: e.g., drugs used in emergency procedures
(antidotes, anesthesia, acute ischemic stroke), actinic keratosis, treatment of
lice
120
Treatment duration of > 1-12 months: e.g., anti-infective therapy with
maximum up to 12 months treatment (HCV), parenteral nutrients,
prophylactic flu drugs (~ 5 months), peptic ulcer, Assisted Reproductive
Technology (ART), pre-term labor, preeclampsia, pre-surgical (hysterectomy)
treatment, fracture healing (these are acute use but with long half-lives)
20
Treatment duration of >1-10 years: e.g., stage of disease with short life
expectancy (severe Alzheimer’s), non-genotoxic anticancer treatment being
used in a patient population with longer term survival (breast cancer, CML),
drugs specifically labeled for less than 10 years of use, drugs administered
intermittently to treat acute recurring symptoms
2
(chronic Herpes, gout
attacks, substance dependence such as smoking cessation), macular
degeneration, HIV
3
10
Treatment duration of >10 years to lifetime: e.g., chronic use indications with
high likelihood for lifetime use across broader age range (hypertension,
dyslipidemia, asthma, Alzheimer’s (except severe AD), hormone therapy (e.g.,
GH, TH, PTH), lipodystrophy, schizophrenia, depression, psoriasis, atopic
dermatitis, COPD, cystic fibrosis, seasonal and perennial allergic rhinitis
1.5
1
This table shows general examples; each example should be examined on a case-by-case basis. For
example, 10 µg/day may be acceptable in cases where the life expectancy of the patient may be
limited e.g., severe Alzheimer’s disease, even though the drug use could exceed 10 year duration.
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2
Intermittent use over a period >10 yrs but based on calculated cumulative dose it falls under the >1-
10 yr category.
3
HIV is considered a chronic indication but resistance develops to the drugs after 5-10 years and the
therapy is changed to other HIV drugs.
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Glossary
Acceptable intake:
In the context of this guideline, an intake level that poses negligible cancer risk, or for serious/life-
threatening indications where risk and benefit are appropriately balanced.
Acceptable limit:
Maximum acceptable concentration of an impurity in a drug substance or drug product derived from
the acceptable intake and the daily dose of the drug.
Acceptance criterion:
Numerical limits, ranges, or other suitable measures for acceptance of the results of analytical
procedures.
Control strategy:
A planned set of controls, derived from current product and process understanding that ensures
process performance and product quality. The controls can include parameters and attributes related
to drug substance and drug product materials and components, facility and equipment operating
conditions, in-process controls, finished product specifications, and the associated methods and
frequency of monitoring and control.
Cumulative intake:
The total intake of a substance that a person is exposed to over time.
Degradation Product: A molecule resulting from a chemical change in the drug molecule brought
about over time and/or by the action of light, temperature, pH, water, or by reaction with an excipient
and/or the immediate container/closure system.
DNA-reactive:
The potential to induce direct DNA damage through chemical reaction with DNA.
Expert knowledge:
In the context of this guideline, expert knowledge can be defined as a review of pre-existing data and
the use of any other relevant information to evaluate the accuracy of an in silico model prediction for
mutagenicity.
Genotoxicity:
A broad term that refers to any deleterious change in the genetic material regardless of the
mechanism by which the change is induced.
Impurity:
Any component of the drug substance or drug product that is not the drug substance or an excipient.
Mutagenic impurity:
An impurity that has been demonstrated to be mutagenic in an appropriate mutagenicity test model,
e.g., bacterial mutagenicity assay.
Periodic verification testing:
ICH guideline M7(R1) on assessment and control of DNA reactive (mutagenic) impurities in
pharmaceuticals to limit potential carcinogenic risk
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Also known as periodic or skip testing in ICH Q6A.
(Q)SAR and SAR:
In the context of this guideline, refers to the relationship between the molecular (sub) structure of a
compound and its mutagenic activity using (Quantitative) Structure-Activity Relationships derived from
experimental data.
Purge factor:
Purge reflects the ability of a process to reduce the level of an impurity, and the purge factor is defined
as the level of an impurity at an upstream point in a process divided by the level of an impurity at a
downstream point in a process. Purge factors may be measured or predicted.
Structural alert:
In the context of this guideline, a chemical grouping or molecular (sub) structure which is associated
with mutagenicity.
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References
1. International Conference on Harmonisation (2006). Q3A(R2): Impurities in New Drug Substances.
2. International Conference on Harmonisation (2006). Q3B(R2): Impurities in New Drug Products.
3. International Conference on Harmonisation (2009). M3(R2): Guidance on Nonclinical Safety
Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals.
4. International Conference on Harmonisation (2009). S9: Nonclinical Evaluation for Anticancer
Pharmaceuticals.
5. International Conference on Harmonisation (1996). Q1B: Stability Testing: Photostability Testing of
New Drug Substances and Products.
6. Sutter A, Amberg A, Boyer S, Brigo A, Contrera JF, Custer LL, Dobo KL, Gervais V, Glowienke S,
van Gompel J, Greene N, Muster W, Nicolette J, Reddy MV, Thybaud V, Vock E, White AT, Müller L
(2013). Use of in silico systems and expert knowledge for structure-based assessment of
potentially mutagenic impurities. Regul Toxicol Pharmacol 2013 67:39-52.
7. International Conference on Harmonisation (2011). Q3C(R5): Impurities: Guideline for Residual
Solvents.
8. International Conference on Harmonisation (2008). Q10: Pharmaceutical Quality System.
9. International Conference on Harmonisation (2005). Q9: Quality Risk Management.
10. International Conference on Harmonisation (2000). Q6A: Test Procedures and Acceptance Criteria
for New Drug Substances and New Drug Products: Chemical Substances.
11. Teasdale A., Elder D., Chang S-J, Wang S, Thompson R, Benz N, Sanchez Flores I, (2013). Risk
assessment of genotoxic impurities in new chemical entities: strategies to demonstrate control.
Org Process Res Dev 17:221−230.
12. International Conference on Harmonisation (2012). Q11: Development and Manufacture of Drug
Substances (Chemical Entities and Biotechnological/Biological Entities).
13. International Conference on Harmonisation (2011). S2(R1): Guidance on Genotoxicity Testing and
Data Interpretation for Pharmaceuticals Intended for Human Use.
14. Test 471. Bacterial Reverse Mutation Test OECD Guideline for Testing of Chemicals Section 4 1997
July
15. Brigo, A. and Müller, L. (2011) Development of the Threshold of Toxicological Concern Concept and
its Relationship to Duration of Exposure, in Genotoxic Impurities (Ed. A. Teasdale), John Wiley &
Sons, Inc., Hoboken, NJ, USA. doi: 10.1002/9780470929377.ch2
16. Müller L., Mauthe R.J., Riley C.M., Andino M.M., De Antonis D., Beels C., DeGeorge J., De Knaep
A.G.M., Ellison D., Fagerland J.A., Frank R., Fritschel B., Galloway S., Harpur E., Humfrey C.D.N.,
Jacks A.S.J., Jagota N., Mackinnon J., Mohan G., Ness D.K., O’Donovan M.R., Smith M.D.,
Vudathala G., Yotti L. (2006). A rationale for determining, testing, and controlling specific
impurities in pharmaceuticals that possess potential for genotoxicity. Regul Toxicol Pharmacol
44:198-211.
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Appendices
Appendix 1: Scope scenarios for application of the ICH M7 guideline
Scenario
Applies to
Drug
Substance
Applies
to Drug
Product
Comments
Registration of new drug
substances and associated
drug product
Yes
Yes
Primary intent of the M7 Guideline
Clinical trial applications
for new drug substances
and associated drug
product
Yes
Yes
Primary intent of the M7 Guideline
Clinical trial applications
for new drug substances
for a anti-cancer drug per
ICH S9
No
No
Out of scope of M7 Guideline
Clinical trial applications
for new drug substances
for an orphan drug
Yes
Yes
There may be exceptions on a case by case
basis for higher impurity limits
Clinical trial application for
a new drug product using
an existing drug substance
where there are no
changes to the drug
substance manufacturing
process
No
Yes
Retrospective application of the M7 Guideline
is not intended for marketed products unless
there are changes made to the synthesis.
Since no changes are made to the drug
substance synthesis, the drug substance
would not require reevaluation. Since the
drug product is new, application of this
guideline is expected.
A new formulation of an
approved drug substance
is filed
No
Yes
See Section 4.2
A product that is
previously approved in a
member region is filed for
the first time in a different
member region. The
product is unchanged.
Yes
Yes
As there is no mutual recognition, an existing
product in one member region filed for the
first time in another member region would be
considered a new product.
A new supplier or new site
of the drug substance is
registered. There are no
changes to the
manufacturing process
used in this registered
application.
No
No
As long as the synthesis of the drug
substance is consistent with previously
approved methods, then reevaluation of
mutagenic impurity risk is not necessary.
The applicant would need to demonstrate
that no changes have been made to a
previously approved process/product. Refer
to Section 4.1.
An existing product
Yes
Yes
Since the patient population and acceptable
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(approved after the
issuance of ICH M7 with
higher limits based on ICH
S9) associated with an
advanced cancer indication
is now registered for use
in a non-life threatening
indication
cancer risk have changed, the previously
approved impurity control strategy and limits
will require reevaluation. See Section 4.3.
New combination product
is filed that contains one
new drug substance and
an existing drug substance
Yes (new
drug
substance)
No (existing
drug
substance)
Yes
M7 would apply to the new drug substance.
For the existing drug substance,
retrospective application of M7 to existing
products is not intended. For the drug
product, this would classify as a new drug
product so the guideline would apply to any
new or higher levels of degradation products.
Appendix 2: Case examples to illustrate potential control approaches
Case 1: Example of an option 3 control strategy
An intermediate X is formed two steps away from the drug substance and impurity A is routinely
detected in intermediate X. The impurity A is a stable compound and carries over to the drug
substance. A spike study of the impurity A at different concentration levels in intermediate X was
performed at laboratory scale. As a result of these studies, impurity A was consistently removed to less
than 30% of the TTC-based limit in the drug substance even when impurity A was present at 1% in
intermediate X. Since this intermediate X is formed only two steps away from the drug substance and
the impurity A level in the intermediate X is relatively high, the purging ability of the process has
additionally been confirmed by determination of impurity A in the drug substance in multiple pilot-scale
batches and results were below 30% of the TTC-based limit. Therefore, control of the impurity A in the
intermediate X with an acceptance limit of 1.0% is justified and no test is warranted for this impurity in
the drug substance specification.
Case 2: Example of an option 3 control strategy: based on predicted purge from a spiking
study using standard analytical methods
A starting material Y is introduced in step 3 of a 5-step synthesis and an impurity B is routinely
detected in the starting material Y at less than 0.1% using standard analytical methods. In order to
determine if the 0.1% specification in the starting material is acceptable, a purge study was conducted
at laboratory scale where impurity B was spiked into starting material Y with different concentration
levels up to 10% and a purge factor of > 500 fold was determined across the final three processing
steps. This purge factor applied to a 0.1% specification in starting material Y would result in a
predicted level of impurity B in the drug substance of less than 2 ppm. As this is below the TTC-based
limit of 50 ppm for this impurity in the drug substance, the 0.1% specification of impurity B in starting
material Y is justified without the need for providing drug substance batch data on pilot scale or
commercial scale batches.
Case 3: Example of an option 2 and 4 control strategy: control of structurally similar
mutagenic impurities
The Step 1 intermediate of a 5-step synthesis is a nitroaromatic compound that may contain low levels
of impurity C, a positional isomer of the step 1 intermediate and also a nitroaromatic compound. The
ICH guideline M7(R1) on assessment and control of DNA reactive (mutagenic) impurities in
pharmaceuticals to limit potential carcinogenic risk
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amount of impurity C in the step 1 intermediate has not been detected by ordinary analytical methods,
but it may be present at lower levels. The step 1 intermediate is positive in the bacterial mutagenicity
assay. The step 2 hydrogenation reaction results in a 99% conversion of the step 1 intermediate to
the corresponding aromatic amine. This is confirmed via in-process testing. An assessment of purge
of the remaining step 1 nitroaromatic intermediate was conducted and a high purge factor was
predicted based on purge points in the subsequent step 3 and 4 processing steps. Purge across the
step 5 processing step is not expected and a specification for the step 1 intermediate at the TTC-based
limit was established at the step 4 intermediate (Option 2 control approach). The positional isomer
impurity C would be expected to purge via the same purge points as the step 1 intermediate and
therefore will always be much lower than the step 1 intermediate itself and therefore no testing is
required and an Option 4 control strategy for impurity C can be supported without the need for any
additional laboratory or pilot scale data.
Case 4: Example of an option 4 control strategy: highly reactive impurity
Thionyl chloride is a highly reactive compound that is mutagenic. This reagent is introduced in step 1
of a 5 step synthesis. At multiple points in the synthesis, significant amounts of water are used. Since
thionyl chloride reacts instantaneously with water, there is no chance of any residual thionyl chloride to
be present in the drug substance. An Option 4 control approach is suitable without the need for any
laboratory or pilot scale data.
Implementation of guideline:
Implementation of M7 is encouraged after publication; however, because of the complexity of the
guideline, application of M7 is not expected prior to 18 months after ICH publication.
The following exceptions to the 18 month timeline apply.
1. Ames tests should be conducted according to M7 upon ICH publication. However, Ames tests
conducted prior to publication of M7 need not be repeated.
2. When development programs have started phase 2b/3 clinical trials prior to publication of M7
these programs can be completed up to and including marketing application submission and
approval, with the following exceptions to M7.
No need for two QSAR assessments as outlined in Section 6.
No need to comply with the scope of product impurity assessment as outlined in Section 5.
No need to comply with the documentation recommendations as outlined in Section 9.
3. Given the similar challenges for development of a commercial manufacturing process,
application of the aspects of M7 listed above to new marketing applications that do not include
Phase 2b/3 clinical trials would not be expected until 36 months after ICH publication of M7.
ICH guideline M7(R1) on assessment and control of DNA reactive (mutagenic) impurities in
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Appendix 3: Addendum to ICH M7
Application of the Principles of the ICH M7 Guideline to Calculation of Compound-Specific
Acceptable Intakes
List of abbreviations .................................................................................. 33
Introduction .............................................................................................. 35
Methods .................................................................................................... 35
Acrylonitrile (CAS# 107-13-1) ................................................................... 43
Aniline (CAS# 62-53-3) and Aniline Hydrochloride (CAS# 142-04-1) ....... 49
Aniline and Aniline HCl – Details of carcinogenicity studies ....................... 51
Benzyl Chloride (α-Chlorotoluene, CAS# 100-44-7) .................................. 56
Bis(chloromethyl)ether (BCME, CAS# 542-88-1)....................................... 62
p-Chloroaniline (CAS# 106-47-8) and p-Chloroaniline HCl (CAS# 20265-
96-7) ......................................................................................................... 65
1-Chloro-4-Nitrobenzene (para-Chloronitrobenzene, CAS# 100-00-5) ...... 69
p-Cresidine (2-Methoxy-5-Methyl Aniline, CAS# 120-71-8)...................... 74
Dimethylcarbamyl Chloride (CAS# 79-44-7) ............................................. 78
Dimethyl Sulfate (CAS# 77-78-1) .............................................................. 82
Ethyl Chloride (Chloroethane, CAS# 75-00-3) ........................................... 86
Glycidol (CAS# 556-52-5) ......................................................................... 89
Hydrazine (CAS# 302-01-2) ...................................................................... 92
Hydrogen Peroxide (CAS# 7722-84-1) ...................................................... 98
Methyl Chloride (Chloromethane, CAS# 74-87-3) ................................... 103
Note 1 ..................................................................................................... 107
Note 2 ..................................................................................................... 109
Note 3 ..................................................................................................... 111
33
List of abbreviations
AI Acceptable Intakes
ATSDR Agency for Toxic Substances & Disease Registry
BC Benzyl Chloride
BCME Bis(chloromethyl)ether
BUA Biodegradable in water Under Aerobic conditions
CAC Cancer Assessment Committee
CCRIS Chemical Carcinogenesis Research Information System
CHL Chinese Hamster Lung fibroblast cell line
CICAD Concise International Chemical Assessment Document
CIIT Chemical Industry Institute of Toxicology
CNS Central Nervous System
CPDB Carcinogenicity Potency Database
CYP Cytochrome P-450
DMCC Dimethylcarbamyl Chloride
DMS Dimethyl Sulfate
DNA Deoxyribose Nucleic Acid
EC European Commission
ECHA European Chemical Agency
EFSA European Food Safety Authority
EMA European Medicines Agency
EPA Environmental Protection Agency
EU European Union
FDA Food and Drug Administration
GRAS Generally Recognized As Safe
HSDB Hazardous Substance Database
IARC International Agency for Research on Cancer
IPCS International Programme on Chemical Safety
IRIS Integrated Risk Information System
JETOC Japan Chemical Industry Ecology-Toxicology & Information Center
JRC Joint Research Centre
LOAEL Lowest-Observed Adverse Effect Level
MTD Maximum Tolerated Dose
34
NA Not applicable
NC Not calculated; individual tumour type incidences not provided in WHO, 2002
NCI National Cancer Institute
NOAEL No-Observed Adverse Effect Level
NOEL No-Observed Effect Level
NSRL No Significant Risk Level
NTP National Toxicology Program
OECD Organisation for Economic Cooperation and Development
PCE Polychromatic Erythrocytes
PDE Permissible Daily Exposure
RfC Reference Concentration
ROS Reactive Oxygen Species
SCCP Scientific Committee on Consumer Products
SCCS Scientific Committee on Consumer Safety
SCE Sister Chromatid Exchanges
SIDS Screening Information Dataset
TBA Tumor Bearing Animal
TD50 Chronic dose-rate in mg/kg body weight/day which would cause tumors in half of the animals at the end
of a standard lifespan for the species taking into account the frequency of that tumor type in control
animals
TTC-based Threshold of Toxicological Concern-based
UDS Unscheduled DNA Synthesis
UNEP United Nations Environmental Programme
US EPA United States Environmental Protection Agency
WHO World Health Organization
35
Introduction
The ICH M7 Guideline discusses the derivation of Acceptable Intakes (AIs) for mutagenic impurities
with positive carcinogenicity data, (Section 7.2.1) and states: “Compound-specific risk
assessments to derive acceptable intakes should be applied instead of the TTC-based (Threshold of
Toxicological Concern-based) acceptable intakes where sufficient carcinogenicity data exist. For a
known mutagenic carcinogen, a compound-specific acceptable intake can be calculated based on
carcinogenic potency and linear extrapolation as a default approach. Alternatively, other
established risk assessment practices such as those used by international regulatory bodies may be
applied either to calculate acceptable intakes or to use already existing values published by
regulatory authorities.”
In this Addendum to ICH M7, AIs or Permissible Daily Exposures (PDEs) have been derived for a
set of chemicals that are considered to be mutagens and carcinogens and are common in
pharmaceutical manufacturing, or are useful to illustrate the principles for deriving compound-
specific intakes described in ICH M7
1
. The set of chemicals include compounds in which the
primary method used to derive AIs for carcinogens with a likely mutagenic mode of action is the
“default approach” from ICH M7 of linear extrapolation from the calculated cancer potency estimate,
the TD
50
. Some chemicals that are mutagens and carcinogens (classified as Class 1 in ICH M7)
may induce tumors through a non-mutagenic mode of action. Therefore, additional compounds are
included to highlight alternative principles to deriving compound-specific intakes (i.e. PDE, see
below). Other compounds (e.g., aniline) are included even though the available data indicates that
they are non-mutagenic; nevertheless, the historical perception has been that they are genotoxic
carcinogens.
ICH M7 states in Section 7.2.2: “The existence of mechanisms leading to a dose response that is
non-linear or has a practical threshold is increasingly recognized, not only for compounds that
interact with non-DNA (Deoxyribose Nucleic Acid) targets but also for DNA-reactive compounds,
whose effects may be modulated by, for example, rapid detoxification before coming into contact
with DNA, or by effective repair of induced damage. The regulatory approach to such compounds
can be based on the identification of a No-Observed Effect Level (NOEL) and use of uncertainty
factors (see ICH Q3C(R5)…) to calculate a Permissible Daily Exposure (PDE) when data are
available."
Examples are included in this Addendum to illustrate assessments of mode of action for some Class
1 chemicals that justify derivation of a PDE calculated using uncertainty factors as described in ICH
Q3C(R5) (Ref. 1). These chemicals include hydrogen peroxide, which induces oxidative stress, and
aniline which induces tumors secondary to hemosiderosis as a consequence of methemoglobinemia.
It is emphasized that the AI or PDE values presented in this Addendum address carcinogenic risk.
Other considerations, such as quality standards, may affect final product specifications. For
example, the ICH M7 guidance (Section 7.2.2) notes that when calculating acceptable intakes from
compound-specific risk assessments, an upper limit would be 0.5%, or, for example, 500 µg in a
drug with a maximum daily dose of 100 mg.
Methods
The general approach used in this addendum for deriving AIs included a literature review, selection
of cancer potency estimate [TD
50
, taken from the CPDB (Carcinogenicity Potency Database (Ref. 2),
1
Some chemicals are included whose properties (including chemical reactivity, solubility, volatility, ionizability) allow
efficient removal during the steps of most synthetic pathways, so that a specification based on an acceptable intake will
not typically be needed.
36
or calculated from published studies using the same method as in the CPDB] and ultimately
calculation of an appropriate AI or PDE in cases with sufficient evidence for a threshold mode of
action (see Section 3). The literature review focused on data relating to exposure of the general
population (i.e., food, water, and air), mutagenicity/genotoxicity, and carcinogenicity. Based on
the description of DNA-reactive mutagens in ICH M7, results from the standard bacterial reverse
mutation assay (Ames test) were used as the main criterion for determining that a chemical was
mutagenic. Other genotoxicity data, especially in vivo, were considered in assessing a likely mode
of action for tumor induction. Any national or international regulatory values for acceptable
exposure levels (e.g., US EPA, US FDA, EMA, ECHA, WHO) are described in the compound-specific
assessments. Toxicity information from acute, repeat-dose, reproductive, neurological, and
developmental studies was not reviewed in depth except to evaluate observed changes that act as
a carcinogenic precursor event (e.g., irritation/inflammation, or methemoglobinemia).
1. Standard Method
1.1. Linear Mode of Action and Calculation of AI
Note 4 of ICH M7 states: “It is possible to calculate a compound-specific acceptable intake based
on rodent carcinogenicity potency data such as TD
50
values (doses giving a 50% tumor incidence
equivalent to a cancer risk probability level of 1:2). Linear extrapolation to a probability of 1 in
100,000 (i.e., the accepted lifetime risk level used) is achieved by simply dividing the TD
50
by
50,000. This procedure is similar to that employed for derivation of the TTC.”
Thus, linear extrapolation from a TD
50
value was considered appropriate to derive an AI for those
Class 1 impurities (known mutagenic carcinogens) with no established “threshold mechanism”, that
is, understanding of a mode of action that results in a non-linear dose-response curve. In many
cases, the carcinogenicity data were available from the CPDB; the conclusions were based either on
the opinion of the original authors of the report on the carcinogenicity study (“author opinion” in
CPDB) or on the conclusions of statistical analyses provided in the CPDB. When a pre-calculated
TD
50
value was identified in the CPDB for a selected chemical, this value was used to calculate the
AI; the relevant carcinogenicity data were not reanalyzed and the TD
50
value was not recalculated.
If robust data were available in the literature but not in the CPDB, then a TD
50
was calculated
based on methods described in the CPDB (Ref. 3). The assumptions for animal body weight,
respiratory volume, and water consumption for calculation of doses were adopted from ICH Q3C
and ICH Q3D (Ref. 1, 4).
1.2. Selection of Studies
The quality of studies in the CPDB is variable, although the CPDB does impose criteria for inclusion
such as the proportion of the lifetime during which test animals were exposed. For the purposes of
this Addendum additional criteria were applied when studies were of lesser quality. Studies of
lesser quality are defined here as those where one or more of the following scenarios were
encountered:
< 50 animals per dose per sex;
< 3 dose levels;
Lack of concurrent controls;
Intermittent dosing (< 5 days per week);
Dosing for less than lifetime.
37
The more robust studies were generally used to derive limits. However studies that did not fulfill
all of the above criteria were in some cases considered adequate for derivation of an AI when other
aspects of the study were robust, for example when treatment was for 3 days per week (e.g.,
benzyl chloride) but there was evidence that higher doses would not have been tolerated, i.e., a
Maximum Tolerated Dose (MTD) as defined by the National Toxicology Program (NTP) or ICH
S1C(R2) (Ref. 5) was attained. Calculations of potency take intermittent or less-than-lifetime
dosing such as that for benzyl chloride into account; for example, in the CPDB the dose levels
shown have been adjusted to reflect the estimated daily dose levels, such that the daily dose given
3 times per week is multiplied by 3/7 to give an average daily dose; a comparable adjustment is
made if animals are treated for less than 24 months. Use of less robust data can sometimes be
considered acceptable when no more complete data exist, given the highly conservative nature of
the risk assessment in which TD
50
was linearly extrapolated to a 1 in 100,000 excess cancer risk.
