M7(R2) Addendum: 
Application of the Principles 
of the ICH M7 Guidance to 
Calculation of Compound-
Specific Acceptable Intakes 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
July 2023 
ICH-Multidisciplinary
   
M7(R2) Addendum: 
Application of the Principles 
of the ICH M7 Guidance to 
Calculation of Compound-
Specific Acceptable Intakes 
Guidance for Industry 
 
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U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
July 2023 
ICH-Multidisciplinary
  Contains Nonbinding Recommendations 
FOREWORD 
 
The International Council for Harmonisation of Technical Requirements for Pharmaceuticals 
for Human Use (ICH) has the mission of achieving greater regulatory harmonization 
worldwide to ensure that safe, effective, and high-quality medicines are developed, 
registered, and maintained in the most resource-efficient manner.  By harmonizing the 
regulatory expectations in regions around the world, ICH guidelines have substantially 
reduced duplicative clinical studies, prevented unnecessary animal studies, standardized 
safety reporting and marketing application submissions, and contributed to many other 
improvements in the quality of global drug development and manufacturing and the products 
available to patients.  
 
ICH is a consensus-driven process that involves technical experts from regulatory authorities 
and industry parties in detailed technical and science-based harmonization work that results 
in the development of ICH guidelines.  The commitment to consistent adoption of these 
consensus-based guidelines by regulators around the globe is critical to realizing the benefits 
of safe, effective, and high-quality medicines for patients as well as for industry.  As a 
Founding Regulatory Member of ICH, the Food and Drug Administration (FDA) plays a 
major role in the development of each of the ICH guidelines, which FDA then adopts and 
issues as guidance to industry.
  Contains Nonbinding Recommendations 
TABLE OF CONTENTS 
 
INTRODUCTION.................................................................................................................... 1 
METHODS ............................................................................................................................... 2 
I.  STANDARD METHOD (1) ........................................................................................ 3 
A.  Linear Mode of Action and Calculation of AI (1.1) .............................................................. 3 
B.  Selection of Studies (1.2) .......................................................................................................... 3 
C.  Selection of Tumor and Site (1.3) ............................................................................................ 4 
D.  Route of Administration (1.4) ................................................................................................. 4 
E.  Calculation of AI From the TD  (1.5) .................................................................................... 5 
50
II.  CONSIDERATION OF ALTERNATIVE METHODS FOR CALCULATION 
OF AI (2) ....................................................................................................................... 5 
A.  Human Relevance of Tumors (2.1) ......................................................................................... 5 
B.  Published Regulatory Limits (2.2) .......................................................................................... 5 
III.  NONLINEAR (THRESHOLD) MODE OF ACTION AND CALCULATION OF 
PDE (3) .......................................................................................................................... 6 
IV.  ACCEPTABLE LIMIT BASED ON EXPOSURE IN THE ENVIRONMENT 
(E.G., IN THE DIET) (4) ............................................................................................. 7 
LIST OF ABBREVIATIONS ................................................................................................. 8 
REFERENCES ....................................................................................................................... 11 
ACCEPTABLE INTAKES OR PERMISSIBLE DAILY EXPOSURES ......................... 12 
ACETALDEHYDE (CAS# 75-07-0) .................................................................................... 15 
REFERENCES (FOR ACETALDEHYDE) ........................................................................ 20 
ACRYLONITRILE (CAS# 107-13-1) .................................................................................. 23 
REFERENCES (FOR ACRYLONITRILE) ....................................................................... 28 
ANILINE (CAS# 62-53-3) AND ANILINE HYDROCHLORIDE (CAS# 142-04-1) ...... 30 
REFERENCES (FOR ANILINE AND ANILINE HYDROCHLORIDE) ....................... 34 
BENZYL CHLORIDE (-CHLOROTOLUENE, CAS# 100-44-7) ................................. 38 
REFERENCES (FOR BENZYL CHLORIDE) .................................................................. 44 
BIS(CHLOROMETHYL)ETHER (BCME, CAS# 542-88-1)............................................ 46 
REFERENCES (FOR BIS(CHLOROMETHYL)ETHER) ............................................... 49 
P-CHLOROANILINE (CAS# 106-47-8) AND P-CHLOROANILINE HCL (CAS# 
20265-96-7).............................................................................................................................. 50 
REFERENCES (FOR P-CHLOROANILINE AND P-CHLOROANILINE HCL) ........ 54 
1-CHLORO-4-NITROBENZENE (PARA-CHLORONITROBENZENE,  
CAS# 100-00-5)....................................................................................................................... 56 
REFERENCES (FOR 1-CHLORO-4-NITROBENZENE) ............................................... 60 
i  Contains Nonbinding Recommendations 
P-CRESIDINE (2-METHOXY-5-METHYL ANILINE, CAS# 120-71-8) ....................... 62 
REFERENCES (FOR P-CRESIDINE)................................................................................ 65 
1,2-DIBROMOETHANE (CAS# 106-93-4) ......................................................................... 67 
REFERENCES (FOR 1,2-DIBROMOETHANE) .............................................................. 71 
DIMETHYLCARBAMYL CHLORIDE (CAS# 79-44-7) .................................................. 73 
REFERENCES (FOR DIMETHYLCARBAMYL CHLORIDE) ..................................... 76 
DIMETHYL SULFATE (CAS# 77-78-1)............................................................................. 77 
REFERENCES (FOR DIMETHYL SULFATE) ................................................................ 81 
EPICHLOROHYDRIN (CAS# 106-89-8)............................................................................ 82 
REFERENCES (FOR EPICHLOROHYDRIN) ................................................................. 86 
ETHYL BROMIDE (CAS# 74-96-4) .................................................................................... 87 
REFERENCES (FOR ETHYL BROMIDE) ....................................................................... 90 
ETHYL CHLORIDE (CHLOROETHANE, CAS# 75-00-3) ............................................. 91 
REFERENCES (FOR ETHYL CHLORIDE) ..................................................................... 94 
FORMALDEHYDE (CAS# 50-00-0) ................................................................................... 95 
REFERENCES (FOR FORMALDEHYDE) ..................................................................... 102 
GLYCIDOL (CAS# 556-52-5) ............................................................................................ 106 
REFERENCES (FOR GLYCIDOL) .................................................................................. 109 
HYDRAZINE (CAS# 302-01-2) .......................................................................................... 110 
REFERENCES (FOR HYDRAZINE) ............................................................................... 114 
HYDROGEN PEROXIDE (CAS# 7722-84-1)................................................................... 116 
REFERENCES (FOR HYDROGEN PEROXIDE) .......................................................... 121 
METHYL CHLORIDE (CHLOROMETHANE, CAS# 74-87-3) ................................... 123 
REFERENCES (FOR METHYL CHLORIDE) ............................................................... 126 
STYRENE (CAS# 100-42-5) ............................................................................................... 127 
REFERENCES (FOR STYRENE) ..................................................................................... 132 
VINYL ACETATE (CAS# 108-05-4) ................................................................................. 135 
REFERENCES (FOR VINYL ACETATE) ...................................................................... 142 
NOTE 1 ................................................................................................................................. 144 
REFERENCES (FOR NOTE 1) ......................................................................................... 146 
NOTE 2 ................................................................................................................................. 147 
REFERENCES (FOR NOTE 2) ......................................................................................... 150 
NOTE 3 ................................................................................................................................. 151 
NOTE 4 ................................................................................................................................. 152 
REFERENCES (FOR NOTE 4) ......................................................................................... 154 
ii  Contains Nonbinding Recommendations 
NOTE 5 ................................................................................................................................. 155 
REFERENCES (FOR NOTE 5) ......................................................................................... 157 
 
iii  Contains Nonbinding Recommendations 
M7(R2) Addendum: Application of the Principles  
of the ICH M7 Guidance to Calculation  
of Compound-Specific Acceptable Intakes 
Guidance for Industry1 
 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) 
on this topic. It does not establish any rights for any person and is not binding on FDA or the public.  
You can use an alternative approach if it satisfies the requirements of the applicable statutes and 
regulations. To discuss an alternative approach, contact the FDA office responsible for this guidance 
as listed on the title page.   
 
 
 
INTRODUCTION 
 
The International Council for Harmonisation (ICH) guidance for industry M7(R2) Assessment 
and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential 
Carcinogenic Risk (ICH M7(R2)) (July 2023) discusses the derivation of acceptable intakes 
(AIs) for mutagenic impurities with positive carcinogenicity data, (section VII.B.1 (7.2.1)) 
and states:2  
 
Compound-specific risk assessments to derive acceptable intakes should be applied 
instead of the [threshold of toxicological concern-based] 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. 
 
In this addendum to ICH M7(R2), 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(R2).3 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(R2) of linear extrapolation 
 
1 This guidance was developed within the Expert Working Group (Multidisciplinary) of the International 
Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use 
(ICH) and has been subject to consultation by the regulatory parties, in accordance with the ICH process. This 
document has been endorsed by the ICH Assembly at Step 4 of the ICH process, April 2023. At Step 4 of the 
process, the final draft is recommended for adoption to the regulatory bodies of the ICH regions. 
2 We update guidances periodically. To make sure you have the most recent version of a guidance, check the 
FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents.  
3 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 AI will not typically be needed. 
1  Contains Nonbinding Recommendations 
from the calculated cancer potency estimate, the TD .4 Some chemicals that are mutagens 
50
and carcinogens (classified as Class 1 in ICH M7(R2)) may induce tumors through a 
nonmutagenic 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 
nonmutagenic; nevertheless, the historical perception has been that they are genotoxic 
carcinogens. 
 
ICH M7(R2) states in section VII.B.2 (7.2.2):  
 
The existence of mechanisms leading to a dose response that is nonlinear 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 (ICH guidance for industry Q3C(R8) Impurities: 
Guidance for Residual Solvents [ICH Q3C(R8)] (December 2021)) 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(R8) (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(R2) guidance (section VII.B.2 (7.2.2)) notes that when calculating 
AIs from compound-specific risk assessments, an upper limit would be 0.5 percent, or, for 
example, 500 micrograms in a drug with a maximum daily dose of 100 milligrams (mg). 
 
METHODS 
 
The general approach used in this addendum for deriving AIs included a literature review, 
selection of cancer potency estimate (TD ), taken from the CPDB (Carcinogenic Potency 
50
Database (Ref. 2), 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 III (3) of this addendum). The literature 
review focused on data relating to exposure of the general population (i.e., food, water, air), 
mutagenicity/genotoxicity, and carcinogenicity. Based on the description of DNA-reactive 
mutagens in ICH M7(R2), 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., U.S. Environmental Protection Agency (EPA), U.S. Food and Drug 
Administration, European Medicines Agency, European Chemicals Agency, World Health 
 
4 TD  is defined as the chronic dose-rate in milligrams per kilograms of body weight per day, which would 
50
cause tumors in half of the animals at the end of a standard life span for the species taking into account the 
frequency of that tumor type in control animals. 
2  Contains Nonbinding Recommendations 
Organization (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, methemoglobinemia). 
 
 
I.  STANDARD METHOD (1)5 
 
A.  Linear Mode of Action and Calculation of AI (1.1) 
 
Note 4 of ICH M7(R2) states:  
 
It is possible to calculate a compound-specific acceptable intake based on rodent 
carcinogenicity potency data such as TD  values (doses giving a 50 percent tumor 
50
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  by 50,000. This procedure is similar to that employed for 
50
derivation of the TTC. 
 
Thus, linear extrapolation from a TD  value was considered appropriate to derive an AI for 
50
those Class 1 impurities (known mutagenic carcinogens) with no established threshold 
mechanism, that is, understanding of a mode of action that results in a nonlinear 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 precalculated TD  value was identified in the CPDB for a 
50
selected chemical, this value was used to calculate the AI; the relevant carcinogenicity data 
were not reanalyzed and the TD  value was not recalculated. 
50
 
If robust data were available in the literature but not in the CPDB, then a TD  was calculated 
50
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(R8) and the ICH guidance for industry Q3D(R2) Elemental Impurities (ICH Q3D(R2)) 
(September 2022) (Ref. 1, 4). 
 
B.  Selection of Studies (1.2) 
 
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: 
 
  Less than 50 animals per dose per sex 
  Less than three dose levels 
  Lack of concurrent controls 
  Intermittent dosing (less than 5 days per week)  
  Dosing for less than lifetime 
 
5 The numbers in parentheses reflect the organizational breakdown of the document endorsed by the ICH 
Assembly at Step 4 of the ICH process, April 2023. 
3  Contains Nonbinding Recommendations 
 
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 as defined by the National Toxicology Program or 
the ICH guidance for industry S1C(R2) Dose Selection for Carcinogenicity Studies of 
Pharmaceuticals (September 2008) (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 three 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  was linearly extrapolated to a 1 in 100,000 excess cancer risk. In these cases, the 
50
rationale supporting the basis for the recommended approach is provided in the compound-
specific assessments. 
 
C.  Selection of Tumor and Site (1.3) 
 
The lowest TD  of a particular organ site for an animal species and sex was selected from 
50
the most robust studies. When more than one study exists, the CPDB provides a calculated 
harmonic mean TD , but in this addendum the lowest TD  was considered a more 
50 50
conservative estimate. Data compiled as all tumor-bearing animals were not considered in 
selecting an appropriate TD  from the CPDB; mixed tumor types (e.g., adenomas and 
50
carcinomas) in one tissue (e.g., liver) were used where appropriate because this often gives a 
more sensitive potency estimate. 
 
D.  Route of Administration (1.4) 
 
Section VII.E (7.5) of ICH M7(R2) states:  
 
The above risk approaches described in section VII (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  from the route 
50
with the lowest TD  value was selected for the AI calculation and is thus usually considered 
50
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 ICH M7(R2). 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  was lower than for other routes, then a separate AI was developed for that 
50
route (e.g., dimethyl carbamoyl chloride, hydrazine). 
 
4  Contains Nonbinding Recommendations 
E.  Calculation of AI From the TD  (1.5) 
50
 
Calculating the AI from the TD  is as follows (see Note 4 of ICH M7(R2) for example):  
50
 
AI = TD /50,000 x 50 kilograms (kg) 
50
 
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 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. 
 
 
II.  CONSIDERATION OF ALTERNATIVE METHODS FOR CALCULATION 
OF AI (2) 
 
A.  Human Relevance of Tumors (2.1) 
 
Note 4 of ICH M7(R2) states:  
 
As an alternative of using the most conservative TD  value from rodent 
50
carcinogenicity studies irrespective of its relevance to humans, an in-depth 
toxicological expert assessment of the available carcinogenicity data can be done 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 nonlinear dose response are not 
relevant to humans at the low, nontoxic 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 nonlinear 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 , were used for the linear extrapolation to calculate the AI because a mutagenic 
50
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. 
 
B.  Published Regulatory Limits (2.2) 
 
Note 4 of ICH M7(R2) also states:  
 
Compound-specific acceptable intakes can also be derived from published 
recommended values from internationally recognized bodies such as the 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. 
 
5  Contains Nonbinding Recommendations 
In this addendum, available regulatory limits are described (omitting occupational health 
limits because they are typically regional and may use different risk levels). However, the 
conservative linear extrapolation from the TD  was generally used as the primary method to 
50
derive the AI, as the default approach of ICH M7(R2), and for consistency across 
compounds. It is recognized that minor differences in methodology for cancer risk 
assessment can result in different recommended limits (e.g., adjusting for body surface area 
in calculations), but the differences are generally quite small when linear extrapolation is the 
basis of the calculation. 
 
 
III.  NONLINEAR (THRESHOLD) MODE OF ACTION AND CALCULATION 
OF PDE (3) 
 
ICH M7(R2) states in section VII.B.2 (7.2.2):  
 
The existence of mechanisms leading to a dose response that is nonlinear 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 (ICH guidance for industry Q3C(R8) Impurities: 
Guidance for Residual Solvents (December 2021)) 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 II.A (2.1) of this addendum) that lack human 
relevance at low doses, based upon their association with a nonlinear dose response for tumor 
induction: 
 
  Chemicals that induce methemoglobinemia, hemosiderin deposits in tissues such as 
spleen, and subsequent inflammation and tumors (e.g., aniline, 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, nonirritating 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 because abundant endogenous protective mechanisms exist (e.g., 
hydrogen peroxide) 
6  Contains Nonbinding Recommendations 
 
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(R8) (Ref. 1) 
and ICH Q3D(R2) (Ref. 4). 
 
 
IV.  ACCEPTABLE LIMIT BASED ON EXPOSURE IN THE ENVIRONMENT 
(E.G., IN THE DIET) (4) 
 
As noted in ICH M7(R2) section VII.E (7.5), Higher acceptable intakes may be justified 
when human exposure to the impurity will be much greater from other sources e.g., food, 
endogenous metabolism (e.g., formaldehyde). 
 
For example, formaldehyde is not a carcinogen orally, so regulatory limits have been based 
on noncancer endpoints. Health Canada (Ref. 8), WHO IPCS (Ref. 9), and the U.S. EPA 
(Ref. 10) recommend an oral limit of 0.2 mg/kg/day, or 10 mg/day for a 50 kg person. 
7  Contains Nonbinding Recommendations 
LIST OF ABBREVIATIONS 
 
AI    acceptable intake 
 
ACGIH  American Conference of Governmental Industrial Hygienists 
 
API    active pharmaceutical ingredient 
 
ATSDR  Agency for Toxic Substances and 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   Carcinogenic Potency Database 
 
CYP    cytochrome P450 
 
DMCC   Dimethylcarbamyl chloride  
 
DMS    dimethyl sulfate 
 
DNA    deoxyribose nucleic acid 
 
DP    drug product 
 
EC    European Commission  
 
ECHA   European Chemicals Agency 
 
EFSA    European Food Safety Authority  
 
EMA    European Medicines Agency 
 
EPA    Environmental Protection Agency 
 
8  Contains Nonbinding Recommendations 
EU    European Union 
 
FDA    Food and Drug Administration  
 
GRAS   generally recognized as safe  
 
HSDB   Hazardous Substance Data Bank 
 
IARC    International Agency for Research on Cancer 
 
i.p.    intraperitoneal 
 
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 
 
NA    not applicable 
 
NC  not calculated; individual tumor type incidences not provided in the World 
Health Organization, 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   Organization for Economic Co-operation and Development  
 
OSHA   Occupational Safety and Health Administration 
 
PCE    polychromatic erythrocytes 
 
PDE    permissible daily exposure 
 
RfC    reference concentration 
 
ROS    reactive oxygen species 
9  Contains Nonbinding Recommendations 
 
s.c.    subcutaneous 
 
SCCP    Scientific Committee on Consumer Products  
 
SCCS    Scientific Committee on Consumer Safety  
 
SCE    sister chromatid exchange 
 
SIDS    Screening Information Dataset 
 
TBA    tumor-bearing animal 
 
TD   Chronic dose-rate in milligrams per kilograms of body weight per day, which 
50
would cause tumors in half of the animals at the end of a standard life span for 
the species taking into account the frequency of that tumor type in control 
animals 
 
TDI    tolerable daily intake 
 
TTC-based  threshold of toxicological concern-based  
 
UDS    unscheduled DNA synthesis 
 
UNEP   United Nations Environment Programme 
 
U.S. EPA  United States Environmental Protection Agency  
 
WHO    World Health Organization 
 
10  Contains Nonbinding Recommendations 
REFERENCES 
 
1.  International Council for Harmonisation (ICH) guidance for industry Q3C(R8) 
Impurities: Guidance for Residual Solvents (December 2021)1 
 
2.  Carcinogenic Potency Database (CPDB), website available at 
https://files.toxplanet.com/cpdb/index.html. 
 
3.  Carcinogenic Potency Database (CPDB), website available at 
https://files.toxplanet.com/cpdb/index.html. 
 
4.  ICH guidance for industry Q3D(R2) Elemental Impurities (September 2022) 
 
5.  ICH guidance for industry S1C(R2): Dose Selection for Carcinogenicity Studies of 
Pharmaceuticals (September 2008) 
 
6.  Muller L, Gocke E, Lave T, and Pfister T, 2009, Ethyl Methanesulfonate Toxicity in 
Viracept-A Comprehensive Human Risk Assessment Based on Threshold Data for 
Genotoxicity, Toxicol Lett, 190(3):317329. 
 
7.  Cao X, Mittelstaedt RA, Pearce MG, Allen BC, Soeteman-Hernandez LG, Johnson GE, et 
al., 2014, Quantitative Dose-Response Analysis of Ethyl Methanesulfonate Genotoxicity 
in Adult gpt-delta Transgenic Mice, Environ Mol Mutagen, 55(5):385399. 
 
8.  Health Canada, 2001 Priority Substances List Assessment Report for Formaldehyde, 
Ottawa, Ministry of Public Works and Government Services, available at 
https://www.canada.ca/en/health-canada/services/environmental-workplace-
health/reports-publications/environmental-contaminants/canadian-environmental-
protection-act-1999-priority-substances-list-assessment-report-formaldehyde.html.  
 
9.  Liteplo RG, Beauchamp R, Chenier R, Meek ME, World Health Organization (WHO), et 
al., 2002,, Concise International Chemical Assessment Document 40: Formaldehyde, 
available at https://apps.who.int/iris/bitstream/handle/10665/42430/a73769.pdf.  
 
10. U.S. Environmental Protection Agency, 1990, Integrated Risk Information System (IRIS), 
available at https://www.epa.gov/iris/. 
 
 
1 We update guidances periodically. To make sure you have the most recent version of a guidance, check the 
FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents.  
11  Contains Nonbinding Recommendations 
ACCEPTABLE INTAKES OR PERMISSIBLE DAILY EXPOSURES 
 
Chemical  AI or PDE 
Compound  CAS#  Comment 
Structure  (g/day) 
Linear Extrapolation From TD
50 
Acrylonitrile  107-13-1    6  TD50 linear 
extrapolation 
Benzyl chloride  100-44-7    41  TD50 linear 
extrapolation 
Bis(chloromethyl)ether  542-88-1    0.004  TD50 linear 
extrapolation 
1-Chloro-4-  100-00-5    117  TD50 linear 
nitrobenzene  extrapolation 
p-Cresidine  120-71-8    45  TD50 linear 
extrapolation 
1,2-Dibromoethane  106-93-4    2  TD50 linear 
extrapolation 
Dimethylcarbamyl  79-44-7    0.6 (inhalation)*  TD50 linear 
chloride  5 (all other  extrapolation 
routes) 
 
Epichlorohydrin  106-89-8    3  TD50 linear 
extrapolation 
Ethyl bromide  74-96-4    32  TD50 linear 
extrapolation 
Ethyl chloride  75-00-3    1,810  TD50 linear 
extrapolation 
Glycidol  556-52-5    4  TD50 linear 
extrapolation 
Hydrazine  302-01-2    0.2 (inhalation)*  TD50 linear 
extrapolation 
39 (all other 
routes) 
Methyl chloride  74-87-3     1,361  TD50 linear 
extrapolation 
Styrene  100-42-5    154  TD50 linear 
extrapolation 
12  Contains Nonbinding Recommendations 
 
Chemical  AI or PDE 
Compound  CAS#  Comment 
Structure  (g/day) 
Threshold-Based PDE 
Aniline  62-53-3    720  PDE based on 
Aniline hydrochloride  142-04-1  threshold mode of 
action 
(hemosiderosis) 
Endogenous and/or Environmental Exposure 
Acetaldehyde  75-07-0    2,000 (oral)*  Oral PDE is based 
185 (all other  on average food 
routes)  intake; all other 
routes based on 
TD  linear 
50
extrapolation from 
an inhalation study 
Formaldehyde  50-00-0    8,000 or 215  Inhalation route 
ppb, whichever  based on TD50 
is lower  linear extrapolation 
(inhalation)*  or local irritation; 
all other routes 
10,000 (all other 
based on average 
routes) 
food intake 
 
Hydrogen peroxide  7722-84-1      68,000 or 0.5%,  68 mg/day is 1% of 
whichever is  estimated 
lower  endogenous 
production 
Vinyl acetate  108-05-4    2,000 (oral)*  Oral PDE is based 
758 (all other  on average food 
routes)  intake for 
acetaldehyde; all 
other routes based 
on TD  linear 
50
extrapolation from 
an inhalation study 
13  Contains Nonbinding Recommendations 
 
Chemical  AI or PDE 
Compound  CAS#  Comment 
Structure  (g/day) 
Other Cases 
p-Chloroaniline  106-47-8    34  AI based on liver 
p-Chloroaniline  20265-96-7  tumors for which 
hydrochloride  mutagenic mode of 
action cannot be 
ruled out 
Dimethyl sulfate  77-78-1    1.5  Carcinogenicity 
data available, but 
inadequate to 
derive AI. Default 
to TTC 
* Route specific limit 
CAS = Chemical Abstracts Service; AI = acceptable intake; PDE = permissible daily exposure; g = 
microgram; TD  = chronic dose-rate in milligrams per kilogram of body weight per day, which would cause 
50
tumors in half of the animals at the end of a standard life span for the species; ppb = parts per billion; mg = 
milligrams; TTC = threshold of toxicological concern. 
 
14  Contains Nonbinding Recommendations 
ACETALDEHYDE (CAS# 75-07-0) 
 
Potential for Human Exposure 
 
Acetaldehyde is formed endogenously in the human body from the metabolism of ethanol 
and carbohydrates as well as from bacteria in the alimentary tract. Humans are exposed to 
acetaldehyde mainly in food, alcoholic beverages, and cigarette smoke and to a lesser extent 
from environmental emissions (Ref. 1, 2). The determination of endogenous acetaldehyde in 
blood, breath, and saliva is challenging as the techniques are prone to artifacts and 
contaminants (Ref. 3, 4). Nevertheless, a daily endogenous production of 360 milligrams 
(mg)/day of acetaldehyde was calculated based on a constant endogenous total acetaldehyde 
concentration in the blood of 2.2 1.1 micromole/liter (L) (Ref. 3) and acetaldehyde 
clearance of 0.95 L/minute (Ref. 5). Average acetaldehyde consumption of up to 48 mg/day 
comes from consumption of alcoholic beverages (Ref. 6). Endogenous acetaldehyde 
concentrations and the associated cancer risk are significantly higher in individuals with an 
acetaldehyde dehydrogenase-2 (ALDH2) genetic polymorphism (Ref. 7). The exogenous 
exposure from food (without alcoholic beverages or added acetaldehyde as flavoring agent) 
was estimated to be around 2 mg/day on average and 8 mg/day at the 95th percentile of the 
German population (Ref. 8). The Joint Food and Agriculture of the United Nations and the 
World Health Organization Expert Committee on Food Additives (JECFA) estimated food 
additive consumption to be 9.7 mg/day in the United States and 11 mg/day in Europe 
although this estimate is restricted to consumers who eat foods in which acetaldehyde is 
added as a flavor (Ref. 9). The Japanese Food Safety Committee (FSC) estimated domestic 
consumption to be 9.6 mg/day in Europe and 19.2 mg/day in the United States (Ref. 10). 
Acetaldehyde is used in synthesis of pharmaceuticals. 
 
Mutagenicity/Genotoxicity 
 
The genotoxicity of acetaldehyde has been previously reviewed by the Chemical Evaluation 
and Research Institute, Japan (Ref 11), and other authors (Ref. 1, 5, 12 to 18). Acetaldehyde 
was negative in Ames mutation assays, but induced increases in mutations at the 
hypoxanthine-guanine-phosphoribosyl transferase (HPRT) locus in mammalian cells, which 
included point mutations as demonstrated by sequencing (Ref. 13). DNA and DNA-protein 
adducts were observed in cultured cells treated with acetaldehyde (Ref. 14, 15), and DNA 
adducts were measured in urine of healthy volunteers and in blood cells from persons who 
abuse alcohol (Ref. 5). Acetaldehyde is primarily an inducer of larger scale chromosomal 
effects. It induces chromosomal aberrations and micronuclei in vitro and was positive in the 
mouse lymphoma L5178Y TK+/- assay (Ref 13). Acetaldehyde induced increases in 
micronuclei in the bone marrow of rats and mice (Ref 17).   
 
Carcinogenicity 
 
Acetaldehyde is an International Agency for Research on Cancer (IARC) Group 2B 
carcinogen, and acetaldehyde associated with the consumption of alcoholic beverages is an 
IARC 1 carcinogen (i.e., carcinogenic to humans). Acetaldehyde was carcinogenic in rats and 
hamsters after inhalation exposure (Ref. 1).  
 
In humans, acetaldehyde is the primary metabolite of alcohol, and both high and low alcohol 
consumption has been correlated with an increased relative risk for certain human cancers 
(e.g., oral cavity, pharynx cancer, breast cancer) (Ref. 19, 20). The relative risk was increased 
15  Contains Nonbinding Recommendations 
in smokers with a high alcohol consumption and a possible contribution of acetaldehyde 
derived from cigarette smoke (Ref. 19). Also, geographical regions with consumption of 
alcoholic beverages containing high acetaldehyde concentrations showed a tendency for 
higher incidence of squamous-cell cancer and cancer of the esophagus (Ref. 21). 
Furthermore, available epidemiological data indicate that there is an increased risk for 
development of alcohol-related cancers for those individuals who are deficient in detoxifying 
acetaldehyde to acetate by ALDH. Especially the genetic variant ALDH2*1/*2 is strongly 
associated with alcohol-related cancers in not only heavy drinkers but those with moderate 
levels of alcohol consumption (Ref. 1, 7, 19). Meta analyses and large cohort studies report 
conflicting conclusions about whether there are increased risks of head, neck, and mammary 
tumors associated with moderate alcohol consumption in the U.S. populations where ALDH 
deficiency is relatively infrequent. The literature on the elevated risk of head and neck 
cancers associated with acetaldehyde exposure in heavy drinkers and smokers and in 
moderate drinkers with ALDH deficiency does not include discussion of whether those 
exposures are also associated with histopathological changes consistent with irritation or 
tissue proliferation (Ref. 22). 
 
In rodents, only inhalation carcinogenicity studies are available in the Carcinogenic Potency 
Database (CPDB) (Ref. 23). The most robust study was conducted with Wistar rats (Ref. 24) 
with whole-body inhalation exposure to 0, 750, 1500, or 3000/1000 parts per million (ppm) 
(reduced after 11 months due to toxicity), 6  hours/day at 5 days/week for up to 28 months. 
The corresponding doses in the CPDB were 0, 70.8, 142, and 147 mg/kilogram (kg) for male 
rats and 0, 101, 202, and 209 mg/kg for female rats. In the high-dose group, 50 percent of the 
male and 42 percent of the female animals had died by week 67 and no high-dose animals 
were alive by week 102. An increased incidence of tumors at the site of contact (i.e., nasal 
squamous cell carcinomas) was observed in males (1/49, 1/52, 10/53, and 15/49 in control 
and low-, mid-, and high-dose groups) and females (0/50, 0/48, 5/53, and 17/53, respectively) 
at the end of the study. There were also increases in nasal adenocarcinomas at all doses, the 
incidences were 0/49, 16/52, 31/53, and 21/49 in males and 0/50, 6/48, 26/53, and 21/53 in 
females, respectively. Based on these data, the TD  value in the CPDB was estimated to be 
50
185 mg/kg for nasal adenocarcinoma in male rats in the most sensitive sex and tissue.  
 
An oral carcinogenicity study (Ref. 25) was conducted in Sprague Dawley rats with 
acetaldehyde administration in drinking water. In this study, 50 rats per group were given 0, 
50, 250, 500, 1500, and 2500 mg/L acetaldehyde in drinking water for 104 weeks, and the 
experiment was terminated when the last animal died at 161 weeks of age. The 
concentrations correspond to 0, 5, 25, 49, 147, and 246 mg/kg/day for male rats and 0, 5, 27, 
53, 155, and 260 mg/kg/day for female rats, respectively. Incidences of adenocarcinomas, 
lymphomas and leukemias, mammary tumors, and cranial osteosarcomas, were described by 
the investigators as significantly greater in at least one group of exposed rats, relative to 
control. There was no increase in malignant tumors at the site of contact organs (i.e. the oral 
cavity and gastrointestinal tract) or in the liver. This study suggests that acetaldehyde may be 
carcinogenic after intake via drinking water. However, there was no clear dose-response 
relationship, and therefore, many evaluators found that no clear conclusion can be drawn 
from this study (Ref. 5, 12, 19). In another evaluation of the same data, two different dose-
response models were used to estimate cancer potency, and the authors concluded that their 
quantitative risk assessment indicates the need to lower acetaldehyde exposure in the general 
population but also acknowledged that naturally occurring acetaldehyde cannot be reduced 
(Ref. 21). In this model, the carcinogenic potency was calculated for all tumor-bearing 
animals because the authors found that there was insufficient statistical power to generate a 
16  Contains Nonbinding Recommendations 
model for any specific cancer site. A TD  related to oral administration of acetaldehyde was 
50
not calculated.  
 
Acetaldehyde  Details of Carcinogenicity Studies 
 
Animals/  Duration/  Most Sensitive  TD50 
Study  Dose  Controls  Doses  Tumor  (mg/kg/ 
Exposure 
Group  Site/Sex  day) 
Ref. 24  55/sex/  28 months,  55  3:   Nasal / Adeno- 185a 
group   inhalation  M: 70.8;  carcinoma / 
Wistar  142; 147  Male 
rat  mg/kg/day 
  F: 101; 202; 
209 
mg/kg/day 
 
Ref. 25  50/sex/  24 months,  50  5:  Not identifiable  NCb 
  group   drinking  M: 5; 25; 49; 
Sprague  water  147; 246 
Dawley    mg/kg/day 
rat    F: 5; 27; 53; 
  155; 260 
mg/kg/day 
 
Ref. 26  30/sex/  52 weeks,  30  1:  Larynx / Mixed  461 
  group   Inhalation  M: 344  tumor type / 
Syrian  mg/kg/day   Male 
golden  F: 391 
hamster  mg/kg/day 
 
Studies listed are in the Carcinogenic Potency Database (CPDB) (Ref. 24). 
mg = milligram; kg = kilogram; NC = not calculated.  
a TD  taken from the CPDB, carcinogenicity study selected for acceptable intake derivation. 
50
b The TD  was not calculated because of lack of dose response and sufficient statistical power; the study is not 
50
presented in the CPDB. 
 
Mode of Action for Carcinogenicity 
 
Acetaldehyde is a strong electrophile and is capable of reacting with strong nucleophiles, for 
example DNA bases or amino acid residues on proteins. Although not mutagenic in the 
standard bacterial reversion assay, evidence for DNA reactivity and mutagenicity was shown 
for acetaldehyde by the presence of DNA and DNA-protein adducts in vitro and in vivo, as 
well as by the positive result in the in vitro HPRT mutagenicity assay in mammalian cells. 
Despite its reactive nature, there is evidence for a nonlinear dose response associated with the 
genotoxicity and carcinogenicity of acetaldehyde (Ref. 14). The dose-response of 
acetaldehyde-induced adducts at concentrations between 1 and 1,000 M has been measured 
in a cell culture system allowing the discrimination between endogenous and exogenous 
adducts induced by added acetaldehyde. These concentrations are comparable to salivary 
acetaldehyde concentrations measured before and after consumption of beverages containing 
alcohol with or without acetaldehyde (Ref. 27, 28). The exogenous adducts only exceeded the 
endogenous background level of adducts above a critical concentration.   
17  Contains Nonbinding Recommendations 
 
ALDH, which efficiently detoxifies acetaldehyde, is responsible for the nonlinear dose-
response relationship. ALDH enzymes are expressed in the mitochondria and cytosol of most 
tissues (e.g., liver, gastrointestinal tract, kidneys, nasal epithelium/olfactory epithelium, 
lung), and they metabolize acetaldehyde to form acetate and one proton (Ref. 29). The release 
of protons can reduce cellular pH and thus cause nonspecific cytotoxicity with subsequent 
proliferative effects. The importance of detoxification was shown in ALDH-deficient animal 
models. For example, acetaldehyde-induced chromosome damage and mutation is observed 
in mice deficient in ALDH2 activity following inhalation and oral (gavage) exposure, but not 
in ALDH2-proficient mice (Ref. 30). Similarly, more acetaldehyde-derived DNA adducts 
were seen in alcoholics with a deficient aldehyde dehydrogenase genotype (allelic variant 
type ALDH2*1/2*2 with about 10 percent residual ALDH activity) compared to those with 
efficient genotype ALDH2*1/2*1 (Ref. 31), and moderate drinkers with the genotype are at 
increased risk of head and neck cancers (Ref. 19).  
 
The inhalation carcinogenicity data and mechanistic study data suggest that acetaldehyde 
cancer risk is highest at and possibly limited to the site of contact. The nasal tumors in 
inhalation carcinogenicity studies were only found at inhalation doses also associated with 
cytotoxicity and severe irritation causing regenerative proliferation consistent with the 
hypothesis that there could be promotion of growth of mutated cells (Ref. 5, 14). 
Detoxification of acetaldehyde by ALDH in airway cells may make tumor induction less 
likely at lower, nonirritating doses. However, there are no published measurements that 
would allow discrimination between the irritating effect and the potential mutagenic effect in 
cancer development.    
 
Regulatory and/or Published Limits 
 
Acetaldehyde is listed in the U.S. Food and Drug Administrations (FDAs) generally 
recognized as safe list for flavoring substances and adjuvants (21 CFR 182.60) (Ref 32). The 
Japanese FSC has no safety concerns when acetaldehyde is used as a flavoring agent because 
it is completely metabolized into nonreactive acetic acid and finally carbon dioxide, and thus, 
its level as a flavoring agent is presumed not to exceed the physiological range (Ref. 10). The 
JECFA evaluation has concluded that there are no safety concerns at current levels of intake 
when used as a flavoring agent (Ref. 9).  
 
The Committee on Emergency and Continuous Exposure Guidance Levels for Selected 
Submarine Contaminants (Ref. 33) recommended a continuous exposure guidance level of 2 
ppm corresponding to 3.6 mg/cubic meter (m3). This represents an exposure of 3.6 mg/m3 
times 28.8 m3 (24 hours in a day  the International Council for Harmonisation (ICH) 
guidance for industry Q3C(R8) Impurities: Guidance for Residual Solvents (December 2021) 
assumption)1 equals 104 mg/day. 
 
The U.S. Environmental Protection Agency (EPA) did not consider a threshold for 
acetaldehyde carcinogenicity and has calculated that a concentration of 5 micrograms (g)/m3 
acetaldehyde represents a 10-5 (1 in 100,000) excess lifetime cancer risk based on the rat 
inhalation carcinogenicity study and application of linear extrapolation (Ref. 34). For a 24-
 
1 We update guidances periodically.  To make sure you have the most recent version of a guidance, check the 
FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 
18  Contains Nonbinding Recommendations 
hour exposure, this represents 5 g/m3 times 28.8 m3 equals 144 g/day. EPA did not 
consider the risk via the oral route. 
 
Permissible Daily Exposure (PDE) for Oral Exposure 
 
Rationale for selection of study for PDE calculation 
 
Given the weight of evidence for a nonlinear dose response for the carcinogenicity of 
acetaldehyde following oral administration and high background exposure from a wide 
variety of foods, a PDE of 2 mg/day is identified for oral limit based on the estimated average 
intake of acetaldehyde from food of around 2 mg/day (Ref. 8).  
 
PDE (oral) = 2 mg/day 
 
Acceptable Intake (AI) for All Other Routes 
 
Rationale for selection of study for AI calculation 
 
The inhalation study in rats by Woutersen et al. (Ref. 24) was used to derive the AI for all 
other routes. This study comprises group sizes of 55/sex/dose, and animals were treated for 
lifetime (i.e., 28 months). According to the ICH guidance for industry M7(R2) Assessment 
and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential 
Carcinogenic Risk recommendations for selecting the most relevant study for deriving an AI, 
this is considered the most appropriate and robust study available for acetaldehyde. The 
inhalation carcinogenicity data and mechanistic study data suggest acetaldehyde cancer risk 
to be associated with cytotoxicity at the site of contact as nasal tumors were only found at 
doses also associated with cytotoxicity and severe irritation causing regenerative proliferation 
a promotion of growth of mutated cells.  
 
Calculation of AI 
 
Lifetime AI = TD /50,000 x 50 kg 
50
 
Lifetime AI = 185 mg/kg/day/50,000 x 50 kg 
 
Lifetime AI (all other routes) = 185 g/day 
 
19  Contains Nonbinding Recommendations 
REFERENCES (FOR ACETALDEHYDE) 
 
1.  International Agency for Research on Cancer (IARC), 1999, Acetaldehyde. In: IARC 
Monographs on the Evaluation of Carcinogenic Risk to Humans, Volume 71, Lyons, 
France: World Health Organization, , 319335.  
 
2.  OBrien PJ, Siraki AG, and Shangari N, 2005, Aldehyde Sources, Metabolism, Molecular 
Toxicity Mechanisms, and Possible Effects on Human Health, Crit Rev Toxicol, 
35(7):609662. 
 
3.  Fukunaga T, Sillanaukee P, and Eriksson CJ, 1993, Problems Involved in the 
Determination of Endogenous Acetaldehyde in Human Blood, Alcohol , 28(5):535541.  
 
4.  Jones AW, 1995, Measuring and Reporting the Concentration of Acetaldehyde in Human 
Breath, Alcohol, 30(3):271285. 
 
5.  Maximum Workplace Concentration Commission (MAK), 2013, The MAK Collection 
for Occupational Health and Safety, Acetaldehyde, 158.  
 
6.  Lachenmeier DW, Gill JS, Chick J, and Rehm J, 2015, The Total Margin of Exposure of 
Ethanol and Acetaldehyde for Heavy Drinkers Consuming Cider or Vodka, Food Chem 
Toxicol, 83:210214. 
 
7.  Vakevainen S, Tillonen J, Agarwal DP, Srivastava N, and Salaspuro M, 2000, High 
Salivary Acetaldehyde After a Moderate Dose of Alcohol in ALDH2-Deficient Subjects: 
Strong Evidence for the Local Carcinogenic Action of Acetaldehyde, Alcohol Clin Exp 
Res, 24(6):873877.  
 
8.  Uebelacker M and Lachenmeier DW, 2011, Quantitative Determination of Acetaldehyde 
in Foods Using Automated Digestion With Simulated Gastric Fluid Followed by 
Headspace Gas Chromatography, J Autom Methods Manag Chem, 2011:907317.  
 