In these cases, the rationale supporting the basis for the recommended approach is provided in the
compound-specific assessments.
1.3. Selection of Tumor and Site
The lowest TD
50
of a particular organ site for an animal species and sex was selected from the most
robust studies. When more than one study exists, the CPDB provides a calculated harmonic mean
TD
50
, but in this Addendum the lowest TD
50
was considered a more conservative estimate. Data
compiled as “all Tumor Bearing Animals” (TBA) were not considered in selecting an appropriate
TD
50
from the CPDB; mixed tumor types (e.g., adenomas and carcinomas) in one tissue (e.g.,
liver) were used where appropriate as this often gives a more sensitive potency estimate.
1.4. Route of Administration
Section 7.5 of ICH M7 states: “The above risk approaches described in Section 7 are applicable to
all routes of administration and no corrections to acceptable intakes are generally warranted.
Exceptions to consider may include situations where data justify route-specific concerns that should
be evaluated case-by-case.”
In this Addendum, when robust data were available from carcinogenicity studies for more than one
route, and the tumor sites did not appear to be route-specific, the TD
50
from the route with the
lowest TD
50
value was selected for the AI calculation and is thus usually considered suitable for all
routes. Exceptions may be necessary case by case; for example, in the case of a potent site-of-
contact carcinogen a route-specific AI or PDE might be necessary. Other toxicities such as irritation
might also limit the AI for a certain route, but only tumorigenicity is considered in this Addendum
similar to M7. Here, if tumors were considered site-specific (e.g., inhalation exposure resulting in
respiratory tract tumors with no tumors at distal sites) and the TD
50
was lower than for other
routes, then a separate AI was developed for that route (e.g., dimethyl carbamoyl chloride,
hydrazine).
1.5. Calculation of AI from the TD
50
Calculating the AI from the TD
50
is as follows (see Note 4 of ICH M7 for example):
AI = TD
50
/ 50,000 x 50 kg
The weight adjustment assumes an arbitrary adult human body weight for either sex of 50 kg. This
relatively low weight provides an additional safety factor against the standard weights of 60 kg or
70 kg that are often used in this type of calculation. It is recognized that some adult patients
38
weigh less than 50 kg; these patients are considered to be accommodated by the inherent
conservatism (i.e., linear extrapolation of the most sensitive organ site) used to determine an AI.
2. Consideration of Alternative Methods for Calculation of
AI
2.1. Human relevance of tumors
Note 4 of ICH M7 states: “As an alternative of using the most conservative TD
50
value from rodent
carcinogenicity studies irrespective of its relevance to humans, an in-depth toxicological expert
assessment of the available carcinogenicity data can be done in order to initially identify the
findings (species, organ, etc.) with highest relevance to human risk assessment as a basis for
deriving a reference point for linear extrapolation.”
Human relevance of the available carcinogenicity data was considered for deriving AIs. Effects in
rodents associated with toxicities that occur with a non-linear dose response are not relevant to
humans at the low, non-toxic concentrations associated with a pharmaceutical impurity. For
example, in the case of p-chloroaniline, the most sensitive site for tumor induction was the spleen,
but these tumors were associated with hemosiderosis, considered to be a mode of action with a
non-linear dose response, and thus not relevant to humans at low doses that do not induce
hemosiderosis. In the case of p-chloroaniline, liver tumors, with a higher TD
50
, were used for the
linear extrapolation to calculate the AI because a mutagenic mode of action could not be ruled out
for liver tumors. A second category of tumors considered not to be relevant to humans is tumors
associated with a rodent-specific mode of action e.g., methyl chloride, with species difference in
metabolism.
2.2. Published regulatory limits
Note 4 of ICH M7 also states: “Compound-specific acceptable intakes can also be derived from
published recommended values from internationally recognized bodies such as World Health
Organization (WHO, International Programme on Chemical Safety (IPCS) Cancer Risk Assessment
Programme) and others using the appropriate 10
-5
lifetime risk level. In general, a regulatory limit
that is applied should be based on the most current and scientifically supported data and/or
methodology.”
In this Addendum, available regulatory limits are described (omitting occupational health limits as
they are typically regional and may use different risk levels). However the conservative linear
extrapolation from the TD
50
was generally used as the primary method to derive the AI, as the
default approach of ICH M7, and for consistency across compounds. It is recognized that minor
differences in methodology for cancer risk assessment can result in different recommended limits
(for example adjusting for body surface area in calculations), but the differences are generally quite
small when linear extrapolation is the basis of the calculation.
3. Non-linear (Threshold) Mode of Action and Calculation
of PDE
ICH M7 states in Section 7.2.2: “The existence of mechanisms leading to a dose response that is
non-linear or has a practical threshold is increasingly recognized, not only for compounds that
interact with non-DNA targets but also for DNA-reactive compounds, whose effects may be
modulated by, for example, rapid detoxification before coming into contact with DNA, or by
effective repair of induced damage. The regulatory approach to such compounds can be based on
39
the identification of a No-Observed Effect Level (NOEL) and use of uncertainty factors (see ICH
Q3C(R5)) to calculate a Permissible Daily Exposure (PDE) when data are available.”
An example of a DNA-reactive chemical for which a threshold has been proposed for mutagenicity
in vitro and in vivo is ethyl methane sulfonate (Ref. 6, 7). A PDE calculation using uncertainty
factors, instead of linear extrapolation is appropriate in such cases where a threshold has been
established.
This threshold approach was considered appropriate in the compound-specific assessments for
carcinogens with modes of action (Section 2.1) that lack human relevance at low doses, based
upon their association with a non-linear dose response for tumor induction:
Chemicals that induce methemoglobinemia, hemosiderin deposits in tissues such as spleen, and
subsequent inflammation and tumors (e.g., aniline and related compounds);
Supporting information includes evidence that mutagenicity was not central to the mode of
action, such as weak evidence for mutagenicity e.g., aniline; and/or lack of correlation
between sites or species in which in vivo genotoxicity (such as DNA adducts) and tumor
induction were seen.
Chemicals that induce tumors associated with local irritation/inflammation (such as rodent
forestomach tumors) and are site-of-contact carcinogens may be considered not relevant to
human exposure at low, non-irritating concentrations as potential impurities in pharmaceuticals
(e.g., benzyl chloride);
Chemicals that act through oxidative damage, so that deleterious effects do not occur at lower
doses since abundant endogenous protective mechanisms exist, (e.g., hydrogen peroxide).
Acceptable exposure levels for carcinogens with a threshold mode of action were established by
calculation of PDEs. The PDE methodology is further explained in ICH Q3C(R5) (Ref. 1) and ICH
Q3D (Ref. 4).
4. 4. Acceptable Limit Based on Exposure in the
Environment, e.g., in the Diet
As noted in ICH M7 Section 7.5, “Higher acceptable intakes may be justified when human exposure
to the impurity will be much greater from other sources e.g., food, or endogenous metabolism (e.g.,
formaldehyde).”
For example, formaldehyde is not a carcinogen orally, so that regulatory limits have been based on
non-cancer endpoints. Health Canada (Ref. 8), WHO IPCS (Ref. 9) and US Environmental
Protection Agency (EPA) (Ref. 10) recommend an oral limit of 0.2 mg/kg/day, or 10 mg/day for a
50 kg person.
40
References
1. International Conference on Harmonisation (2011). Q3C(R5): Impurities: Guideline for Residual
Solvents
2. Carcinogenicity Potency Database (CPDB). [Online]. Available from: URL:
https://toxnet.nlm.nih.gov/cpdb/
3. Carcinogenicity Potency Database (CPDB): [Online]. Available from: URL:
https://toxnet.nlm.nih.gov/cpdb/td50.html
4. International Conference on Harmonisation (2014). Q3D: Impurities: Guideline for Elemental
Impurities
5. International Conference on Harmonisation (2008). S1C(R2): Dose Selection for
Carcinogenicity Studies of Pharmaceuticals
6. Müller L, Gocke E, Lave T, Pfister T. Ethyl methanesulfonate toxicity in Viracept-A
comprehensive human risk assessment based on threshold data for genotoxicity. Toxicol Lett
2009;190:317-29.
7. Cao X, Mittelstaedt RA, Pearce MG, Allen BC, Soeteman-Hernández LG, Johnson GE, et al.
Quantitative dose-response analysis of ethyl methanesulfonate genotoxicity in adult gpt-delta
transgenic mice. Environ Mol Mutagen 2014;55:385-99.
8. Health Canada. 2001 Priority substances list assessment report: Formaldehyde. Ottawa.
Ministry of Public Works and Government Services. February. [Online]. Available from: URL:
http://www.hc-sc.gc.ca/ewh-semt/pubs/contaminants/psl2-lsp2/index_e.html
9. World Health Organization (WHO). International Programme on Chemical Safety (IPCS). 2002.
Concise International Chemical Assessment Document 40. Formaldehyde. [Online]. Available
from: URL: http://www.who.int/ipcs/publications/cicad/en/cicad40.pdf
10. US Environmental Protection Agency.Integrated Risk Information System (IRIS). [Online].
1990; Available from: URL: http://www.epa.gov/iris/
.
41
Acceptable Intakes (AIs) or Permissible Daily Exposures (PDEs)
Compound
CAS#
Chemical
Structure
AI or PDE
(µg/day)
Comment
Linear extrapolation from TD
50
Acrylonitrile
107-13-1
6
TD
50
linear
extrapolation
Benzyl Chloride
100-44-7
41
TD
50
linear
extrapolation
Bis(chloromethyl)ether
542-88-1
0.004
TD
50
linear
extrapolation
1-Chloro-4-nitrobenzene
100-00-5
117
TD
50
linear
extrapolation
p-Cresidine
120-71-8
45
TD
50
linear
extrapolation
Dimethylcarbamoyl
chloride
79-44-7
5
0.6
(Inhalation)*
TD
50
linear
extrapolation
Ethyl chloride
75-00-3
1,810
TD
50
linear
extrapolation
Glycidol
556-52-5
4
TD
50
linear
extrapolation
Hydrazine
302-01-2
39
0.2
(Inhalation)*
TD
50
linear
extrapolation
Methyl Chloride
74-87-3
Cl-CH
3
1,361
TD
50
linear
extrapolation
Threshold-based PDE
Aniline
Aniline HCl
62-53-3
142-04-1
720
PDE based on
threshold mode of
action
(Hemosiderosis)
Endogenous and/or Environmental Exposure
Hydrogen peroxide
7722-84-1
68,000 or
0.5%
whichever is
lower
68 mg/day is 1% of
estimated
endogenous
production
Other Cases
p-Chloroaniline
p-Chloroaniline HCl
106-47-8
20265-96-7
34
AI based on liver
tumors for which
mutagenic mode of
action cannot be
ruled out
42
Compound
CAS#
Chemical
Structure
AI or PDE
(µg/day)
Comment
Dimethyl Sulfate
77-78-1
1.5
Carcinogenicity data
available, but
inadequate to derive
AI. Default to TTC
*
Route specific limit
43
Acrylonitrile (CAS# 107-13-1)
Potential for human exposure
No data are available for exposure of the general population.
Mutagenicity/Genotoxicity
Acrylonitrile is mutagenic and genotoxic in vitro and potentially positive in vivo.
The World Health Organization (WHO) Concise International Chemical Assessment Document
(CICAD, Ref. 1), provided a thorough risk assessment of acrylonitrile. In this publication, oxidative
metabolism was indicated as a critical step for acrylonitrile to exert genotoxic effects, implicating
cyanoethylene oxide as a DNA-reactive metabolite. A detailed review of genotoxicity testing in a
range of systems is provided (Ref. 1) with references, so only a few key conclusions are
summarized here.
Acrylonitrile is mutagenic in:
Microbial reverse mutation assay (Ames) in Salmonella typhimurium TA 1535 and TA 100 only in
the presence of rat or hamster S9 and in several Escherichia coli strains in the absence of
metabolic activation;
Human lymphoblasts and mouse lymphoma cells, reproducibly with S9, in some cases without S9;
Splenic T cells of rats exposed via drinking water.
In vivo genotoxicity studies are negative or inconclusive, and reports of DNA binding are
consistently positive in liver, but give conflicting results in brain.
Carcinogenicity
Acrylonitrile is classified by IARC as a Group 2B carcinogen, possibly carcinogenic to humans (Ref.
2).
Acrylonitrile is a multi-organ carcinogen in mice and rats, with the brain being the primary target
organ in rat. There are four oral carcinogenicity studies cited in the CPDB (Ref. 3) and the results
from three additional oral studies are summarized in Ref. 1. Of these seven studies only one is
negative but this study tested only a single dose administered for short duration (Ref. 4).
The NCI/NTP (National Cancer Institute) study in the CPDB of acrylonitrile in mice (Ref. 5) was
selected for derivation of the oral AI, based on robust study design and the most conservative TD
50
value. In this 2 year-study, 3 doses of acrylonitrile were administered by oral gavage to male and
female mice. There were statistically significant increases in tumors of the Harderian gland and
forestomach.
In the 1980 study of Quast et al (Ref. 6), cited in the CPDB as a report from Dow Chemical, it
appears that the most sensitive TD
50
is for astrocytomas in female rats (5.31 mg/kg/day).
However, this same study was later described in detail (Ref. 7) and the calculated doses in that
published report are higher than those listed in the CPDB. Quast (Ref. 7) describes the derivation
of doses in mg/kg/day from the drinking water concentrations of 35, 100 and 300 ppm, adjusting
for body weight and the decreased water consumption in the study. The TD
50
for astrocytomas
derived from these numbers is 20.2 mg/kg/day for males and 20.8 for females, in contrast to the
calculated values in the CPDB of 6.36 and 5.31 mg/kg/day. (The TD
50
’s calculated from the dose
estimates by Quast (Ref. 7) for forestomach tumors are also higher than those in the CPDB based
on the same study, as shown in the Table below). Central Nervous System (CNS), tumors are
44
described (Ref. 7), but the most sensitive TD
50
was for stomach tumors, as shown in the Table
below.
Studies considered less robust included three rat drinking water studies. The largest study (Ref. 8)
included five acrylonitrile treated groups with 100 animals per dose and 200 control animals, but
serial sacrifices of 20 animals per treatment group occurred at 6, 12, 18 and 24 months. Data
summaries by WHO (Ref. 1) and by US EPA (Ref. 9) present tumor incidence based on data from
all time points combined. Therefore, the incidence of tumors reported may be an underestimate of
the total tumors that would be observed if all animals were kept on study for 2 years. Two studies
(Ref. 10, 11) each had only two dose levels and individual tumor types are not reported (Ref. 1),
although tumors of stomach, Zymbal gland and brain were observed.
Acrylonitrile has also been studied by the inhalation route. Fifty rats per sex per dose were
exposed for 2 years to acrylonitrile, and brain tumors were observed (Ref. 12). This study however,
tested only 2 dose levels. The other inhalation studies were deficient in number of animals per
group, duration of exposure, or administration of a single dose, although brain tumors were
observed.
Acrylonitrile – Details of carcinogenicity studies
Study
Animals/
dose group
Duration/
Exposure
Controls
Doses
Most
sensitive
tumor
site/type/s
ex
TD
50
(mg/kg/d
)
Ref. 5
*
50 B6C3F1
Mice (F)
2 years
Gavage
50
3: 1.79;7.14;
14.3 mg/kg/d
Forestomach
6.77
+
50 B6C3F1
Mice (M)
2 years
Gavage
50
3: 1.79;7.14;
14.3 mg/kg/d
Forestomach
5.92
+
Ref. 6
~50 SD Spartan
rats
(F)
2 years
Drinking
water
~80
3:
2.00;5.69;
15.4 mg/kg/d
Astrocytoma
5.31
++
(20.8)
~50 SD Spartan
rats
(M)
2 years
Drinking
water
~80
3:
1.75;4.98;
14.9 mg/kg/d
Stomach,
non-glandular
6.36
++
(9.0)
Ref 7
(report
of Ref.
6)
~50 female SD
Spartan rats
2 years
Drinking
water
~80
3:
4.4;10.8; 25
mg/kg/d
Stomach,
non-glandular
19.4
~50 SD male
Spartan rats
2 years
Drinking
water
~80
3:
3.4;8.5;
21.3 mg/kg/d
Stomach,
non-glandular
9.0
Ref. 8
¥
100 male rats
~2 years
Drinking
water
~200
5:
0.1-8.4
mg/kg/d
Brain
astrocytoma
(22.9)
+
100 female rats
~2 years
Drinking
water
~200
5:
0.1-10.9
mg/kg/d
Brain
astrocytoma
(23.5)
+
Ref. 11
¥
100/sex
Rats
19-22 mo
Drinking
water
~98
2:
~0.09; 7.98
mg/kg/d
Stomach,
Zymbal’s
gland, brain,
spinal cord
NC
Ref. 10
¥
50/sex
Rats
18 mo
Drinking
water
No
2:
14;70 mg/kg/d
Brain,
Zymbal’s
gland,
forestomach
NC
^
45
Study
Animals/
dose group
Duration/
Exposure
Controls
Doses
Most
sensitive
tumor
site/type/s
ex
TD
50
(mg/kg/d
)
Ref. 13
20
male CD rats
2 years
Drinking
water
No
3:
1; 5; 25
mg/kg/d
Zymbal’s
gland
30.1
Ref. 4
40/sex
SD rats
1 year
3d/wk
Gavage
75/sex
1:
1.07 mg/kg/d
Neg in both
sexes
NA
Ref. 12
100/sex
SD Spartan rat
2 years
6 h/d;
5d/wk
Inhalation
100
2:
M: 2.27; 9.1
F: 3.24; 13.0
mg/kg/d
Brain
Astrocytoma
Male
32.4
Ref. 4
30/sex
SD rats
1 year
5d/wk
Inhalation
30
4:
M: 0.19; 0.38;
0.76; 1.52
F:
0.27;0.54;1.0;
2.17
mg/kg/d
Brain glioma
Male
19.1
Ref. 4
54 female SD
rats
2 years
5d/wk
Inhalation
60
1:
11.1 mg/kg/d
Brain glioma
(132)
Studies listed are in CPDB (Ref. 3) unless otherwise noted.
The TD
50
values represent the TD
50
from the most sensitive tumor site.
TD
50
values in parentheses are considered less reliable as explained in footnotes.
*
Carcinogenicity study selected for AI calculation; in CPDB.
^
NC= Not calculated as individual tumor type incidences not provided in WHO (Ref. 1).
+
TD
50
calculated based on astrocytoma incidence implied as most significant site by WHO (Ref. 1).
Serial sampling reduced number of animals exposed for 2 years, so tumor incidences may be
underestimates.
++
Taken from the CPDB. Note that based on the dose calculations by the author (Ref. 7) the TD
50
for astrocytomas and stomach tumors in Spartan rats (20.8 and 9.0) are higher than those in the
CPDB.
NA= Not applicable.
¥
Not in CPDB. Summarized in Refs. 1 and 9.
Single dose-level study.
Mode of action for carcinogenicity
Although the mechanism of carcinogenesis remains inconclusive, a contribution of DNA interaction
cannot be ruled out (Ref. 1). CNS tumors were seen in multiple carcinogenicity studies in rats, in
addition to forestomach tumors; forestomach tumors were also the most sensitive tumor type in
mice.
Forestomach tumors are associated with local irritation and inflammation, and Quast (Ref. 7) notes
the typical association between these tumors in rats and hyperplasia and/or dyskeratosis, with
other inflammatory and degenerative changes. Forestomach tumors in rodents administered high
concentrations orally, a type of site-of-contact effect, may not be relevant to human exposure at
low concentrations that are non-irritating (Ref. 14). Acrylonitrile is not only a site-of-contact
carcinogen. Tumors were seen in the CNS, in addition to tissues likely to be exposed directly such
as the gastrointestinal tract and tongue. Forestomach tumors were seen after administration of
acrylonitrile to rats in drinking water, and to mice by gavage. The AI for acrylonitrile was derived
based on mouse forestomach tumors.
46
Regulatory and/or published limits
The US EPA (Ref. 9) calculated an oral slope factor of 0.54 /mg/kg/day and a drinking water limit
of 0.6 µg/L at the 1/100,000 risk level, based on the occurrence of multi-organ tumors in a
drinking water study in rats. This drinking water limit equates to a daily dose of ~1 µg/day for a
50 kg human.
47
Acceptable intake (AI)
Rationale for selection of study for AI calculation
Both inhalation and oral studies (gavage and drinking water) are available. Tumors of the CNS
were seen by both routes of administration, and acrylonitrile is rapidly absorbed via all routes of
exposure and distributed throughout examined tissues (Ref. 1), so that a specific inhalation AI was
not considered necessary. All of the carcinogenicity studies that were used by the US EPA (Ref. 9)
in the derivation of the drinking water limit for acrylonitrile were reviewed when selecting the most
robust carcinogenicity study for the derivation of an AI. The NCI/NTP study (Ref. 5) was selected
to calculate the AI based on the TD
50
derived from administering acrylonitrile by oral gavage to
male and female mice since the tumor type with the lowest TD
50
was forestomach tumors in male
mice, with a TD
50
value of 5.92 mg/kg/day. As discussed in the Methods Section 2.2, linear
extrapolation from the TD
50
was used here to derive the AI, and it is expected that minor
differences in methodology can result in different calculated limits; thus the AI calculated below for
potential pharmaceutical impurities is slightly higher than that derived by US EPA (Ref. 9) for
drinking water.
Calculation of AI
Lifetime AI = TD
50
/50,000 x 50kg
Lifetime AI = 5.92 (mg/kg/day)/50,000 x 50 kg
Lifetime AI = 5.9 µg/day (6 µg/day)
References
1. World Health Organization (WHO). Concise International Chemical Assessment Document
(CICAD) 39. Acrylonitrile. [Online]. Geneva. 2002; Available from: URL:
http://www.inchem.org/documents/cicads/cicads/cicad39.htm
2. International Agency for Research on Cancer (IARC). IARC Monographs on the Evaluation of
Carcinogenic Risks to Humans. International Agency for Research on Cancer, World Health
Organization, Lyon. Acrylonitrile 1999; Vol. 71, 43.
3. Carcinogenicity Potency Database (CPDB). [Online]. Available from: URL:
http://toxnet.nlm.nih.gov/cpdb/
4. Maltoni C, Ciliberti A, Cotti G, Perino G. Long-term carcinogenicity bioassays on acrylonitrile
administered by inhalation and by ingestion to Sprague-Dawley rats. Annals of the New York
Academy of Sciences 1988;534:179–202.
5. National Toxicology Program (NTP) Toxicology and Carcinogenesis Studies of Acrylonitrile (CAS
No. 107-13-1) in B6C3F1 Mice (Gavage Studies). NTP TR 506 NIH Publication No. 02-4440.
2001;198.
6. Quast JF, Wade CE, Humiston CG, Carreon RM, Hermann EA, Park CN et al, Editors. A Two-
Year Toxicity and Oncogenicity Study with Acrylonitrile Incorporated in the Drinking Water of
Rats, Final Report. Dow Chemical USA, Midland, MI; 1980.
7. Quast, JF Two-year toxicity and oncogenicity study with acrylonitrile incorporated in the
drinking water of rats. Toxicol Lett 2002;132:153-96.
8. Bio/Dynamics Inc. Monsanto Company. 1980. A twenty-four month oral toxicity/carcinogenicity
study of acrylonitrile administered in the drinking water to Fischer 344 rats. Final report. Four
volumes. St. Louis, MO. Project No. 77-1744; BDN-77-27.
48
9. US EPA. Acrylonitrile (CAS# 107-13-1). Integrated Risk Information System (IRIS)
[Online].1987. Available from: URL:
https://cfpub.epa.gov/ncea/iris2/chemicalLanding.cfm?substance_nmbr=206
10. Bigner DD, Bigner SH, Burger PC, Shelburne JD, Friedman HS. Primary brain tumors in Fischer
344 rats chronically exposed to acrylonitrile in their drinking water. Food and Chemical
Toxicology 1986;24:129–37.
11. Bio/Dynamics Inc. Monsanto Company, Division of Biology and Safety evaluation. 1980. A
twenty-four month oral toxicity/carcinogenicity study of acrylonitrile administered to Spartan
rats in the drinking water. Final report. Two volumes. St. Louis, MO. Project No. 77-1745; BDN-
77-28.
12. Quast JF, Schuetz DJ, Balmer MF, Gushow TS, Park CN, McKenna MJ, editors. A Two-Year
Toxicity and Oncogenicity Study with Acrylonitrile Following Inhalation Exposure of Rats, Final
Report. Dow Chemical USA, Midland, MI; 1980.
13. Gallagher GT, Maull EA, Kovacs K, Szab S. Neoplasms in rats ingesting acrylonitrile for two
years. J Am Col Toxicol 1988;7:603-15.
14. Proctor DM, Gatto NM, Hong SJ, Allamneni KP. Mode-of-action framework for evaluation of the
relevance of rodent forestomach tumors in cancer risk assessment. Toxicol. Sci 2007;98:313-
26.
49
Aniline (CAS# 62-53-3) and Aniline Hydrochloride (CAS#
142-04-1)
Potential for human exposure
Aniline occurs naturally in some foods (i.e., corn, grains, beans, and tea), but the larger source of
exposure is in industrial settings.
Mutagenicity/genotoxicity
Aniline is not mutagenic in the microbial reverse mutation assay (Ames) in Salmonella. Aniline is
included in this Addendum because of the historical perception that aniline is a genotoxic
carcinogen, since some in vitro and in vivo genotoxicity tests are positive.
Aniline is not mutagenic in the 5 standard strains of Salmonella or in E.Coli WP2 uvrA, with or
without S9 (Ref. 1, 2, 3, 4, 5, 6, 7, 8).
Aniline was positive in the mouse lymphoma L5178Y cell tk assay with and without S9 at quite high
concentrations, such as 0.5 to 21 mM (Ref. 9, 10, 11).
Chromosomal aberration tests gave mixed results, with some negative reports and some positive
results in hamster cell lines at very high, cytotoxic concentrations, e.g., about 5 to 30 mM, with or
without S9 metabolic activation (Ref. 1, 12, 13, 14, 15).
In vivo, chromosomal aberrations were not increased in the bone marrow of male CBA mice after
two daily intraperitoneal (i.p.) doses of 380 mg/kg (Ref. 16), but a small increase in chromosomal
aberrations 18 h after an oral dose of 500 mg/kg to male PVR rats was reported (Ref. 17).
Most studies of micronucleus induction are positive in bone marrow after oral or i.p. treatment of
mice (Ref. 18, 19, 20, 21) or rats (Ref. 17, 22), and most commonly at high doses, above 300
mg/kg. Dietary exposure to 500, 1000 and 2000 ppm for 90 days was associated with increases in
micronuclei in peripheral blood of male and female B6C3F1 mice (Ref. 23).
In vivo, a weak increase in Sister Chromatid Exchanges (SCE), reaching a maximum of 2-fold
increase over the background, was observed in the bone marrow of male Swiss mice 24 h after a
single i.p. dose of 61 to 420 mg/kg aniline (Ref. 24, 25). DNA strand breaks were not detected in
the mouse bone marrow by the alkaline elution assay in this study.
50
Carcinogenicity
Aniline is classified by IARC as Group 3, not classifiable as to its carcinogenicity in humans (Ref. 4).
Bladder cancers in humans working in the dye industry were initially thought to be related to
aniline exposure but were later attributed to exposures to intermediates in the production of aniline
dyes, such as -naphthylamine, benzidine, and other amines.
The Chemical Industry Institute of Toxicology (CIIT, Ref. 26) performed a study in which aniline
hydrochloride was administered in the diet for 2 years to CD-F rats (130 rats/sex/group) at levels
of 0, 200, 600, and 2000 ppm. An increased incidence of primary splenic sarcomas was observed
in male rats in the high dose group only. This study was selected for derivation of the PDE for
aniline based on the robust study design with 3 dose groups and a large group size
(130/sex/group).
The results of the CIIT study are consistent with those of the dietary study by the US National
Cancer Institute (Ref. 27) of aniline hydrochloride in which male rats had increases in
hemangiosarcomas in multiple organs including spleen, and a significant dose-related trend in
incidence of malignant pheochromocytoma. In mice (Ref. 27), no statistically significant increase
in any type of tumor was observed at very high doses.
Aniline itself did not induce tumors in rats when tested in a less robust study design (Ref. 28).
51
Aniline and Aniline HCl – Details of carcinogenicity studies
Study
Animals/
dose group
Duration/
Exposure
Controls
Doses
Most sensitive
tumor
site/type/sex
TD
50
(mg/kg/d
)
Ref. 26
*
Aniline
HCl
130/sex/
group, CD-F
rats
2 years
Diet
130
3:
200, 600 and
2000 ppm in
diet
(M; 7.2; 22;
72 mg/kg/d)
Spleen sarcoma
(high dose).
NOEL at low
dose
Not
reported
Ref. 27
**
Aniline
HCl
50/sex/group,
F344 rats
103 weeks
(107-110
wk study)
Diet
50
2:
3000 and
6000 ppm in
diet
(F: 144;268
M: 115;229
mg/kg/d)
Spleen
hemangio-
sarcoma/Male
160 (Male)
Ref. 27
**
Aniline
HCl
50/sex/group
B6C3F1 mice
103 weeks
(107-110
wk study)
Diet
50
2:
6000 and
12000 ppm
in diet
(F: 741;1500
M: 693;1390
mg/kg/d)
Negative
NA
Ref. 28
**
Aniline
10-18/group,
male Wistar
rats
80 weeks
Diet
Yes
3:
0.03, 0.06
and 0.12%
in diet
(15;30;60
mg/kg/d)
Negative
NA
*
Carcinogenicity study selected for PDE calculation. Not in CPDB.
**
Taken from CPDB (Ref. 29). The TD
50
values represent the TD
50
from the most sensitive tumor
site.
NA = Not applicable
Mode of action for carcinogenicity
In animal studies, aniline caused methemoglobinemia and hemolysis at high doses, the latter of
which could indirectly lead to increases in micronuclei by inducing erythropoiesis (Ref. 19, 30, 31).
Micronuclei are induced in both rats and mice, while aniline-induced tumors are seen in rats but not
mice, adding to the evidence that genotoxicity is not key to the mode of action for aniline-induced
tumors.
Aniline-induced toxicity in the spleen appears to be a contributory factor for its carcinogenicity via
free radical formation and tissue injury (Ref. 32). High doses (>10 mg/kg) of aniline lead to iron
accumulation in the spleen resulting from the preferential binding of aniline to red blood cells and
damaged cells accumulating in the spleen. Iron-mediated oxidative stress in the spleen appears to
induce lipid peroxidation, malondialdehyde-protein adducts, protein oxidation, and up-regulation of
Transforming Growth Factor-β 1, all of which have been detected in the rat spleen following aniline
exposure (Ref. 33). Increased oxidative stress may be a continual event during chronic exposure
to aniline and could contribute to the observed cellular hyperplasia, fibrosis, and tumorigenesis in
rats (Ref. 32, 34). The lack of tumorigenicity in mice may be due to less severe toxicity observed
in spleen compared to that in rats (Ref. 17, 35).
In support of this toxicity-driven mode of action for carcinogenicity, the dose response for aniline-
induced tumorigenicity in rats is non-linear (Ref. 36). When considering the NCI and CIIT studies
which both used the same rat strain, no tumors were observed when aniline hydrochloride was
administered in the diet at a concentration of 0.02% (equal to approximately 7.2 mg/kg/day aniline
52
in males). This, together with studies evaluating the pattern of accumulation of bound radiolabel
derived from aniline in the spleen (Ref. 37) support the conclusion that a threshold exists for
aniline carcinogenicity (Ref. 36). The weight of evidence supports the conclusion that these tumors
do not result from a primary mutagenic mode of action (Ref. 38).
Regulatory and/or published limits
The US EPA (Ref. 39) outlines a quantitative cancer risk assessment for aniline based on the CIIT
study (Ref. 26) and use of a linearised multistage. The resulting cancer potency slope curve was
0.0057/mg/kg/day and the dose associated with a 1 in 100,000 lifetime cancer risk is calculated to
be 120 µg/day. However, the assessment states that this procedure may not be the most
appropriate method for the derivation of the slope factor as aniline accumulation in the spleen is
nonlinear (Ref. 39). Minimal accumulation of aniline and no hemosiderosis is observed at doses
below 10 mg/kg and as already described, hemosiderosis may be important in the induction of the
splenic tumors observed in rats.
Permissible daily exposure (PDE)
It is considered inappropriate to base an AI for aniline on linear extrapolation for spleen tumors
observed in rats, since these have a non-linear dose response, aniline is not mutagenic, and
genotoxicity is not central to the mode of action of aniline-induced carcinogenicity. The PDE is
derived using the process defined in ICH Q3C (Ref. 40).
Rationale for selection of study for PDE calculation
Data from the CIIT 2-year rat carcinogenicity study (Ref. 26) have been used. Dose levels of 200,
600, and 2000 ppm for aniline hydrochloride in the diet were equivalent to dose levels of aniline of
7.2, 22 and 72 mg/kg/day. Tumors were observed in high dose males and one stromal sarcoma of
the spleen was identified at 22 mg/kg/day. Based on these data the lowest dose of 7.2 mg/kg/day
was used to define the No-Observed Effect Level for tumors (NOEL).
The PDE calculation is: (NOEL x body weight adjustment (kg)) / F1 x F2 x F3 x F4 x F5
The following safety factors as outlined in ICH Q3C have been applied to determine the PDE for
aniline:
F1 = 5 (rat to human)
F2 = 10 (inter- individual variability)
F3 = 1 (study duration at least half lifetime)
F4 = 10 (severe toxicity – non-genotoxic carcinogenicity)
F5 = 1 (using a NOEL)
Lifetime PDE = 7.2 mg/kg/day x 50 kg / (5 x 10 x 1 x 10 x 1)
Lifetime PDE = 720 µg/day
References
1. Chung KT, Murdock CA, Zhou Y, Stevens SE, Li YS, Wei CI, et al. Effects of the nitro-group on
the mutagenicity and toxicity of some benzamines. Environ Mol Mutagen 1996;27:67-74.
2. IARC. Some aromatic amines, anthraquinones and nitroso compounds, and inorganic fluorides
used in drinking water and dental preparations. Monographs on the Evaluation of the
Carcinogenic Risk of Chemicals to Humans. International Agency for Research on Cancer, World
Health Organization, Lyon. 1982; 27:39.
53
3. IARC. Genetic and related effects: An update of selected IARC Monographs from volumes 1 to
42. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans.
International Agency for Research on Cancer, World Health Organization, Lyon.1987.
Addendum 6: 68.
4. IARC. Overall evaluation of carcinogenicity: An update of IARC monographs volumes 1 to 42.
Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. International
Agency for Research on Cancer, World Health Organization, Lyon. 1987. Addendum 7: pp 99
and 362.
5. Jackson MA, Stack HF, Waters MD. The genetic toxicology of putative nongenotoxic carcinogens.
Mutat Res 1993;296:241-77.
6. Brams A, Buchet JP, Crutzen-Fayt MC, De Meester C, Lauwerys R, Leonard A. A Comparative
Study, With 40 Chemicals, of The Efficiency of ohe Salmonella Assay and the SOS Chromotest
(Kit Procedure). Toxicol Lett 1987;38:123-33.
7. Rashid KA, Arjmand M, Sandermann H, Mumma RO. Mutagenicity of chloroaniline / lignin
metabolites in the Salmonella/microsome assay. J Environ Sci Health 1987;Part B B22(6):721-
9.
8. Gentile JM, Gentile GJ and Plewa M. Mutagenicity of selected aniline derivatives to Salmonella
following plant activation and mammalian hepatic activation. Mutat Res 1987;188:185-96.
9. Wangenheim J, Bolcsfoldi G. Mouse lymphoma L5178Y thymidine kinase locus assay of 50
compounds; Mutagenesis 1988;3(3):193-205.
10. Amacher DE, Paillet SC, Turner GN, Ray VA, Salsburg DS. Point mutations at the thymidine
kinase locus in L5178Y mouse lymphoma cells. Mutat Res 1980;72:447-74.
11. McGregor DB, Brown AG, Howgate S, Mcbride D, Riach C, Caspary WJ. Responses of the
L5178y mouse lymphoma cell forward mutation assay. V: 27 Coded Chemicals. Environ Mol
Mutagen 1991;17:196-219.
12. Abe S, Sasaki M. Chromosome aberrations and sister chromatic exchanges in Chinese hamster
cells exposed to various chemicals. J Natl Cancer Inst 1977;58:1635-41.
13. Ishidate M, Jr, Odashima S. Chromosome tests with 134 compounds on Chinese hamster cells
in vitro – A screening for chemical carcinogens. Mutat Res 1977;48:337-54.
14. Ishidate M Jr. The data book of chromosomal aberration tests in vitro on 587 chemical
substances using Chinese hamster fibroblast cell line (CHL cells). Tokyo . The Realize Inc.
1983;p26.
15. Galloway SM, Armstrong MJ, Reuben C, Colman S, Brown B, Cannon C, et al. Chromosome
aberrations and sister chromatid exchanges in Chinese Hamster Ovary cells: Evaluations Of 108
Chemicals. Environ Mol Mutagen 1987;10 Suppl 10:1-175.
16. Jones E, Fox V. Lack of clastogenicity activity of aniline in the mouse bone marrow.
Mutagenesis 2003;18:283-6.
17. Bomhard EM. High-dose clastogenic activity of aniline in the rat bone marrow and its
relationship to the carcinogenicity in the spleen of rats. Arch Toxicol 2003;77:291-7.
18. Westmoreland C, Gatehouse DG. Effects of aniline hydrochloride in the mouse bone marrow
micronucleus test after oral administration. Carcinogenesis 1991;12:1057-9.
54
19. Ashby J, Vlachos DA, Tinwell H. Activity of aniline in the mouse bone marrow micronucleus
assay. Mutat Res 1991;263:115-7.
20. Sicardi SM, Martiarena JL, Iglesian MT. Mutagenic and analgesic activities of aniline derivatives.
J Pharm Sci 1991;80:761-4.
21. Ress NB, Witt KL, Xu J, Haseman JK, Bucher JR. Micronucleus induction in mice exposed to
diazoaminobenzene or its metabolites, benzene and aniline: implications for
diazoaminobenzene carcinogenicity. Mutat Res 2002;521:201-8.
22. George E, Andrews M, and Westmoreland C. Effects of azobenzene and aniline in the rodent
bone marrow micronucleus test. Carcinogenesis 1990;11:1551-5.
23. Witt KL, Knapton A, Wehr CM, Hook GJ, Mirsalis J, Shelby MD et al. Micronucleated erythrocyte
frequency in peripheral blood of B6C3F1 mice from short-term, prechronic and chronic studies
of the NTP carcinogenesis bioassay program. Environ Mol Mutagen 2000;36:163–94.
24. Parodi S, Pala M, Russo P, Zunino A, Balbi C, Albini A, et al. DNA damage in liver, kidney, bone
marrow, and spleen of rats and mice treated with commercial and purified aniline as
determined by alkaline elution assay and sister chromatid exchange induction. Cancer Res
1982;42:2277-83.
25. Parodi S, Zunino A, Ottaggio L, De Ferrari M, Santi L. Lack of correlation between the capability
of inducing sister chromatid exchanges in vivo and carcinogenic potency for 16 aromatic
amines and azo derivatives. Mutat Res 1983;108:225-38.
26. CIIT. 1982. 104-week chronic toxicity study in rats with aniline hydrochloride. Final report.
Report prepared for CIIT by Hazleton Laboratories America, Inc. CIIT Docket No. 11642. CIIT,
Research Triangle Park, NC.
27. NCI (National Cancer Institute) National Toxicology Program. Technical report on the bio-assay
for Aniline hydrochloride for possible carcinogenicity. (CAS No., 142-04-1). NCI-CG-TR-130.
1978. Available from: URL: https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr130.pdf
28. Hagiwara A, Arai M, Hirose M, Nakanowatari J-I, Tsuda H and Ito N. Chronic effects of
norharman in rats treated with aniline. Toxicol Lett 1980;6:71-5.
29. Carcinogenicity Potency Database (CPDB). [Online]. Available from: URL:
http://toxnet.nlm.nih.gov/cpdb/
30. Steinheider G, Neth R, Marguardt H. Evaluation of nongenotoxic and genotoxic factors
modulating the frequency of micronucleated erythrocytes in the peripheral blood of mice. Cell
Biol Toxicol 1985;1:197-211.
31. Tweats D, Blakey D, Heflich RH, Jacobs A, Jacobsen SD, Nohmi TT, et al. Report of the IWGT
working group on strategies and interpretation of regulatory in vivo tests. I. Increases in
micronucleated bone marrow cells in rodents that do not indicate genotoxic hazards. Mutat Res
2007;627:78-91.
32. Khan MF, Wu X, Boor PJ, Ansari GAS. Oxidative modification of lipids and proteins in aniline
induced splenic toxicity. Toxicol Sci 1999;48:134-40.
33. Khan MF, Wu X, Wang JL. Upregulation of transforming growth factor-beta 1 in the spleen of
aniline-induced rats. Toxicol Appl Pharmacol 2003;187:22-8.
55
34. Weinberger MA, Albert RH, Montgomery SB. Splenotoxicity associated with splenic sarcomas in
rats fed high doses of D & C Red No. 9 or aniline hydrochloride. J Natl Cancer Inst 1985;
5:681-7.
35. Smith RP, Alkaitis AA, Shafer PR. Chemically induced methemoglobinemias in the mouse.
Biochem. Pharmacol 1967;16:317-28.
36. Bus JS, Popp JA. Perspectives on the mechanism of action of the splenic toxicity of aniline and
structurally-related compounds. Food Chem Toxicol 1987;25:619-26.
37. Robertson O, Cox MG, Bus JS. Response of the erythrocyte and spleen to aniline insult in
Fischer 344 rats. Toxicologist 1983;3:128.
38. Bomhard EM, Herbold BA. Genotoxic activities of aniline and its metabolites and their
relationship to the carcinogenicity of aniline in the spleen of rats. Crit Rev Toxicol 2005;35:783-
835.
39. US Environmental Protection Agency. Aniline (CAS No 62-53-3). Integrated Risk Information
System (IRIS). [Online]. 1988. Available from: URL:
https://cfpub.epa.gov/ncea/iris/iris_documents/documents/subst/0350_summary.pdf
40. International Conference on Harmonisation (2011). Q3C(R5): Impurities: Guideline for Residual
Solvents.
56
Benzyl Chloride (α-Chlorotoluene, CAS# 100-44-7)
Potential for human exposure
Human exposure is mainly occupational via inhalation while less frequent is exposure from
ingesting contaminated ground water.
Mutagenicity/genotoxicity
Benzyl chloride is mutagenic and genotoxic in vitro but not in mammalian systems in vivo.
The International Agency for Research on Cancer (IARC) published a monograph performing a
thorough review of the mutagenicity/genotoxicity data for benzyl chloride (Ref. 1). Some of the
key conclusions are summarized here.
Benzyl chloride is mutagenic in:
Microbial reverse mutation assay (Ames) in Salmonella typhimurium strain TA100. Results of the
standard assay are inconsistent across and within laboratories, but clear increases are obtained
when testing in the gaseous phase (Ref. 2);
Chinese hamster cells (Ref. 1).
Benzyl chloride did not induce micronuclei in vivo in mouse bone marrow following oral,
intraperitoneal or subcutaneous administration, but did form DNA adducts in mice after i.v.
administration (Ref. 1).
Carcinogenicity
Benzyl chloride is classified as Group 2A, probably carcinogenic to humans (Ref. 3).
Benzyl chloride was administered in corn oil by gavage 3 times/week for 104 weeks to F-344 rats
and B6C3F1 mice (Ref. 4). Rats received doses of 0, 15, or 30 mg/kg (estimated daily dose: 0, 6.4,
12.85 mg/kg); mice received doses of 0, 50, or 100 mg/kg (estimated daily dose: 0, 21.4, 42.85
mg/kg). In rats, the only statistically significant increase in the tumor incidence was for thyroid C-
cell adenoma/carcinoma in the female high-dose group (27% versus 8% for control). A discussion
of whether these thyroid tumors were treatment-related is included below. Several toxicity studies
were conducted but C-cell hyperplasia was noted only in this lifetime study and only in female rats.
In mice (Ref. 4), there were statistically significant increases in the incidence of forestomach
papillomas and carcinomas (largely papillomas) at the high dose in both males and females (62%
and 37%, respectively, compared with 0% in controls). Epithelial hyperplasia was observed in the
stomachs of animals without tumors. There were also statistically significant increases in male but
not female mice in hemangioma or hemangiosarcoma (10% versus 0% in controls) at the high
dose and in carcinoma or adenoma in the liver but only at the low dose (54% versus 33% in
controls). In female, but not male, mice there were significant increases in the incidence of
alveolar-bronchiolar adenoma or carcinoma at the high dose (12% versus 1.9% in controls).
Additional studies to assess carcinogenic potential were conducted but were not considered of
adequate study design for use in calculating an AI. In one of three topical studies (Ref. 5) skin
carcinomas were increased, although not statistically significantly (15% versus 0% in benzene
controls). Initiation-promotion studies to determine the potential of benzyl chloride to initiate skin
cancer, using croton oil and the phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate) as
promoters (Ref. 6, 7, 8) were of limited duration and the published reports were presented as
preliminary findings, but no final results have been located in the literature. Injection site
sarcomas were seen after subcutaneous administration (Ref. 9).
57
Benzyl chloride – Details of carcinogenicity studies
Study
Animals/dose
group
Duration/
Exposure
Controls
Doses
Most
sensitive
tumor
site/type/sex
or tumor
observations
TD
50
(mg/kg/d)
Ref. 4
*
52/sex/group
F344 rat
2 year
3 times/wk
Gavage
52
2:
15 and 30
mg/kg
(6 and 12
mg/kg/d)
Thyroid
C-cell
neoplasm/
Female
40.6
Ref. 4
52/sex/group
B6C3F1 mouse
2 year
3 times/wk
Gavage
52
2:
50 and
100
mg/kg
(21 and
42
mg/kg/d)
Forestomach
papilloma,
carcinoma/
Male
49.6
Ref. 5
11/group
female ICR
mouse
9.8 mo
3 times/wk
for 4 wks, 2
times/wk
Dermal
Yes
(benzene
treated)
1:
10 µL
No skin tumors
NC
^
Ref. 5
20/group
female ICR
mouse
50 weeks
2 times/wk
Dermal
20
(benzene
treated)
1:
2.3 µL
Skin
squamous cell
carcinoma
NC
^
Ref. 6
20/group
male ICI Swiss
albino mouse
>7 mo
2 times/wk
Dermal, in
toluene
20
1:
100
µg/mouse
No skin tumors
NC
^
Ref. 9
14 (40 mg/kg),
and 8 (80
mg/kg)
BD rat
51 weeks
1 time/wk
Subcutaneous
Yes
2:
40 and 80
mg/kg/wk
Injection site
sarcoma
NC
^
Ref. 7
40/sex/group
Theiler's
Original mouse
10 mo
1 dose (in
toluene); wait
1 wk
Promoter
(croton oil)
2 times/wk
40
1:
1 mg/
mouse
No skin tumors
NC
^
Ref. 8
Sencar mice
6 mo
1 dose;
Promoter
(TPA)
2 times/wk
Yes
3: 10;
100 and
1000 µg/
mouse
20% skin
tumors [5% in
TPA controls]
(DMBA controls
had skin
tumors by 11
weeks)
NC
^
Studies listed are in CPDB (Ref. 10) unless otherwise noted.
*
Carcinogenicity study selected for AI calculation.
^
NC= Not calculated; small group size, limited duration. Not included in CPDB as route with
greater likelihood of systemic exposure is considered more relevant.
Mode of action for carcinogenicity
The tumor types with the lowest calculated TD
50
(highest potency) in the CPDB (Ref. 10) for benzyl
chloride are forestomach tumors in mice and thyroid C-cell tumors in female rats. The relevance of
58
the forestomach tumors to human risk assessment for low, non-irritating doses such as those
associated with a potential impurity is highly questionable.
Forestomach tumors in rodents have been the subject of much discussion in assessment of risk to
humans. With non-mutagenic chemicals, it is recognized that after oral gavage administration,
inflammation and irritation related to high concentrations of test materials in contact with the
forestomach can lead to hyperplasia and ultimately tumors. Material introduced by gavage can
remain for some time in the rodent forestomach before discharge to the glandular stomach, in
contrast to the rapid passage through the human esophagus. Such tumor induction is not relevant
to humans at non-irritating doses. The same inflammatory and hyperplastic effects are also seen
with mutagenic chemicals, where it is more complex to determine relative contribution to mode of
action of these non-mutagenic, high-dose effects compared with direct mutation induction.
However, often a strong case can be made for site-of-contact tumorigenesis that is only relevant at
concentrations that cause irritation/inflammation, potentially with secondary mechanisms of
damage. Cell proliferation is expected to play an important role in tumor development such that
there is a non-linear dose response and the forestomach (or other site-of-contact) tumors are not
relevant to low-dose human exposure.
Proctor et al (Ref. 11) proposed a systematic approach to evaluating relevance of forestomach
tumors in cancer risk assessment, taking into account whether any known genotoxicity is
potentially relevant to human tissues (this would include whether a compound is genotoxic in vivo),
whether tumors after oral administration of any type are specific to forestomach, and whether
tumors are observed only at doses that irritate the forestomach or exceed the MTD.