9.  Joint FAO/WHO Expert Committee on Food Additives (JECFA), 1999, Evaluation of 
Certain Food Additives and Contaminants, Forty-Ninth Report of the Joint FAO/WHO 
Expert Committee on Food Additives, World Health Organization Technical Report 
Series, 884:30p. 
 
10. Food Safety Commission (FSC), 2005, Evaluation of Food Additives: Acetaldehyde, 
Tokyo, Japan. 
 
11. Chemicals Evaluation and Research Institute (CERI) Japan, 2007, Hazard Assessment 
Report Acetaldehyde, Japan.  
 
12. European Commission (EC), 2012, Scientific Committee on Consumer Safety: Opinion 
On: Acetaldehyde, SCCS/1468/12. 
 
13. Scientific Committee on Cosmetic Products and Non-Food Products (SCCNFP), 2004, 
Opinion of the Scientific Committee on Cosmetic Products and Non-Food Products for 
Consumers Concerning Acetaldehyde, SCCNFP/0821/04. 
 
20  Contains Nonbinding Recommendations 
14. Albertini RJ, 2013, Vinyl Acetate Monomer (VAM) Genotoxicity Profile: Relevance for 
Carcinogenicity, Crit Rev in Toxicol, 43(8):671706. 
 
15. Grafstrom RC, Dypbukt JM, Sundqvist K, Atzori L, Nielsen I, Curren RD, and Harris 
CC, 1994, Pathobiological Effects of Acetaldehyde in Cultured Human Epithelial Cells 
and Fibroblasts, Carcinogenesis, 15(5):985990. 
 
16. Moeller BC, Recio L, Green A, Sun W, Wright FA, Bodnar WM, and Swenberg JA, 
2013, Biomarkers of Exposure and Effect in Human Lymphoblastoid TK6 Cells 
Following [13C2]-Acetaldehyde Exposure, Toxicol Sci, 133(1):112. 
 
17. Morita T, Asano N, Awogi T, Sasaki YF, Sato S-I, Shimada H, Sutou S, Suzuki T, 
Wakata A, Sofuni T, and Hayashi M, 1997, Evaluation of the Rodent Micronucleus 
Assay in the Screening of IARC Carcinogens (Groups 1, 2A and 2B). The Summary 
Report of the 6th Collaborative Study by CSGMT/JEMS MMS, Mutation Research, 
389:3122. 
 
18. Speit G, Froehler-Keller M, Schuetz P, and Neuss S, 2008, Low Sensitivity of the Comet 
Assay to Detect Acetaldehyde-Induced Genotoxicity, Mutation Research, 657:9397. 
 
19. IARC, 2012, IARC Monographs on the Evaluation of the Carcinogenic Risk of 
Chemicals to Humans: Personal Habits and Indoor Combustions, Volume 100 E, Lyon, 
France: World Health Organization. 
 
20. Shield KD, Soerjomataram I, and Rehm J, 2016, Alcohol Use and Breast Cancer: A 
Critical Review, Alcohol Clin Exp Res, 40(6):11661181.  
 
21. Lachenmeier DW, Kanteres F, and Rehm J, 2009, Carcinogenicity of Acetaldehyde in 
Alcoholic Beverages: Risk Assessment Outside Ethanol Metabolism, Addiction, 
104(4):533-550. 
 
22. Bagnardi V, Rota M, Botteri E, Tramacere I, Islami F, Fedirko V, Scotti L, Jenab M, 
Turati F, Pasquali E, Pelucchi C, Galeone C, Bellocco R, Negri E, Corrao G, Boffetta P, 
and La Vecchia C, 2015, Alcohol Consumption and Site-Specific Cancer Risk: A 
Comprehensive Dose-Response Meta-Analysis, Br J Cancer, 112(3):580593.  
 
23. Carcinogenic Potency Database (CPDB), website available at 
https://files.toxplanet.com/cpdb/index.html.   
 
24. Woutersen RA, Appelman LM, Van Garderen-Hoetmer A, and Feron VJ, 1986, 
Inhalation Toxicity of Acetaldehyde in Rats. III. Carcinogenicity Study, Toxicology, 
41(2):213231.  
 
25. Soffritti M, Belpoggi F, Lambertini L, Lauriola M, Padovani M, and Maltoni C, 2002, 
Results of Long-Term Experimental Studies on the Carcinogenicity of Formaldehyde and 
Acetaldehyde in Rats, Ann N Y Acad Sci, 982:87105. 
 
26. Feron VJ, Kruysse A, and Woutersen RA, 1982, Respiratory Tract Tumours in Hamsters 
Exposed to Acetaldehyde Vapour Alone or Simultaneously to Benzo(a)pyrene or 
Diethylnitrosamine, Eur J Clin Oncol, 18(1):1331. 
21  Contains Nonbinding Recommendations 
 
27. Homann N, Jousimies-Somer H, Jokelainen K, Heine R, and Salaspuro M, 1997, High 
Acetaldehyde Levels in Saliva After Ethanol Consumption: Methodological Aspects and 
Pathogenetic Implications, Carcinogenesis, 18(9):17391743. 
 
28. Salaspuro V, Hietala J, Kaihovaara P, Pihlajarinne L, Marvola M, and Salaspuro M, 2002, 
Removal of Acetaldehyde From Saliva by a Slow-Release Buccal Tablet of L-cysteine, 
Int J Cancer, 97(3):361364. 
 
29. Sladek NE, 2003, Human Aldehyde Dehydrogenases: Potential Pathological, 
Pharmacological and Toxicological Impact, J Biochem Mol Toxicol,17(1):723. 
 
30. Kunugita N, Isse T, Oyama T, Kitagawa K, Ogawa M, Yamaguchi T, Kinaga T, and 
Kawamoto T, 2008, Increased Frequencies of Micronucleated Reticulocytes and T-Cell 
Receptor Mutation in ALDH2 Knockout Mice Exposed to Acetaldehyde, J Toxicol 
Sci,33(1):3136. 
 
31. Matsuda T, Yabushita H, Kanaly RA, Shibutani S, and Yokoyama A, 2006, Increased 
DNA Damage in ALDH2-Deficient Alcoholics, Chem Res Toxicol, 19(10):13741378. 
 
32. U.S. Food and Drug Administration, 21 CFR part 182, Substances Generally Recognized 
as Safe, and 21 CFR 182.60, Synthetic flavoring substances and adjuvants. 
 
33. National Research Council (U.S) Committee on Emergency and Continuous Exposure 
Guidance Levels for Selected Submarine Contaminants, 2009, Emergency and 
Continuous Exposure Guidance Levels for Selected Submarine Contaminants, Volume 3, 
Washington, D.C.: National Academies Press.  
 
34. U.S. Environmental Protection Agency, 1988, Integrated Risk Information System 
(IRIS): Chemical Assessment Summary: Acetaldehyde (CASRN 75-07-0) 
Carcinogenicity Assessment, available at 
https://cfpub.epa.gov/ncea/iris/iris_documents/documents/subst/0290_summary.pdf. 
 
22  Contains Nonbinding Recommendations 
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 (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 the International Agency for Research on Cancer as a Group 2B 
carcinogen, possibly carcinogenic to humans (Ref. 2). 
Acrylonitrile is a multiorgan carcinogen in mice and rats, with the brain being the primary 
target organ in rat. There are four oral carcinogenicity studies cited in the Carcinogenic 
Potency Database (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/National Toxicology Program) study in the CPDB 
of acrylonitrile in mice (Ref. 5) was selected for derivation of the oral acceptable intake (AI), 
based on robust study design and the most conservative TD  value. In this 2-year study, 
50
three 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  is for astrocytomas in female rats (5.31 milligrams 
50
(mg)/kilogram (kg)/day). However, this same study was later described in detail (Ref. 7), and 
23  Contains Nonbinding Recommendations 
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 parts per million, adjusting for body weight and the 
decreased water consumption in the study. The TD  for astrocytomas derived from these 
50
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 s calculated from the dose 
50
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 described (Ref. 7), but the most sensitive TD  was for stomach tumors, as shown 
50
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 the U.S. Environmental Protection Agency 
(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 two 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 
 
Most 
Sensitive 
Animals/  Duration/  TD50 
Study  Controls  Doses  Tumor 
dose group  Exposure  (mg/kg/d) 
Site/Type/S
ex 
  50 B6C3F1  2 years,  50  3: 1.79; 7.14;  Forestomach  6.77c 
Mice (F)  gavage  14.3 mg/kg/d 
Ref. 5a 
50 B6C3F1  2 years,  50  3: 1.79; 7.14;  Forestomach  5.92c 
Mice (M)  gavage  14.3 mg/kg/d 
  ~50 SD  2 years,  ~80  3:  Astrocytoma  5.31d 
  Spartan rats  drinking  2.00; 5.69;  (20.8) 
(F)  water  15.4 mg/kg/d 
Ref. 6 
~50 SD  2 years,  ~80  3:  Stomach,  6.36d 
Spartan rats  drinking  1.75; 4.98;  non-  (9.0) 
(M)  water  14.9 mg/kg/d  glandular 
24  Contains Nonbinding Recommendations 
Most 
Sensitive 
Animals/  Duration/  TD50 
Study  Controls  Doses  Tumor 
dose group  Exposure  (mg/kg/d) 
Site/Type/S
ex 
  ~50 female SD  2 years,  ~80  3:  Stomach,  19.4 
Ref 7  Spartan rats  drinking  4.4; 10.8; 25  non- 
(report  water  mg/kg/d  glandular 
of Ref.  ~50 SD male  2 years,  ~80  3:  Stomach,  9.0 
6)  Spartan rats  drinking  3.4; 8.5;  non- 
water  21.3 mg/kg/d  glandular 
  100 male rats  ~2 years,  ~200  5:  Brain  (22.9)c 
Ref. 8e  drinking  0.1-8.4  astrocytoma 
water  mg/kg/d 
  100 female rats  ~2 years,  ~200  5:  Brain  (23.5)c 
drinking  0.1-10.9  astrocytoma 
water  mg/kg/d 
  100/sex  19-22  ~98  2:  Stomach,  NC 
Rats  months,  ~0.09; 7.98  Zymbals 
Ref. 11e 
drinking  mg/kg/d  gland, brain, 
water  spinal cord 
  50/sex  18  No  2:  Brain,  NCb 
Rats  months,  14; 70  Zymbals 
Ref. 10e 
drinking  mg/kg/d  gland, 
water  forestomach 
  20  2 years,  No  3:  Zymbals  30.1 
Ref. 13  male CD rats  drinking  1; 5; 25  gland 
water  mg/kg/d 
  40/sex  1 year  75/sex  1:  Neg in both  NA 
Ref. 4  SD rats  3d/wk,  1.07 mg/kg/d  sexes 
gavage 
  100/sex  2 years  100  2:  Brain  32.4 
SD Spartan rat  6 h/d;  M: 2.27; 9.1  astrocytoma 
Ref. 12 
5d/wk,  F: 3.24; 13.0  Male 
inhalation  mg/kg/d 
  30/sex  1 year  30  4:  Brain glioma  19.1 
  SD rats  5d/wk,  M: 0.19; 0.38;  Male 
  inhalation  0.76; 1.52 
Ref. 4  F: 
0.27;0.54;1.0; 
2.17 
mg/kg/d 
  54 female SD  2 years  60  1:  Brain glioma  (132)f 
Ref. 4  rats  5d/wk,  11.1 mg/kg/d 
inhalation 
Studies listed are in the Carcinogenic Potency Database (CPDB) (Ref. 3) unless otherwise noted. 
The TD  values represent the TD  from the most sensitive tumor site. 
50 50
TD  values in parentheses are considered less reliable as explained in footnotes. 
50
mg = milligram; kg = kilogram; d = day; wk = week; F = female; M = Male; NA = not applicable. 
a Carcinogenicity study selected for acceptable intake (AI) calculation; in CPDB. 
25  Contains Nonbinding Recommendations 
b NC = Not calculated as individual tumor type incidences not provided in the World Health Organization 
(WHO) (Ref. 1). 
c TD  calculated based on astrocytoma incidence implied as most significant site by WHO (Ref. 1). Serial 
50
sampling reduced number of animals exposed for 2 years, so tumor incidences may be underestimates. 
d Taken from the CPDB. Note that based on the dose calculations by the author (Ref. 7) the TD  for 
50
astrocytomas and stomach tumors in Spartan rats (20.8 and 9.0) are higher than those in the CPDB. 
e Not in CPDB. Summarized in Refs. 1 and 9. 
f 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 nonirritating (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. 
 
Regulatory and/or Published Limits 
 
The U.S. EPA (Ref. 9) calculated an oral slope factor of 0.54/mg/kg/day and a drinking water 
limit of 0.6 micrograms (g)/liter at the 1 in 100,000 risk level, based on the occurrence of 
multiorgan tumors in a drinking water study in rats. This drinking water limit equates to a 
daily dose of about1 g/day for a 50 kg human. 
 
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 U.S. 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  derived from 
50
administering acrylonitrile by oral gavage to male and female mice because the tumor type 
with the lowest TD  was forestomach tumors in male mice, with a TD  value of 5.92 
50 50
mg/kg/day. As discussed in the Methods section II.B (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 U.S. EPA (Ref. 9) for drinking water. 
26  Contains Nonbinding Recommendations 
 
Calculation of AI 
 
Lifetime AI = TD /50,000 x 50 kg 
50
 
Lifetime AI = 5.92 (mg/kg/day)/50,000 x 50 kg 
 
Lifetime AI = 5.9 g/day (6 g/day)
27  Contains Nonbinding Recommendations 
REFERENCES (FOR ACRYLONITRILE) 
 
1.  World Health Organization (WHO), 2002, Concise International Chemical Assessment 
Document 39: Acrylonitrile, Geneva, available at 
http://www.inchem.org/documents/cicads/cicads/cicad39.htm. 
 
2.  International Agency for Research on Cancer (IARC), 1999, Acrylonitrile. In: IARC 
Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 71, Lyon, 
France: World Health Organization, 43. 
 
3.  Carcinogenic Potency Database, website available at 
https://files.toxplanet.com/cpdb/index.html. 
 
4.  Maltoni C, Ciliberti A, Cotti G, and Perino G, 1988, Long-Term Carcinogenicity 
Bioassays on Acrylonitrile Administered by Inhalation and by Ingestion to Sprague-
Dawley Rats, Ann N Y Acad Sciences, 534:179202. 
 
5.  National Toxicology Program (NTP), 2001, NTP Technical Report on Toxicology and 
Carcinogenesis Studies of Acrylonitrile (CAS No. 107-13-1) in B6C3F1 Mice (Gavage 
Studies), Research Triangle Park: NIH Publication, No. 02-4440, 198. 
 
6.  Quast JF, Wade CE, Humiston CG, Carreon RM, Hermann EA, Park CN, et al., editors, 
1980, A Two-Year Toxicity and Oncogenicity Study With Acrylonitrile Incorporated in 
the Drinking Water of Rats, Final Report, Midland, MI: Dow Chemical USA. 
 
7.  Quast JF, 2002, Two-Year Toxicity and Oncogenicity Study With Acrylonitrile 
Incorporated in the Drinking Water of Rats, Toxicol Lett, 132(3):153196. 
 
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: Division of Biology and 
Safety Evaluation, Project No. 77-1744; BDN-77-27. 
 
9.  U.S. Environmental Protection Agency, 1987, Integrated Risk Information System 
(IRIS): Chemical Assessment Summary: Acrylonitrile (CASRN 107-13-1), available at 
https://cfpub.epa.gov/ncea/iris2/chemicalLanding.cfm?substance_nmbr=206. 
 
10. Bigner DD, Bigner SH, Burger PC, Shelburne JD, and Friedman HS, 1986, Primary Brain 
Tumors in Fischer 344 rats Chronically Exposed to Acrylonitrile in Their Drinking 
Water, Food and Chem Toxicol, 24(2):129137. 
 
11. Bio/Dynamics Inc. Monsanto Company, 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: Division of Biology and 
Safety Evaluation, Project No. 77-1745; BDN-77-28. 
 
12. Quast JF, Schuetz DJ, Balmer MF, Gushow TS, Park CN, and McKenna MJ, editors, 
1980, A Two- Year Toxicity and Oncogenicity Study With Acrylonitrile Following 
Inhalation Exposure of Rats, Final Report, Midland, MI: Dow Chemical USA. 
 
28  Contains Nonbinding Recommendations 
13. Gallagher GT, Maull EA, Kovacs K, and Szab S, 1988, Neoplasms in Rats Ingesting 
Acrylonitrile for Two Years, J Am Col Toxicol, 7(5):603615. 
 
14. Proctor DM, Gatto NM, Hong SJ, and Allamneni KP, 2007, Mode-of-Action Framework 
for Evaluation of the Relevance of Rodent Forestomach Tumors in Cancer Risk 
Assessment, Toxicol Sci, 98(2):313326. 
29  Contains Nonbinding Recommendations 
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, 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 because some in vitro and in vivo genotoxicity tests are positive. 
 
Aniline is not mutagenic in the five standard strains of Salmonella or in Escherichia coli 
WP2uvrA, 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 millimolar (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 milligrams (mg)/kilogram (kg) (Ref. 16), 
but a small increase in chromosomal aberrations 18 hours 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, 1,000, and 2,000 parts per million (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, reaching a maximum of twofold 
increase over the background, was observed in the bone marrow of male Swiss mice 24 hours 
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. 
 
Carcinogenicity 
 
Aniline is classified by the International Agency for Research on Cancer (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. 
 
30  Contains Nonbinding Recommendations 
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 2,000 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 permissible daily exposure (PDE) for aniline based on the 
robust study design with three 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 U.S. 
National Cancer Institute (NCI) (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).  
 
Aniline and Aniline Hydrochloride (HCl)  Details of Carcinogenicity Studies 
 
Most 
Animals/ 
Duration/  Sensitive  TD50 
Study  Dose  Controls  Doses 
Exposure  Tumor  (mg/kg/d) 
Group 
Site/Type/Sex 
  130/sex/  2 years,   130  3:  Spleen sarcoma  Not 
group, CD-F  diet  200, 600,  (high dose).  reported 
Ref. 26a 
rats  and 2,000  NOEL at low 
Aniline 
ppm in diet  dose 
HCl 
(M; 7.2; 22; 
72 mg/kg/d) 
  50/sex/group,  103 weeks  50  2:  Spleen  160 
  F344 rats  (107-110  3,000 and  hemangio-  (Male) 
Ref. 27b  week  6,000 ppm  sarcoma/Male 
Aniline  study),  in diet 
HCl  diet  (F: 144;268 
M: 115;229 
mg/kg/d) 
  50/sex/group  103 weeks  50  2:  Negative  NA 
  B6C3F1  (107-110  6,000 and 
mice  week  12,000 ppm 
Ref. 27b  study),  in diet 
Aniline  Diet  (F: 
HCl    741, 1,500 
  M:  
693, 1,390 
Mg/kg/d) 
   
31  Contains Nonbinding Recommendations 
Most 
Animals/ 
Duration/  Sensitive  TD50 
Study  Dose  Controls  Doses 
Exposure  Tumor  (mg/kg/d) 
Group 
Site/Type/Sex 
  10-18/group,  80  Yes  3:  Negative  NA 
  male Wistar  weeks,  0.03%, 
Ref. 28b  rats  diet  0.06%, 
Aniline  and 0.12% 
in diet 
(15; 30 
;60  
mg/kg/d) 
a Carcinogenicity study selected for permissible daily exposure calculation. Not in the Carcinogenic Potency 
Database (CPDB). 
b Taken from CPDB (Ref. 29). The TD  values represent the TD  from the most sensitive tumor site.  
50 50
HCl = hydrochloride; mg = milligram; kg = kilogram; d = day; ppm = parts per million; NOEL = no-observed 
effect level; M = male; F = female; 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, and 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 (greater 
than10 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- 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 nonlinear (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 percent (equal to 
approximately 7.2 mg/kg/day aniline 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). 
 
32  Contains Nonbinding Recommendations 
Regulatory and/or Published Limits 
 
The U.S. Environmental Protection Agency (Ref. 39) outlines a quantitative cancer risk 
assessment for aniline based on the CIIT study (Ref. 26). 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 micrograms (g)/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 
 
It is considered inappropriate to base an acceptable intake for aniline on linear extrapolation 
for spleen tumors observed in rats; because these have a nonlinear 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 the ICH guidance for 
industry Q3C(R8) Impurities: Guidance for Residual Solvents (ICH Q3C(R8)) (December 
2021) (Ref. 40).1 
 
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 2,000 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 (NOEL) for tumors. 
 
The PDE calculation is (NOEL times body weight adjustment (kg)) / F1 times F2 times F3 
times F4 times F5. 
 
The following safety factors as outlined in ICH Q3C(R8) have been applied to determine the 
PDE for aniline: 
 
F1 = 5 (rat to human) 
F2 = 10 (interindividual variability) 
F3 = 1 (study duration at least half lifetime) 
F4 = 10 (severe toxicity  nongenotoxic 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 g/day
 
1 We update guidances periodically.  To make sure you have the most recent version of a guidance, check the 
FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents.  
33  Contains Nonbinding Recommendations 
REFERENCES (FOR ANILINE AND ANILINE HYDROCHLORIDE) 
 
1.  Chung KT, Murdock CA, Zhou Y, Stevens SE, Li YS, Wei CI, et al., 1996, Effects of the 
Nitro-Group on the Mutagenicity and Toxicity of Some Benzamines, Environ Mol 
Mutagen, 27(1):6774. 
 
2.  International Agency for Research on Cancer (IARC), 1982, IARC Monographs on the 
Evaluation of the Carcinogenic Risk of Chemicals to Humans: Some Aromatic Amines, 
Anthraquinones and Nitroso Compounds, and Inorganic Fluorides Used in Drinking 
Water and Dental Preparations, Volume 27, Lyon, France: World Health Organization, 
27:39. 
 
3.  IARC, 1987, Monographs on the Evaluation of the Carcinogenic Risk to Humans: 
Genetic and Related Effects: An Updating of Selected IARC Monographs From Volumes 
1 to 42, Lyon, France: World Health Organization, Addendum 6, 68. 
 
4.  IARC, 1987, IARC Monographs on the Evaluation of the Carcinogenic Risk to Humans: 
Overall Evaluations of Carcinogenicity: An Updating of IARC Monographs Volumes 1 to 
42, Lyon, France: World Health Organization, Addendum 7, 99 and 362. 
 
5.  Jackson MA, Stack HF, and Waters MD, 1993, The Genetic Toxicology of Putative 
Nongenotoxic Carcinogens, Mutat Res, 296(3):24177. 
 
6.  Brams A, Buchet JP, Crutzen-Fayt MC, De Meester C, Lauwerys R, and Leonard A, 
1987, A Comparative Study, With 40 Chemicals, of the Efficiency of the Salmonella 
Assay and the SOS Chromotest (Kit Procedure), Toxicol Lett, 38(12):123133. 
 
7.  Rashid KA, Arjmand M, Sandermann H, and Mumma RO, 1987, Mutagenicity of 
Chloroaniline/Lignin Metabolites in the Salmonella/Microsome Assay, J Environ Sci 
Health, 22(6):721729. 
 
8.  Gentile JM, Gentile GJ, and Plewa MJ, 1987, Mutagenicity of Selected Aniline 
Derivatives to Salmonella Following Plant Activation and Mammalian Hepatic 
Activation, Mutat Res, 188(3):185196. 
 
9.  Wangenheim J and Bolcsfoldi G, 1988, Mouse Lymphoma L5178Y Thymidine Kinase 
Locus Assay of 50 Compounds, Mutagenesis, 3(3):193205. 
 
10. Amacher DE, Paillet SC, Turner GN, Ray VA, and Salsburg DS, 1980, Point Mutations at 
the Thymidine Kinase Locus in L5178Y Mouse Lymphoma Cells, Mutat Res, 72(3):447
474. 
 
11. McGregor DB, Brown AG, Howgate S, Mcbride D, Riach C, and Caspary WJ, 1991, 
Responses of the L5178y Mouse Lymphoma Cell Forward Mutation Assay, V: 27 Coded 
Chemicals, Environ Mol Mutagen, 17(3):196219. 
 
12. Abe S and Sasaki M, 1977, Chromosome Aberrations and Sister Chromatid Exchanges in 
Chinese Hamster Cells Exposed to Various Chemicals, J Natl Cancer Inst, 58(6):1635
1641. 
 
34  Contains Nonbinding Recommendations 
13. Ishidate M, Jr, and Odashima S, 1977, Chromosome Tests With 134 Compounds on 
Chinese Hamster Cells In Vitro  A Screening for Chemical Carcinogens, Mutat Res, 
48(34):337354. 
 
14. Ishidate M, Jr., 1983, 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., 26. 
 
15. Galloway SM, Armstrong MJ, Reuben C, Colman S, Brown B, Cannon C, et al., 1987, 
Chromosome Aberrations and Sister chromatid Exchanges in Chinese Hamster Ovary 
Cells: Evaluations of 108 Chemicals, Environ Mol Mutagen, 10 Suppl 10:1175. 
 
16. Jones E and Fox V, 2003, Lack of Clastogenicity Activity of Aniline in the Mouse Bone 
Marrow, Mutagenesis, 18(3):283285. 
 
17. Bomhard EM, 2003, High-Dose Clastogenic Activity of Aniline in the Rat Bone Marrow 
and Its Relationship to the Carcinogenicity in the Spleen of Rats, Arch Toxicol, 
77(5):291297. 
 
18. Westmoreland C and Gatehouse DG, 1991, Effects of Aniline Hydrochloride in the 
Mouse Bone Marrow Micronucleus Test After Oral Administration, Carcinogenesis, 
12(6):10571059. 
 
19. Ashby J, Vlachos DA and Tinwell H, 1991, Activity of Aniline in the Mouse Bone 
Marrow Micronucleus Assay, Mutat Res, 263(2):115117. 
 
20. Sicardi SM, Martiarena JL, and Iglesias MT, 1991, Mutagenic and Analgesic Activities of 
Aniline Derivatives, J Pharm Sci, 80(8):761764. 
 
21. Ress NB, Witt KL, Xu J, Haseman JK, and Bucher JR, 2002, Micronucleus Induction in 
Mice Exposed to Diazoaminobenzene or Its Metabolites, Benzene and Aniline: 
Implications for Diazoaminobenzene Carcinogenicity, Mutat Res, 521(12):201208. 
 
22. George E, Andrews M, and Westmoreland C, 1990, Effects of Azobenzene and Aniline in 
the Rodent Bone Marrow Micronucleus Test, Carcinogenesis, 11(9):15511556. 
 
23. Witt KL, Knapton A, Wehr CM, Hook GJ, Mirsalis J, Shelby MD, et al., 2000, 
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, 36(3):163194. 
 
24. Parodi S, Pala M, Russo P, Zunino A, Balbi C, Albini A, et al., 1982, 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, 42(6):227722783. 
 
25. Parodi S, Zunino A, Ottaggio L, De Ferrari M, and Santi L, 1983, 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, 108(1
3):225238. 
35  Contains Nonbinding Recommendations 
 
26. Chemical Industry Institute of Technology (CIIT), 1982, 104-Week Chronic Toxicity 
Study in Rats With Aniline Hydrochloride: Final Report, Research Triangle Park, NC: 
CIIT. 
 
27. National Cancer Institute, 1978, Bio-Assay of Aniline Hydrochloride for Possible 
Carcinogenicity (CAS No. 142-04-1) NCI-CG-TR-130, Bethesda, MD: Department of 
Health, Education, and Welfare, available at 
https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr130.pdf. 
 
28. Hagiwara A, Arai M, Hirose M, Nakanowatari JI, Tsuda H, and Ito N, 1980, Chronic 
Effects of Norharman in Rats Treated With Aniline, Toxicol Lett, 6(2):7175. 
 
29. Carcinogenic Potency Database, website available at 
https://files.toxplanet.com/cpdb/index.html. 
 
30. Steinheider G, Neth R, and Marguardt H, 1985, Evaluation of Nongenotoxic and 
Genotoxic Factors Modulating the Frequency of Micronucleated Erythrocytes in the 
Peripheral Blood of Mice, Cell Biol Toxicol, 1(3):197211. 
 
31. Tweats DJ, Blakey D, Heflich RH, Jacobs A, Jacobsen SD, Morita T, Nohmi TT, et al., 
2007, 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, 627(1):7891. 
 
32. Khan MF, Wu X, Boor PJ, and Ansari GA, 1999, Oxidative Modification of Lipids and 
Proteins in Aniline Induced Splenic Toxicity, Toxicol Sci, 48(1):134140. 
 
33. Khan MF, Wu X, and Wang J, 2003, Upregulation of Transforming Growth Factor-Beta 
1 in the Spleen of Aniline-Induced Rats, Toxicol Appl Pharmacol, 187(1):2228. 
 
34. Weinberger MA, Albert RH, and Montgomery SB, 1985, Splenotoxicity Associated With 
Splenic Sarcomas in Rats Fed High Doses of D & C Red No. 9 or Aniline Hydrochloride, 
J Natl Cancer Inst, 75(4):681687. 
 
35. Smith RP, Alkaitis AA, and Shafer PR, 1967, Chemically Induced Methemoglobinemias 
in the Mouse, Biochem Pharmacol, 16(2):317328. 
 
36. Bus JS and Popp JA, 1987, Perspectives on the Mechanism of Action of the Splenic 
Toxicity of Aniline and Structurally-Related Compounds, Food Chem Toxicol, 
25(8):619626. 
 
37. Robertson O, Cox MG, and Bus JS, 1983, Response of the Erythrocyte and Spleen to 
Aniline Insult in Fischer 344 Rats, Toxicologist, 3:128. 
 
38. Bomhard EM and Herbold BA, 2005, Genotoxic Activities of Aniline and Its Metabolites 
and Their Relationship to the Carcinogenicity of Aniline in the Spleen of Rats, Crit Rev 
Toxicol, 35(10):783835.  
 
36  Contains Nonbinding Recommendations 
39. U.S. Environmental Protection Agency, 1988, Integrated Risk Information System: 
Chemical Assessment Summary: Aniline (CAS No 62-53-3), available at 
https://cfpub.epa.gov/ncea/iris/iris_documents/documents/subst/0350_summary.pdf. 
 
40. International Council for Harmonisation guidance for industry Q3C(R8) Impurities: 
Guidance for Residual Solvents (December 2021)1 
 
1 We update guidances periodically.  To make sure you have the most recent version of a guidance, check the 
FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents.  
37  Contains Nonbinding Recommendations 
BENZYL CHLORIDE (-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 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 
intravenous 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 three times a week for 104 weeks to 
F-344 rats and B6C3F1 mice (Ref. 4). Rats received doses of 0, 15, or 30 milligrams 
(mg)/kilogram (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 percent versus 8 percent 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 percent and 37 percent, respectively, compared with 0 percent 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 percent versus 0 percent in controls) at the high dose and in carcinoma 
or adenoma in the liver but only at the low dose (54 percent versus 33 percent in controls). In 
female, but not male, mice there were significant increases in the incidence of alveolar-
38  Contains Nonbinding Recommendations 
bronchiolar adenoma or carcinoma at the high dose (12 percent versus 1.9 percent in 
controls). 
 
Additional studies to assess carcinogenic potential were conducted but were not considered of 
adequate study design for use in calculating an acceptable intake (AI). In one of three topical 
studies (Ref. 5) skin carcinomas were increased, although not statistically significantly (15 
percent versus 0  percent 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). 
 
Benzyl Chloride  Details of Carcinogenicity Studies 
 
Most 
Sensitive 
Tumor 
Animals/Dose  Duration/ TD50 
Study  Controls  Doses  Site/Type/Se
Group  Exposure  (mg/kg/d) 
x or Tumor 
Observation
s 
  52/sex/group  2 year  52  2:  Thyroid  40.6 
F344 rat  3 times/  15 and 30  C-cell 
Ref. 4a  wk, gavage  mg/kg  neoplasm/ 
(6 and 12  Female 
mg/kg/d) 
  52/sex/group  2 year  52  2:  Forestomach  49.6 
  B6C3F1  3 times/  50 and  papilloma, 
  mouse  wk, gavage  100  carcinoma/ 
Ref. 4  mg/kg  Male 
(21 and 
42 
mg/kg/d) 
  11/group  9.8 months  Yes  1:  No skin  NC b 
  female ICR  3 times/wk  (benzen 10 L  tumors 
Ref. 5  mouse  for 4 wks, 2  e 
times/wk  treated) 
dermal 
  20/group  50 weeks  20  1:  Skin  NC b 
female ICR  2 times/wk  (benzen 2.3 L  squamous 
Ref. 5 
mouse  Dermal  e  cell 
treated)  carcinoma 
39  Contains Nonbinding Recommendations 
Most 
Sensitive 
Tumor 
Animals/Dose  Duration/ TD50 
Study  Controls  Doses  Site/Type/Se
Group  Exposure  (mg/kg/d) 
x or Tumor 
Observation
s 
  20/group  >7 months  20  1:  No skin  NC b 
male ICI  2 times/wk,  100  tumors 
Ref. 6 
Swiss albino  dermal,  in  g/mouse 
mouse  toluene 
  14 (40  51 weeks  Yes  2:  Injection site  NC b 
  mg/kg), and 8  1 time/wk,  40 and 80  sarcoma 
Ref. 9  (80 mg/kg)  subcutaneous  mg/kg/wk 
BD rat 
  40/sex/group  10 months  40  1:  No skin  NC b 
  Theiler's  1 dose (in  1  tumors 
Original  toluene); wait  mg/m
Ref. 7 
mouse  1 wk  ouse 
Promoter 
(croton oil) 
2 times/wk 
  Sencar mice  6 months  Yes  3: 10; 100  20% skin  NC b 
  1 dose;  and  tumors (5% 
  Promoter  1000 g/  in TPA 
(TPA)  mouse  controls) 
Ref. 8 
2 times/wk  (DMBA 
controls had 
skin tumors 
by 11 weeks) 
Studies listed are in Carcinogenic Potency Database (CPDB) (Ref. 10) unless otherwise noted. 
a Carcinogenicity study selected for acceptable intake calculation. 
b NC= Not calculated; small group size, limited duration. Not included in CPDB as route with greater likelihood 
of systemic exposure is considered more relevant. 
mg = milligram; kg = kilogram; d = day; wk = week; L = microliter; g = microgram; DMBA = 
dimethylbenzanthracene. 
 
Mode of Action for Carcinogenicity 
 
The tumor types with the lowest calculated TD  (highest potency) in the Carcinogenic 
50
Potency Database (Ref. 10) for benzyl chloride are forestomach tumors in mice and thyroid 
C-cell tumors in female rats. The relevance of the forestomach tumors to human risk 
assessment for low, nonirritating 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 nonmutagenic 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 
40  Contains Nonbinding Recommendations 
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 nonirritating 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 nonmutagenic, 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 nonlinear 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 maximum tolerated dose (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 Organization for Economic Co-operation and 
Development (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 to 3 weeks from severe acute and chronic gastritis of the forestomach, 
often with ulcers, following oral administration three times a week of doses greater than 250 
mg/kg for males and greater than125 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 (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  454 mg/kg/day), and in thyroid C-cell adenomas or 
50
carcinomas in the female rats (TD  40.6 mg/kg/day). The levels of thyroid C-cell tumors in 
50
41  Contains Nonbinding Recommendations 
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 National Toxicology Program (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 percent). 
 
Regulatory and/or Published Limits 
 
The U.S. Environmental Protection Agency (EPA) (Ref. 15) derived an oral slope factor of 
1.7 times 10-1 per (mg/kg)/day, which corresponds to a 1 in 100,000 risk level of 2 
micrograms (g)/liter or approximately 4 g a day using U.S. EPA assumptions. 
 
Acceptable Intake  
 
Rationale for selection of study for AI calculation 
 
The most robust evaluation of the carcinogenic potential of benzyl chloride was the Lijinsky 
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 National Cancer Institute /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 10 rats of each sex given 125 or 
250 mg/kg (3 days per week) died within 2 to 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 (three times per week). At this dose, there was no 
difference from controls in survival in the 2-year carcinogenicity study, but three 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 II.B (2.2), linear extrapolation from the TD  was used 
50
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 because 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 because 
they were associated with the lowest TD : 40.6 mg/kg/day. 
50
 
42  Contains Nonbinding Recommendations 
Calculation of AI 
 
Lifetime AI = TD /50,000 x 50 kg 
50
 
Lifetime AI = 40.6 (mg/kg/day)/50,000 x 50 kg 
 
Lifetime AI = 40.6 g/day (41 g/day)
43  Contains Nonbinding Recommendations 
REFERENCES (FOR BENZYL CHLORIDE) 
 
1.  International Agency for Research on Cancer (IARC), 1999, IARC Monographs on the 
Evaluation of the Carcinogenic Risks to Humans: Re-evaluation of Some Organic 
Chemicals, Hydrazine, and Hydrogen Peroxide (Part 1, Part 2, Part 3), Lyon, France: 
World Health Organization, available at 
http://monographs.iarc.fr/ENG/Monographs/vol71/mono71-19.pdf. 
 
2.  Fall M, Haddouk H, Morin JP, and Forster R, 2007, Mutagenicity of Benzyl Chloride in 
the Salmonella/Microsome Mutagenesis Assay Depends on Exposure Conditions, Mutat 
Res, 633(1):1320. 
 
3.  IARC, 1987, IARC Monographs on the Evaluation of the Carcinogenic Risk to Humans: 
Overall Evaluations of Carcinogenicity: An Updating of Selected IARC Monographs 
Volumes 1 to 42, Lyon, France: World Health Organization, Suppl. 7:126127; 148149. 
 
4.  Lijinsky W, 1986, Chronic Bioassay of Benzyl Chloride in F344 Rats and (C57BL/6J X 
BALB/c) F1 Mice, J Natl Cancer Inst, 76(6):12311236. 
 
5.  Fukuda K, Matsushita H, Sakabe H, and Takemoto K, 1981, Carcinogenicity of Benzyl 
Chloride, Benzal Chloride, Benzotrichloride and Benzoyl Chloride in Mice by Skin 
Application, Gan, 72(5):655664. 
 
6.  Ashby J, Gaunt C, and Robinson M, 1982, Carcinogenicity Bioassay of 4-
Chloromethylbiphenyl (4CMB), 4-Hydroxymethylbiphenyl (4HMB) and Benzyl Chloride 
(BC) on Mouse Skin: Interim (7 Month) Report, Mutat Res, 100(14):399-401. 
 
7.  Coombs MM, 1982, 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, 100:403405. 
 
8.  Coombs MM, 1982, 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, 100(14):407409. 
 
9.  Druckrey H, Kruse H, Preussmann R, Ivankovic S, and Landschuetz C, 1970, 
Cancerogenic Alkylating Substances. III. Alkyl-halogenides, -Sulfates, -Sulfonates and 
Strained Heterocyclic Compounds, 74(3):241273. 
 
10. Carcinogenic Potency Database, website available at 
https://files.toxplanet.com/cpdb/index.html. 
 
11. Proctor DM, Gatto NM, Hong SJ, and Allamneni KP, 2007, Mode-of-Action Framework 
for Evaluation of the Relevance of Rodent Forestomach Tumors in Cancer Risk 
Assessment, Toxicol Sci, 98(2):313326. 
 
12. Organization for Economic Co-operation and Development, Chemicals Screening 
Information Dataset (SIDS) for High Volume Chemicals Benzyl Chloride Report, 
published by the United Nations Environmental Programme (UNEP).  
 
44  Contains Nonbinding Recommendations 
13. Haseman JK, Huff J, and Boorman GA, 1984, Use of Historical Control Data in 
Carcinogenicity Studies in Rodents, Toxicol Pathol, 12(2):126135. 
 
14. Haseman JK, Hailey JR, and Morris RW, 1998, Spontaneous Neoplasm Incidence in 
Fischer 344 Rats and B6C3F1 Mice in Two-Year Carcinogenicity Studies: A National 
Toxicology Program Update, Toxicol Pathol, 26(3):428441. 
 
15. U.S. Environmental Protection Agency, 1989, Integrated Risk Information System 
(IRIS): Chemical Assessment Summary: Benzyl chloride (CAS 100-44-7), available at 
https://cfpub.epa.gov/ncea/iris/iris_documents/documents/subst/0393_summary.pdf. 
45  Contains Nonbinding Recommendations 
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 
bis(chloromethyl)ether (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 6 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 the U.S. Environmental Protection Agency (EPA) as a Group A, 
known human carcinogen (Ref. 5), and by the International Agency for Research on Cancer 
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 acceptable intake (AI) 
based on the most robust study design and the lowest TD  value. Groups of male Sprague-
50
Dawley rats and Ha/ICR mice were exposed by inhalation to 1, 10, and 100 parts per billion 
(ppb) of BCME 6 hours a day, 5 days a week for 6 months and subsequently observed for the 
duration of their natural life span (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 
percent of the surviving rats, which had been exposed to 100 ppb (7780 nanograms 
(ng)/kilogram (kg)/day, or about 8 micrograms (g)/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 percent 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 parts per million  (100 ppb) 6 hours a day, 5 days a week for 10, 20, 40, 60, 80, or 
46  Contains Nonbinding Recommendations 
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 
subcutaneous application (Ref. 10). 
 