As described above and in the table, benzyl chloride predominantly induces tumors at the site-of-
contact in rats and mice following exposure to high doses by gavage (forestomach tumors), by
injection (injection site sarcoma) and by topical application in a skin tumor initiation-promotion
model in sensitive Sencar mice. An OECD report in the Screening Information Dataset (SIDS) for
high volume chemicals describes benzyl chloride as intensely irritating to skin, eyes, and mucous
membranes in acute and repeat dose studies (Ref. 12). Groups of 10 Fischer 344 rats of both
sexes died within 2-3 weeks from severe acute and chronic gastritis of the forestomach, often with
ulcers, following oral administration 3 times/week of doses > 250 mg/kg for males and >125
mg/kg for females (Ref. 4). Proliferative changes observed in female rats at lower doses included
hyperplasia of the forestomach (62 mg/kg), and hyperkeratosis of the forestomach (30 mg/kg).
The incidence of forestomach tumors was high in mice in the carcinogenicity study, and Lijinsky et
al (Ref. 4) also observed non-neoplastic lesions in the forestomach of the rat in the subchronic
range-finding study, but few forestomach neoplasms developed in the rat carcinogenicity assay.
Due to the steepness of the dose-response curve and the difficulty establishing the MTD for rats,
the author speculates that it was possible that the dose used in the rat study was marginally too
low to induce a significant carcinogenic effect in rats.
In the case of benzyl chloride, other tumor types were discussed as possibly treatment-related
besides those at the site-of-contact. In the mouse oral bioassay, Lijinsky characterized the
carcinogenic effects other than forestomach tumors as “marginal”, comprising an increase of
endothelial neoplasms in males, alveolar-bronchiolar neoplasms of the lungs only in female mice
(neither of these is statistically significant) and hepatocellular neoplasms only in low dose male
mice (this tumor type was discounted as not dose related). It is of note that OECD SIDS (Ref. 12)
reports observations of severe to moderate dose-related liver hyperplasia in a 26-week oral toxicity
study in mice.
Statistically significant increases were reported in hemangiomas/hemangiosarcomas of the
circulatory system in the male mice (TD
50
454 mg/kg/day), and in thyroid C-cell adenomas or
59
carcinomas in the female rats (TD
50
40.6 mg/kg/day). The levels of thyroid C-cell tumors in female
rats in the high dose group, while higher than female concurrent controls, (14/52 versus 4/52 in
controls) were similar to the levels in the male concurrent controls (12/52). In males, thyroid C-
cell tumor levels were lower in treated than in control rats. In a compilation of historical control
data from Fisher 344 rats in the NTP studies (Ref. 13, 14), males and females show comparable
levels of C-cell adenomas plus carcinomas in this rat strain, although the range is wider in males.
Thus it is likely justifiable to compare the thyroid tumor levels in female rats treated with benzyl
chloride with the concurrent controls of both sexes, and question whether the female thyroid
tumors are treatment-related, although they were higher than the historical control range cited at
the time (10%).
Regulatory and/or published limits
The US EPA (Ref. 15) derived an Oral Slope Factor of 1.7×10
-1
per (mg/kg)/day, which corresponds
to a 1 in 100,000 risk level of 2 μg/L or approximately 4 μg/day using US EPA assumptions.
Acceptable intake (AI)
Rationale for selection of study for AI calculation
The most robust evaluation of the carcinogenic potential of benzyl chloride was the Lijinsky et al
study (Ref. 4) that utilized oral (gavage) administration. In this study, the animals were treated 3
days a week rather than 5 days a week as in a typical NCI/NTP study. Overall, however, the rat
study is considered adequate for calculation of an AI because there was evidence that the top dose
was near the maximum tolerated dose. In a 26-week range finding study described in the same
report (Ref. 4), all ten rats of each sex given 125 or 250 mg/kg (3 days per week) died within 2-3
weeks. The cause of death was severe gastritis and ulcers in the forestomach; in many cases there
was also myocardial necrosis. At 62 mg/kg, only 4 of 26 females survived to 26 weeks, and
myocardial necrosis and forestomach hyperplasia were seen; hyperkeratosis of the forestomach
was seen in some females at 30 mg/kg. At 62 mg/kg benzyl chloride, there was a decrease in
body weight gain in both sexes, which was statistically significant in males. Thus, the high dose
chosen for the carcinogenicity study was 30 mg/kg (3 times per week). At this dose, there was no
difference from controls in survival in the 2-year carcinogenicity study, but 3 male rats had
squamous cell carcinomas and papillomas of the forestomach, so it is unlikely that a lifetime study
could have been conducted at a higher dose.
As described in the Methods Section 2.2, linear extrapolation from the TD
50
was used to derive the
AI. As described above, it is highly unlikely that benzyl chloride poses a risk of site-of-contact
tumors in humans exposed to low concentrations as impurities in pharmaceuticals, well below
concentrations that could cause irritation/inflammation. Therefore, the observed forestomach
tumors in male mice are not considered relevant for the AI calculation. The significance of the
thyroid C-cell tumors in female rats is also questionable since these tumors occur commonly in
control rats. However, given the uncertain origin of these tumors, the thyroid C-cell tumors were
used to derive the AI since they were associated with the lowest TD
50:
40.6 mg/kg/day.
Calculation of AI
Lifetime AI = TD
50
/50,000 x 50 kg
Lifetime AI = 40.6 (mg/kg/day)/50,000 x 50 kg
Lifetime AI = 40.6 µg/day (41 µg/day)
60
References
1. IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man.
International Agency for Research on Cancer, World Health Organization, Lyon. [Online] 1972-
PRESENT. (Multivolume work). 1999. Available from: URL:
http://monographs.iarc.fr/ENG/Monographs/vol71/mono71-19.pdf
2. Fall M, Haddouk H, Morin JP, Forster R. Mutagenicity of benzyl chloride in the
Salmonella/microsome mutagenesis assay depends on exposure conditions. Mutat Res
2007;633:13-20.
3. IARC. An update of selected IARC Monographs from volumes 1 to 42. Monographs on the
Evaluation of the Carcinogenic Risk of Chemicals to Humans. International Agency for Research
on Cancer, World Health Organization, Lyon.1987. Suppl. 7: 126–7; 148–9.
4. Lijinsky W. Chronic Bioassay of Benzyl Chloride in F344 Rats and (C57BL/6J X BALB/c) F1 Mice.
J Natl Cancer Inst 1986;76:1231-6.
5. Fukuda K, Matsushita H, Sakabe H, Takemoto K. Carcinogenicity of benzyl chloride, benzal
chloride, benzotrichloride and benzoyl chloride in mice by skin application. Gann
1981;72(5):655-64.
6. Ashby J, Gaunt C, Robinson M. Carcinogenicity bioassay of 4-chloromethylbiphenyl (4CMB), 4-
hydroxymethylbiphenyl (4HMB) and benzyl chloride (BC) on mouse skin: Interim (7 month)
report. Mutat Res 1982;100:399-401.
7. Coombs MM. Attempts to initiate skin tumors in mice in the 2-stage system using 4-
chloromethylbiphenyl (4CMB), -hydroxymethylbiphenyl (4HMB) and benzyl chloride (BC),
Report of the experiment at 10 months. Mutat Res 1982;100:403-5.
8. Coombs MM. The UKEMS Genotoxicity Trial: A summary of the assays for skin tumour induction
in mice, the subcutaneous implant test and the sebaceous gland suppression test. Mutat Res
1982;100:407-9.
9. Druckrey H, Kruse H, Preussmann R, Ivankovic S, Landschuetz C. Cancerogenic alkylating
substances. III. Alkyl-halogenides, - sulfates, - sulfonates and strained heterocyclic compounds.
1970;74(3):241-73.
10. Carcinogenicity Potency Database (CPDB). [Online]. Available from: URL:
http://toxnet.nlm.nih.gov/cpdb/
11. Proctor DM, Gatto NM, Hong SJ, Allamneni KP. Mode-of-action framework for evaluation of the
relevance of rodent forestomach tumors in cancer risk assessment. Toxicol Sci 2007;98:313-26.
12. OECD Chemicals Screening Information Dataset (SIDS) for high volume chemicals benzyl
chloride report published by the United Nations Environmental Programme (UNEP). [Online].
Available from: URL:http://www.chem.unep.ch/irptc/sids/OECDSIDS/100447.pdf
13. Haseman JK, Huff J, Boorman GA. Use of historical control data in carcinogenicity studies in
rodents., Toxicol Pathol 1984;12:126-35.
14. Haseman JK, Hailey JR, Morris RW. Spontaneous neoplasm incidence in Fischer 344 rats and
B6C3F1 mice in two-year carcinogenicity studies: A National Toxicology Program update,
Toxicol Pathol 1998;26:428-41.
62
Bis(chloromethyl)ether (BCME, CAS# 542-88-1)
Potential for human exposure
Industrial use, mainly via inhalation with minimal environmental exposure as result of rapid
degradation in the environment, which is supported by the reported absence of BCME in ambient
air or water (Ref. 1).
Mutagenicity/genotoxicity
BCME is mutagenic and genotoxic in vitro and in vivo.
BCME is mutagenic in:
Microbial reverse mutation assay (Ames), Salmonella typhimurium (Ref. 2).
In vivo, BCME did not cause chromosomal aberrations in bone-marrow cells of rats exposed by
inhalation for six months (Ref. 3). A slight increase in the incidence of chromosomal aberrations
was observed in peripheral lymphocytes of workers exposed to BCME (Ref. 4).
Carcinogenicity
BCME is classified by US EPA as a Group A, known human carcinogen (Ref. 5), and by IARC as a
Group 1 compound, carcinogenic to humans (Ref. 6).
As described in the above reviews, numerous epidemiological studies have demonstrated that
workers exposed to BCME (via inhalation) have an increased risk for lung cancer. Following
exposure by inhalation, BCME is carcinogenic to the respiratory tract of rats and mice as described
in the following studies:
The study of Leong et al (Ref. 3) was selected for derivation of the AI based on the most robust
study design and the lowest TD
50
value. Groups of male Sprague-Dawley rats and Ha/ICR mice
were exposed by inhalation to 1, 10, and 100 ppb of BCME 6 h/day, 5 days/week for 6 months and
subsequently observed for the duration of their natural lifespan (about 2 years). Evaluation of
groups of rats sacrificed at the end of the 6-month exposure period revealed no abnormalities in
hematology, exfoliative cytology of lung washes, or cytogenetic parameters of bone marrow cells.
However, 86.5% of the surviving rats which had been exposed to 100 ppb (7780 ng/kg/day, or ~8
µg/kg/day) of BCME subsequently developed nasal tumors (esthesioneuroepitheliomas, tumors of
the olfactory epithelium, which are similar to the rare human neuroblastoma) and approximately
4% of the rats developed pulmonary adenomas. Tumors were not observed in rats exposed to 10
or 1 ppb of BCME. Mice exposed to 100 ppb of BCME did not develop nasal tumors, but showed a
significant increase in incidence of pulmonary adenomas over the control mice. Mice exposed to 10
or 1 ppb of BCME did not show a significant increase in incidence of pulmonary adenomas.
In an inhalation study, male Sprague-Dawley rats were exposed to BCME at a single dose level of
0.1 ppm (100 ppb) 6 h/day, 5 days/week for 10, 20, 40, 60, 80, or 100 days, then observed for
the remainder of their lifetimes (Ref. 7). There was a marked increase in the incidence of several
types of respiratory tract tumors in the treated animals compared with the controls.
BCME is a site-of-contact carcinogen, producing injection site sarcomas (Ref. 8) and skin tumors in
mice, (Ref. 9); it also induces lung adenomas in newborn mice following sub-cutaneous application
(Ref. 10).
Bis(chloromethyl)ether (BCME) – Details of carcinogenicity studies
Study
Animals/dos
e group
Duration/
Exposure
Controls
Doses
Most sensitive
tumor
site/type/sex
TD
50
(mg/kg/
d)
63
Ref. 3
*
~104/group
Rat, male
Sprague-
Dawley.
28 weeks
6 h/d, 5
d/wk
Inhalation
104
3:
1; 10; 100
ppb
(53;528;
7780
ng/ kg/d)
Nasal passage -
esthesioneuro-
epitheliomas
0.00357
Ref. 3
138-144/
group
Mouse, male
ICR/Ha.
25 weeks
6 h/d,
5 d/wk
Inhalation
157
3:
1; 10; 100
ppb
(0.295;
2.95;33.6
ng/kg/d)
Lung adenomas
No
significant
increases
Ref. 7
30-50 treated
for different
durations with
same
concentration,
male Sprague
Dawley rats.
6h/d,
5d/wk, for
10, 20, 40,
60, 80,
and 100
exposures.
Inhalation
240
1:
0.1 ppm
Lung and nasal
cancer
NC
^
Ref. 7
100/group
male Golden
Syrian
Hamsters.
Lifetime
6h/d,
5d/wk,
Inhalation
NA
1:
1 ppm
One
undifferentiated
in the lung
NC
^
Ref. 9
50/group
female ICR/Ha
Swiss mice.
424-456
days,
once
weekly
Intra-
peritoneal
50
1:
0.114
mg/kg/d
Sarcoma (at the
injection site)
0.182
Studies listed are in CPDB (Ref. 11) unless otherwise noted.
*
Carcinogenicity study selected for AI calculation
^
NC= Not calculated due to non-standard carcinogenicity design. Not in CPDB.
NA= Not available since controls were not reported in the study
Mode of action for carcinogenicity
BCME is a mutagenic carcinogen, and the acceptable intake is calculated by linear extrapolation
from the TD50.
Regulatory and/or published limits
The US EPA (Ref. 5), calculated an oral cancer slope factor of 220 per mg/kg/day based on
linearised multistage modelling of the inhalation study data by Kuschner et al (Ref. 7). The inhaled
(and oral) dose associated with a 1 in 100,000 lifetime cancer risk is 3.2 ng/day (1.6 x 10
-8
mg/m
3
for inhalation, 1.6 x 10
-6
mg/L for oral exposure).
Acceptable intake (AI)
Rationale for selection of study for AI calculation
BCME is an in vitro mutagen, causes cancer in animals and humans and is classified as a known
human carcinogen. Oral carcinogenicity studies were not conducted, so that intraperitoneal
injection and inhalation studies are considered as a basis for setting an AI. The most sensitive
endpoint was an increase in nasal tumors (esthesioneuroepitheliomas) in male rats in the inhalation
carcinogenicity study (Ref. 3), with a TD
50
of 3.57µg/kg/day. The AI derived by linear
extrapolation from that TD
50,
~4ng/day, is essentially the same as the 3.2 ng/day recommendation
of the US EPA. The study (Ref. 3) had a reliable design with multiple dose levels and >50 animals
per dose group.
Evidence for tumors at other sites than those exposed by inhalation is lacking; the study cited
above (Ref. 10) that describes lung tumors in newborn mice following skin application may not be
64
definitive if inhalation may have occurred as a result of skin application. However, the AI derived
here from inhalation data is considered applicable to other routes, because it is highly conservative
(orders of magnitude below the default TTC of 1.5 µg/day). The AI is also similar to the limit
derived by US EPA (based on inhalation data) that is recommended both for inhalation and
ingestion (drinking water) of BCME (4 ng/day vs 3.2 ng/day).
Calculation of AI
Lifetime AI = TD
50
/50,000 x 50 kg
Lifetime AI = 3.57 µg/kg/day/50,000 x 50
Lifetime AI = 0.004 μg/day or 4 ng/day
References
1. NIH ROC. National Institutes of Health. Report on Carcinogens, Twelfth Edition [Online]. 2011.
Available from: URL:
http://ntp.niehs.nih.gov/ntp/roc/twelfth/profiles/bis(chloromethyl)ether.pdf
2. Nelson N. The chloroethers - occupational carcinogens: A summary of laboratory and
epidemiology studies. Ann. NY Acad Sci 1976;271:81-90.
3. Leong BKJ, Kociba RI, Jersey GC. A lifetime study of rats and mice exposed to vapors of
bis(chloromethy1) ether. Toxicol Appl Pharmacol 1981;58:269-81.
4. IARC. Bis(chloromethyl)ether and chloromethyl methyl ether (technical-grade). Monographs on
the Evaluation of Carcinogenic Risk of Chemicals to Humans. International Agency for Research
on Cancer, World Health Organization. Lyon. 1987;Addendum 7: 131-3.
5. US Environmental Protection Agency. Bis(chloromethyl)ether (CAS# 542-88-1). Integrated Risk
Information System (IRIS). [Online]. 1999. Available from: URL:
http://www.epa.gov/iris/subst/0375.htm
6. IARC. Chemicals, industrial processes and industries associated with cancer in humans.
Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. International
Agency for Research on Cancer, World Health Organization, Lyon. 1982;Volumes 1 to 29,
Addendum 4.
7. Kuschner M, Laskin S, Drew RT, Cappiello V, Nelson N. Inhalation carcinogenicity of alpha halo
ethers. III. Lifetime and limited period inhalation studies with bis(chloromethyl)ether at 0.1
ppm. Arch Environ Health 1975;30:73-7.
8. Van Duuren BL, Sivak A, Goldschmidt BM, Katz C, Melchionne S. Carcinogenicity of halo-ethers.
J Nat Cancer Inst 1969; 43: 481-6.
9. Van Duuren BL, Goldschmidt BM, Seidman I. Carcinogenic activity of di- and trifunctional -
chloro ethers and of 1,4-dichlorobutene-2 in ICR/HA swiss mice. Cancer Res 1975;35:2553-7.
10. Gargus JL, Reese WH Jr., Rutter, HA. 1969. Induction of lung adenomas in newborn mice by
bis(chloromethyl)ether. Toxicol Appl Pharmacol 1969;15:92-96.
11. Carcinogenicity Potency Database (CPDB). [Online]. Available from: URL:
http://toxnet.nlm.nih.gov/cpdb/
65
p-Chloroaniline (CAS# 106-47-8) and p-Chloroaniline HCl
(CAS# 20265-96-7)
Potential for human exposure
Industrial exposure is primarily derived from the dye, textile, rubber and other industries (Ref. 1).
If released into the environment, it is inherently biodegradable in water under aerobic conditions
(Ref. 2).
Mutagenicity/Genotoxicity
p-Chloroaniline is mutagenic in vitro, with limited evidence for genotoxicity in vivo.
A detailed review of genotoxicity testing in a range of systems is provided by WHO (Ref. 3) with
references, so only key conclusions are summarized here.
p-Chloroaniline is mutagenic in:
Microbial reverse mutation assay (Ames); 2 to 3-fold increase in revertants was seen in some
laboratories but not in others.
Positive results reported in the mouse lymphoma L5178Y cell tk assay (Ref. 3) are small increases,
associated with substantial cytotoxicity, and do not meet the current criteria for a positive assay
using the “global evaluation factor” (Ref. 4).
Small increases in chromosomal aberrations in Chinese hamster ovary cells were not consistent
between two laboratories.
In vivo, a single oral treatment did not induce micronuclei in mice at 180 mg/kg, but a significant
increase was reported at 300 mg/kg/day after 3 daily doses in mice.
Carcinogenicity
p-Chloroaniline is classified by IARC as Group 2B, possibly carcinogenic to humans with adequate
evidence of carcinogenicity in animals and inadequate evidence in humans (Ref. 5).
Carcinogenicity studies in animals have been conducted for p-chloroaniline or its hydrochloride salt,
p-Chloroaniline HCl.
The NTP (Ref. 6) oral gavage study was used to calculate the AI, where p-chloroaniline HCl was
carcinogenic in male rats, based on the increased incidence of spleen tumors: (Combined incidence
of sarcomas: vehicle control, 0/49; low dose, 1/50; mid dose, 3/50; high dose, 38/50). Fibrosis of
the spleen, a preneoplastic lesion that may progress to sarcomas, was seen in both sexes (Ref. 6,
7). In female rats, splenic neoplasms were seen only in one mid-dose rat and one high-dose rat.
Increased incidences of pheochromocytoma of the adrenal gland in male and female rats may have
been related to p-chloroaniline administration; malignant pheochromocytomas were not increased.
In male mice, the incidence of hemangiosarcomas of the liver or spleen in high dose group was
greater than that in the vehicle controls (4/50 in 0 mg/kg/day; 4/49 in 2.1 mg/kg/day; l/50 in 7.1
mg/kg/day; 10/50 in 21.4 mg/kg/day). The incidences of hepatocellular adenomas or carcinomas
(combined) were increased in dosed male mice; of these, the numbers of hepatocellular carcinomas
were (3/50 in 0 mg/kg/day; 7/49 in 2.1 mg/kg/day; 11/50 in 7.1 mg/kg/day; 17/50 in 21.4
mg/kg/day). The female mouse study was negative. The final conclusion of NTP (Ref. 6) was that
there was clear evidence of carcinogenicity in male rats, equivocal evidence of carcinogenicity in
female rats, some evidence of carcinogenicity in male mice, and no evidence of carcinogenicity in
female mice.
66
An earlier study used p-chloroaniline administered in feed to rats and mice (Ref. 8). Splenic
neoplasms were found in dosed male rats and hemangiomatous tumors in mice. While the
incidences of these tumors are strongly suggestive of carcinogenicity, NCI concluded that sufficient
evidence was not found to establish the carcinogenicity of p-chloroaniline in rats or mice under the
conditions of these studies. Since p-chloroaniline is unstable in feed, the animals may have
received the chemical at less than the targeted concentration (Ref. 3). Therefore, this study is
deemed inadequate.
p-Chloroaniline and p-Chloroaniline HCl – Details of carcinogenicity studies
Study
Animals/
dose
group
Duration/
Exposure
Controls
Doses
Most sensitive
tumor
site/type/sex
TD
50
(mg/kg/d
)
Ref. 6
*
p-chloroaniline
HCl
50/group
male
B6C3F1
mice
103 weeks
5 times/
wk
Gavage
50
3:
3; 10; 30
mg/kg
(2.1; 7.1;
21.4
mg/kg/d)
Hepatocellular
adenomas or
carcinomas
33.8
Ref. 6
p-chloroaniline
HCl
50/group
female
B6C3F1
mice
103 weeks
5 times/
wk
Gavage
50
3:
3; 10; 30
mg/kg
(2.1; 7.1;
21.4
mg/kg/d)
Negative
NA
Ref. 6
p-chloroaniline
HCl
50/group
male
Fischer
344 rat
103 weeks
5 times/
wk
Gavage
50
3:
2; 6;18
mg/kg
(1.4; 4.2;
12.6
mg/kg/d)
Spleen
fibrosarcoma,
haemangiosarcoma,
osteosarcoma
7.62
Ref. 6
p-chloroaniline
HCl
50/group
female
Fischer
344 rat
103 weeks
5 times/
wk
Gavage
50
3:
2; 6; 18
mg/kg
(1.4; 4.2;
12.6
mg/kg/d)
No significant
increases; equivocal
NA
Ref. 8
50/group
male
Fischer
344 rat
78 weeks
(study
duration:
102 wk)
Diet
20
2:
250; 500
ppm
(7.7;
15.2
mg/kg/d)
Mesenchymal tumors
(fibroma,
fibrosarcoma,
haemangiosarcoma,
osteosarcoma,
sarcoma not
otherwise specified)
of the spleen or
splenic capsule
72
Ref. 8
50/group
female
Fischer
344 rat
78 weeks
(study
duration:
102 wk)
Diet
20
2:
250; 500
ppm
(9.6, 19
mg/kg/d)
Negative
NA
Ref. 8
50/group
male
B6C3F1
mice
78 weeks
(study
duration:
91 wk)
Diet
20
2:
2500; 5000
ppm
(257;275
mg/kg/d)
Haemangiosarcomas
(subcutaneous tissue,
spleen, liver, kidney).
Increased incidence of
all vascular tumors
Not
significant
(CPDB)
Ref. 8
50/group
female
B6C3F1
mice
78 weeks
(study
duration:
102 wk)
Diet
20
2:
2500; 5000
ppm
(278, 558
mg/kg/d)
Haemangiosarcomas
(liver and spleen).
Increased incidence of
combined vascular
tumors
1480
67
Studies listed are in CPDB (Ref. 9)
*
Carcinogenicity study selected for AI calculation.
NA = Not applicable
Mode of action for carcinogenicity
p-Chloroaniline induced tumors in male rats, such as spleen fibrosarcomas and osteosarcomas,
typical for anline and related chemicals. Repeated exposure to p-chloroaniline leads to
cyanosis and methemoglobinemia, followed by effects in blood, liver, spleen, and kidneys,
manifested as changes in hematological parameters, splenomegaly, and moderate to severe
hemosiderosis in spleen, liver, and kidney, partially accompanied by extramedullary
hematopoiesis (Ref. 6, 8). These effects occur secondary to excessive compound-induced
hemolysis and are consistent with a regenerative anemia (Ref. 3). The evidence supports an
indirect mechanism for tumorigenesis, secondary to methemoglobinemia, splenic fibrosis and
hyperplasia (Ref. 10), and not tumor induction related to a direct interaction of p-chloroaniline
or its metabolites with DNA. Similarly, the reported induction of micronuclei in vivo is likely
to be secondary to regenerative anemia/altered erythropoeisis, as with aniline (Ref. 11,12).
The tumor type with the lowest TD
50
was spleen tumors in male rats. However, since this
tumor type is associated with a non-linear dose relation, spleen tumors were not used to
calculate the acceptable intake. Based on non-neoplastic (hematotoxic) effects, WHO (Ref. 3)
recommends a level of 2 µg/kg/day, i.e., 100 µg/day for a 50 kg human.
Although the in vitro mutagenicity data for p-chloroaniline indicate small increases in
mutations that are not reproducible across laboratories, a mutagenic component to a mode of
action for liver tumors cannot be ruled out.
Regulatory and/or published limits
No regulatory limits have been published for p-chloroaniline or the hydrochloride salt.
Acceptable intake (AI)
Because a mutagenic component to the mode of action for male mouse liver tumors cannot be
ruled out, the AI was derived by linear extrapolation from the TD
50
of 33.8 mg/kg/day for
combined numbers of adenomas and carcinomas.
Calculation of AI
Based on male mouse liver tumors for p-chloroaniline HCl
Lifetime AI = TD
50
/50,000 x 50kg
Lifetime AI = 33.8mg/kg/day /50,000 x 50 kg
Lifetime AI = 34 µg/day
References
1. Beard RR, Noe JT. Aromatic nitro and amino compounds, Clayton GD, Clayton FE,
editors. Patty's Industrial Hygiene and Toxicology. New York. John Wiley 1981;
2A:2413–89.
2. BUA. p-Chloroaniline. Beratergremium für Umweltrelevante Altstoffe (BUA) der
Gesellschaft Deutscher Chemiker. Weinheim, VCH, 1995;171. (BUA Report 153).
3. World Health Organization (WHO). International Programme on Chemical Safety (IPCS).
2003. Concise International Chemical Assessment Document 48. 4-chloroaniline. [Online].
Available from: URL: http://www.inchem.org/documents/cicads/cicads/cicad48.htm
68
4. Moore, MM, Honma, M, Clements J, Bolcsfoldi G, Burlinson B, Cifone M, et al.Mouse
Lymphoma Thymidine Kinase GeneMutation Assay: Follow-upMeeting of the
International Workshop on Genotoxicity Testing_Aberdeen, Scotland, 2003_Assay
Acceptance Criteria, Positive Controls, and Data Evaluation. Environ Mol Mutagen
2006;47:1-5.
5. IARC. Para-chloroaniline. In: Occupational exposures of hairdressers and barbers and
personal use of hair colourants; some hair dyes, cosmetic colourants, industrial dyestuffs
and aromatic amines. Monographs on the Evaluation of the Carcinogenic Risk of
Chemicals to Humans. International Agency for Research on Cancer, World Health
Organization, Lyon. 1993;57: 305-21.
6. NTP. Technical report on the toxicology and carcinogenesis studies of para-chloroaniline
hydrochloride (CAS No. 20265-96-7) in F344/N rats and B6C3F1 mice (gavage studies).
National Toxicology Program, Research Triangle Park, NC. 1989. NTP TR 351...
7. Goodman DG, Ward JM, Reichardt WD. Splenic fibrosis and sarcomas in F344 rats fed
diets containing aniline hydrochloride, p-Chloroaniline, azobenzene, o-toluidine
hydrochloride, 4,4'-sulfonyldianiline, or D & C Red No. 9. J Natl Cancer Inst 1984;3:265-
73.
8. NCI. Bioassay of p-Chloroaniline for possible carcinogenicity, CAS No. 106-47-8. US
National Cancer Institute, Bethesda, MD. 1979;NCI-CG-TR-189.
9. Carcinogenicity Potency Database (CPDB). [Online]. Available from: URL:
http://toxnet.nlm.nih.gov/cpdb/
10. Bus JS, Popp JA. Perspectives on the mechanism of action of the splenic toxicity of
aniline and structurally-related compounds. Food Chem Toxicol 1987;25:619–26.
11. Ashby J, Vlachos DA, Tinwell H. Activity of aniline in the mouse bone marrow
micronucleus assay. Mutat Res 1991;263:115-7.
12. Tweats D, Blakey D, Heflich RH, Jasobs A, Jacobsen SD, Nohmi TT, et al. Report of the
IWGT working group on strategies and interpretation of regulatory in vivo tests. I.
Increases in micronucleated bone marrow cells in rodents that do not indicate genotoxic
hazards. Mutat Res 2007;627:78-91.
69
1-Chloro-4-Nitrobenzene (para-Chloronitrobenzene, CAS#
100-00-5)
Potential for human exposure
Potential for exposure is in industrial use. No data are available for exposure of the general
population.
Mutagenicity/genotoxicity
Chloro-4-nitrobenzene is mutagenic and genotoxic in vitro and in vivo.
Chloro-4-nitrobenzene was mutagenic in:
Microbial reverse mutation assay (Ames) Salmonella typhimurium strains TA100 and TA1535 in the
presence of S9 metabolic activation, and was negative in TA1537, TA1538, TA98, and E.coli
WP2uvrA (Ref. 1, 2, 3, 4). It was also weakly positive without metabolic activation in TA1535 in 2
of 4 studies (Ref. 4).
In vivo, DNA strand breaks were induced in the liver, kidney, and brain of male Swiss mice when
chloro-4-nitrobenzene was administered intraperitoneally (Ref. 5, 6).
Carcinogenicity
1-Chloro-4-nitrobenzene is classified by IARC as a Group 2 carcinogen, not classifiable as to its
carcinogenicity in humans (Ref. 7) and US EPA considers it to be a Group B2 carcinogen or
probable human carcinogen (Ref. 8).
Animal carcinogenicity studies have been conducted with 1-chloro-4-nitrobenzene by administration
in the feed to rats and mice (Ref. 9, 10) or by gavage in male rats (Ref. 12).
In a 2-year diet study (Ref. 9), there were significant increases in spleen tumors (fibroma,
fibrosarcoma, osteosarcoma and sarcoma) in rats of both sexes, and there were increases in spleen
hemangiosarcomas in both sexes, that were statistically significant in males at the mid and high
doses (7.7 and 41.2 mg/kg/day). Non-neoplastic changes of the spleen such as fibrosis, and
capsule hyperplasia were seen. An increase in adrenal medullary pheochromocytomas was seen at
the high dose that was statistically significant in females (53.8 mg/kg/day). In mice, the only
significant increase in tumors was in liver hemangiosarcomas at the high dose in females (275.2
mg/kg/day). Hematologic disturbances such as decreases in red blood cell numbers and
haematocrit, and extramedullary hematopoiesis, were seen both in rats and in mice.
In another diet study (Ref. 10), 1-chloro-4-nitrobenzene did not induce tumors in male CD-1 rats
when fed in the diet for 18 months. The concentration in the diet was adjusted during the 18-
month period due to toxicity as follows: The low dose group received 2000 ppm for the first 3
months, 250 ppm for next 2 months, and 500 ppm from 6 to 18 months; the high dose group
received 4000 ppm for the first 3 months, 500 ppm for next 2 months, and 1000 ppm from 6 to 18
months. The average daily exposure was approximately 17 and 33 mg/kg for the low and high
dose groups, respectively. Rats were sacrificed 6 months after the last dose and examined for
tumors. No treatment-related increases in tumors were observed in the 11 tissues examined (lung,
liver, spleen, kidney, adrenal, heart, bladder, stomach, intestines, testes and pituitary).
The same laboratory (Ref. 10) also investigated the carcinogenic potential of 1-chloro-4-
nitrobenzene in male and female CD-1 mice, given in the diet for 18 months. Mice were sacrificed
3 months after the last exposure and 12 tissues (lung, liver, spleen, kidney, adrenal, heart, bladder,
stomach, intestines, and reproductive organs) were examined for tumors. A dose-dependent
70
increase in vascular tumors (hemangiomas or hemangiosarcomas) of liver, lung, and spleen was
observed in both male and female mice.
In an oral study (Ref. 11), male and female Sprague-Dawley rats (n = 60) were given 1-chloro-4-
nitrobenzene by gavage 5 days/week for 24 months. In both sexes, toxicity was observed:
methemoglobinemia in mid- and high-dose groups, and hemosiderin and anemia in the high-dose
group.
1-Chloro-4-nitrobenzene – Details of carcinogenicity studies
Study
Animals/
dose group
Duration/
Exposure
Controls
Doses
Most sensitive
tumor
site/type/sex
TD
50
(mg/kg/d
)
Ref. 9
*+
50/group
male F344
rats (SPF)
2 years
(Diet)
50
3:
40; 200;
1000
ppm.
(1.5;
7.7; 41.2
mg/kg/d)
Spleen
hemangiosarcomas
7.7 mg/kg/d
173.5
50/group
female F344
rats (SPF)
2 years
(Diet)
50
3:
40; 200;
1000
ppm.
(1.9;
9.8;53.8
mg/kg/d)
Pheochromo-
cytoma/Female
53.8 mg/kg/d
116.9
**
50/group
male
Crj:BDF1
(SPF)
2 years
(Diet)
50
3:
125;500;
2000
ppm.
(15.3;
60.1;240
.1
mg/kg/d)
NA
50/group
female
Crj:BDF1
(SPF)
2 years
(Diet)
50
3:
125;500;
2000
ppm.
(17.6;
72.6;
275.2
mg/kg/d)
Hepatic
hemangiosarcomas
275.2 mg/kg/d
1919.9
Ref. 10
14-15/
group
male CD-1
rats
18 mo
Diet;
sacrificed 6
mo after
last dose
16
2:
Average
17 and
33
mg/kg;
(see
text)
(22.6
and 45.2
mg/kg/d)
NA
Negative
˄
14-20/sex
group
CD-1 mice
18 mo
Diet;
sacrificed 3
mo after
last dose
15/sex
2:
M: 341;
720.
F: 351;
780
mg/kg/d
Vascular
(hemangiomas/
hemangio-
sarcomas)/Male
430
˄
Ref. 11
+
60/sex/
group
Sprague
24 mo
5 d/
wk,
Yes
3:
0.1; 0.7;
5
NA
Negative
71
Dawley rat
Gavage
mg/kg/d
Studies listed are in CPDB (Ref. 12) unless otherwise noted..
*
Carcinogenicity study selected for AI/PDE calculation.
**
TD50 calculated based on carcinogenicity data (see Note 1)
+
Not in CPDB.
˄
Histopathology limited to 11-12 tissues.
NA = Not applicable
Mode of action for carcinogenicity
1-Chloro-4-nitrobenzene is significantly metabolized by reduction to 4-chloroaniline (p-
chloroaniline) in rats (Ref. 13), rabbits (Ref. 14) and humans (Ref. 15). p-Chloroaniline has
been shown to produce hemangiosarcomas and spleen tumors in rats and mice, similar to 1-
chloro-4-nitrobenzene (Ref. 16). Like aniline, an indirect mechanism for vascular
tumorigenesis in liver and spleen was indicated, secondary to oxidative erythrocyte injury and
splenic fibrosis and hyperplasia, both for 4-chloroaniline (Ref. 16) and 1-chloro-4-
nitrobenzene (Ref. 17). Methemoglobinemia and associated toxicity is a notable effect of 1-
chloro-4-nitrobenzene. A non-linear mechanism for tumor induction is supported by the fact
that in the oral gavage study (Ref. 11), carried out at lower doses than the diet studies (Ref. 9,
10), methemoglobinemia and hemosiderin were seen but there was no increase in tumors.
The tumor type with the lowest TD
50
was adrenal medullary pheochromocytomas in female
rats (Ref. 9). This tumor type is common as a background tumor in F344 rats, especially
males, and is seen after treatment with a number of chemicals, many of them non-mutagenic
(Ref. 18). It has been proposed that these tumors are associated with various biochemical
disturbances, and the mode of action for induction of pheochromocytomas by chemicals such
as aniline and p-chloroaniline that are toxic to red blood cells may be secondary to uncoupling
of oxidative phosphorylation (Ref. 18) or perhaps hypoxia.
Overall, there is substantial evidence for a non-mutagenic mode of action as follows:
The most notable types of tumors induced were those associated with methemoglobinemia,
(spleen and vascular tumors);
Adrenal medullary pheochromocytomas may be associated with the same perturbations;
There is clearly a non-linear dose relation (based on no-effect doses and on the negative
results of the lower-dose study (Ref. 11).
However, in mutagenicity studies in Salmonella, 1-chloro-4-nitrobenzene was mutagenic in
Salmonella TA100 and TA1535 (but not TA98 and other strains). This may indicate a
mutagenic component to the mode of action for tumor induction by 1-chloro-4-nitrobenzene,
and the pattern of mutagenicity is different from its metabolite p-chloroaniline, which was not
consistently detected as mutagenic across laboratories, and was reproducibly mutagenic only
in Salmonella TA98 with rat liver S9 (Ref. 19) indicating differences in mutagenic
metabolites or mechanism. In vivo genotoxicity data are lacking to help assess potential for a
mutagenic mode of action.
Since 1-chloro-4-nitrobenzene is mutagenic, and a mutagenic mode of action cannot be ruled
out, an AI calculation was performed.
Regulatory and/or published limits
No regulatory limits have been published, for example by US EPA, WHO, or Agency for
Toxic Substances & Disease Registry (ATSDR).
72
Calculation of AI
The most sensitive TD
50
is that for adrenal medullary pheochromocytomas in female rats (Ref.
9).
Lifetime AI = TD
50
/50,000 x 50 kg
Lifetime AI = 117 mg/kg/day /50,000 x 50 kg
Lifetime AI = 117 µg/day
References
1. Haworth S, Lawlor T, Mortelmans K, Speck W, Zeiger E. Salmonella mutagenicity test
results for 250 chemicals. Environ Mutagen 1983;5 Suppl 1:1-142
2. Japan Chemical Industry Ecology-Toxicology & information Center (JETOC). Japan:
Mutagenicity test data of existing chemical substances based on the toxicity investigation
system of the Industrial Safety and Health law. 2005 Addendum 3.
3. Kawai A, Goto S, Matsumoto Y, Matsushita H. Mutagenicity of aliphatic and aromatic
nitro compounds. Sangyoigaku 1987; 29: 34-55.
4. NTP. Technical Report on Toxicity Studies on 2-Chloronitrobenzene and 4-
Chloronitrobenzene (CAS Nos. 88-73-3 and 100-00-5) Administered by Inhalation to
F344/N Rats and B6C4F1 Mice. National Toxicology Program, Research Triangle Park,
NC. 1993;NTP TR 33.
5. Cesarone CF, Bolognesi C, Santi L. DNA damage induced in vivo in various tissues by
nitrobenzene derivatives. Mutat Res 1983;116:239-46.
6. Cesarone CF, Fugassa E, Gallo G, Voci A, Orunesu M. Influence of the culture time on
DNA damage and repair in isolated rat hepatocytes exposed to nitrochlorobenzene
derivatives. Mutat Res 1984;131:215-22.
7. IARC. Printing processes and printing inks, carbon black and some nitro compounds.
Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. World
Health Organization, Lyon. 1996.Vol. 65.
8. US Environmental Protection Agency (USEPA). Health Effects Assessment Summary
Tables. Office of Solid Waste and Emergency Response, US Environmental Protection
Agency, Washington DC. 1995; No. PB95-921199.
9. Matsumoto M., Aiso S, Senoh H, Yamazaki K, Arito H, Nagano K, et al. Carcinogenicity
and chronic toxicity of para-chloronitrobenzene in rats and mice by two-year feeding. J.
Environ Pathol Toxicol Oncol 2006;25:571-84.
10. Weisburger EK, Russfield AB, Homburger F, Weisburger JH, Boger E, Van Dongen, et al.
Testing of twenty-one environmental aromatic amines or derivatives for long-term toxicity
or carcinogenicity. J Environ Pathol Toxicol 1978;2:325-56.
11. Schroeder RE, Daly JW. A chronic oral gavage study in rats with p-nitrochlorobenzene.
Biodynamics Inc. 1984. Project No. 80-2487. NTIS/OTS 0536382.
12. Carcinogenicity Potency Database (CPDB). [Online]. Available from: URL:
http://toxnet.nlm.nih.gov/cpdb/
73
13. Yoshida T, Andoh K, Tabuchi T. Identification of urinary metabolites in rats treated with
p-chloronitrobenzene. Arch Toxicol 1991;65: 52-8.
14. Bray HG, James SP, Thorpe WV. The metabolism of the monochloronitrobenzenes in the
rabbit. Biochem J 1956;64:38-44.
15. Yoshida T, Tabuchi T, Andoh K. Pharmacokinetic study of p-chloronitrobenzene in
humans suffering from acute poisoning. Drug Metab Dispos 1993;21:1142-6.
16. IARC. Occupational exposures of hairdressers and barbers and personal use of hair
colourants; some hair dyes, cosmetic colourants, industrial dyestuffs and aromatic amines.
Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans.
International Agency for Research on Cancer, World Health Organization, Lyon. 1993;57.
17. Travlos GS, Mahler J, Ragan HA, Chou BJ, Bucher JR. Thirteen-week inhalation toxicity
of 2- and 4-chloronitrobenzene in F344/N rats and B6C3F1 mice. Fundam Appl Toxicol
1996;30:75-92.
18. Greim H, Hartwig A, Reuter U, Richter-Reichel HB, Thielman HW. Chemically induced
pheochromocytomas in rats: mechanisms and relevance for human risk assessment. Crit
Rev Toxicol 2009;39:695-718.
19. WHO. CICAD 48: Concise International Chemical Assessment Document 48 p-
Chloroaniline. Geneva. [Online]. 2003; Available from: URL:
http://www.inchem.org/documents/cicads/cicads/cicad48.htm
74
p-Cresidine (2-Methoxy-5-Methyl Aniline, CAS# 120-71-8)
Potential for human exposure
Potential for exposure is in industrial use. No data are available for exposure of the general
population.
Mutagenicity/Genotoxicity
p-Cresidine is mutagenic/genotoxic in vitro with equivocal evidence for genotoxicity in vivo.
p-Cresidine is mutagenic in:
Several Salmonella strains in the presence of metabolic activation (Ref. 1, 2, 3).
Big Blue transgenic mouse model with the lamda cII gene; p-cresidine was administered a
diet of 0.25 and 0.5%, comparable to the doses in the carcinogenicity study, for 180 days (Ref.
4).
In vivo, p-cresidine did not induce micronuclei in bone marrow of mice (Ref. 5. 6, 7), or in
p53 heterozygous or nullizygous mice (Ref. 8). Increases in micronuclei in another study in
p53 heterozygous mice may be secondary to methemobolinemia and regenerative anemia as
with aniline and related compounds (Ref. 9).
DNA strand breaks were not observed using the alkaline elution method in several tissues
including bladder (Ref. 6; 7) but DNA strand breaks assessed by the Comet assay were
reported in bladder mucosa, but not other tissues, after oral treatment of mice with p-cresidine
(Ref. 10).
Carcinogenicity
p-Cresidine is classified by IARC as a Group 2B carcinogen, or possibly carcinogenic in
humans (Ref. 11).
There is only one set of carcinogenicity studies in the standard rodent model. In NTP studies
(Ref. 5) p-cresidine induced tumors in lifetime studies in Fischer 344 rats and B6C3F1 mice,
with p-cresidine administered in the feed. No carcinogenicity data are available for other
routes of exposure.
p-Cresidine was administered in the feed, to groups of 50 male and 50 female animals of each
species. There were also 50 control animals of each sex. The concentrations of p-cresidine
were 0.5 or 1.0 percent in the diet, but in mice the concentrations administered were reduced
after 21 weeks to 0.15 and 0.3 percent. The dose levels, converted to mg/kg/day in the CPDB
(Ref. 12), were 198 and 368 mg/kg/day for male rats; 245 and 491 mg/kg/day for female rats;
260 and 552 mg/kg/day for male mice and 281 and 563 mg/kg/day for female mice.
All dosed animals, except for high dose male mice, were administered p-cresidine in the diet
for 104 weeks and observed for an additional period of up to 2 weeks. All high dose male
mice were dead by the end of week 92. Mortality rates were dose-related for both sexes of
both species. That incidences of certain tumors were higher in low dose than in high dose
groups was probably due to accelerated mortality in the high dose groups.
In dosed rats of both sexes, statistically significant incidences of bladder carcinomas
(combined incidences of papillary carcinomas, squamous-cell carcinomas, transitional-cell
papillomas, transitional-cell carcinomas, and undifferentiated carcinomas) and olfactory
neuroblastomas were observed. The combined incidence of neoplastic nodules of the liver,
75
hepatocellular carcinomas, or mixed hepato/cholangio carcinomas was also significant in low
dose male rats. In both male and female dosed mice, the incidence of bladder carcinomas
(combined incidence of carcinomas, squamous-cell carcinomas, and transitional-cell
carcinomas) was significant. The incidence of hepatocellular carcinomas was significant in
dosed female mice.
In summary, p-cresidine was carcinogenic to Fischer 344 rats, causing increased incidences of
carcinomas and of papillomas of the urinary bladder in both sexes, increased incidences of
olfactory neuroblastomas in both sexes, and of liver tumors in males. p-Cresidine was also
carcinogenic in B6C3F1 mice, causing carcinomas of the urinary bladders in both sexes and
hepatocellular carcinomas in females.
Induction of bladder tumors was also seen in a short-term carcinogenicity model in p53+/-
hemizygous mice. p-Cresidine was used as a positive control in a large inter-laboratory
assessment of the mouse model (Ref. 13). Increases in bladder tumors were seen in 18 of 19
studies in which p-cresidine was administered by gavage at 400 mg/kg/day for 26 weeks, and
in the single study where compound was given in feed.
76
p-Cresidine – Details of carcinogenicity studies
Study
Animals/
dose
group
Duration/
Exposure
Controls
Doses
Most
sensitive
tumor
site/type
/sex
TD
50
(mg/kg/d
)
Ref. 5
*
50/sex/
group
B6C3F1
mice
2 year
Feed
50
2:
0.5 and 1%
Reduced after
21 wk to 0.15
and 0.3%.
M: 260:552.
F: 281; 563
mg/kg/d
Urinary
bladder
/Male
44.7
Ref. 5
50/sex/
group
Fisher 344
rats
2 year
Feed
50
0.5 and 1%
M: 198;396.
F: 245;491
mg/kg/d
Urinary
bladder
/Male
88.4
*
Carcinogenicity study selected for AI calculation.
Studies listed are in CPDB (Ref. 12).
Mode of action for carcinogenicity
p-cresidine is a mutagenic carcinogen, and the acceptable intake is calculated by linear
extrapolation from the TD50.
Regulatory and/or published limits
No regulatory limits have been published
Acceptable intake (AI)
Rationale for selection of study for AI calculation:
The only adequate carcinogenicity studies of p-cresidine were those reported in the CPDB and
conducted by NCI/NTP (Ref. 5). The study in mice was selected for derivation of the AI
since the most sensitive TD
50
was based on urinary bladder tumors in male mice.
Calculation of AI
The most sensitive TD
50
values from the NCI/NTP studies are for the urinary bladder in both
sexes of rats and mice; in rats the TD
50
was 110 mg/kg/day for females and 88.4 mg/kg/day
for males; in mice the TD
50
was 69 mg/kg/day for females and 44.7 mg/kg/day for males. The
most conservative value is that identified for male mice.
The lifetime AI is calculated as follows:
Lifetime AI = TD
50
/50,000 x 50 kg
Lifetime AI = 44.7 mg/kg/day /50,000 x 50 kg
Lifetime AI = 45 μg/day
References
1. Zeiger E, Anderson B, Haworth S, Lawlor T, Mortelmans K. Salmonella mutagenicity
tests: IV. Results from the testing of 300 chemicals. Environ Mol Mutagen 1988;11 Suppl
12:1-158.
77
2. Dunkel VC, Zeiger E, Brusick D, McCoy E, McGregor D, Mortelmans K, et al.
Reproducibility of microbial mutagenicity assays: II. Testing of carcinogens and
noncarcinogens in Salmonella typhimurium and Escherichia coli. Environ Mutagen
1985;7 Suppl 5:1-248.
3. Japan Chemical Industry Ecology-Toxicology and Information Center (JETOC);
Mutagenicity test data of existing chemical substances based on the toxicity investigation
of the Industrial Safety and Health law; 1997; Suppl.
4. Jakubczak JL, Merlino G, French JE, Muller WJ, Paul B, Adhya S et al. Analysis of
genetic instability during mammary tumor progression using a novel selection-based assay
for in vivo mutations in a bacteriophage transgene target. Proc Natl Acad Sci (USA)
1996; 93(17):9073-8.