Bis(chloromethyl)ether  Details of Carcinogenicity Studies 
 
Most Sensitive 
Animals/Dose  Duration/  TD50 
Study  Controls  Doses  Tumor 
Group  Exposure  (mg/kg/d) 
Site/Type/Sex 
  ~104/group  28 weeks  104  3:  Nasal passage -  0.00357 
  Rat, male  6 h/d, 5  1; 10; 100  esthesioneuro- 
Sprague-  d/wk.  ppb  epitheliomas 
Ref. 3a 
Dawley.  inhalation  (53;528; 
7780 
ng/kg/d) 
  138-144/  25 weeks  157  3:  Lung adenomas  No 
  group Mouse,  6 h/d,  1; 10; 100  significant 
male ICR/Ha.  5 d/wk,  ppb  increases 
Ref. 3 
inhalation  (0.295; 
2.95;33.6 
ng/kg/d) 
  30-50 treated  6h/d,  240  1:  Lung and nasal  NCb 
  for different  5d/wk, for  0.1 ppm  cancer 
  durations with  10, 20, 40, 
Ref. 7  same  60, 80, 
concentration,  and 100 
male Sprague  exposures, 
Dawley rats.  inhalation 
  100/group  Lifetime  NA  1:  One  NCb 
male Golden  6h/d,  1 ppm  undifferentiated 
Ref. 7 
Syrian  5d/wk,  in the lung 
hamsters.  inhalation 
  50/group  424-456  50  1:  Sarcoma (at the  0.182 
  female ICR/Ha  days,  0.114  injection site) 
Swiss mice.  once  mg/kg/d 
Ref. 9 
weekly
, intra 
peritoneal 
Studies listed are in Carcinogenic Potency Database (CPDB) (Ref. 11) unless otherwise noted. 
a Carcinogenicity study selected for acceptable intake calculation. 
b NC= Not calculated due to nonstandard carcinogenicity design. Not in CPDB. NA= Not available since 
controls were not reported in the study. 
mg = milligram; kg = kilogram; d = day; h = hour; wk = week; ppb = parts per billion; ng = nanogram; ppm = 
parts per million; NA = not applicable. 
47  Contains Nonbinding Recommendations 
 
Mode of Action for Carcinogenicity 
 
BCME is a mutagenic carcinogen, and the AI is calculated by linear extrapolation from the 
TD . 
50
 
Regulatory and/or Published Limits 
 
The U.S. EPA (Ref. 5) calculated an oral cancer slope factor of 220 per milligram 
(mg)/kg/day based on linearized 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 times 10-8 mg/cubic meter for inhalation, 1.6 times 10-6 mg/liter 
for oral exposure). 
 
Acceptable Intake 
 
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  of 3.57g/kg/day. The AI 
50
derived by linear extrapolation from that TD , about 4 ng/day, is essentially the same as the 
50
3.2 ng/day recommendation of the U.S. EPA. The study (Ref. 3) had a reliable design with 
multiple dose levels and greater than 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 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 threshold of toxicological concern 
of 1.5 g/day). The AI is also similar to the limit derived by the U.S. EPA (based on 
inhalation data) that is recommended both for inhalation and ingestion (drinking water) of 
BCME (4 ng/day versus 3.2 ng/day). 
 
Calculation of AI 
 
Lifetime AI = TD /50,000 x 50 kg  
50
 
Lifetime AI = 3.57 g/kg/day/50,000 x 50 kg  
 
Lifetime AI = 0.004 g/day or 4 ng/day
48  Contains Nonbinding Recommendations 
REFERENCES (FOR BIS(CHLOROMETHYL)ETHER) 
 
1.  National Institutes of Health, 2011, Report on Carcinogens, 12th Edition.  
 
2.  Nelson N, 1981, The Chloroethers  Occupational Carcinogens: A Summary of 
Laboratory and  Epidemiology Studies, Ann N Y Acad Sci, 271:8190. 
 
3.  Leong BK, Kociba RJ, and Jersey GC, 1981, A Lifetime Study of Rats and Mice Exposed 
to Vapors of Bis(chloromethy1)ether, Toxicol Appl Pharmacol, 58(2):269281. 
 
4.  International Agency for Research on Cancer (IARC), 1987, Monographs on the 
Evaluation of Carcinogenic Risk to Humans: Bis(chloromethyl)ether and chloromethyl 
methyl ether (technical-grade).. International Agency for Research on Cancer, World 
Health Organization. Lyon, France: World Health Organization, Addendum 7:131133. 
 
5.  U.S. Environmental Protection Agency, 1999,  Integrated Risk Information System 
(IRIS): Chemical Assessment Summary: Bis(chloromethyl)ether (CAS# 542-88-1. 
 
6.  IARC, 1982, Monographs on the Evaluation of the Carcinogenic Risk to Humans: 
Chemicals, Industrial Processes and Industries Associated With Cancer in Humans, 
Volumes 1 to 29, Lyon, France: World Health Organization, Addendum 4. 
 
7.  Kuschner M, Laskin S, Drew RT, Cappiello V, and Nelson N, 1975, Inhalation 
Carcinogenicity of Alpha Halo Ethers. III. Lifetime and Limited Period Inhalation Studies 
With Bis(chloromethyl)ether at 0.1 ppm, Arch Environ Health, 30(2):7377. 
 
8.  Van Duuren BL, Sivak A, Goldschmidt BM, Katz C, and Melchionne S, 1969, 
Carcinogenicity of Halo-Ethers, J Natl Cancer Inst, 43(2):481486. 
 
9.  Van Duuren BL, Goldschmidt BM, and Seidman I, 1975, Carcinogenic Activity of Di- 
and Trifunctional -chloro Ethers and of 1,4-dichlorobutene-2 in ICR/HA Swiss Mice, 
Cancer Res, 35:25532557. 
 
10. Gargus JL, Reese WH, Jr., and Rutter HA, 1969, Induction of Lung Adenomas in 
Newborn Mice by Bis(chloromethyl)ether, Toxicol Appl Pharmacol, 15:9296. 
 
11. Carcinogenic Potency Database (CPDB), website available at 
https://files.toxplanet.com/cpdb/index.html. 
49  Contains Nonbinding Recommendations 
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 the World 
Health Organization (WHO) (Ref. 3) with references, so only key conclusions are 
summarized here. 
 
p-Chloroaniline is mutagenic in: 
 
  Microbial reverse mutation assay (Ames); two- to three-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 milligrams 
(mg)/kilogram (kg), but a significant increase was reported at 300 mg/kg/day after three daily 
doses in mice. 
 
Carcinogenicity 
 
p-Chloroaniline is classified by the International Agency for Research on Cancer 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 National Toxicology Program (NTP) (Ref. 6) oral gavage study was used to calculate the 
acceptable intake (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 
50  Contains Nonbinding Recommendations 
increased. In male mice, the incidence of hemangiosarcomas of the liver or spleen in high-
dose groups 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; and 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. 
 
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. Although the 
incidences of these tumors are strongly suggestive of carcinogenicity, the National Cancer 
Institute concluded that sufficient evidence was not found to establish the carcinogenicity of 
p-chloroaniline in rats or mice under the conditions of these studies. Because 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 Hydrochloride  Details of Carcinogenicity Studies 
 
Most 
Animals/ 
Duration/  Sensitive  TD50 
Study  Dose  Controls  Doses 
Exposure  Tumor  (mg/kg/d) 
Group 
Site/Type/Sex 
50/group  103 weeks  50  3:  Hepatocellular   
Ref. 6a 
male  5 times/  3; 10; 30  adenomas or   
p- 
B6C3F1  wk,  mg/kg  carcinomas. 
chloroaniline  33.8 
mice  gavage  (2.1; 7.1; 
HCl 
21.4 
mg/kg/d) 
50/group  103 weeks  50  3:  Negative.   
Ref. 6 
female  5 times/  3; 10; 30   
p- 
B6C3F1  wk,  mg/kg 
chloroaniline  NA 
mice  gavage  (2.1; 7.1; 
HCl 
21.4 
mg/kg/d) 
50/group  103 weeks  50  3:  Spleen   
Ref. 6 
male  5 times/  2; 6;18  fibrosarcoma,   
p- 
Fischer  wk,  mg/kg  hemangiosarcoma, 
chloroaniline  7.62 
344 rat  gavage  (1.4; 4.2;  osteosarcoma. 
HCl 
12.6 
mg/kg/d) 
  50/group  103 weeks  50  3:  No significant   
Ref. 6  female  5 times/  2; 6; 18  increases;   
p-  Fischer  wk,  mg/kg  equivocal.   
chloroaniline  344 rat  gavage  (1.4; 4.2;  NA 
HCl  12.6 
mg/kg/d) 
51  Contains Nonbinding Recommendations 
Most 
Animals/ 
Duration/  Sensitive  TD50 
Study  Dose  Controls  Doses 
Exposure  Tumor  (mg/kg/d) 
Group 
Site/Type/Sex 
  50/group  78 weeks  20  2:  Mesenchymal   
  male  (study  250; 500  tumors (fibroma,   
  Fischer  duration:  ppm  fibrosarcoma,   
Ref. 8  344 rat  102 wk),  (7.7;  hemangiosarcoma,  72 
diet  15.2  osteosarcoma, 
mg/kg/d)  sarcoma not 
otherwise specified) 
of the spleen or 
splenic 
capsule. 
  50/group  78 weeks  20  2:  Negative.   
  female  (study  250; 500   
Ref. 8  Fischer  duration:  ppm  NA 
344 rat  102 wk),  (9.6, 19 
diet  mg/kg/d) 
  50/group  78 weeks  20  2:  Hemangiosarcomas   
  male  (study  2500;  (subcutaneous   
  B6C3F1  duration:  5000 ppm  tissue, spleen, liver,  Not 
Ref. 8  mice  91 wk),  (257; 275  kidney).  significant 
diet  mg/kg/d)  Increased incidence  (CPDB) 
of all vascular 
tumors. 
  50/group  78 weeks  20  2:  Hemangiosarcomas   
  female  (study  2500;  (liver and spleen).   
Ref. 8  B6C3F1  duration:  5000 ppm  Increased incidence  1480 
mice  102 wk),  (278, 558  of combined 
diet  mg/kg/d)  vascular tumors. 
Studies listed are in the Carcinogenic Potency Database (Ref. 9). 
a Carcinogenicity study selected for acceptable intake calculation.  
mg = milligram; kg = kilogram; d = day; HCl = hydrochloride; wk = week; NA = not applicable; ppm = parts 
per million; CPDB = Carcinogenic Potency Database. 
 
Mode of Action for Carcinogenicity 
 
p-Chloroaniline-induced tumors in male rats, such as spleen fibrosarcomas and 
osteosarcomas, typical for aniline 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 does not support tumor 
induction related to a direct interaction of p-chloroaniline or its metabolites with DNA. 
52  Contains Nonbinding Recommendations 
Similarly, the reported induction of micronuclei in vivo is likely to be secondary to 
regenerative anemia/altered erythropoiesis, as with aniline (Ref. 11,12). 
 
The tumor type with the lowest TD  was spleen tumors in male rats. However, since this 
50
tumor type is associated with a nonlinear dose relation, spleen tumors were not used to 
calculate the AI. Based on nonneoplastic (hematotoxic) effects, WHO (Ref. 3) recommends a 
level of 2 g/kg/day (i.e., 100 g/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 
 
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  of 33.8 mg/kg/day for 
50
combined numbers of adenomas and carcinomas. 
 
Calculation of AI 
 
Based on male mouse liver tumors for p-chloroaniline HCl 
 
Lifetime AI = TD /50,000 x 50 kg 
50
 
Lifetime AI = 33.8mg/kg/day /50,000 x 50 kg 
 
Lifetime AI = 34 g/day
53  Contains Nonbinding Recommendations 
REFERENCES (FOR P-CHLOROANILINE AND P-CHLOROANILINE HCL) 
 
1.  Beard RR, Noe JT, 1981, Aromatic Nitro and Amino Compounds. In: Clayton GD and 
Clayton FE, editors. Pattys Industrial Hygiene and Toxicology, New York: John Wiley 
and Sons, 2A:24132489. 
 
2.  Beratergremium fur Umweltrelevante Altstoffe (BUA), 1995, p-Chloroaniline. 
Beratergremium fur Umweltrelevante Altstoffe (BUA) der Gesellschaft Deutscher 
Chemiker (BUA Report 153). Weinheim: VCH, 171.  
 
3.  World Health Organization and International Programme on Chemical Safety, 2003, 
Concise International Chemical Assessment Document 48. 4-chloroaniline, available at 
http://www.inchem.org/documents/cicads/cicads/cicad48.htm. 
 
4.  Moore MM, Honma M, Clements J, Bolcsfoldi G, Burlinson B, Cifone M, et al., 2006, 
Mouse Lymphoma Thymidine Kinase Gene Mutation Assay: Follow-up Meeting of the 
International Workshop on Genotoxicity Testing  Aberdeen, Scotland, 2003  Assay 
Acceptance Criteria, Positive Controls, and Data Evaluation, Environ Mol Mutagen, 
47:15. 
 
5.  International Agency for Research on Cancer, 1993, Para-Chloroaniline. In: Monographs 
on the Evaluation of the Carcinogenic Risks to Humans: Volume 57: Occupational 
Exposures of Hairdressers and Barbers and Personal Use of Hair Colourants; Some Hair 
Dyes, Cosmetic Colourants, Industrial Dyestuffs and Aromatic Amines, Lyon, France: 
World Health Organization,  305321. 
 
6.  National Toxicology Program (NTP), 1989, 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),  Research Triangle Park, NC: National 
Institutes of Health . 
 
7.  Goodman DG, Ward JM, and Reichardt WD, 1984, 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, 
73(1):265273. 
 
8.  National Cancer Institute, 1979, Bioassay of p-Chloroaniline for Possible Carcinogenicity 
(CAS No. 106-47-8) NCI-CG-TR-189, Bethesda, MD: Department of Health, Education, 
and Welfare,  available at https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr189.pdf.  
 
9.  Carcinogenic Potency Database, website available at 
https://files.toxplanet.com/cpdb/index.html. 
 
10. Bus JS and Popp JA, 1987, Perspectives on the Mechanism of Action of the Splenic 
Toxicity of Aniline and Structurally-Related Compounds, Food Chem Toxicol, 
25(8):619626. 
 
11. Ashby J, Vlachos DA, and Tinwell H, 1991, Activity of Aniline in the Mouse Bone 
Marrow Micronucleus Assay, Mutat Res, 263(2):115117. 
 
54  Contains Nonbinding Recommendations 
12. Tweats DJ, Blakey D, Heflich RH, Jacobs A, Jacobsen SD, Morita T, Nohmi T, et al., 
2007, 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, 627(1):7891. 
55  Contains Nonbinding Recommendations 
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 Escherichia coli WP2uvrA (Ref. 1, 2, 3, 4). It was also weakly 
positive without metabolic activation in TA1535 in two of four 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 the International Agency for Research on Cancer as 
a Group 2 carcinogen, not classifiable as to its carcinogenicity in humans (Ref. 7), and the 
U.S. Environmental Protection Agency (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, which were statistically significant in males at the 
mid and high doses (7.7 and 41.2 milligrams (mg)/kilogram (kg)/day). Nonneoplastic 
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 hematocrit, 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 2,000 parts per 
million (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 4,000 ppm for the first 3 months, 500 ppm for next 2 
months, and 1,000 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 
56  Contains Nonbinding Recommendations 
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 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 a 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 
 
Most 
Animals/ 
Duration/  Sensitive  TD50 
Study  Dose  Controls  Doses 
Exposure  Tumor  (mg/kg/d) 
Group 
Site/Type/Sex 
  50/group  2  50  3:  Spleen  173.5 
  male F344  years,  40; 200;  hemangiosarcomas 
  rats (SPF)  diet  1,000  7.7 mg/kg/d 
ppm. 
 
(1.5; 7.7; 
 
41.2 
 
mg/kg/d) 
 
50/group  2  50  3:  Pheochromo-  116.9b 
 
female F344  years,  40; 200;  cytoma/Female 
 
rats (SPF)  diet  1,000  53.8 mg/kg/d 
  ppm. 
  (1.9; 
  9.8;53.8 
  mg/kg/d) 
50/group  2  50  3:  NA   
Ref. 9ac 
male  years,  125;500; 
 
Crj:BDF1  diet  2,000 
 
(SPF)  ppm. 
 
(15.3; 
 
60.1; 240 
 
.1 
 
mg/kg/d) 
57  Contains Nonbinding Recommendations 
Most 
Animals/ 
Duration/  Sensitive  TD50 
Study  Dose  Controls  Doses 
Exposure  Tumor  (mg/kg/d) 
Group 
Site/Type/Sex 
  50/group  2  50  3:  Hepatic  1919.9 
Ref. 9ac  female  years,  125;500;  hemangiosarcomas 
Crj:BDF1  diet  2,000  275.2 mg/kg/d 
(SPF)  ppm. 
(17.6; 
72.6; 
275.2 
mg/kg/d) 
14-15/  18  16  2:  NA  Negatived 
 
group  months,  Average 
male CD-1  diet;  17 and 
rats  sacrificed  33 
6 months  mg/kg; 
 
after last  (see 
 
dose  text) 
Ref. 10 
(22.6 
and 45.2 
mg/kg/d) 
14-20/sex  18  15/sex  2:  Vascular  430d 
group  months,  M: 341;  (hemangiomas/ 
CD-1 mice  diet;  720.  hemangio- 
sacrificed  F: 351;  sarcomas)/Male 
3 months  780 
after last  mg/kg/d 
dose 
  60/sex/  24 months  Yes  3:  NA  Negative 
group  5 d/  0.1; 0.7; 
Ref. 11c  Sprague  wk,  5 
Dawley rat  gavage  mg/kg/d 
Studies listed are in the Carcinogenic Potency Database (CPDB) (Ref. 12) unless otherwise noted. 
a Carcinogenicity study selected for acceptable intake/permissible daily exposure calculation. 
b TD  calculated based on carcinogenicity data (see Note 1). 
50
c Not in CPDB. 
d Histopathology limited to 11 to 12 tissues.  
mg = milligram; kg = kilogram; d = day; wk = week; ppm = parts per million; NA = not applicable; M = male; F 
= female. 
 
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 
58  Contains Nonbinding Recommendations 
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 nonlinear 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  was adrenal medullary pheochromocytomas in female 
50
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 nonmutagenic 
(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 nonmutagenic 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 nonlinear 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. 
 
Because 1-chloro-4-nitrobenzene is mutagenic, and a mutagenic mode of action cannot be 
ruled out, an acceptable intake (AI) calculation was performed. 
 
Regulatory and/or Published Limits 
 
No regulatory limits have been published, for example by the U.S. EPA, the World Health 
Organization, or the Agency for Toxic Substances and Disease Registry. 
 
Calculation of AI 
 
The most sensitive TD  is for adrenal medullary pheochromocytomas in female rats (Ref. 9). 
50
 
Lifetime AI = TD /50,000 x 50 kg 
50
 
Lifetime AI = 117 mg/kg/day /50,000 x 50 kg 
 
Lifetime AI = 117 g/day
59  Contains Nonbinding Recommendations 
REFERENCES (FOR 1-CHLORO-4-NITROBENZENE) 
 
1.  Haworth S, Lawlor T, Mortelmans K, Speck W, and Zeiger E, 1983, Salmonella 
Mutagenicity Test Results for 250 Chemicals, Environ Mutagen, 5 Suppl 1:1142. 
 
2.  Japan Chemical Industry Ecology-Toxicology & information Center (JETOC), 2005, 
Mutagenicity Test Data of Existing Chemical Substances Based on the Toxicity 
Investigation  System of the Industrial Safety and Health Law, Addendum 3, Tokyo, 
Japan: JETOC. 
 
3.  Kawai A, Goto S, Matsumoto Y, and Matsushita H, 1987, Mutagenicity of Aliphatic and 
Aromatic Nitro compounds, Sangyo Igaku, 29(1): 3455. 
 
4.  National Toxicology Program (NTP), 1993, NTP Technical Report on Toxicity Studies of 
2-Chloronitrobenzene and 4- Chloronitrobenzene (CAS Nos. 88-73-3 and 100-00-5) 
Administered by Inhalation to F344/N Rats and B6C4F  Mice, Research Triangle Park, 
1
NC: National Institutes of Health. 
 
5.  Cesarone CF, Bolognesi C, and Santi L, 1983, DNA Damage Induced In Vivo in Various 
Tissues by Nitrobenzene Derivatives, Mutat Res, 116(34):239246.
 
 
6.  Cesarone CF, Fugassa E, Gallo G, Voci A, and Orunesu M, 1984, Influence of the Culture 
Time on DNA Damage and Repair in Isolated Rat Hepatocytes Exposed to 
Nitrochlorobenzene  Derivatives, Mutat Res, 131(56):21522. 
 
7.  International Agency for Research on Cancer (IARC), 1996, IARC Monographs on the 
Evaluation of the Carcinogenic Risks to Humans: Volume 65: Printing Processes and 
Printing Inks, Carbon Black and Some Nitro Compounds, Lyon, France: World Health 
Organization.  
 
8.  U.S.  Environmental  Protection  Agency  (EPA).  Health  Effects  Assessment  Summary 
Tables, No. PB95-921199, Washington, DC: EPA . 
 
9.  Matsumoto M., Aiso S, Senoh H, Yamazaki K, Arito H, Nagano K, et al., 2006, 
Carcinogenicity and Chronic Toxicity of para-Chloronitrobenzene in Rats and Mice by 
Two-Year Feeding, J Environ Pathol Toxicol Oncol, 25(3):571584. 
 
10. Weisburger EK, Russfield AB, Homburger F, Weisburger JH, Boger E, Van Dongen CG, 
et al., 1978, Testing of Twenty-One Environmental Aromatic Amines or Derivatives for 
Long-Term Toxicity or Carcinogenicity, J Environ Pathol Toxicol, 2(2):32556. 
 
11. Schroeder RE and Daly JW, 1984, A Chronic Oral Gavage Study in Rats with p-
Nitrochlorobenzene,  Biodynamics Inc., Project No. 80-2487, NTIS/OTS 0536382. 
 
12. Carcinogenic Potency Database, website available at 
https://files.toxplanet.com/cpdb/index.html. 
 
13. Yoshida T, Andoh K, and Tabuchi T, 1991, Identification of Urinary Metabolites in Rats 
Treated With p-Chloronitrobenzene, Arch Toxicol, 65(1): 5258. 
 
60  Contains Nonbinding Recommendations 
14. Bray HG, James SP, and Thorpe WV, 1956, The Metabolism of the 
Monochloronitrobenzenes in the Rabbit, Biochem J, 64(1):3844. 
 
15. Yoshida T, Tabuchi T, and Andoh K, 1993, Pharmacokinetic Study of p-
Chloronitrobenzene in Humans Suffering From Acute Poisoning, Drug Metab Dispos, 
21(6):11426. 
 
16. IARC, 1993, IARC Monographs on the Evaluation of the Carcinogenic Risks to Humans: 
Volume 57: Occupational Exposures of Hairdressers and Barbers and Personal Use of 
Hair Colourants; Some Hair Dyes, Cosmetic Colourants, Industrial Dyestuffs and 
Aromatic Amines, Lyon, France: World Health Organization. 
 
17. Travlos GS, Mahler J, Ragan HA, Chou BJ, and Bucher JR, 1996, Thirteen-Week 
Inhalation Toxicity of 2- and 4-Chloronitrobenzene in F344/N Rats and B6C3F1 Mice, 
Fundam Appl Toxicol, 30(1):7592. 
 
18. Greim H, Hartwig A, Reuter U, Richter-Reichel HB, and Thielman HW, 2009, 
Chemically Induced Pheochromocytomas in Rats: Mechanisms and Relevance for Human 
Risk Assessment, Crit  Rev Toxicol, 39(8):695718. 
 
19. World Health Organization, 2003, Concise International Chemical Assessment Document 
48: 4-Chloroaniline, Geneva, available at 
http://www.inchem.org/documents/cicads/cicads/cicad48.htm. 
61  Contains Nonbinding Recommendations 
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 percent, 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 the International Agency for Research on Cancer 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 the National 
Toxicology Program (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 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 milligram (mg) per 
kilogram (kg) per day in the Carcinogenic Potency Database CPDB (Ref. 12), were 198 and 
396 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 
62  Contains Nonbinding Recommendations 
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, 
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 increased instances 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 interlaboratory 
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. 
 
p-Cresidine  Details of Carcinogenicity Studies 
 
Most 
Animals/  Sensitive 
Duration/  TD50 
Study  Dose  Controls  Doses  Tumor 
Exposure  (mg/kg/d) 
Group  Site/Typ
e/Sex 
  50/sex/  2  50  2:  Urinary  44.7 
  group  year,  0.5 and 1%  bladder 
  B6C3F1  feed  reduced after  /Male 
mice  21 weeks to 
Ref. 5a  0.15 and 
0.3%. 
M: 260:552. 
F: 281; 563 
mg/kg/d 
  50/sex/  2  50  0.5 and 1%  Urinary  88.4 
group  year,  M: 198;396.  bladder 
Ref. 5 
Fisher 344  feed  F: 245;491  /Male 
rats  mg/kg/d 
63  Contains Nonbinding Recommendations 
a Carcinogenicity study selected for acceptable intake calculation. 
Studies listed are in the Carcinogenic Potency Database (Ref. 12). 
mg = milligram; kg = kilogram; M = male; F = female. 
 
Mode of Action for Carcinogenicity 
 
p-Cresidine is a mutagenic carcinogen, and the acceptable intake (AI) is calculated by linear 
extrapolation from the TD . 
50
 
Regulatory and/or Published Limits 
 
No regulatory limits have been published 
 
Acceptable Intake 
 
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 the National Cancer Institute (NCI)/NTP (Ref. 5). The study in mice was 
selected for derivation of the AI because the most sensitive TD  was based on urinary 
50
bladder tumors in male mice. 
 
Calculation of AI 
 
The most sensitive TD  values from the NCI/NTP studies are for the urinary bladder in both 
50
sexes of rats and mice; in rats the TD  was 110 mg/kg/day for females and 88.4 mg/kg/day 
50
for males; in mice the TD  was 69 mg/kg/day for females and 44.7 mg/kg/day for males. The 
50
most conservative value is that identified for male mice. The lifetime AI is calculated as 
follows:  
 
Lifetime AI = TD /50,000 x 50 kg 
50
 
Lifetime AI = 44.7 mg/kg/day /50,000 x 50 kg 
 
Lifetime AI = 45 g/day 
64  Contains Nonbinding Recommendations 
REFERENCES (FOR P-CRESIDINE) 
 
1.  Zeiger E, Anderson B, Haworth S, Lawlor T, and Mortelmans K, 1988, Salmonella 
Mutagenicity Tests: IV. Results From the Testing of 300 Chemicals, Environ Mol 
Mutagen, 1988,11 Suppl 12:1157. 
 
2.  Dunkel VC, Zeiger E, Brusick D, McCoy E, McGregor D, Mortelmans K, et al., 1985, 
Reproducibility of Microbial Mutagenicity Assays: II. Testing of Carcinogens and 
Noncarcinogens in Salmonella typhimurium and Escherichia coli, Environ Mutagen, 7 
Suppl 5:1248. 
 
3.  Japan Chemical Industry Ecology-Toxicology and Information Center (JETOC), 1997, 
Mutagenicity Test Data of Existing Chemical Substances Based on the Toxicity 
Investigation  of the Industrial Safety and Health Law, Supplement, Tokyo, Japan: 
JETOC. 
 
4.  Jakubczak JL, Merlino G, French JE, Muller WJ, Paul B, Adhya S, and Gargas S, 1996, 
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, 93(17):90739078. 
 
5.  National Cancer Institute, 1979, Bioassay of p-Cresidine for Possible Carcinogenicity 
(CAS No. 120-71-8) NCI-CG-TR-142, Bethesda, MD: Department of Health, Education, 
and Welfare, available at https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr142.pdf.  
 
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, 250(12):115133. 
 
7.  Morita T, Asano N, Awogi T, Sasaki YF, Sato S-I, Shimada H, et al., 1997, Evaluation of 
the Rodent Micronucleus Assay in the Screening of IARC Carcinogens (Groups 1, 2A and 
2B). The Summary Report of the 6th Collaborative Study by CSGMT/JEMS.MMS, Mutat 
Res, 389:3122. 
 
8.  Delker DA, Yano BL, and Gollapudi BB, 2000, Evaluation of Cytotoxicity, Cell 
Proliferation, and Genotoxicity Induced by p-Cresidine in Hetero- and Nullizygous 
Transgenic p53 mice, Toxicol Sci, 55(2):361369. 
 
9.  Stoll RE, Blanchard KT, Stoltz JH, Majeska JB, Furst S, Lilly PD, et al., 2006, 
Phenolphthalein and Bisacodyl: Assessment of Genotoxic and Carcinogenic Responses in 
Heterozygous p53  (+/-) Mice and Syrian Hamster Embryo (SHE) Assay, Toxicol Sci, 
90(4):440450. 
 
10. Sasaki YF, Nishidate E, Su YQ, Matsusaka N, Tsuda S, Susa N, et al., 1998, Organ-
Specific Genotoxicity of the Potent Rodent Bladder Carcinogens o-Anisidine and p-
Cresidine, Mutat  Res, 412:155160. 
 
11. International Agency for Research on Cancer (IARC), 1982, para-Cresidine. In: IARC 
Monographs on the Evaluation of the Carcinogenic Risks to Humans: Overall Evaluations 
65  Contains Nonbinding Recommendations 
of Carcinogenicity: An Updating of IARC Monographs Volumes 1 to 42: Supplement 7, 
Lyon, France: World Health Organization, 27:92.. 
 
12. Carcinogenic Potency Database, website, available at 
https://files.toxplanet.com/cpdb/index.html. 
 
13. Storer RD, French JE, Haseman J, Hajian G, LeGrand EK, Long GG, et al., 2001, p53+/- 
Hemizygous Knockout Mouse: Overview of Available Data, Toxicol Pathol, 29 
Suppl:3050. 
66  Contains Nonbinding Recommendations 
1,2-DIBROMOETHANE (CAS# 106-93-4) 
 
Potential for Human Exposure 
 
1,2-Dibromoethane was previously used as an insect fumigant and soil nematicide but was 
banned by the U.S. Environmental Protection Agency  and the European Commission  due to 
toxicity concerns (Ref. 1, 2). 1,2-Dibromoethane is used in the synthesis of active 
pharmaceutical ingredients. 
 
Mutagenicity/Genotoxicity 
 
1,2-Dibromoethane is mutagenic/genotoxic in vitro and in vivo. 1,2-Dibromoethane was 
mutagenic in the Ames mutation assay both in the presence and absence of added metabolic 
activation (Ref. 37). 1,2-Dibromoethane was positive in the mouse lymphoma assay, with 
and without metabolic activation (Ref. 8). It caused a dose-dependent increase in DNA repair 
in both spermatocytes and hepatocytes in vitro (Ref. 9) and induced mutations in Chinese 
hamster ovary (CHO) cells (Ref. 10). 1,2-Dibromoethane increased the frequencies of 
chromosome aberrations in a dose-dependent manner in CHO cells (Ref. 11). In vivo in the 
comet assay in rats, positive results were obtained in liver and glandular stomach following 
treatment with 1,2-dibromoethane at 100 (milligrams) (mg)/kilogram (kg). 1,2-
Dibromoethane was negative in the bone marrow erythrocyte micronucleus test in rats when 
tested up to 100 mg/kg (Ref. 12).  
 
Carcinogenicity 
 
1,2-Dibromoethane is classified by the International Agency for Research on Cancer as 
probably carcinogenic to humans (Group 2A) (Ref. 13). Inhalation and oral carcinogenicity 
studies are cited in the Carcinogenic Potency Database (Ref. 14). 1,2-Dibromoethane was 
carcinogenic following both routes of administration in male and female rats and mice (Ref. 
1621). The most sensitive tumor sites were forestomach following oral administration 
(gavage or drinking water) and nasal cavity following inhalation. Other tumor sites include 
blood vessels, lung, liver, and mammary glands. There was more than one positive 
experiment in both species.  
 
1,2-Dibromoethane  Details of Carcinogenicity Studies 
 
Most 
Animals/ 
Duration/  Sensitive  TD  
Study  Dose  Controls  Dosesa  50
Exposure  Tumor  (mg/kg/d)b 
Group 
Site/Type/Sex 
Ref. 16  30/sex/  M: 65  50  1:   Forestomach/  11.8 
  group  weeks  4 mmol/L  Squamous 
B6C3F1  F: 73  M: 116   carcinoma/Ma
mice  weeks,  F: 103  le 
  drinking  mg/kg/day 
water 
Ref. 17  50/sex/  78 weeks,  100  1:   Forestomach/  9.44 
  group  drinking  M: 46.7  Squamous 
B6C3F1  water  F: 48  carcinoma/Ma
mice  mg/kg/day  le 
67  Contains Nonbinding Recommendations 
Most 
Animals/ 
Duration/  Sensitive  TD  
Study  Dose  Controls  Dosesa  50
Exposure  Tumor  (mg/kg/d)b 
Group 
Site/Type/Sex 
Ref. 18  50/sex/  53 weeks,  20  2:   Forestomach/  2.38 
  group  gavage  M: 30, 53   Squamous 
B6C3F1    F: 26, 52  cell 
mice  mg/kg/day  carcinoma/ 
    Male 
Ref. 18  50/sex/  M: 40  20  2:   Stomach/  1.26 
  group  weeks  M: 27.4,  Squamous   
Osborne- F: 50  29.2   cell 
Mendel  weeks,  F: 26.7, 28.1  carcinoma/ 
rats  gavage  mg/kg/day  Female 
 
Ref. 19  50/sex/  M: 78  50  2:   Lung/  18.2 
  group  weeks, F:  M: 19.9,  Multiple   
B6C3F1  96 weeks,  79.5   tumor types/ 
mice  inhalation  F: 23.9, 95.6  Male 
  mg/kg/day 
Ref. 19c  50/sex/  M: 95  50  2:   Nasal cavity/  2.33 
   group   weeks  M: 4.0, 15.9   Carcinomas   
F344 rats   F: 97  F: 5.71, 22.8  and 
weeks,  mg/kg/day  adenocarcino
inhalation      ma/ Female 
Ref. 20  48/sex/  78 weeks,  48  1:   Nasal  1.19 
  group  inhalation  M: 9.39   cavity/Multipl
Sprague-   F: 13.4  e tumor 
Dawley  mg/kg/day  types/Male 
rats  
 
Ref. 21d  50/sex/  103 weeks  50  2:   Nasal  NC 
  group  (10 ppm) /  M and F:  cavity/Multipl
B6C3F1  90 weeks  3.55, 14.18  e tumor 
mice    (40 ppm),  mg/kg/day  types/Female 
  inhalation 
mg = milligram; kg = kilogram; d = day; M = male; F = female; mmol = millimole; L = liter; ppm = parts per 
million; NC = not calculated. 
a mg/kg/day values stated in the Carcinogenic Potency Database (CPDB) (Ref. 14).  
b Individual TD  values are the CPDB TD  values as reported in the Lhasa carcinogenicity database (Ref. 15). 
50 50
TD  values represent the TD  from the most sensitive tumor site.  
50 50
c Carcinogenicity study selected for acceptable intake derivation. 
d This study was conducted specifically to evaluate the types of tumors formed in the nasal cavity of mice 
following inhalation exposure. No other tissues were evaluated for carcinogenicity.  
 
The most robust carcinogenicity study is the inhalation study conducted by the National 
Toxicology Program (NTP) (Ref. 19) in F344 rats. This study (duration 95 weeks in males 
and 97 weeks in females) included two test article treatment groups with adequate dose 
spacing (male: 4.0, 15.9 mg/kg/day, female: 5.71, 22.8 mg/kg/day with 50 rats/sex/group) 
and a control group (50/sex). The TD  from the most sensitive sex and site is 2.33 
50
mg/kg/day. Another study with inhalation exposure conducted in Sprague Dawley rats (Ref. 
68  Contains Nonbinding Recommendations 
20) resulted in a lower TD ; however, the study comprised only one dose group, only 78 
50
weeks exposure duration, and 48 animals/dose; therefore, this study was considered inferior 
to the NTP study with respect to estimating the TD .  
50
 
The study in B6C3F1 mice with 1,2-dibromoethane administered by gavage for 53 weeks 
(Ref. 18) is the most robust study using the oral route of exposure. This study employed two 
test article dose groups (50/sex/group) in addition to a control group (20/sex). The TD  from 
50
the most sensitive sex and site is 2.38 mg/kg/day. Another oral study was conducted in 
Osborne-Mendel rats included  two dose groups; however, due to insufficient dose spacing 
(Ref. 18) and less than 1 year exposure, the study is considered less useful as it limits 
characterization of the dose-response relationship and estimation of the TD  (Ref. 18).  
50
 
Mode of Action for Carcinogenicity 
 
1,2-Dibromoethane is a mutagenic carcinogen, which is expected to be mutagenic based on 
an alkylating mechanism of action. Therefore, the acceptable intake (AI) can be calculated by 
linear extrapolation from the TD . The tumor types with the lowest calculated TD  (highest 
50 50
potency) for 1,2-dibromoethane following oral exposure are forestomach tumors in mice and 
rats (Ref. 18).  Following inhalation exposure, the lowest calculated TD  values are 
50
associated with the lung and nasal cavity for mice and rats, respectively. High concentrations 
of orally dosed nonmutagenic chemicals have been shown to cause inflammation and 
irritation after contact with the forestomach leading to hyperplasia and ultimately tumors. 
Substances that are dosed 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. Hence, such tumor induction is considered not relevant to humans at nonirritating 
doses (Ref. 22, 23). The same inflammatory and hyperplastic effects are also seen with 
mutagenic chemicals. However, in the case of 1,2-dibromoethane, which is a directly DNA-
reactive alkylating agent and a reported multisite, multispecies carcinogen, it is difficult to 
discriminate between the contribution to the mode of action of these nonmutagenic, high-
dose effects compared with direct mutation induction. 
 
Regulatory and/or Published Limits 
 
No regulatory limits have been published. 
 
Acceptable Intake  
 
Rationale for selection of study for AI calculation 
 
1,2-Dibromoethane is a mutagenic carcinogen via the inhalation and oral routes of exposure. 
1,2-Dibromoethane is considered to be a carcinogen in both mice and rats. The available 
toxicological data indicate that absorption of inhaled 1,2-dibromoethane occurs in several 
animal species. In rats, oral absorption is nearly complete within 30 minutes (Ref. 1). 
Therefore, it can be reasonably assumed that complete systemic exposure to 1,2-
dibromoethane occurs following oral and inhalation exposure. This assumption is also 
supported by the observation of distal tumors in animals exposed to 1,2-dibromoethane by 
both routes of exposure. TD  values tend to be similar across species and routes of 
50
administration.  
 
69  Contains Nonbinding Recommendations 
Taking into consideration the carcinogenicity data generated by NTP in both mice and rats, 
the TD  for the most sensitive sex/site from the most appropriate study is 2.33 mg/kg/day. 
50
This is the TD  value derived from F344 female rats based on the incidence of nasal cavity 
50
tumors.  
 
Given that the TD  values recommended for the derivation of an inhalation AI and an oral 
50
AI are very similar (2.33 and 2.38 mg/kg/day, respectively), a single AI for both routes of 
administration is calculated below using a TD  value of 2.3 mg/kg/day. 
50
 
Calculation of AI 
 
Lifetime AI = TD /50,000 x 50 kg 
50
 
Lifetime AI = 2.3 mg/kg/day/50,000 x 50 kg 
 
Lifetime AI = 2 micrograms (g)/day
70  Contains Nonbinding Recommendations 
REFERENCES (FOR 1,2-DIBROMOETHANE) 
 
1.  Agency for Toxic Substances and Disease Registry, 2018, Toxicological Profile for 1,2-
Dibromoethane, U.S. Department of Health and Human Services. 
 
2.  European Council Directive, 1987, (87/ 1 81 /EEC) amending the Annex to Directive 
79/117/EEC prohibiting the placing on market and use of plant protection products 
containing certain active substances, Official Journal of the European Communities, L 
71/33. 
 
3.  Moriya M, Ohta T, Watanabe K, Miyazawa T, Kato K, and Shirasu Y, 1983, Further 
Mutagenicity Studies on Pesticides in Bacterial Reversion Assay Systems, Mutat Res, 
116(34):185216. 
 
4.  McCann J, Choi E, Yamasaki E, and Ames BN, 1975, Detection of Carcinogens as 
Mutagens in the Salmonella/Microsome Test: Assay of 300 Chemicals, Proc Natl Acad 
Sci USA, 72(12):51355139. 
 
5.  Rannug U, Sundvall A, and Ramel C, 1978, The Mutagenic Effect of 1,2-dichloroethane 
on Salmonella typhimurium 1. Activation Through Conjugation With Glutathione in 
Vitro, Chem Biol Interact, 20(1):116. 
 
6.  Strobel K and Grummt T, 1987, Aliphatic and Aromatic Halocarbons as Potential 
Mutagens in Drinking Water. III. Halogenated Ethanes and Ethenes, Toxicol Environ 
Chem, 15:101128. 
 
7.  Dunkel VC, Zeiger E, Brusick D, McCoy E, McGregor D, Mortelmans K, Rosenkranz 
HS, and Simmon VF, 1985, Reproducibility of Microbial Mutagenicity Assays: II. 
Testing of Carcinogens and Noncarcinogens in Salmonella typhimurium and Escherichia 
coli, Environ Mutagen, 7(Suppl. 5):1248. 
 
8.  Clive D, McCuen R, Spector JF, Piper C, and Mavournin KH, 1983, Specific Gene 
Mutations in L5178Y Cells in Culture: A Report of the U.S. Environmental Protection 
Agency Gene-Tox Program, Mutat Res, 115(2):225251. 
 
9.  Working PK, Smith-Oliver T, White RD, and Butterworth BE, 1986, Induction of DNA 
Repair in Rat Spermatocytes and Hepatocytes by 1,2-Dibromoethane: The Role of 
Glutathione Conjugation, Carcinogenesis, 7(3):467472. 
 
10. Tan EL and Hsie AW, 1981, Mutagenicity and Cytotoxicity of Haloethanes as Studied in 
the CHO/HGPRT System, Mutat Res, 90(2):183191. 
 
11. Asita A, 1989, A Comparative Study of the Clastogenic Activity of Ethylating Agents, 
Mutagenesis, 4:432436. 
 
12. Takasawa H, Takashima R, Narumia K, Kawasako K, Hattoria A, Kawabata M, and 
Hamada S, 2015, Results of the International Validation of the In Vivo Rodent Alkaline 
Comet Assay for the Detection of Genotoxic Carcinogens: Individual Data for 1,2-
Dibromoethane, p-Anisidine, and o-Anthranilic Acid in the 2nd Step of the 4th Phase 
71  Contains Nonbinding Recommendations 
Validation Study Under the JaCVAM Initiative, Mutat Res Genet Toxicol Environ 
Mutagen, 786788:144150. 
 
13. International Agency for Research on Cancer, 1999, IARC Monographs on the 
Evaluation of Carcinogenic Risks to Humans: Volume 71: Re-Evaluation of Some 
Organic Chemicals, Hydrazine and Hydrogen Peroxide, Lyon, France: World Health 
Organization, 641. 
 