5. NCI. Technical report on the Bioassay of p-cresidine for possible carcinogenicity.
National Toxicology Program, Research Triangle Park, NC. 1979; TR 142.
6. Ashby J, Lefevre PA, Tinwell H, Brunborg G, Schmezer P, Pool-Zobel B, et al. The non-
genotoxicity to rodents of the potent rodent bladder carcinogens o-anisidine and p-
cresidine. Mutat Res 1991;250:115-133.
7. Morita T, Norihide A, Awogi T, Sasaki Yu F, Sato-S-I, Shimada H, et al. Evaluation of
the rodent micronucleus assay in the screening of IARC carcinogens (Groups 1, 2A and
2B). The summary report of the 6
th
collaborative study by CSGMT/JEMS.MMS. Mutat
Res 1997;389:3-122.
8. Delker DA, Yano BL, Gollapudi BB. Evaluation of cytotoxicity, cell proliferation, and
genotoxicity induced by p-cresidine in hetero- and nullizygous transgenic p53 mice.
Toxicol Sci 2000;55:361-9.
9. Stoll RE, Blanchard KT, Stoltz JH, Majeski JB, Furst S, Lilly PD et al. Phenolphthalein
and nisacodyl: Assessment of genotoxic and carcinogenic responses in heterozygous p53
(+/-)
mice and Syrian Hamster Embryo (SHE) assay. Toxicol Sci 2006;90:440-50.
10. Sasaki YF, Nishidate E, Su YQ, Matsusaka N, Tsuda S, Susa N, et al. Organ-specific
genotoxicity of the potent rodent bladder carcinogens o-anisidine and p-cresidine. Mutat
Res 1998;412:155-60.
11. IARC. para-Cresidine. Monographs on the Evaluation of the Carcinogenic Risk of
Chemicals to Humans. World Health Organization, Lyon. 1982;27:92. reviewed in Suppl
7 1987.
12. Carcinogenicity Potency Database (CPDB). [Online]. Available from: URL:
http://toxnet.nlm.nih.gov/cpdb/
13. Storer RD, French JE, Haseman J, Hajian G, LeGran EK, Long GD, et al. p53
+/-
hemizygous knockout mouse: Overview of available data. Toxicologic Pathol 2001; 29
Suppl:30-50.
78
Dimethylcarbamyl Chloride (CAS# 79-44-7)
Potential for human exposure
Potential for exposure is in industrial use. No data are available for exposure of the general
population.
Mutagenicity/genotoxicity
Dimethylcarbamyl chloride (DMCC) is considered mutagenic and genotoxic in vitro and in vivo.
DMCC was mutagenic in:
Salmonella typhimurium TA100, TA1535, TA1537, TA98 and TA1538 with and without metabolic
activation (Ref. 1, 2);
In vivo, positive results were seen in the micronucleus assay (Ref. 3).
Carcinogenicity
DMCC is classified by IARC as a Group 2A compound, or probably carcinogenic to humans (Ref. 4).
No deaths from cancer were reported in a small study of workers exposed for periods ranging from
6 months to 12 years, and there is inadequate evidence in humans for the carcinogenicity of DMCC.
There is evidence that DMCC induced tumors in rodents.
Since oral studies are lacking, the studies considered for AI derivation used inhalation and
intraperitoneal administration.
Syrian golden hamsters were exposed to 1 ppm DMCC by inhalation for 6 hours/day, 5 days/week
until the end of their lives or sacrifice due to moribundity (Ref. 5). Squamous cell carcinoma of the
nasal cavity was seen in 55% of the animals whereas no spontaneous nasal tumors were seen in
the controls or historical controls. When early mortality was taken into consideration, the
percentage of tumor bearing animals was calculated to be 75% (Ref. 5).
DMCC was tested for carcinogenic activity in female ICR/Ha Swiss mice by skin application,
subcutaneous injection and intraperitoneal (i.p.) injection (Ref. 6; this study was selected to
calculate the AI). In the skin application, 2 mg of DMCC was applied 3 times a week for 492 days;
this was seen to induce papillomas in 40/50 mice and carcinomas in 30/50 mice. Subcutaneous
injection once weekly was continued for 427 days at a dose of 5 mg/week. Sarcomas and
squamous cell carcinomas were seen in 36/50 and 3/50 mice, respectively, after the subcutaneous
injection. In the i.p. experiment, the mice were injected weekly with 1 mg DMCC for a total
duration of 450 days. The treatment induced papillary tumors of the lung in 14/30 animals and
local malignant tumors in 9/30 animals (8/30 were sarcomas). In the control groups, no tumors
were seen by skin application, 1/50 sarcoma by subcutaneous injection, and 1/30 sarcoma and
10/30 papillary tumors of lung by i.p. injection. Overall, only the local (injection site) tumors were
significantly increased; tumors at distant sites were not statistically significantly increased
compared with controls.
Dimethylcarbamyl chloride – Details of carcinogenicity studies
Study
Animals/
dose
group
Duration/
Exposure
Controls
Doses
Tumor
observations
TD
50
(mg/kg/d
)
Ref. 6
*
30
female
ICR/Ha
Swiss mice
64 weeks
Once/wk
Intra-
peritoneal
30
1:
1 mg
5.71
mg/kg/d
Injection site:
malignant
tumors/Female
4.59
˄˄˄
79
Study
Animals/
dose
group
Duration/
Exposure
Controls
Doses
Tumor
observations
TD
50
(mg/kg/d
)
Ref. 5
**
99
male Syrian
golden
hamsters
Lifetime
6 h/d,
5 d/wk
Inhalation
50 sham
treated
200
untreated
1:
1 ppm
0.553
mg/kg/d
Squamous cell
carcinoma of nasal
cavity
0.625
Ref. 6
50
female
ICR/Ha
Swiss mice
70 weeks
3 times/wk
Skin
50
1:
2 mg
Skin: Papillomas
and carcinomas/
Female
NA
˄
Ref. 6
50
female
ICR/Ha
Swiss mice
61 weeks
Once/wk
Subcutaneous
50
1:
5 mg
Injection site:
Fibrosarcomas;
Squamous cell
carcinomas/
Female
NA
˄
Ref. 7
Male
Sprague-
Dawley rats
6 weeks
6 h/d,
5 d/wk
Inhalation;
examined at
end of life
Yes
1:
1 ppm
Nasal tumors/Male
NA
˄˄˄˄
Ref. 8
30-50
female
ICR/Ha
Swiss mice
18-22 mo
3 times/wk
Skin
Yes
2:
2 and 4.3
mg
Skin.
Mainly skin
squamous
carcinoma/Female
NA
˄
Ref. 8
Female
ICR/Ha
Swiss mice
18-22 mo
Once/wk
Subcutaneous
Yes
1:
4.3 mg
Site of
administration.
Mainly sarcoma.
Hemangioma,
squamous
carcinoma and
papilloma also
seen/Female
NA
˄˄
Ref. 8
Female
ICR/Ha
Swiss mice
12 mo
Once/wk
Subcutaneous;
examined at
end of life
Yes
2:
0.43
and 4.3
mg
NA
˄˄
Studies listed are in CPDB (Ref. 9) unless otherwise noted.
*
Carcinogenicity study selected for non-inhalation AI.
**
Carcinogenicity study selected for inhalation AI.
NA= Not applicable
˄
Did not examine all tissues histologically. Subcutaneous and skin painting studies are not included
in CPDB as route with greater likelihood of whole body exposure is considered more valuable.
˄˄
Subcutaneous and skin painting studies are not included in CPDB as route with greater likelihood
of whole body exposure is considered more valuable.
˄˄˄
Histopathology only on tissues that appeared abnormal at autopsy.
˄˄˄˄
Examined only for nasal cancer. Does not meet criteria for inclusion in CPDB of exposure for at
least one fourth of the standard lifetime.
Regulatory and/or published limits
No regulatory limits have been published.
Acceptable intake (AI)
Based on the above data, DMCC is considered to be a mutagenic carcinogen. As a result, linear
extrapolation from the most sensitive TD
50
in carcinogenicity studies is an appropriate method with
which to derive an acceptable risk dose. Since DMCC appears to be a site-of-contact carcinogen, it
80
was appropriate to derive a separate AI for inhalation exposure compared with other routes of
exposure.
No information from oral administration is available, so that for routes of exposure other than
inhalation, the study by Van Duuren et al (Ref. 6), with administration by i.p. injection, was used.
The TD
50
was 4.59 mg/kg/day based on mixed tumor incidences (CPDB).
The lifetime AI is calculated as follows:
Lifetime AI = TD
50
/50,000 x 50 kg
Lifetime AI = 4.59 mg/kg/day /50,000 x 50 kg
Lifetime AI = 5 µg/day
Inhalation AI
The inhalation AI is calculated as follows:
After inhalation of DMCC, nasal cancer in hamsters is the most sensitive endpoint and the TD
50
was
0.625 mg/kg/day.
Lifetime AI = TD
50
/50,000 x 50 kg
Lifetime AI = 0.625 mg/kg/day /50,000 x 50 kg
Lifetime inhalation AI = 0.6 µg/day
References
1. Dunkel V, Zeiger E, Brusick D, McCoy E, McGregor D, Mortelmans K, et al. Reproducibility of
microbial mutagenicity assays. I. Tests with Salmonella typhimurium and Escherichia coli using
a standardized protocol. Environ Mutagen 1984;6 Suppl 2:1-251.
2. Kier LD, Brusick DJ, Auletta AE, Von Halle ES, Brown MM, Simmon VF, et al. The Salmonella
typhimurium/mammalian microsomal assay. A report of the U.S. Environmental Protection
Agency Gene-Tox Program. Mutat Res 1986;168:69-240.
3. Heddle JA, Hite M, Kirkhart B, Mavournin K, MacGregor JT, Newell GW, et al. The induction of
micronuclei as a measure of genotoxicity. A report of the U.S. Environmental Protection Agency
Gene-Tox Program. Mutat Res 1983;123:61-118.
4. IARC. Monographs on the evaluation of the Carcinogenic Risk of Chemicals to Man. Geneva:
International Agency for Research on Cancer, World Health Organization. [Online] 1972-
PRESENT. (Multivolume work). 1999;71:539. Available from: URL:
http://monographs.iarc.fr/index.php
5. Sellakumar AR, Laskin S, Kuschner M, Rush G, Katz GV, Snyder CA, et al. Inhalation
carcinogenesis by dimethylcarbamoyl chloride in Syrian golden hamsters. J Environ Pathol
Toxicol 1980;4:107-15.
6. Van Duuren BL, Goldschmidt BM, Katz C, Seidman I, Paul JS. Carcinogenic activity of alkylating
agents. J Natl Cancer Inst 1974;53:695-700.
7. Snyder CA, Garte SJ, Sellakumar AR, Albert RE. Relationships between the levels of binding to
DNA and the carcinogenic potencies in rat nasal mucosa for three alkylating agents, Cancer Lett
1986;33:175-81.
82
Dimethyl Sulfate (CAS# 77-78-1)
Potential for human exposure
Dimethyl sulfate (DMS) is found in ambient air with mean concentration of 7.4 µg per cubic meter
or 1.4 ppb based on 1983 data compiled from a single site by the US EPA (Ref. 1).
Mutagenicity/genotoxicity
DMS is mutagenic/genotoxic in vitro and in vivo (Ref. 2).
DMS is mutagenic in:
The microbial reverse mutation assay (Ames), Salmonella typhimurium strains TA98, TA100,
TA1535, TA1537 and TA1538 with and without activation (Ref. 3).
In vivo, DMS forms alkylated DNA bases and is consistently positive in genotoxicity assays (Ref. 4).
Elevated levels of chromosomal aberrations have been observed in circulating lymphocytes of
workers exposed to DMS (Ref. 4).
Carcinogenicity
DMS is classified by IARC as a Group 2A carcinogen, probably carcinogenic to humans (Ref. 4).
No epidemiological studies were available for DMS although a small number of cases of human
exposure and bronchial carcinoma have been reported. DMS is carcinogenic in animals by chronic
and subchronic inhalation, and single and multiple subcutaneous injections; however, DMS has not
been tested by the oral route of exposure. DMS is carcinogenic in rats, mice, and hamsters (Ref.
4). The carcinogenicity studies for DMS were limited for a variety of reasons and this is likely why
DMS is not listed on the Carcinogenicity Potency Database (CPDB). The studies evaluating
carcinogenicity of DMS are described below (excerpted from US EPA, Ref. 5).
83
DMS- Details of carcinogenicity studies
Study
Animals
Duration/
Exposure
Controls
Doses
Tumor
observations
TD
50
(mg/kg/d
)
Ref. 6
Golden
hamsters,
Wistar
rats, and
NMRI
mice
male and
female
(number
not clearly
specified)
15 mo
6 h/d,
2 d/wk followed
by 15 mo
observation
period
Inhalation
Yes
2:
0.5; 2.0 ppm
Tumors in lungs,
thorax and nasal
passages at both
doses
NA
˄
Ref. 7
20-27 BD
rats
Sex not
specified
130 days
1 h/d, 5 d/wk
followed by 643
day observation
period
Inhalation
No
2:
3; 10 ppm
Squamous cell
carcinoma in
nasal epithelium
at 3 ppm.
Squamous cell
carcinomas in
nasal epithelium
and lympho-
sarcoma in the
thorax with
metastases to
the lung at 10
ppm.
NA
˄˄
Ref. 8
8-17 BD
Rats
Sex not
specified
394 days
The duration of
the study was
not reported but
mean tumor
induction time
was 500 days
Subcutaneous
No
2:
8; 16
mg/kg/wk
Injection-site
sarcomas in 7/11
at low dose and
4/6 at high dose;
occasional
metastases to
the lung. One
hepatic
carcinoma.
NA
˄˄˄
Ref. 7
15 BD
Rats
Sex not
specified
Up to 740 day
evaluation
Following single
injection
Subcutaneous
No
1:
50 mg/kg
Local sarcomas
of connective
tissue in 7/15
rats; multiple
metastases to
the lungs in
three cases
NA
˄˄˄
Ref. 7
12 BD rats
Sex not
specified
800 days
Once/wk
Intravenous
No
2:
2; 4 mg/kg
No tumors
reported
NA
˄˄˄
Ref. 7
8 BD rats
(pregnant
females)
1 year offspring
observation
following
single dose,
gestation day 15
Intravenous
No
1:
20 mg/kg
4/59 offspring
had malignant
tumors of the
nervous system
while 2/59 had
malignant
hepatic tumors.
NA
˄˄˄˄
Ref. 9
90
female
CBAX57Bl
/6 mice
Duration not
reported
4 h/d, 5 d/wk
Inhalation
Not
indicated
3:
0.4; 1; 20
mg/m
3
Increase in lung
adenomas at
high dose
NA*
Ref. 10
20 ICR/Ha
Swiss
mice
¥
475 days
3 times/wk
Dermal
Not
indicated
1:
0.1 mg
No findings
NA**
84
Studies listed are in not in CPDB.
NA = Not applicable
˄
Control data not reported. Tumor incidences not tabulated by species or dose.
˄˄
Small group size. No concurrent control group. One rat at high dose had a cerebellar tumor and
two at low dose had nervous system tumors which are very rare and distant from exposure.
˄˄˄
Small group size, no concurrent control group.
˄˄˄˄
No concurrent control group.
* Duration not reported
** Limited number of animals. Only one dose tested. Even when DMS was combined with tumor
promoters no tumors were noted.
¥
Sex not specified
Mode of action for carcinogenicity
Dimethyl Sulfate is a mutagenic carcinogen, and the acceptable intake is calculated by linear
extrapolation from the TD50.
Regulatory and/or published limits
The European Union (EU) Institute for Health and Consumer Protection (ECHA, Ref.11) developed a
carcinogenicity slope curve based on the inhalation carcinogenicity data for DMS. ECHA calculated
a T
25
(dose that resulted in a 25% increase in tumors) using the rat inhalation study (Ref. 7).
Systemic effects (nervous system) and local nasal tumors were observed in this limited
carcinogenicity study. However, as with other studies listed, this study was severely limited with
high mortality, no control animals, only 2 dose groups and minimal pathological evaluations;
therefore, the study was not suitable for linear extrapolation.
Acceptable intake (AI)
While DMS is considered to be a likely oral carcinogen and probable human carcinogen, there are
no oral carcinogenicity studies from which to derive a TD
50
value. Moreover, the inhalation studies
that are available are limited for a variety of reasons and are not suitable for TD
50
extrapolation.
Given this, it is reasonable to limit DMS to the threshold of toxicological concern (TTC) lifetime level
of 1.5 µg/day.
Lifetime AI = 1.5 µg/day
References
1. US EPA. Health and Environmental effects profile for dimethyl sulfate. Prepared by the Office of
Health and Environmental Assessment, Environmental Criteria and Assessment Office,
Cincinnati, OH for the Office of Solid Waste and Emergency Response, Washington, DC. 1985.
2. Hoffmann GR. Genetic effects of dimethyl sulfate, diethyl sulfate, and related compounds.
Mutat Res 1980;75:63-129.
3. Skopek TR, Liber HL, Kaden DA, Thilly WG. Relative sensitivities of forward and reverse
mutation assays in Salmonella typhimurium. Proc Natl Acad Sci USA 1978;75:4465-9.
4. IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans.
International Agency for Research on Cancer, World Health Organization, Lyon. 1999;71:575
5. US Environmental Protection Agency. Dimethyl sulfate (CASRN 77-78-1). Integrated Risk
Information System (IRIS). [Online]. 1988. Available from: URL:
https://cfpub.epa.gov/ncea/iris/iris_documents/documents/subst/0365_summary.pdf#namedd
est=woe
85
6. Schlogel FA, Bannasch P. Carcinogenicity and Chronic Toxicity of Inhaled Dimethyl Sulfate. (In
German) (Inaugural Dissertation) Julius-Maximilians University, Würzburg 1972. (data in Ref.
11).
7. Druckrey H. Carcinogenic alkylating compounds: III. Alkyl halogenids, sulfates, sulfonates, and
heterocyclics. (Article in German) Z. Krebsforsch 1970;74:241–273.
8. Druckrey H. Carcinogenic alkylating compounds: I. Dimethyl sulfate, carcinogenic effect in rats
and probable cause of occupational cancer. (Article in German) Z. Krebsforsch 1966; 68:103–
111.
9. Fomenko VN, Katasova LD, Domshlak MG (1983); USSR Minist Health All-Union Sci Soc Med
Genet 1:348-49 as cited in WHO; Environ Health Criteria 1985; Dimethyl Sulfate p.36
10. Van Duuren BL, Goldschmidt BM, Katz C, Seidman I, Paul JS. Carcinogenic activity of alkylating
agents. J Natl Cancer Inst 1974;53:695-700.
11. ECHA (European Chemical Agency). European Union Risk Assessment Report: Institute for
Health and Consumer Protection. Dimethyl Sulphate. [Online]. 2002 Vol. 12. Available from:
URL: http://echa.europa.eu/documents/10162/3d2e4243-8264-4d09-a4ab-92dde5abfadd
86
Ethyl Chloride (Chloroethane, CAS# 75-00-3)
Potential for human exposure
Low levels (parts-per-trillion) from contaminated ambient air and drinking water. Dermal contact
as a topical anesthetic.
Mutagenicity/genotoxicity
Ethyl chloride is mutagenic and genotoxic in vitro but not in vivo. IARC (Ref. 1) has reviewed the
mutagenicity data for ethyl chloride; key points are summarized here.
Ethyl chloride was mutagenic in:
Microbial reverse mutation assay (Ames), Salmonella typhimurium strains TA100 and TA1535 and
in Escherichia coli WP2uvrA with and without metabolic activation when tested in conditions that
enable exposure to gas (Ref. 2, 3, 4);
CHO cell hprt assay with and without metabolic activation.
In vivo ethyl chloride was negative in a mouse bone marrow micronucleus test after inhalation at
approximately 25,000 ppm for 3 days, and in an Unscheduled DNA Synthesis (UDS) assay in
female mouse liver (Ref. 5).
Carcinogenicity
Ethyl chloride was designated by IARC as Class 3, or not classifiable as to its carcinogenicity (Ref.
1).
Only one carcinogenicity study was found for ethyl chloride, NTP studies (Ref. 6) in rats and mice of
both sexes via inhalation for 6 h/day, 5 days/week for 100 weeks. The single exposure
concentration (15,000 ppm) tested was limited by safety concern (explosion risk) and on the lack
of obvious effect in a 3 month range-finding study up to 19,000 ppm. These data were later
assessed by US EPA (Ref. 7), comparing ethyl chloride with ethyl bromide. Ethyl chloride was
notable because, along with structurally similar ethyl bromide, it induced very high numbers of
uncommon uterine tumors (endometrial carcinomas) in mice, but not rats. Ethyl chloride produced
clear evidence of carcinogenicity in female mice (uterus) and equivocal evidence of carcinogenicity
in male and female rats. Due to poor survival, the male mouse study was considered inadequate
although there was an increased incidence of lung tumors.
Ethyl Chloride – Details of carcinogenicity studies
Study
Animals/
dose group
Duration/
Exposure
Controls
Doses
Most sensitive
tumor
site/sex
TD
50
(mg/kg/d)
Ref. 6, 7
*
50/sex/
group
B6C3F1
mice
100 weeks
6 h/d,
5 d/wk
Inhalation
50
1:
M: 10.4
F: 12.4
g/kg/d
Uterus/Female
1810
Ref. 6, 7
50/sex/
group
Fischer 344
rats
100 weeks
6 h/d,
5 d/wk
Inhalation
50
1:
M: 2.01 F:
2.88
g/kg/d
Negative
NA
*
Carcinogenicity study selected for AI calculation. Studies listed are in CPDB (Ref. 8).
NA = Not applicable
87
Mode of action of carcinogenicity
Holder (Ref. 7) proposes reactive metabolites may contribute to carcinogenicity, but notes female
mice have a marked stress response to ethyl chloride exposure at the high concentrations used in
the carcinogenicity study; such stress has been shown to lead to adrenal stimulation. It was
proposed that high corticosteroid production could promote development of endometrial cancers in
mice.
Regulatory and/or published limits
The US EPA established an inhalation Reference Concentration (RfC) for non-carcinogenic effects of
10 mg/m
3
, or 288 mg/day assuming a respiratory volume of 28,800 L/day (Ref. 9).
Acceptable intake (AI)
Rationale for selection of study for AI calculation
Although the studies are not robust in design (having a single dose group), the high level of a
specific rare type of uterine carcinoma of endometrial original in mice (43/50 affected compared
with 0/49 controls) suggest a strong carcinogenic response. The observation is supported by the
fact that the same type of tumors (mouse uterine tumors) was seen with a comparator molecule
ethyl bromide, in a more robust carcinogenicity study with 3 doses and a control (Ref. 10).
Ethyl chloride is considered to be a mutagenic carcinogen. Based on the NTP inhalation study the
most sensitive species/site is female mouse uterus. Since the number of tumors is high, it is
possible to calculate a TD
50
even though only one dose was tested. The authors of the CPDB (Ref.
8) converted 0 and 15,000 ppm to doses of 0 and 12.4 g/kg and calculated a TD
50
of 1810
mg/kg/day for mouse uterine tumors.
Lifetime AI = TD
50
/50,000 x 50 kg
Lifetime AI = 1810 mg/kg/day /50,000 x 50 kg
Lifetime AI = 1,810 µg/day
88
References
1. IARC. Chloroethane. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to
Humans. International Agency for Research on Cancer, World Health Organization, Lyon.
1999;71:1345.
2. Goto S, Shiraishi F, Tanabe K, Endo O, Machii K, Tezuka Y, et al. Mutagenicity Detection
Method for Vinyl Chloride and Vinylidene Chloride Gases. Kankyo Kagaku 1995; 5(2):235-40.
3. Zeiger E, Anderson B, Haworth S, Lawlor T, Mortelmans K. Salmonella mutagenicity tests. V.
Results from the testing of 311 chemicals. Environ Mol Mutagen 1992; 19 Suppl 21:2-141.
4. Araki A, Noguchi T, Kato F, Matsushima T. Improved method for mutagenicity testing of
gaseous compounds by using a gas sampling bag. Mutat Res 1994; 307(1):335-44.
5. Ebert R, Fedtke N, Certa H, Wiegand HJ, Regnier JF, Marshall R, et al. SW. Genotoxicity Studies
With Chloroethane. Mutat Res 1994; 322(1):33-43.
6. NCI/NTP Technical Report on the toxicology and carcinogenesis studies of chloroethane.
National Toxicology Program, Research Triangle Park, NC. NTP TR 346 1989. [Online]. 1989;
Available from: URL: https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr346.pdf
7. Holder JW. Analysis of Chloroethane Toxicity and Carcinogenicity Including a Comparison With
Bromoethane. Toxicology and Industrial Health 2008; 24(10):655-675.