14. Carcinogenic Potency Database, website available at 
https://files.toxplanet.com/cpdb/index.html. 
 
15. Lhasa Carcinogenicity Database, website available at 
https://carcdb.lhasalimited.org/carcdb-frontend/. 
 
16. van Duuren BL, Melchionne S, Kline SA, and Seidman I, 1985, Carcinogenicity 
Bioassays of Bromoacetaldehyde and Bromoethanol  Potential Metabolites of 
Dibromoethane, Teratog Carcinog Mutagen, 5(6):393403. 
 
17. Van Duuren BL, Melchionne S, Seidman I, and Pereira MA, 1986, Chronic Bioassays of 
Chlorinated Humic Acids in B6C3F1 Mice, Environmental Health Perspectives, 69:109
117. 
 
18. National Toxicology Program, 1978, Bioassay of 1,2-Dibromoethane for Possible 
Carcinogenicity (CAS No. 106-93-4) (National Toxicology Program Technical Report 
TR-86), Bethesda, Maryland: National Institutes of Health. 
 
19. National Toxicology Program (NTP), 1982, NTP Technical Report on the Carcinogenesis 
Bioassay of 1,2-dibromoethane (CAS No. 106-93-4) in F344 Rats and B6C3F1 Mice 
(Inhalation Study), TR-210, Research Triangle Park, NC: National Institutes of Health. 
 
20. Wong LCK, Winston JM, Hong CB, and Plotnick H, 1982, Carcinogenicity and Toxicity 
of 1,2-Dibromoethane in the Rat, Toxicology and Applied Pharmacology, 63:155165. 
 
21. Stinson SF, Reznik G, and Ward JM, 1981, Characteristics of Proliferative Lesions in the 
Nasal Cavities of Mice Following Chronic Inhalation of 1,2-Dibromoethane, Cancer Lett, 
12(12):121129. 
 
22. Proctor DM, Suh M, Chappell G, Borghoff SJ, Thompson CM, Wiench K, Finch L, and 
Ellis-Hutchings R, 2018, An Adverse Outcome Pathway (AOP) for Forestomach Tumors 
Induced by Non-genotoxic Initiating Events, Regul Toxicol Pharmacol, 96:3040. 
 
23. Proctor DM, Gatto NM, Hong SJ, and Allamneni KP, 2007, Mode-of-Action Framework 
for Evaluating the Relevance of Rodent Forestomach Tumors in Cancer Risk Assessment, 
Toxicol Sci, 98:313326. 
72  Contains Nonbinding Recommendations 
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 the International Agency for Research on Cancer 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 acceptable intake (AI) derivation 
used inhalation and intraperitoneal administration. 
 
Syrian golden hamsters were exposed to 1 parts per million 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 percent 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 percent (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 milligrams (mg) of DMCC was applied three 
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. 
73  Contains Nonbinding Recommendations 
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 
 
Animals/  Tumor 
Duration/  TD50 
Study  Dose  Controls  Doses  Observation
Exposure  (mg/kg/d) 
Group  s 
  30  64 weeks  30  1:  Injection site :  4.59 e 
female  once/wee 1 mg  malignant 
Ref. 6a 
ICR/Ha  k, intra-  5.71  tumors/Female 
Swiss mice  peritoneal  mg/kg/d 
  99  Lifetime  50 sham  1:  Squamous cell  0.625 
male  6 h/d,  treated  1 ppm  carcinoma of nasal 
Ref. 5b  Syrian  5 d/week,  200  0.553  cavity 
golden  inhalation  untreated  mg/kg/d 
hamsters 
  50  70 weeks  50  1:  Skin: Papillomas  NAc 
Ref. 6  female  3  2 mg  and carcinomas/ 
ICR/Ha  times/wee Female 
Swiss mice  k, Skin 
  50  61 weeks  50  1:  Injection site:  NAc 
female  once/week,  5 mg  Fibrosarcomas; 
Ref. 6 
ICR/Ha  subcutaneous  Squamous cell 
Swiss mice  carcinomas/ 
Female 
  Male  6 weeks  Yes  1:  Nasal tumors/Male  NAf 
  Sprague-  6 h/d,  1 ppm 
Dawley  5 d/week, 
Ref. 7 
rats  inhalation; 
examined at 
end of life 
  30-50  18-22 months  Yes  2:  Skin.  NAc 
female  3 times  2 and  Mainly skin 
Ref. 8 
ICR/Ha  /week,  4.3 mg  squamous 
Swiss mice  skin  carcinoma/Female 
  Female  18-22 months  Yes  1:  Site of  NAd 
  ICR/Ha  once/week,  4.3 mg  administration. 
  Swiss mice  subcutaneous  Mainly sarcoma. 
Hemangioma, 
Ref. 8 
squamous 
carcinoma and 
papilloma also 
seen/Female 
  Female  12 months  Yes  2:    NAd 
  ICR/Ha  once/week,  0.43   
Ref. 8  Swiss mice  subcutaneous;  and 4.3   
examined at  mg 
end of life 
Studies listed are in the Carcinogenic Potency Database (CPDB) (Ref. 9) unless otherwise noted. 
74  Contains Nonbinding Recommendations 
a Carcinogenicity study selected for noninhalation acceptable intake (AI). 
b Carcinogenicity study selected for inhalation AI.  
c 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. 
d Subcutaneous and skin painting studies are not included in CPDB as route with greater likelihood of whole 
body exposure is considered more valuable. 
e Histopathology only on tissues that appeared abnormal at autopsy. 
f Examined only for nasal cancer. Does not meet criteria for inclusion in CPDB of exposure for at least one 
fourth of the standard lifetime. 
mg = milligram; kg = kilogram; d = day; h = hour; ppm = parts per million; NA = not applicable. 
 
Regulatory and/or Published Limits 
 
No regulatory limits have been published. 
 
Acceptable Intake 
 
Based on the above data, DMCC is considered to be a mutagenic carcinogen. As a result, 
linear extrapolation from the most sensitive TD  in carcinogenicity studies is an appropriate 
50
method with which to derive an acceptable risk dose. Since DMCC appears to be a site-of- 
contact carcinogen, it 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  was 4.59 mg/kg/day based on mixed tumor incidences (Carcinogenic 
50
Potency Database). 
 
The lifetime AI is calculated as follows:  
 
Lifetime AI = TD /50,000 x 50 kg 
50
 
Lifetime AI = 4.59 mg/kg/day /50,000 x 50 kg 
 
Lifetime AI = 5 micrograms (g)/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  was 0.625 mg/kg/day. 
50
 
Lifetime AI = TD /50,000 x 50 kg 
50
 
Lifetime AI = 0.625 mg/kg/day /50,000 x 50 kg 
 
Lifetime inhalation AI = 0.6 g/day
75  Contains Nonbinding Recommendations 
REFERENCES (FOR DIMETHYLCARBAMYL CHLORIDE) 
 
1.  Dunkel V, Zeiger E, Brusick D, McCoy E, McGregor D, Mortelmans K, et al., 1984, 
Reproducibility of Microbial Mutagenicity Assays. I. Tests With Salmonella typhimurium 
and Escherichia coli Using a Standardized Protocol, Environ Mutagen, 6 Suppl 2:1251. 
 
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, 168:69240. 
 
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, 123:61118. 
 
4.  International Agency for Research on Cancer, 1999, Monographs on the Evaluation of the 
Carcinogenic Risks to Humans: Volume 71, Lyon, France: World Health Organization 
539. Available at http://monographs.iarc.fr/index.php. 
 
5.  Sellakumar AR, Laskin S, Kuschner M, Rush G, Katz GV, Snyder CA, et al., 1980, 
Inhalation Carcinogenesis by Dimethylcarbamoyl Chloride in Syrian Golden Hamsters, J 
Environ  Pathol Toxicol, 4:107115. 
 
6.  Van Duuren BL, Goldschmidt BM, Katz C, Seidman I, and Paul JS, 1974, Carcinogenic 
Activity of Alkylating Agents, J Natl Cancer Inst, 53:695700. 
 
7.  Snyder CA, Garte SJ, Sellakumar AR, and Albert RE, 1986, Relationships Between the 
Levels of Binding to DNA and the Carcinogenic Potencies in Rat Nasal Mucosa for Three 
Alkylating Agents, Cancer Lett, 33:17581. 
 
8.  Van Duuren BL, Melchionne S, and Seidman I, 1987, Carcinogenicity of Acylating 
Agents: Chronic Bioassays in Mice and Structure-Activity Relationships (SARC), J Am 
Col Toxicol, 6:479487. 
 
9.  Carcinogenic Potency Database, website available at 
https://files.toxplanet.com/cpdb/index.html. 
76  Contains Nonbinding Recommendations 
DIMETHYL SULFATE (CAS# 77-78-1) 
 
Potential for Human Exposure 
 
Dimethyl sulfate (DMS) is found in ambient air with mean concentration of 7.4 (micrograms) 
g per cubic meter or 1.4 parts per billion based on 1983 data compiled from a single site by 
the U.S. Environmental Protection Agency (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 the International Agency for Research on Cancer 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 Carcinogenic 
Potency Database. The studies evaluating carcinogenicity of DMS are described below 
(excerpted from U.S. EPA, Ref. 5). 
 
77  Contains Nonbinding Recommendations 
Dimethyl Sulfate (DMS) - Details of Carcinogenicity Studies 
 
Tumor 
Duration/  TD50 
Study  Animals  Controls  Doses  Observation
Exposure  (mg/kg/d) 
s 
  Golden  15 months  Yes  2:  Tumors in  NAa 
  hamsters,  6 h/d,  0.5; 2.0 ppm  lungs, thorax 
  Wistar  2 d/wk followed  and nasal 
  rats, and  by 15  passages at both 
  NMRI  months  doses 
mice  observation 
Ref. 6 
male and  period, 
female  inhalation 
(number 
not 
clearly 
specified) 
  20-27 BD  130 days  No  2:  Squamous cell  NAb 
  rats  1 h/d, 5 d/wk  3; 10 ppm  carcinoma in 
  Sex not  followed by  nasal epithelium 
  specified  643-day  at 3 ppm. 
  observation  Squamous cell 
  period,  carcinomas in 
Ref. 7  inhalation  nasal epithelium 
and lympho- 
sarcoma in the 
thorax with 
metastases to 
the lung at 10 
ppm. 
  8-17 BD  394 days  No  2:  Injection-site  NAc 
  Rats  The duration of  8; 16  sarcomas in 
  Sex not  the study was  mg/kg/wk  7/11 at low dose 
  specified  not reported but  and 4/6 at high 
Ref. 8  mean tumor  dose; occasional 
induction time  metastases to 
was 500 days,  the lung. One 
subcutaneous  hepatic 
carcinoma. 
  15 BD  Up to 740 day  No  1:  Local sarcomas  NAc 
  Rats  evaluation  50 mg/kg  of connective 
  Sex not  Following  tissue in 7/15 
Ref. 7  specified  single  rats; multiple 
injection,  metastases to 
subcutaneous  the lungs in 
three cases 
  12 BD  800 days  No  2:  No tumors  NAc 
rats  Once/wk,  2; 4 mg/kg  reported 
Ref. 7 
Sex not  intravenous 
specified 
78  Contains Nonbinding Recommendations 
Study  Animals  Duration/  Controls  Doses  Tumor  TD50 
Exposure  observations  (mg/kg/d) 
  8 BD rats  1 year offspring  No  1:  4/59 offspring  NAd 
  (pregnant  observation  20 mg/kg  had malignant 
  females)  following  tumors of the 
Ref. 7  single dose,  nervous system 
gestation day  while 2/59 had 
15, intravenous  malignant 
hepatic tumors. 
  90  Duration not  Not  3:  Increase in lung  NAe 
female  reported  indicated  0.4; 1; 20  adenomas at 
Ref. 9 
CBAX57  4 h/d, 5  mg/m3  high dose 
Bl/6 mice  d/wk, 
inhalation 
  20  475 days  Not  h  No findings  NAf 
ICR/Ha  3  indicated  0.1 mg 
Ref. 10 
Swiss  times/wk, 
miceg  dermal 
Studies listed are in not in the Carcinogenic Potency Database.  
a Control data not reported. Tumor incidences not tabulated by species or dose. 
b 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. 
c Small group size, no concurrent control group. 
d No concurrent control group. 
e Duration not reported 
f Limited number of animals. Only one dose tested. Even when DMS was combined with tumor promoters no 
tumors were noted. 
g Sex not specified. 
mg = milligram; kg = kilogram; d = day; h = hour; wk = week; ppm = parts per million; NA = not applicable; m 
= meter. 
 
Mode of Action for Carcinogenicity 
 
DMS is a mutagenic carcinogen, and the acceptable intake (AI) is calculated by linear 
extrapolation from the TD . 
50
 
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  (dose that resulted in a 25 percent increase in tumors) using the 
25
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 two dose groups, and 
minimal pathological evaluations; therefore, the study was not suitable for linear 
extrapolation. 
 
Acceptable Intake 
 
Although 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  value. Moreover, the 
50
79  Contains Nonbinding Recommendations 
inhalation studies that are available are limited for a variety of reasons and are not suitable for 
TD  extrapolation. Given this, it is reasonable to limit DMS to the threshold of toxicological 
50
concern lifetime level of 1.5 g/day. 
 
Lifetime AI = 1.5 g/day  
80  Contains Nonbinding Recommendations 
REFERENCES (FOR DIMETHYL SULFATE) 
 
1.  U.S. Environmental Protection Agency, 1985, 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. 
 
2.  Hoffmann GR, 1980, Genetic Effects of Dimethyl Sulfate, Diethyl Sulfate, and Related 
Compounds, Mutat Res, 75:63129. 
 
3.  Skopek TR, Liber HL, Kaden DA, and Thilly WG, 1978, Relative Sensitivities of 
Forward and Reverse Mutation Assays in Salmonella typhimurium, Proc Natl Acad Sci 
USA, 75:44654469. 
 
4.  International Agency for Research on Cancer, 1999, Monographs on the Evaluation of the 
Carcinogenic Risks to Humans: Volume 71, Lyon, France: World Health Organization, 
575. 
 
5.  U.S. Environmental Protection Agency, 1988, Integrated Risk Information System 
(IRIS): Chemical Assessment Summary: Dimethyl Sulfate (CASRN 77-78-1), available 
at 
https://cfpub.epa.gov/ncea/iris/iris_documents/documents/subst/0365_summary.pdf#nam
e. 
 
6.  Schlogel FA and Bannasch P, 1972, Carcinogenicity and Chronic Toxicity of Inhaled 
Dimethyl Sulfate. (In German) (Inaugural Dissertation) Julius-Maximilians University, 
Wurzburg (data in Ref. 11). 
 
7.  Druckrey H, 1970, Carcinogenic Alkylating Compounds: III. Alkyl Halogenids, Sulfates, 
Sulfonates, and Heterocyclics, (Article in German) Z. Krebsforsch, 74:241273. 
 
8.  Druckrey H, 1966, Carcinogenic Alkylating Compounds: I. Dimethyl Sulfate, 
Carcinogenic Effect in Rats and Probable Cause of Occupational Cancer, (Article in 
German) Z. Krebsforsch, 68:103111. 
 
9.  Fomenko VN, Katasova LD and Domshlak MG, 1983, USSR Minist Health All-Union 
Sci Soc Med Genet 1:34849 as cited in World Health Organization, Environ Health 
Criteria, Dimethyl Sulfate:36. 
 
10. Van Duuren BL, Goldschmidt BM, Katz C, Seidman I, and Paul JS, 1974, Carcinogenic 
Activity of Alkylating Agents, J Natl Cancer Inst, 53:695700. 
 
11. ECHA (European Chemical Agency), 2002, European Union Risk Assessment Report:  
Institute for Health and Consumer Protection.  Dimethyl Sulphate, [Online] 2002 Vol. 12, 
available at https://echa.europa.eu/documents/10162/3d2e4243-8264-4d09-a4ab-
92dde5abfadd.  
81  Contains Nonbinding Recommendations 
EPICHLOROHYDRIN (CAS# 106-89-8) 
 
Potential for Human Exposure 
 
Epichlorohydrin is used in the synthesis of active pharmaceutical ingredients.  
 
Mutagenicity/Genotoxicity 
 
Epichlorohydrin is mutagenic and genotoxic in vitro, with mixed results of genotoxicity tests 
in vivo. Although genotoxicity in vitro is seen both with and without liver S9 metabolic 
activation, activity tends to be suppressed by S9 (Ref. 1, 2, 3). Epichlorohydrin is mutagenic 
in the Ames test in several strains of Salmonella typhimurium and in Escherichia coli 
WP2uvrA with and without metabolic activation using both plate incorporation and 
preincubation protocols (Ref. 4). In vitro, epichlorohydrin is positive in mammalian cells for 
mutation, and for chromosome and DNA damage.  
 
Carcinogenicity 
 
Epichlorohydrin is classified by the International Agency for Research on Cancer as a Group 
2A carcinogen, probably carcinogenic to humans (Ref. 1). Epichlorohydrin is a site-of-
contact carcinogen, by oral, subcutaneous, and inhalation routes.  
 
In an oral study, Wester et al. (Ref. 5) treated rats by oral gavage with epichlorohydrin, five 
times per week for lifetime at 2 and 10 milligrams (mg)/kilogram (kg); when converted to an 
average daily dose for seven days per week, the doses shown in the Carcinogen Potency 
Database (CPDB) (Ref. 6) are 1.43 and 7.14 mg/kg/day, respectively. In the surviving rats at 
the end of the study, squamous cell carcinomas were found in the forestomach of all 24 
females and 35 of 43 males at the high dose, and in 2 of 27 females and 6 of 43 males at the 
low dose. The tumors were considered low grade, and there was no evidence of metastasis; 
no increase in tumors was found at other sites. At both dose levels, there were proliferative 
changes in the forestomach mucosa with ulceration and necrosis observed in some cases at 
the high dose. A TD  of 2.55 mg/kg/day is reported in the CPDB. The findings are consistent 
50
with the squamous cell carcinomas seen in the forestomach of male Wistar rats treated with 
epichlorohydrin in drinking water for up to 81 weeks (Ref. 7). The Konishi et al. study is not 
included in the CPDB and not considered in this monograph because of technical 
deficiencies, and poor condition of the animals.  
 
In an inhalation study, Laskin et al. (Ref. 8) treated male Sprague Dawley rats with 
epichlorohydrin by inhalation, 6 hours/day, 5 days/week, either for a short-term regimen (30 
exposures at 100 parts per million (ppm)) with lifetime observation (140 rats per group), or 
throughout lifetime at lower doses, 10 and 30 ppm (100 rats per group). After the shorter term 
and high-dose exposure, squamous cell carcinomas of the nasal cavity in 15/140 rats and 
respiratory tract papillomas in 3/140 rats were observed and were associated with severe 
inflammation in the nasal turbinates, the larynx, and the trachea. After lifetime exposure, 
tumors were seen in 2/100 animals exposed to a dose of 30 ppm and only in the nasal cavity 
(one nasal carcinoma and one nasal papilloma). Despite the low tumor incidence, a TD  of 
50
421 mg/kg/day is reported in the CPDB.   
 
82  Contains Nonbinding Recommendations 
In a subcutaneous study, Van Duuren et al. (Ref. 9) found sarcomas at the injection site after 
subcutaneous injection of epichlorohydrin in mice, but no increase in tumor incidence after 
dermal application, and weekly intraperitoneal injections for over 64 weeks. 
 
Epichlorohydrin  Details of Carcinogenicity Studies 
 
Most 
Animals/ 
Duration/  Sensitive  TD  
50
Study  Dose  Controls  Doses 
Exposure  Tumor  (mg/kg/day) 
Group 
Site/Type/Sex 
Ref. 5a  50/sex  104 weeks,  50  2:  Forestomach 
   Wistar rat   g avage  1.43, 7.14  /Squamous 
mg/kg/day  cell  2.55b,c 
carcinomas / 
Female 
Ref. 7  18/ group  81 weeks,  18  3:  Forestomach / 
  Male  drinking  375, 750,  Squamous 
  Wistar rat   water  1500 ppm.   cell 
  375, 750,  carcinomas  NCd 
1500 
mg/kg/day 
 
140   30 days,  140  1:  Nasal / 
Male  inhalation  100 ppm.  Squamous 
Ref. 8 
Sprague    10.2  cell  
  NCe 
Dawley  mg/kg/day  Carcinomas / 
 
rat   Male 
 
Ref. 8  100  136 weeks,  150  2:  Nasal / 
  Male  inhalation   10, 30 ppm.   Squamous 
Sprague  0.729, 2.88  cell 
421b 
Dawley  mg/kg/day  carcinoma / 
rat    Male 
  
Ref. 9  50 Female   61 weeks, s.c.   150   1:  Injection site 
  ICR/Ha    1 mg/once a  sarcomas  
NCf 
Swiss  week    
mouse    
Ref. 9  50 Female   70 weeks,  150  1:  No skin 
  ICR/Ha  skin  2 mg/3  papillomas or 
NCf 
Swiss    times/week  carcinomas  
mouse   
Ref. 9  50 Female   64 weeks, i.p.  30  1:  No tumors 
  ICR/Ha    5.71  (including no 
NCg 
Swiss  mg/kg/day  injection site 
mouse    sarcomas) 
mg = milligram; kg = kilogram; d = day; ppm = parts per million; NC = not calculated; i.p. = intraperitoneal; s.c. 
= subcutaneous. 
a Carcinogenicity study selected for acceptable intake calculation.  
b The TD  values are taken from the Carcinogenic Potency Database (CPDB) (Ref. 6). 
50
c The TD  value represents the TD  from the most sensitive tumor site. 
50 50
83  Contains Nonbinding Recommendations 
d Not calculated due to short term exposure. 
e Not calculated due to limitations of the study design (injection, single dose level, and did not examine all 
tissues histologically). The skin painting studies showed no increase in skin papillomas or carcinomas. 
f Not calculated: Although TD  is listed in CPDB, there was no increase in tumors. 
50
g Not calculated because the group size was small, the rats were in poor condition, dosing had to be stopped 
intermittently, and there was body weight loss in all dose groups. 
 