8. Carcinogenicity Potency Database (CPDB). [Online]. Available from: URL:
http://toxnet.nlm.nih.gov/cpdb/
9. US Environmental Protection Agency. Ethyl Chloride (CAS# 75-00-3). Integrated Risk
Information System (IRIS).. [Online] 1991. Available from: URL:
https://cfpub.epa.gov/ncea/iris2/chemicalLanding.cfm?substance_nmbr=523
10. NTP. Technical Report on the toxicology and carcinogenesis studies of Ethyl Bromide. National
Toxicology Program, Research Triangle Park, NC. NTP TR 363. [Online]. 1989; Available from:
URL: http://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr363.pdf
89
Glycidol (CAS# 556-52-5)
Potential for human exposure
Heating of glycerol and sugars causes the formation of glycidol. Glycidol is a metabolite of
3-monochloropropane-1, 2-diol, a chloropropanol found in many foods and food ingredients,
including soy sauce and hydrolyzed vegetable protein. Potential daily glycidol exposure in food has
been estimated at 20-80 µg/day (Ref. 1).
Mutagenicity/genotoxicity
Glycidol is mutagenic/genotoxic in vitro and in vivo.
IARC (Ref. 2) and CCRIS (Ref. 3) contain reviews of the mutagenicity/genotoxicity data for
glycidol; key conclusions are summarized here.
Glycidol is mutagenic in:
Microbial reverse mutation assay (Ames), Salmonella strains TA100, TA1535, TA98, TA97 and
TA1537 both with and without rat liver S9 activation and in standard plate and preincubation
assays.
Escherichia coli strain WP2uvrA/pKM101 in a preincubation assay with and without rat liver S9.
In vivo, glycidol was positive in a mouse micronucleus assay by oral gavage in male and female
P16Ink4a/p19Arf haploinsufficient mice.
Carcinogenicity
Glycidol is classified by IARC as Group 2A, or probably carcinogenic in humans (Ref. 2).
In NTP studies (Ref. 4, 5), glycidol was administered by gavage in water to male and female
F344/N rats and B6C3F1 mice. Rats received 0, 37.5, or 75 mg/kg and mice received 0, 25, or 50
mg/kg daily, 5 days per week for 2 years. The average daily doses were calculated by multiplying
the administered dose by 5/7 to account for the 5 days per week dosing schedule and 103/104 to
account for the less-than-lifetime duration of dosing. The resulting average daily doses were 0,
26.5, and 53.1 mg/kg/day in male and female rats, and 0, 17.7, and 35.4 mg/kg/day in male and
female mice.
Exposure to glycidol was associated with dose-related increases in the incidences of neoplasms in
various tissues in both rats (mammary gland tumors in females), and mice (Harderian gland).
Survival of treated rats and mice was markedly reduced compared to controls because of the early
induction of neoplastic disease.
The oral gavage study in hamsters was less robust due to small group size, single dose levels and
shorter duration. Further oral gavage chronic studies with glycidol were conducted by the NTP in
genetically modified mice lacking two tumor suppressor genes (i.e., haploinsufficient
p16Ink4a/p19Arf mice) (Ref. 6). Although there was clear evidence of carcinogenic activity in
males (based on the occurrence of histiocytic sarcomas and alveolar/bronchiolar adenomas) and
some evidence of carcinogenic activity in female mice (based on the occurrence of
alveolar/bronchiolar adenomas), these studies are considered less suitable for dose-response
assessment than the two-year bioassays (Ref. 5) for reasons including the short duration, the small
number of animals used per treatment group, and limited understanding of how dose-response
relationships observed in genetically modified animals correspond with those observed in standard
long-term carcinogenicity bioassays (Ref. 7).
90
Glycidol – Details of carcinogenicity studies
Study
Animals/
dose
group
Duration/
Exposure
Controls
Doses
Most sensitive
tumor
site/sex
TD
50
(mg/kg/
d)
Ref. 5
*
50/sex/
group
F344/N
rats
2 years
5 days/wk
Oral gavage
50
2:
26.5; 53.8
mg/kg/d
Mammary
gland/Female
4.15
Ref. 5
50/sex/
group
B6C3F1
mice
2 years
5 days/wk
Oral gavage
50
2:
17.7; 35.4
mg/kg/d
Harderian gland
/Female
32.9
Ref. 8
12-20/
sex/group
Syrian
Golden
Hamsters
60 weeks
Twice/wk
Gavage
Yes
1:
M: 15.8
F: 17.9
mg/kg/d
Spleen/Female
56.1
˄
Ref. 9
(
**
Cited in
Ref. 2)
20
ICR/Ha
Swiss mice
520 days
3 times/wk
Skin Painting
Yes
1:
5%
No Tumors
NA
˄
Studies listed are in CPDB (Ref. 10) unless otherwise noted.
*
Carcinogenicity study selected for AI calculation.
**
Not in CPDB.
NA= Not applicable.
˄
Not a standard carcinogenicity design. Only one dose, intermittent dosing, and small sample size
(Ref.7).
Mode of action of carcinogenicity
Glycidol is a mutagenic carcinogen, and the acceptable intake is calculated by linear extrapolation
from the TD50.
Regulatory and/or published limits
No regulatory limits have been published, for example by US EPA, WHO, or ATSDR.
91
Acceptable intake (AI)
Rationale for selection of study for AI calculation
The most suitable carcinogenicity data for human cancer potency assessment come from the two-
year oral studies conducted in F344/N rats and B6C3F1 mice by NTP (Ref. 5). The most sensitive
organ site was female mammary glands with a TD
50
of 4.15 mg/kg/day.
Calculation of AI
Lifetime AI = TD
50
/50,000 x 50 kg
Lifetime AI = 4.15 (mg/kg/day)/50,000 x 50 kg
Lifetime AI = 4 µg/day
References
1. Bakhiya N, Abraham K, Gürtler R, Appel KE, Lampen A. Toxicological assessment of 3-
chloropropane-1,2-diol and glycidol fatty acid esters in food. Mol Nutr Food Res 2011;55:509-
21.
2. IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man. Geneva:
International Agency for Research on Cancer, World Health Organization. [Online]. 1972-
PRESENT. (Multivolume work). 2000; 77:469; Available from: URL:
http://monographs.iarc.fr/index.php.
3. CCRIS. Chemical Carcinogenesis Research Information System. National Library of Medicine.
[Online]. 2013. Available from: URL: http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?CCRIS and
search on CAS number.
4. Irwin RD, Eustis SL, Stefanski S, Haseman JK. Carcinogenicity of Glycidol in F344 rats and
B6C3F
1
mice. J Appl Toxicol 1996;16 (3):201-9.
5. NTP. Technical report on the toxicology and carcinogenesis studies of glycidol (CAS No. 556-52-
5) in F344/N Rats and B6C3F1 Mice (Gavage Studies). National Toxicology Program, Research
Triangle Park, NC. 1990. NTP TR 374.
6. NTP. Toxicology and Carcinogenesis Studies of Glycidol (CAS No. 556-52-5) in genetically
modified haploinsufficient p16 (Ink4a)/p19 (Arf) mice (gavage study). Natl Toxicol Program
Genet Modif Model Rep 2007;13:1-81.
7. California Environmental Protection Agency (CalEPA). No Significant Risk Level (NSRL) for the
Proposition 65 carcinogen Glycidol. [Online]. 2010. Available from: URL:
http://www.oehha.ca.gov/prop65/CRNR_notices/pdf_zip/GlycidolNSRL073010.pdf
8. Lijinsky W, Kovatch RM. A study of the carcinogenicity of glycidol in Syrian hamsters. Toxicol
Ind Health 1992;8(5):267-71.
9. Van Duuren BL, Langseth L, Goldschmidt BM, Orris L. Carcinogenicity of epoxides, lactones,
and peroxy compounds. VI. Structure and carcinogenic activity. J Natl Cancer Inst
1967;39:1217–28.
10. Carcinogenicity Potency Database (CPDB). [Online]. Available from: URL:
http://toxnet.nlm.nih.gov/cpdb/
92
Hydrazine (CAS# 302-01-2)
Potential for human exposure
Hydrazine is used in the synthesis of pharmaceuticals, pesticides and plastic foams (Ref. 1).
Hydrazine sulphate has been used in the treatment of tuberculosis, sickle cell anemia and other
chronic illnesses (Ref. 2). There is limited information on the natural occurrence of hydrazine and
derivatives (Ref. 3). Humans may be exposed to hydrazine from environmental contamination of
water, air and soil (Ref. 1); however, the main source of human exposure is in the workplace (Ref.
4). Small amounts of hydrazine have also been reported in tobacco products and cigarette smoke
(Ref. 1, 5).
Mutagenicity/genotoxicity
Hydrazine is mutagenic and genotoxic in vitro and in vivo.
IARC (Ref. 6) has reviewed the mutagenicity of hydrazine. Key observations are summarized here.
Hydrazine was mutagenic in:
Microbial reverse mutation assay (Ames), Salmonella typhimurium strains TA 1535, TA 102, TA 98
and TA 100, and in Escherichia coli strain WP2 uvrA, with and without activation;
In vitro mouse lymphoma L5178Y cells, in tk and hprt genes.
In vivo, (Ref. 6) hydrazine induced micronuclei but not chromosome aberrations in mouse bone
marrow. DNA adducts have been reported in several tissues in vivo.
Carcinogenicity
Hydrazine is classified by IARC as Group 2B, or possibly carcinogenic to humans (Ref. 6) and by US
EPA as Group B2 or a probable human carcinogen (Ref. 7).
There are seven hydrazine carcinogenicity studies cited in the CPDB (Ref. 8): Three inhalation
studies that included 1-year dosing duration, three studies in drinking water and one by oral
gavage. Five of the seven hydrazine carcinogenicity studies were deemed positive by the authors
of the original reports.
The main target organs for oral carcinogenicity of hydrazine in rodents are the liver and lungs. The
most robust oral studies based on group size and dose levels were published in Refs. 9 and 10.
The most robust inhalation study with the lowest TD
50
is in Ref. 11. The most sensitive tumor
targets for inhalation carcinogenicity of hydrazine in rodents are sites of initial contact such as the
nasal cavity and lungs.
The studies done on hydrazine sulphate in the CPDB (Ref. 8) are not shown here as they included
<50 animals per group (and a single dose level in one case), and the calculated TD
50
values were
higher (less potent) than those for the drinking water study of hydrazine (Ref. 9). Given the
similarity between the outcomes from the two robust drinking water studies (Ref. 9, 10), the more
recent study with the higher tested doses (Ref. 10) was selected for the non-inhalation AI
calculation for hydrazine.
Hydrazine – Details of carcinogenicity studies
Study
Animals/
dose group
Duration/
Exposure
Controls
Doses
Most
sensitive
tumor
TD
50
(mg/kg/
d)
93
site/type/se
x
Ref. 9
50/sex/ group
Wistar rats
Lifetime
Drinking
water
50
3:
M: 0.1; 1.5,
2.5.
F: 0.11,
0.57, 2.86
mg/kg/d
Liver/Female
41.6
Ref. 11
*
100/sex/
group
F344 rats
1 year with
18 mo
observation
Inhalation
150
4:
M:1.37,
6.87, 27.5,
137
F: 1.96,
9.81, 39.3,
196
µg/kg/d
Nasal
adenamatous
polyps/Male
0.194
Ref. 12
50/sex/ group
Bor:NMRI,
SPF-bred
NMRI mice
2 year
Drinking
water
50
3:
M: 0.33,
1.67, 8.33.
F: 0.4, 2.0,
10.0
mg/kg/d
Negative
NA,
negative
study
Ref. 11
200
male Golden
Syrian
hamsters
1 year with
12 mo
observation
Inhalation
Yes
3:
0.02, 0.08,
0.41
mg/kg/d
Nasal
adenomatous
polyps/Male
4.16
Ref. 11
400 female
C57BL/6
Mice
1 year with
15 mo
observation
Inhalation
Yes
1:
0.18
mg/kg/d
Negative
NA
Ref. 13
50/sex/ group
Swiss mice
Lifetime
Drinking
water
Not
concurren
t
1:
~1.7-2
mg/kg/d
Lung/Male
2.20
¥
Ref. 14
25
female Swiss
mice
40 weeks
5d/wk
Gavage
85
Untreated
1:
~5 mg/kg/d
Lung/Female
5.67
¥¥
Ref. 10
**^
50/sex/
F344/DuCrj
rats
Lifetime
Drinking
water
Yes
3:
M: 0.97,
1.84, 3.86
F:1.28, 2.50,
5.35
mg/kg/d
Liver/Female
38.7
Ref. 10
^
50/sex
Crj:BDF1 mice
Lifetime
Drinking
water
3:
M: 1.44,
2.65, 4.93
F: 3.54,
6.80, 11.45
mg/kg/d
Liver/Female
52.4
Studies listed are in CPDB (Ref. 8).
*
Carcinogenicity study selected for inhalation AI calculation.
**
Carcinogenicity study selected for non-inhalation TD
50
(see Note 2) and AI calculations.
NA= Not applicable.
¥
Excluded by US EPA (Ref. 7); no concurrent controls. Liver negative.
¥¥
Animal survival affected. Liver negative.
^
Not in CPDB
Mode of action of carcinogenicity
Not defined. DNA adducts have been detected in vivo, (Ref. 15, 16, 17, 18, 19, 20) although they
are reported in tissues that do not develop tumors, so their contribution to tumorigenicity is not
known.
94
Regulatory and/or published limits
The US EPA (Ref. 7) has published an oral slope factor of 3.0 per mg/kg/day and a drinking water
unit risk of 8.5 x 10
-5
per µg/L. At the 1 in 100,000 risk level, this equates to a concentration of
0.1 µg of hydrazine/L of water or ~0.2 µg/day for a 50 kg/human. This limit is a linearized
multistage extrapolation based on the observation of hepatomas in a multi-dose gavage study (Ref.
21) where hydrazine sulfate was administered to mice for 25 weeks followed by observation
throughout their lifetime (Ref. 7). Additional studies were identified that were published after the
oral slope factor was calculated (Ref. 9, 10, 17, 22). These studies could potentially produce a
change in the oral slope factor but it has not yet been re-evaluated by US EPA.
The US EPA (Ref. 7) has also published an inhalation slope factor of 17 per mg/kg/day and an
inhalation unit risk of 4.9x10
-3
per µg/m
3
. At the 1 in 100,000 risk level, this equates to an air
concentration of 2 x 10
-3
µg/m
3
of hydrazine or 0.04 µg/day assuming a person breathes
20 m
3
/day. This limit is a linearized multistage extrapolation based on the observation of nasal
cavity adenoma or adenocarcinoma in male rats in a multi-dose inhalation study where hydrazine
was administered 6 hours/day, 5 days/week for 1 year followed by an 18-month observation period
(cited in Ref. 7). Only the US EPA review of this data was accessible; however, the results appear
to be very similar to, if not the same as, those of Vernot et al (Ref. 11).
Acceptable intake (AI)
Rationale for selection of study for AI calculation
Both oral and inhalation carcinogenicity studies for hydrazine were reviewed to determine if a
separate limit is required specific for inhalation carcinogenicity. Given the more potent
carcinogenicity specific to the first site-of-contact observed in inhalation studies, it was determined
that a separate AI for inhalation exposure was appropriate.
For oral hydrazine, carcinogenicity has been reported in 4 mouse studies and 2 rat studies. The
most sensitive effect in the oral studies was based on hepatocellular adenomas and carcinomas of
the liver in female rats (Ref. 10).
All of the inhalation carcinogenicity studies that were used by the US EPA in the derivation of the
inhalation carcinogenicity limit for hydrazine were taken into consideration when selecting the most
robust carcinogenicity study for the derivation of an AI for inhaled pharmaceuticals. The critical
study by MacEwen et al used by US EPA (Ref. 7) was proprietary but is likely the same one
described in Vernot et al (Ref. 11). Given that the TTC was derived via linear extrapolation from
TD
50
values for hundreds of carcinogens, that same approach was used in the derivation of a
compound-specific AI for hydrazine. The methodology used by the US EPA and the method used
here are both highly conservative in nature. However, given that the methodologies do differ, it is
reasonable to expect some slight differences. The AI was calculated based on the TD
50
derived
from a study in which male and female rats were administered hydrazine via inhalation for one year
with an 18-month observation period (Ref. 11). While a 1-year study is not a standard design for
carcinogenicity, a positive response was observed demonstrating that the window for
carcinogenicity was not missed. The most sensitive target tissue was the male nasal region, with a
TD
50
value of 0.194 mg/kg/day, after being adjusted, as standard practice, to account for 1 vs 2
years of exposure.
Calculation of AI
Lifetime AI = TD
50
/50,000 x 50 kg
Lifetime AI = 38.7 (mg/kg/day)/50,000 x 50 kg
95
Lifetime AI = 39 µg/day
Calculation of inhalation AI
Lifetime AI = TD
50
/50,000 x 50 kg
Lifetime AI = 0.194 (mg/kg/day)/50,000 x 50 kg
Lifetime inhalation AI = 0.2 µg/day
96
References
1. Choudary G, Hansen H. Human health perspective on environmental exposure to hydrazines: A
review. Chemosphere 1998;37:801-43.
2. Von Burg R, Stout T. Hydrazine. J Appl Toxicol 1991;11:447–50.
3. Toth B. A review of the natural occurrence, synthetic production and use of carcinogenic
hydrazines and related chemicals. In vivo. 2000;14(2):299-319.
4. Hazardous Substance Database (HSDB): Hydrazine (302-01-2); [Online]. 2005 June 24 [cited
2013 February 27]; Available from: URL: http://toxnet.nlm.nih.gov/
5. Liu YY, Schmeltz I, Hoffman D. Chemical studies on tobacco smoke. Quantitative analysis of
hydrazine in tobacco and cigarette smoke. Anal Chem 1974;46: 885–9.
6. IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man. Geneva.
International Agency for Research on Cancer, World Health Organization, [Online] 1972-
PRESENT. (Multivolume work). 1999; Available from: URL: http://monographs.iarc.fr/index.php
p. V71 1006.
7. US Environmental Protection Agency. Hydrazine/Hydrazine sulfate (302-01-2). Integrated Risk
Information System (IRIS). [Online]. 1991. Available from: URL:
https://cfpub.epa.gov/ncea/iris/iris_documents/documents/subst/0352_summary.pdf
8. Carcinogenicity Potency Database (CPDB). [Online]. Available from: URL:
http://toxnet.nlm.nih.gov/cpdb/
9. Steinhoff D, Mohr U. The question of carcinogenic effects of hydrazine. Exp Pathol
1988;33:133-40.
10. Matsumoto M, Kano H, Suzuki M, Katagiri T, Umeda Y, Fukushima S. Carcinogenicity and
chronic toxicity of hydrazine monohydrate in rats and mice by two-year drinking water
treatment. Regul Toxicol Pharmacol 2016;76:63-73.
11. Vernot EH, MacEwen JD, Bruner RH, Haun CC, Kinkead ER, Prentice DE, et al. Long-term
inhalation toxicity of hydrazine. Fundam Appl Toxicol 1985;5:l050-64.
12. Steinhoff D, Mohr U, Schmidt WM. On the question of the carcinogenic action of hydrazine -
evaluation on the basis of new experimental results. Exp Pathol 1990;39:1-9.
13. Toth B. Hydrazine, methylhydrazine and methylhydrazine sulfate carcinogenesis in Swiss mice.
Failure of ammonium hydroxide to interfere in the development of tumors. Int J Cancer
1972;9:109-18.
14. Roe FJC, Grant GA, Millican DM. Carcinogenicity of hydrazine and 1,1-dimethylhydrazine for
mouse lung. Nature 1967;16:375-6.
15. Becker RA, Barrows LR, Shank RC. Methylation of liver DNA guanine in hydrazine
hepatotoxicity: dose-response and kinetic characteristics of 7-methylguanine and O
6
-
methylguanine formation and persistence in rats. Carcinogenesis 1981;2:1181-8.
16. Bosan WS, Shank RC. Methylation of liver DNA guanine in hamsters given hydrazine. Toxicol
Appl Pharmacol 1983;70:324-34.
97
17. Bosan WS, Shank RC, MacEwen JD, Gaworski CL, Newberne PM. .Methylation of DNA guanine
during the course of induction of liver cancer in hamsters by hydrazine or dimethylnitrosamine.
Carcinogenesis 1987;8:439-44.
18. Saffhill R, Fida S, Bromley M, O'Connor PJ. Promutagenic alkyl lesions are induced in the tissue
DNA of animals treated with isoniazid. Human Toxicol 1988;7:311-7.
19. Leakakos T, Shank RC. Hydrazine genotoxicity in the neonatal rat. Toxicol Appl Pharmacol
1994;126:295-300.
20. Mathison B, Murphy SE, Shank RC. Hydralazine and other hydrazine derivatives and the
formation of DNA adducts. Toxicol Appl Pharmacol 1994;127:91-8.
21. Biancifiori, C. Hepatomas in CBA/Cb/Se mice and liver lesions in golden hamsters induced by
hydrazine sulfate. J Natl Cancer Inst 1970;44:943.
22. FitzGerald BE, Shank RC. Methylation status of DNA cytosine during the course of induction of
liver cancer in hamsters by hydrazine sulphate. Carcinogenesis 1996;17:2703-9.
98
Hydrogen Peroxide (CAS# 7722-84-1)
Potential for human exposure
Hydrogen peroxide can be present in green tea and instant coffee, in fresh fruits and vegetables
and naturally produced in the body (Ref. 1). It is estimated up to 6.8 g is produced endogenously
per day (Ref. 2). Other common sources of exposure are from disinfectants, some topical cream
acne products, and oral care products which can contain up to 4% hydrogen peroxide (Ref. 2).
Mutagenicity/genotoxicity
Hydrogen peroxide is mutagenic and genotoxic in vitro but not in vivo.
IARC (Ref. 3) and European Commission Joint Research Centre (Ref. 4) reviewed the mutagenicity
data for hydrogen peroxide, and key observations are summarized here.
Hydrogen peroxide is mutagenic in:
Salmonella typhimurium strains TA96, TA97, SB1106p, SB1106, and SB1111 and Escherichia coli
WP2 in the absence of exogenous metabolic activation;
L5178Y mouse lymphoma cell sublines at the hprt locus;
Chinese hamster V79 cells at the hprt locus, in only one of six studies.
In vivo, micronuclei were not induced after administration of hydrogen peroxide to mice
intraperitoneally at up to 1,000 mg/kg, or to catalase-deficient C57BL/6NCr1BR mice in drinking
water at 200, 1,000, 3,000, and 6,000 ppm for two weeks.
Carcinogenicity
Hydrogen peroxide is classified by IARC as Group 3, not classifiable as to its carcinogenicity to
humans (Ref. 3).
There is only one carcinogenicity report (Ref. 5) cited in the CPDB (Ref. 6), in which mice were
treated with hydrogen peroxide in drinking water at 0.1 or 0.4% for approximately 2 years. The
study included two treatment groups and about 50 animals per dose group. Statistically significant
increases in tumors of the duodenum (p<0.005) were observed in both dose groups in the mouse
carcinogenicity study (Ref. 5) although only the duodenal tumors at the high dose in females are
noted as significant in the CPDB (Ref. 6). Thus, 0.1% hydrogen peroxide administered in drinking
water was defined as the Lowest Observed Adverse Effect Level (LOAEL), equivalent to an average
daily dose-rate per kg body weight per day of 167 mg/kg/day.
Studies of 6-month duration or longer are summarised in the following table (adapted from Ref. 2);
they are limited in the numbers of animals and used a single dose level. Most studies did not meet
the criteria for inclusion with a TD
50
calculation in the CPDB. DeSesso et al (Ref. 2) noted that, out
of 14 carcinogenicity studies (2 subcutaneous studies in mice, 2 dermal studies in mice, 6 drinking
water studies [2 in rats and 4 in mice], 1 oral intubation study in hamsters, and 3 buccal pouch
studies), only 3 mouse drinking water studies (Ref. 5, 8, 9) demonstrated increases in tumors (of
the proximal duodenum) with hydrogen peroxide. These mouse studies were thoroughly evaluated
by the Cancer Assessment Committee (CAC) of the US FDA (Ref. 10). The conclusion was that the
studies did not provide sufficient evidence that hydrogen peroxide is a carcinogen (Ref. 10).
In Europe, the Scientific Committee on Consumer Products reviewed the available data for
hydrogen peroxide and concluded that hydrogen peroxide did not meet the definition of a mutagen
(Ref.11) They also stated that the weak potential for local carcinogenic effects has an unclear
mode of action, but a genotoxic mechanism could not be excluded (Ref. 11). In contrast, DeSesso
99
et al (Ref. 2) suggested that dilute hydrogen peroxide would decompose before reaching the target
site (duodenum) and that the hyperplastic lesions seen were due to irritation from food pellets
accompanying a decrease in water consumption, which is often noted with exposure to hydrogen
peroxide in drinking water. The lack of a direct effect is supported by the lack of tumors in tissues
directly exposed via drinking water (mouth, oesophagus and stomach), and the fact that in studies
up to 6 months in the hamster (Ref. 14), in which hydrogen peroxide was administered by gastric
intubation (water intake was not affected), the stomach and duodenal epithelia appeared normal;
this was the basis for the US FDA conclusion above (Ref. 10).
Hydrogen Peroxide – Details of oral carcinogenicity studies
Study
Animals/
dose group
Duration/
Exposure
Controls
Doses
Notes
Ref. 5
*
48-51/sex/ group
C57BL/6J mice
100 weeks
Drinking
water
Yes
2:
0.1; 0.4%
M: 167; 667
F: 200; 800
mg/kg/d
TD
50
7.54 g/kg/d
for female duodenal
carcinoma
Ref. 7
29 mice
C57BL/6J
total male &
female
(additional groups
sampled at
intervals from 7 to
630 days of
treatment; or 10 –
30 days after
cessation of
treatment at 140
days)
700 days
Drinking
water
No
1:
0.4%
No tumors reported.
Time-dependent induction
of erosions and nodules in
stomach and nodules and
plaques in duodenum.
After a recovery period
following 140 days of
H
2
O
2
treatment, by 10 to
30 days without
treatment there were
fewer mice with lesions.
Ref. 8
18 C3H/HeN
mice
total male &
female
6 mo
Drinking
water
No
1:
0.4%
2 mice with duodenal
tumors (11.1%)
Ref. 8
22
B6C3F1 mice
total male &
female
6 mo
Drinking
water
No
1:
0.4%
7 mice with duodenal
tumors (31.8%)
Ref. 8
21 C57BL/6N
¢
mice
total male &
female
7 mo
Drinking
water
No
1:
0.4%
21 mice with duodenal
tumors (100%)
Ref. 8
24 C3HCb/s
¢
mice
total male &
female
6 mo
Drinking
water
No
0.4% only
22 mice with duodenal
tumors (91.7%)
Ref. 9
21 female
C3H/HeN mice
6 mo
Drinking
water
11
1:
0.4%
2 mice with duodenal
tumors (9.5%).
None in controls
Ref. 9
22 female B6C3F1
Mice
6 mo
Drinking
water
12
1:
0.4%
7 mice with duodenal
tumors (31.8%)
None in controls
Ref. 9
24 female
C3HCb/s
¢
mice
6 mo
Drinking
water
28
1:
0.4%
22 mice with duodenal
tumors (91.7%).
None in controls
Ref. 12
3 male rats
21 weeks
Drinking
water
3
1:
1.5%
No tumorigenic effect
observed
Ref. 13
Male and female
rats
2 years
Drinking
Yes
2:
0.3%
No tumorigenic effect
observed
100
(50/sex/group)
water
0.6%
Ref. 14
Hamsters, sex not
reported
(20/group)
15 weeks
and 6 mo
Oral gavage
(5 d/wk)
Yes
1:
70 mg/kg/d
No tumorigenic effect
observed
*
Carcinogenicity study selected for PDE calculation; in CPDB (Ref. 6).
All other studies are not in the CPDB but are summarized in Ref. 2
¢
Catalase deficient
Mode of action for carcinogenicity
Hydrogen peroxide is one of the reactive oxygen species (ROS) that is formed as part of normal
cellular metabolism (Ref. 4). The toxicity of hydrogen peroxide is attributed to the production of
ROS and subsequent oxidative damage resulting in cytotoxicity, DNA strand breaks and
genotoxicity (Ref. 15). Due to the inevitable endogenous production of ROS, the body has evolved
defense mechanisms to limit their levels, involving catalase, superoxide dismutases and glutathione
peroxidase.
Oxidative stress occurs when the body's natural antioxidant defense mechanisms are exceeded,
causing damage to macromolecules such as DNA, proteins and lipids. ROS also inactivate
antioxidant enzymes, further enhancing their damaging effects (Ref. 16). During mitochondrial
respiration, oxygen undergoes single electron transfer, generating the superoxide anion radical.
This molecule shows limited reactivity but is converted to hydrogen peroxide by the enzyme
superoxide dismutase. Hydrogen peroxide is then reduced to water and oxygen by catalase and
glutathione peroxidase (Ref. 17). However, in the presence of transition metals, such as iron and
copper, hydrogen peroxide is reduced further to extremely reactive hydroxyl radicals. They are so
reactive they do not diffuse more than one or two molecular diameters before reacting with a
cellular component (Ref. 16). Therefore, they must be generated immediately adjacent to DNA to
oxidize it. Antioxidants provide a source of electrons that reduce hydroxyl radicals back to water,
thereby quenching their reactivity. Clearly, antioxidants and other cellular defenses that protect
against oxidative damage are limited within an in vitro test system. Consequently, following
treatment with hydrogen peroxide these protective mechanisms are readily overwhelmed inducing
cytotoxicity and genotoxicity in bacterial and mammalian cell lines. Diminution of the in vitro
response has been demonstrated by introducing elements of the protective mechanisms operating
in the body; for example, introducing hydrogen peroxide degrading enzymes, such as catalase or
adjusting the level of transition metals (Ref. 11). Unsurprisingly, in vivo, where the cellular
defense mechanisms are intact, hydrogen peroxide is not genotoxic following short-term exposure.
This suggests that a threshold exists below which the cellular defense mechanisms can regulate
ROS maintaining homeostasis.
Based on the comprehensive European Commission (EC, Ref. 4) risk assessment, the weight of
evidence suggests hydrogen peroxide is mutagenic in vitro when protective mechanisms are
overwhelmed. However, it is not genotoxic in standard assays in vivo. Its mode of action has a
non-linear, threshold effect.
Regulatory and/or published limits
Annex III of the European Cosmetic Regulation (Ref. 18) provided acceptable levels of hydrogen
peroxide in oral hygiene and tooth whitening products. For oral products sold over the counter,
including mouth rinse, toothpaste and tooth whitening or bleaching products, the maximum
concentrations of hydrogen peroxide allowed (present or released) is 0.1%. Higher levels up to 6%
are also permitted providing products are prescribed by dental practitioners to persons over 18
years old. The EC SCCP (Ref. 11) estimated that 3 g of mouthwash or 0.48 g of toothpaste could
be ingested per day. With 0.1% hydrogen peroxide in the product, the amount of hydrogen
101
peroxide potentially ingested would be 3 mg from mouthwash or 0.48 mg from toothpaste. These
values may overestimate ingestion as it is likely that most of the hydrogen peroxide is decomposed
during use of oral care products and is not ingested (Ref. 4).
US FDA - hydrogen peroxide is Generally Recognized As Safe (GRAS) up to 3% for long-term over
the counter use as an anti-gingivitis/anti-plaque agent (Ref. 19).
Permissible daily exposure (PDE)
Hydrogen peroxide is genotoxic via a mode of action with a threshold (i.e., oxidative stress) and is
endogenously produced in the body at high levels that exceed the levels encountered in oral care
and other personal care products. Therefore it was not considered appropriate to derive a PDE
based on carcinogenicity data. Even an intake 1% of the estimated endogenous production of 6.8
g/day, that is, 68 mg/day (or 68,000 µg/day) would not significantly add to background exposure,
but would usually exceed limits based on quality, in a pharmaceutical. The ICH M7 guideline notes
that when calculating acceptable intakes from compound-specific risk assessments, an upper limit
would be determined by a quality limit of 0.5%, or, for example, 500 µg in a drug with a maximum
daily dose of 100 mg.
References
1. Halliwell B, Clement MV, Long LH. Hydrogen peroxide in the human body. FEBS Lett
2000;486:10-13.
2. DeSesso JM, Lavin AL, Hsia SM, Mavis RD. Assessment of the carcinogenicity associated with
oral exposures to hydrogen peroxide. Food and Chem Toxicol 2000;38:1021-41.
3. IARC. Re-evaluation of some organic chemicals, hydrazine and hydrogen peroxide. Monographs
on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. International Agency for
Research on Cancer, World Health Organization, Lyon. 1999 Vol. 71.
4. European Commission Joint Research Center. EU Risk Assessment report. Hydrogen Peroxide.
CASRN 7722-84-1). 38. [Online]2003. Available from: URL:
https://echa.europa.eu/documents/10162/a6f76a0e-fe32-4121-9d9d-b06d9d5f6852
5. Ito A, Watanabe H, Naito M, Naito Y. Induction of duodenal tumors in mice by oral
administration of hydrogen peroxide. Gann the Japanese Journal of Cancer Research 1981;72:
174-5.
6. Carcinogenicity Potency Database (CPDB). [Online]. Available from: URL:
http://toxnet.nlm.nih.gov/cpdb/
7. Ito A, Naito M, Naito Y, Watanabe H. Induction and characterization of gastro-duodenal lesions
in mice given continuous oral administration of hydrogen peroxide. Gann the Japanese Journal
of Cancer Research 1982;73: 315-322.
8. Ito A, Watanabe H, Naito M, Naito Y, Kawashima K. Correlation between induction of duodenal
tumor by hydrogen peroxide and catalase activity in mice. Gann the Japanese Journal of Cancer
Research 1984;75: 17-21.
9. Ito A, Watanabe H, Aoyama H, Nakagawa Y, Mori M. Effect of 1,2-dimethylhydrazine and
hydrogen peroxide for the duodenal tumorigenesis in relation to blood catalase activity in mice.
Hiroshima Journal of Medical Science 1986;35:197-200.
10. US FDA. Irradiation in the production, processing, and handling of food. Food and Drug
Administration. Federal Register 1988; Vol. 53, No. 251:53198-9.
102
11. SCCP. European Commission. Scientific Committee on Consumer Products. Opinion on
Hydrogen peroxide, in its free form or when released, in oral hygiene products and tooth
whitening products. SCCP/1129/07 [Online] 2007. Avalable from: URL:
https://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/sccp_o_122.pdf
12. Hiroto N. and Yokoyama T. Enhancing effeect of hydrogen peroxide upon duodenal and upper
jejunal carcinogenesis in rats. Gann 1981; 72: 811-812. Cited in Ref. 2.
13. Ishikawa T. and Takayama S. (1984) Hydrogen peroxide. In Information Bulletin on the Survey
of Chemicals being Tested for Carcinogenicity. International Agency for Research on Cancer,
Lyon. 1984; 11:86. (Cited in Ref. 2).
14. Li Y, Noblitt T, Zhang A, Origel A, Kafrawy A, Stookey G. Effect of long-term exposure to a
tooth whitener [Abstract]. Journal of Dental Research 1993;72:1162. (Cited in Ref. 2).
15. Tredwin CJ, Naik S, Lewis NJ, Scully C. Hydrogen peroxide tooth-whitening (bleaching)
products: Review of adverse effects and safety issues. British Dental Journal 2006;200:371-6.
16. De Bont R, Larebeke N. Endogenous DNA damage in humans: a review of quantitative data.
Mutagenesis 2004;19:169-85.
17. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature
2000;408:239-47.
18. Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November
2009 on cosmetic products.
19. US FDA. Oral health care drug products for over-the-counter human use;
antigingivitis/antiplaque drug products; establishment of a monograph. Federal Register 2003;
68:32232-86.
103
Methyl Chloride (Chloromethane, CAS# 74-87-3)
Potential for human exposure
Low levels of methyl chloride occur in the environment, since thousands of tons of methyl chloride
are produced naturally every day, e.g., by marine phytoplankton, by microbial fermentation, and
from biomass fires (burning in grasslands and forest fires) and volcanoes, greatly exceeding
release from human activities.
WHO (Ref. 1) reports that the methyl chloride concentration in the air in rural sites is in general
below 2.1 µg/m
3
(1.0 ppb) while in urban cities it is equal to 0.27 to 35 µg/m
3
(0.13-17 ppb),
corresponding to approximately 20-700 µg daily intake (human respiratory volume of 20 m
3
per
day). A wide range of concentrations is reported in rivers, ocean water, ground water and drinking
water, with the maximum drinking water level reported at 44 µg/L in a well sample (Ref. 1).
Mutagenicity/Genotoxicity
Methyl chloride is mutagenic and genotoxic in vitro but equivocal in vivo. WHO (Ref. 1) and US
EPA (Ref. 2) reviewed the mutagenicity data for methyl chloride; key observations are summarized
here.
Methyl chloride is mutagenic in:
Microbial reverse mutation assay (Ames), Salmonella typhimurium TA100, TA1535 and in
Escherichia coli WP2uvrA both in the presence and absence of metabolic activation;
TK6 human lymphoblasts.
In vivo, WHO (Ref. 1) concluded that “though data from standard in vivo genotoxicity studies are
not available, methyl chloride might be considered a very weak mutagen in vivo based on some
evidence of DNA–protein crosslinking at higher doses”.
Carcinogenicity
Methyl chloride is classified by IARC as Group 3: “Inadequate evidence for the carcinogenicity to
humans” (Ref. 3), and by US EPA as a Category D compound not classifiable as to human
carcinogenicity (Ref. 2).
In animals, the only evidence of carcinogenicity comes from a single 2-year bioassay that used the
inhalation route of administration in rats and mice (Ref. 4). A statistically significant increased
incidence of renal benign and malignant tumors was observed only in male B6C3F1 mice at the
high concentration (1,000 ppm). Although not of statistical significance, cortical adenoma was also
seen at 464 mg/m
3
(225 ppm), and development of renal cortical microcysts in mice was seen in
the 103 mg/m
3
(50 ppm) dose group and to some extent in the 464 mg/m
3
(225 ppm) group (Ref.
4). However, no concentration–response relationship could be established. Renal cortical
tubuloepithelial hyperplasia and karyomegaly were also confined to the 1,000-ppm group of male
mice. Neoplasias were not found at lower concentrations or at any other site in the male mouse, or
at any site or concentration in female mice or F-344 rats of either sex. Renal adenocarcinomas
have been shown to occur only in male mice at a level of exposure unlikely to be encountered by
people.
These renal tumors of the male mouse are not likely to be relevant to humans. Methyl chloride is
metabolized by glutathione conjugation and to a lesser extent by p450 oxidation (Ref. 1, 2). Renal
tumors in male mouse are thought to be related to the production of formaldehyde during methyl
chloride metabolism. The cytochrome P-450 (CYP) isozyme believed to be responsible, CYP2E1, is
present in male mouse kidney and is androgen-dependent; female mice had CYP2E1 levels only 20-
25% of those in males. Generation of formaldehyde has been demonstrated in renal microsomes
104
of male CD-1 mice that exceed that of naive (androgen-untreated) female mice, whereas kidney
microsomes from the rat did not generate formaldehyde. Additionally, species-specific metabolic
differences in how the kidney processes methyl chloride strongly suggest that renal mouse
neoplasms via P-450 oxidation are not biologically relevant to humans given that human kidney
lacks the key enzyme (CYP2E1) known to convert methyl chloride to toxic intermediates having
carcinogenic potential. In the rat, renal activity of CYP2E1 was very low. No CYP2E1 activity was
detected in human kidney microsomal samples (Ref. 2), nor was it detected in freshly isolated
proximal tubular cells from human kidney. CYP4A11 was detected in human kidney, but its ability
to metabolize methyl chloride is unknown. In addition to CYP4A11, the only other P-450 enzymes
found at significant levels in human renal microsomes are CYP4F2 and CYP3A. Moreover no
commonly known environmental chemicals appear to be metabolized by the CYP4A family. The
lack of detectable CYP2E1 protein in human kidney (in contrast to mice, which have high levels)
suggests that the metabolism of methyl chloride by P450 (presumably leading to elevated
formaldehyde concentrations) that is likely responsible for the induction of male mouse kidney
tumors are not likely relevant to humans.
However, as highlighted by the US EPA (Ref. 2) and WHO (Ref. 1), the role of hepatic (and/or
kidney) metabolism (leading to potential genotoxic metabolites) via the predominant glutathione
(GSH)-dependent pathway (metabolism of methyl chloride to formate in liver is GSH-dependent,
via the GSH-requiring formaldehyde dehydrogenase that oxidizes formaldehyde to formate) or even
by P450 isozymes other than CYP2E1 in this regard cannot be discounted. Nonetheless, production
of formaldehyde via low doses of methyl chloride would be negligible compared with the basal
formation of formaldehyde in the body (i.e., 878–1310 mg/kg/day; Ref. 5). In addition, based on
the limitations of human relevance, US EPA classified methyl chloride as a group D compound, that
is “Not Classifiable as to Human Carcinogenicity".
105
Methyl Chloride – Details of carcinogenicity studies (only inhalation studies available)
Study
Animals/
dose
group
Duration/
Exposure
Controls
Doses
Most sensitive
tumor
site/sex
TD
50
(mg/kg/d)
Ref. 4
(summarized
in Ref. 1 and
Ref. 2)*
120/sex/
group
B6C3F1
mice
24 mo
6h/d,
5d/wk
Inhalation
Yes
3:
103; 464;
2064 mg/m
3
(50; 225;
1000 ppm)
Kidney tumors
in males only.
No finding in
females.
1,360.7**
Ref. 4
(summarized
in Ref. 1 and
Ref. 2)
120/sex/
group
Fisher 344
rats
24 mo
6h/d,
5d/wk
Inhalation
Yes
3:
103; 464;
2064 mg/m
3
(50; 225;
1000 ppm)
No findings in
males and
females
NA
Note: Studies not listed in CPDB.
*
Carcinogenicity study selected for AI calculation.
**
TD
50
calculated based on carcinogenicity data (see Note 3).
NA = Not applicable
Regulatory and/or published Limits
WHO (Ref. 1) developed a guideline value for the general population of 0.018 mg/m
3
and US EPA
(Ref. 2) developed a reference concentration of 0.09 mg/m
3
. Both were based on the potential for
adverse CNS effects following inhaled methyl chloride.
Acceptable intake (AI)
While the data indicate the tumors observed in male mice are likely not relevant to humans, an AI
was developed because of the uncertainties in data.
Lifetime AI = TD
50
/50,000 x 50 kg
Lifetime AI = 1,360.7 mg/kg/day /50,000 x 50 kg
Lifetime AI = 1,361 μg/day
106
References
1. World Health Organization (WHO). Concise International Chemical Assessment Document
(CICAD) 28. Methyl chloride. [Online]. 2000; Available from: URL:
http://www.inchem.org/documents/cicads/cicads/cicad28.htm
2. US EPA. Methyl chloride. (CAS No. 74-87-3). Integrated Risk Information System (IRIS).
[Online]. 2001; Available from: URL:
https://cfpub.epa.gov/ncea/iris/iris_documents/documents/toxreviews/1003tr.pdf
3. IARC. Methyl Chloride. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to
Humans. International Agency for Research on Cancer, World Health Organization, Lyon. 1999
Vol. 71.
4. CIIT. Final report on a chronic inhalation toxicology study in rats and mice exposed to methyl
chloride. Report prepared by Battelle Columbus Laboratories for the CIIT. 1981 EPA/OTS Doc
#878212061, NTIS/OTS0205952.
5. EFSA. European Food Safety Authority. Endogenous formaldehyde turnover in humans
compared with exogenous contribution from food sources. EFSA Journal 2014; 12 Suppl 2:3550.
107
Note 1
The calculated TD
50
for 1-chloro-4-nitrobenzene is illustrated below since it was not listed in the
CPDB. 1-Chloro-4-nitrobenzene calculations were based on the most sensitive tumor type: female
rat pheochromocytoma (Ref. 1). The doses and incidences are listed below.
ppm
Dose (mg/kg/day)
Number of Positive
Animals
Total Number of
Animals
0
0
3
50
50
1.9
6
50
225
9.8
4
50
1000
53.8
16
50
\
The TD
50
is calculated from crude summary data of tumor incidence over background with the
following equation (Ref. 2, 3):
Where P is the proportion of animals with the specified tumor type observed at a certain dose (D in
the equation) and P
0
is the proportion of animals with the specified tumor type for the control.
Converting β and D into a simple linear equation results in the following:
Plotting the results and using the slope to represent β results in the following graph for the dose-
response and β = 0.0059302912.
y = 0.0059302912x
R² = 0.9353736173
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 20 40 60
108
The TD
50
can then be calculated as follows.
Solving for TD
50
results in in the following equation.
Therefore, the TD
50
= 0.693 / 0.0059302912 or 116.9 mg/kg/day.
References
1. Matsumoto M., Aiso S, Senoh H, Yamazaki K, Arito H, Nagano K, et al. Carcinogenicity and
chronic toxicity of para-chloronitrobenzene in rats and mice by two-year feeding. J. Environ
Pathol Toxicol Oncol 2006; 25:571-84.
2. Gaylor DW, Gold LS. Quick estimate of the regulatory virtually safe dose based on the
maximum tolerated dose for rodent bioassays. Regul Toxicol Pharmacol.1995; 22:57-63
3. Sawyer C, Peto R, Bernstein L, Pike MC. Calculation of carcinogenic potency from long-term
animal carcinogenesis experiments. Biometrics 1984; 40: 27-40.
109
Note 2
The calculated TD
50
for hydrazine is illustrated below since it was not listed in the CPDB. Hydrazine
calculations were based on the most sensitive tumor type: female rats, hepatocellular adenoma
and/or carcinoma (Ref. 1). The doses and incidences are listed below
ppm
Dose (mg/kg/day)
Number of Positive
Animals
Total Number of
Animals
0
0
1
50
20
1.28
0
50
40
2.50
3
50
80
5.35
6
50
The TD
50
is calculated from crude summary data of tumor incidence over background with the
following equation (Ref. 2, 3):
Where P is the proportion of animals with the specified tumor type observed at a certain dose (D in
the equation) and P
0
is the proportion of animals with the specified tumor type for the control.
Converting β and D into a simple linear equation results in the following:
Plotting the results and using the slope to represent β results in the following graph for the dose-
response and β = 0.0179164668.
y = 0.0179164668x
R² = 0.7898920304
-0.04
-0.02
0
0.02
0.04
0.06
0.08
0.1
0.12
0 2 4 6
110
The TD
50
can then be calculated as follows.
Solving for TD
50
results in in the following equation.
Therefore, the TD
50
= 0.693 / 0.0179164668 or 38.7 mg/kg/day.
References
1. Matsumoto M, Kano H, Suzuki M, Katagiri T, Umeda Y, Fukushima S. Carcinogenicity and
chronic toxicity of hydrazine monohydrate in rats and mice by two-year drinking water
treatment. Regul Toxicol Pharmacol 2016;76:63-73.
2. Gaylor DW, Gold LS. Quick estimate of the regulatory virtually safe dose based on the
maximum tolerated dose for rodent bioassays. Regul Toxicol Pharmacol.1995; 22:57-63.
3. Sawyer C, Peto R, Bernstein L, Pike MC. Calculation of carcinogenic potency from long-term
animal carcinogenesis experiments. Biometrics 1984; 40: 27-40.
111
Note 3
The calculated TD
50
for methyl chloride is illustrated below since it was not listed in the CPDB.
Since the methyl chloride study (Ref. 1, 2) is based on inhalation, the inhaled ppm concentrations
need to be converted to dose.
ppm
Dose (mg/kg/day)
1
Number of Positive
Animals
Total Number of
Animals
0
0
0
67
50
28
0
61
225
127
2
57
1000
566
22
86
1. ppm to mg/kg/day conversion – X ppm x 50.5 g/mol (mol weight)/24.45 x 0.043 (breathing
volume) x 6/24 hours x 5/7 days / 0.028 kg (mouse weight) = dose mg/kg/day
The TD
50
is calculated from crude summary data of tumor incidence over background with the
following equation (Ref. 3, 4):
Where P is the proportion of animals with the specified tumor type observed at a certain dose (D in
the equation) and P
0
is the proportion of animals with the specified tumor type for the control.
Converting β and D into a simple linear equation results in the following:
Plotting the results and using the slope to represent β results in the following graph for the dose-
response and β = 0.0005092936.
The TD
50
can then be calculated as follows.
y = 0.0005092936x
R² = 0.9821098671
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 200 400 600
Dose (mg/kg/day)
112
Solving for TD
50
results in in the following equation.
Therefore, the TD
50
= 0.693 / 0.0005092936 or 1360.7 mg/kg/day.
References
1. CIIT. Final report on a chronic inhalation toxicology study in rats and mice exposed to methyl
chloride. Report prepared by Battelle Columbus Laboratories for the CIIT. 1981 EPA/OTS Doc
#878212061, NTIS/OTS0205952.
2. US EPA. Toxicological review of methyl chloride. (CAS No. 74-87-3). In Support of Summary
Information on the IRIS. EPA/635/R01/003. 2001.
3. Gaylor DW, Gold LS. Quick estimate of the regulatory virtually safe dose based on the
maximum tolerated dose for rodent bioassays. Regul Toxicol Pharmacol.1995; 22:57-63.
4. Sawyer C, Peto R, Bernstein L, Pike MC. Calculation of carcinogenic potency from long-term
animal carcinogenesis experiments. Biometrics 1984; 40: 27-40.