Mode of Action for Carcinogenicity 
 
Epichlorohydrin caused tumors only at the site of contact; forestomach and oral cavity tumors 
after oral exposure, nasal tumors after inhalation and injection site sarcomas after 
subcutaneous injection.   
 
Epichlorohydrin is mutagenic in vitro in bacteria and mammalian cells (Ref. 4). It is highly 
irritating to the exposed tissues. For example, dose-related lesions of the forestomach were 
observed in rats given epichlorohydrin by gavage at 3, 7, 19 and 46 mg/kg/day for 10 days, or 
1, 5 and 25 mg/kg/day for 90 days (Ref. 11). There were a range of inflammatory and 
epithelial alterations; most pronounced were dose-related increases in mucosal hyperplasia 
and hyperkeratosis. Data indicate that epichlorohydrin is absorbed, and its metabolites enter 
systemic circulation; however, tumors are seen only at sites of direct contact. For more details 
on relevance of forestomach tumors, see acrylonitrile and benzyl chloride monographs.  
 
Regulatory and/or Published Limits  
 
The World Health Organization (Ref. 12) published a provisional total daily intake of 0.14 
micrograms (g)/kg/day or 8.4 g/day (for a 60 kg adult), based on the assumption of a 
nonlinear dose-response for this site-of-contact carcinogen. The U.S. Environmental 
Protection Agency (EPA) used linear extrapolation to derive a drinking water level (1 in 105 
risk of excess cancer) of 30 g/liter or about 60 g/day (Ref. 13), using data from Konishi et 
al. (Ref. 7). The U.S. EPA also calculated an inhalation concentration of 8 g/cubic meter for 
a 1 in 105 excess cancer risk, or 230 g/day, using the International Council for 
Harmonisation guidance for industry Q3C(R8) Impurities: Guidance for Residual Solvents 
(December 2021)1 assumptions for human daily breathing volume (Ref. 13). 
 
The Food and Drug Administration/Center for Food Safety and Applied Nutrition calculated 
the unit cancer risk of 2.7 x 10-3 (mg/kg/day)-1 using the data in Konishi et al. cited in the 
table above (Ref. 14). A food additive contaminant migrating into human food at an exposure 
of over 0.37 g/kg or 22 g/day would result in an estimated cancer risk over 1:1,000,000. 
 
Acceptable Intake (AI) 
 
Rationale for selection of study for AI calculation 
 
The oral gavage study of Wester et al. (Ref. 5) is the most robust study for calculation of the 
AI and the most sensitive species and tissue is rat forestomach in the gavage carcinogenicity 
study.  The study included an appropriate dose range for measuring tumor incidence, 
demonstrated a clear dose-response, and provided sufficient data for the calculation of a 
compound specific AI.    
 
1 We update guidances periodically.  To make sure you have the most recent version of a guidance, check the 
FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents.  
84  Contains Nonbinding Recommendations 
 
Calculation of AI 
 
Lifetime AI = TD /50,000 x 50 kg 
50
 
Lifetime AI = 2.55 mg/kg/day/50,000 x 50 kg 
 
Lifetime AI = 3 micrograms (g)/day 
85  Contains Nonbinding Recommendations 
REFERENCES (FOR EPICHLOROHYDRIN) 
 
1.  International Agency for Research on Cancer (IARC), 1987, IARC Monographs on the 
Evaluation of Carcinogenic Risks to Humans: Volume 71, Lyon, France: World Health 
Organization, 603628, available at https://publications.iarc.fr/89. 
 
2.  Sram RJ, Landa L, et al., 1983, Effects of Occupational Exposure to Epichlorohydrin on 
the Frequency of Chromosome Aberrations in Peripheral Lymphocytes, Mutat Res, 
122:5964. 
 
3.  Giri AK, 1977, Genetic Toxicology of Epichlorohydrin: A Review. Mutat Res, 386:25
38. 
 
4.  Canter DA, Zeiger E, et al., 1986, Comparative Mutagenicity of Aliphatic Epoxides in 
Salmonella, Mutat Res, 172:10538.  
 
5.  Wester PW, van der Heijden CA, et al., 1985, Carcinogenicity Study With 
Epichlorohydrin (CEP) by Gavage in Rats, Toxicology, 36:325339. 
 
6.  Carcinogenic Potency Database, website available at 
https://files.toxplanet.com/cpdb/index.html.   
 
7.  Konishi Y, Kawabata A, et al., 1980, Forestomach Tumors Induced by Orally 
Administered Epichlorohydrin in Male Wistar Rats, Gann, 71:922923. 
 
8.  Laskin S, Sellakumar AR, et al., 1980, Inhalation Carcinogenicity Study of 
Epichlorohydrin in Noninbred Sprague-Dawley Rats, J Natl Cancer Inst, 65:751757. 
 
9.  Van Duuren BL, Goldschmidt BM, et al., 1974, Carcinogenic Activity of Alkylating 
Agents, J Natl Cancer Inst, 53:695700. 
 
10. Stoner GD, Conran PB, Greisiger EA, Stober J, Morgan M, and Pereira MA, 1986, 
Comparison of Two Routes of Chemical Administration on the Lung Adenoma Response 
in Strain A/J Mice, Toxicol Appl Pharmacol, 82:1931. 
 
11. Daniel FB, Robinson M, et al., 1996, Toxicity Studies of Epichlorohydrin in Sprague-
Dawley Rats, Drug Chem Toxicol, 19:4158. 
 
12. World Health Organization (WHO), 2004, Epichlorohydrin in drinking-water. 
Background document for development of WHO Guidelines for Drinking-water Quality, 
WHO/SDE/WSH/03.04/94. 
 
13. U.S. Environmental Protection Agency, 1988, Integrated Risk Information System 
(IRIS): Epichlorohydrin (CASRN 106-89-8): Cancer Assessment, available at 
https://iris.epa.gov/ChemicalLanding/&substance_nmbr=50.   
 
14. U.S. Food and Drug Administration, 21 CFR Part 176 (Indirect Food Additives: Paper 
and Paperboard Components), available at https://www.ecfr.gov/current/title-21/chapter-
I/subchapter-B/part-176. 
 
86  Contains Nonbinding Recommendations 
ETHYL BROMIDE (CAS# 74-96-4) 
 
Potential for Human Exposure 
 
Ethyl bromide (bromoethane) is a colorless volatile and flammable liquid. It is an alkylating 
agent used primarily as a reagent in synthesis of pharmaceuticals. Its close analog, ethyl 
chloride, which also has a monograph in the International Council for Harmonisation M7(R2) 
Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to 
Limit Potential Carcinogenic Risk,1 is a mutagenic carcinogen. 
 
Mutagenicity/Genotoxicity 
 
Ethyl bromide was mutagenic in the Ames test in Salmonella typhimurium TA98, TA100, 
TA104 with metabolic activation and mutagenic in TA97 with and without metabolic 
activation using the plate incorporation method (Ref.1). As ethyl bromide is a volatile and 
hydrophobic compound, it was also tested in a modified Ames mutation assay as vapor in a 
closed desiccator. In this system, ethyl bromide was mutagenic in TA100 and TA1535, but 
not in TA98, with and without metabolic activation (Ref. 2, 3, 4).  Other Ames mutation 
assays with rat and hamster S9 using the preincubation method showed negative results, most 
likely due to the volatile nature of ethyl bromide (Ref. 4, 5, 6). 
 
In cultured Chinese hamster ovary cells, ethyl bromide induced sister chromatid exchanges 
but not chromosomal aberrations in both the presence and absence of exogenous metabolic 
activation (Ref. 7).  
 
Carcinogenicity 
 
The International Agency for Research on Cancer determined that ethyl bromide is not 
classifiable as to its carcinogenicity to humans (Ref. 8). There are no epidemiological data 
relevant to carcinogenicity and limited evidence in experimental animals for the 
carcinogenicity of ethyl bromide. 
 
In animals, evidence of carcinogenicity was identified from a 2-year bioassay from the 
National Toxicology Program that evaluated ethyl bromide by inhalation administration in 
rats and mice. A variety of effects (dependent on species and sex) were seen with exposures 
of 100, 200, or 400 parts per million (ppm) 6 hours/day, 5 days/week (Ref. 9).  
 
There was some evidence of carcinogenic activity of ethyl bromide for male F344/N rats, as 
indicated by increased incidences of pheochromocytomas and malignant 
pheochromocytomas, combined, of the adrenal medulla (control, 8/40; 100 ppm, 23/45; 200 
ppm, 18/46; 400 ppm, 21/46). In female rats, the incidences of gliomas in the brain and 
adenomas in the lung were increased. However, the incidence of the former was within 
historical control and the incidence of the latter  was not statistically significant by trend test 
or pairwise comparisons. For male B6C3F1 mice, there was equivocal but statistically 
significant increase in incidences of neoplasms of the lung (alveolar/bronchiolar adenomas or 
carcinomas). There was clear evidence of carcinogenic activity for female B6C3F1 mice, as 
 
1 We update guidances periodically.  To make sure you have the most recent version of a guidance, check the 
FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents.  
87  Contains Nonbinding Recommendations 
indicated by neoplasms of the uterus (adenomas or adenocarcinomas), likely due to the same 
mechanism as proposed for ethyl chloride.   
 
Ethyl Bromide  Details of Carcinogenicity Studies 
 
Most 
Animals/  Sensitive 
Duration/  TD  
Study  Dose  Controls  Doses a  Tumor  50
Exposure  (mg/kg/d)b 
Group  Site/Type/S
ex 
Ref. 9  50/sex/  105 weeks,  50  3:  Uterus /  535 
  group  inhalation   M: 115, 229,  Female 
  B6C3F1  458  
mice  F: 137, 275, 
  550 
mg/kg/day 
Ref. 9  50/sex/  106 weeks,  50  3:   Adrenal /  149 c 
  group  inhalation  M: 22.9,  Male 
  F344/N  45.8, 91.7 F:    
  Rats  32.7, 65.5, 
  131 
mg/kg/day 
Ref. 9  50/sex/  106 weeks,  50  3:   Liver  670 
  group  inhalation   M: 22.9,    
  F344/N  45.8, 91.7 F: 
  Rats  32.7, 65.5, 
  131 
mg/kg/day 
a The milligrams per kilogram per day values stated in the Carcinogenic Potency Database (CPDB) (Ref. 10) 
and calculated by method used to standardize average daily dose levels from variety of routes of administration, 
dosing schedules, species, strains and sexes; values stated in CPDB accounted for exposure duration of 24 hours 
per day for 7 days per week. (Dose rate = (administered dose  intake/day  number of doses/week) / (animal 
weight  7 days/week)) 
b TD  calculated in CPDB. 
50
c Carcinogenicity study selected for acceptable intake calculation.  
mg = milligram; kg = kilogram; d = day; M = male; F = female. 
 
Mode of Action for Carcinogenicity 
 
Ethyl bromide is an alkylating agent. It is a mutagenic carcinogen, and the acceptable intake 
(AI) is calculated by linear extrapolation from the TD . 
50
 
Regulatory and/or Published Limits 
 
For ethyl bromide, the American Conference of Governmental Industrial Hygienists 
(ACGIH) threshold limit value-time-weighted average (TLV-TWA) for ethyl bromide is 5 
ppm (22 milligram (mg)/cubic meter (m3)), while Occupational Safety and Health 
Administration (OSHA) and National Institute for Occupational Safety and Health (NIOSH) 
indicate the TWA as 200 ppm (890 mg/m3) (Ref. 11). The ACGIH estimates this value with a 
notation for skin absorption, but OSHA and NIOSH estimates are based on inhalation studies.  
 
88  Contains Nonbinding Recommendations 
Acceptable Intake 
 
Rationale for selection of study for AI calculation 
 
Ethyl bromide is a mutagenic carcinogen via the inhalation route of exposure. Although no 
information on the inhaled bioavailability of ethyl bromide was found, organic solvents have 
high inhalation bioavailability values (Ref. 12), and systemic exposure via inhalation route 
has been demonstrated in multiple studies by clinical observations (Ref. 13). Neoplastic 
lesions were observed in multiple organs where systemic exposure is indicated in mice and 
rats in addition to the site-of-contact tissues (e.g., lung). Therefore, it is reasonable to apply 
the AI derived from inhalation studies for other routes of administration. 
 
Considering all the available data from the inhalation studies in rats and mice, the most 
sensitive tumor endpoint was the combined pheochromocytoma and malignant 
pheochromocytomas of the adrenal gland in male F344/N rats. The TD  calculated by the 
50
Carcinogenic Potency Database for this endpoint was based on tumor incidences, which were 
not statistically significant by trend test . However, the tumor incidence of each dose was 
statistically different from the tumor incidence of the control Therefore, the effect is 
considered relevant. The calculated TD  values for each dose are 32.2 mg/kilogram (kg)/day 
50
for low dose, 115 mg/kg/day for mid dose, 162 mg/kg/day for high dose (Note 2). The lowest 
TD  value of 32.2 mg/kg/day is used because it is considered to conservatively yield the 
50
most sensitive potency estimate for calculating the AI.  
 
Calculation of AI 
 
Lifetime AI = TD /50,000 x 50 kg 
50
 
Lifetime AI = 32.2 mg/kg/day/50,000 x 50 kg 
 
Lifetime AI = 32 micrograms (g)/day
89  Contains Nonbinding Recommendations 
REFERENCES (FOR ETHYL BROMIDE) 
 
1.  Strubel K and Grummt T, 1987, Aliphatic and Aromatic Halocarbons as Potential 
Mutagens in Drinking Water. III. Halogenated Ethanes and Ethenes, Toxicol Environ 
Chem 15:101128. 
 
2.  Barber ED, Donish, WH, and Mueller KR, 1981, A Procedure for the Quantitative 
Measurement of the Mutagenicity of Volatile Liquids in the Ames Salmonella/Microsome 
Assay, Mutat Res, 90:3148. 
 
3.  Simmon VF, 1981, Applications of the Salmonella/Microsome Assay. In: Stich HF and 
San RHC, editors, Short-Term Tests for Chemical Carcinogens. Topics in Environmental 
Physiology and Medicine, 120126. 
 
4.  Zeiger E, Anderson B, Haworth S, Lawlor T, and Mortelmans K, 1992, Salmonella 
Mutagenicity Tests:V. Results from the Testing of 311 Chemicals, Environ Molec 
Mutagen, 19:Suppl 21:2141 
 
5.  Haworth S, Lawlor T, Mortelmans K, Speck W, and Zeiger E, 1983, Salmonella 
Mutagenicity Test Results for 250 Chemicals, Environ Mut, Suppl 1:3142 
 
6.  World Health Organization, 2002, Concise International Chemical Assessment Document 
42: Bromoethane. 
 
7.  Loveday KS, Lugo MH, Resnick MA, Anderson BE, and Zeiger E, 1989, Chromosome 
Aberration and Sister Chromatid Exchange Tests In Vitro in Chinese Hamster Ovary 
Cells In Vitro: II. Results With 20 chemicals, Environ Molec Mutagen, 13: 6094. 
 
8.  National Toxicology Program NTP, 1989, Toxicology and Carcinogenesis Studies of 
Bromoethane (EthylBromide) (CAS NO. 74-96-4) in F344/N Rats and B6C3F Mice 
(Inhalation Studies) (NTP-TR 363), U.S. Department of Health and Human Services. 
 
9.  International Agency for Research on Cancer, 1999, IARC Monographs on the Evaluation 
of Carcinogenic Risks to Humans: Volume 71, 13051307. 
 
10. Carcinogenic Potency Database, website, available at 
https://files.toxplanet.com/cpdb/index.html. 
 
11. National Institute for Occupational Safety and Health, The National Institute for 
Occupational Safety and Health (NIOSH) Publications and Products: Ethyl Bromide, 
available at https://www.cdc.gov/niosh/idlh/74964.html. 
 
12. Fiserova-Bergerova V, 1985, Toxicokinetics of Organic Solvents, Scand J Work Environ 
Health, 11:suppl 1, 721. 
 
13. Sayers RR, Yant WP, Thomas BGH, and Berger LB, 1929, Physiological Response 
Attending Exposure to Vapors of Methyl Bromide, Methyl Chloride, Ethyl Bromide and 
Ethyl Chloride, Public Health Bull, 185:15. 
90  Contains Nonbinding Recommendations 
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. The International Agency 
for Research on Cancer (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); 
 
  Chinese hamster ovary 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 parts per million (ppm) for 3 days, and in an unscheduled 
DNA synthesis 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, National Toxicology Program 
(NTP) studies (Ref. 6) in rats and mice of both sexes via inhalation for 6 hours/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 the U.S. Environmental 
Protection Agency (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. 
 
91  Contains Nonbinding Recommendations 
Ethyl Chloride  Details of Carcinogenicity Studies 
 
Most 
Animals/ 
Duration/  Sensitive  TD50 
Study  Dose  Controls  Doses 
Exposure  Tumor  (mg/kg/d) 
Group 
Site/Sex 
  50/sex/  100 weeks  50  1:  Uterus/Female  1810 
group  6 h/d,  M: 10.4 
Ref. 6, 7a 
B6C3F1  5 d/wk,  F: 12.4 
mice  inhalation  g/kg/d 
  50/sex/  100 weeks  50  1:  Negative  NA 
group  6 h/d,  M: 2.01 
Ref. 6, 7 
Fischer 344  5 d/wk,  F: 2.88 
rats  inhalation  g/kg/d 
a Carcinogenicity study selected for AI calculation. Studies listed are in CPDB (Ref. 8). 
Mg = milligram; kg = kilogram; d = day; h = hour; wk = week; M = male; F = female; g = gram; NA = not 
applicable. 
 
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 U.S. EPA established an inhalation reference concentration for noncarcinogenic effects 
of 10 milligrams (mg)/cubic meter, or 288 mg/day assuming a respiratory volume of 28,800 
liters /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 three 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. Because the number of tumors is 
high, it is possible to calculate a TD  even though only one dose was tested. The authors of 
50
the Carcinogenic Potency Database (Ref. 8) converted 0 and 15,000 ppm to doses of 0 and 
12.4 grams /kilogram (kg) and calculated a TD  of 1810 mg/kg/day for mouse uterine 
50
tumors. 
92  Contains Nonbinding Recommendations 
 
Lifetime AI = TD /50,000 x 50 kg 
50
 
Lifetime AI = 1,810 mg/kg/day /50,000 x 50 kg 
 
Lifetime AI = 1,810 micrograms (g)/day 
93  Contains Nonbinding Recommendations 
REFERENCES (FOR ETHYL CHLORIDE) 
 
1.  International Agency for Research on Cancer, 1999, Chloroethane. In: Monographs on the 
Evaluation of the Carcinogenic Risks to Humans: Volume 71, Lyon, France: World 
Health Organization, 1345. 
 
2.  Goto S, Shiraishi F, Tanabe K, Endo O, Machii K, Tezuka Y, et al., 1995, Mutagenicity 
Detection Method for Vinyl Chloride and Vinylidene Chloride Gases, Kankyo Kagaku, 
5(2):235240. 
 
3.  Zeiger E, Anderson B, Haworth S, Lawlor T, and Mortelmans K, 1992, Salmonella 
Mutagenicity Tests. V. Results From the Testing of 311 Chemicals, Environ Mol 
Mutagen, 19 Suppl 21:2141. 
 
4.  Araki A, Noguchi T, Kato F, and Matsushima T, 1994, Improved Method for 
Mutagenicity Testing of Gaseous Compounds by Using a Gas Sampling Bag, Mutat Res, 
307(1):335344. 
 
5.  Ebert R, Fedtke N, Certa H, Wiegand HJ, Regnier JF, Marshall R, et al., 1994, SW. 
Genotoxicity Studies With Chloroethane, Mutat Res, 322(1):3343. 
 
6.  National Toxicology Program (NTP), 1989, NTP Technical Report on the Toxicology 
and Carcinogenesis Studies of Chloroethane (NTP TR 346 1989), Research Triangle 
Park, NC: National Institutes of Health, available at 
https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr346.pdf. 
 
7.  Holder JW, 2008, Analysis of Chloroethane Toxicity and Carcinogenicity Including a 
Comparison With Bromoethane, Toxicol Ind Health, 24(10):655675. 
 
8.  Carcinogenic Potency Database, website available at 
https://files.toxplanet.com/cpdb/index.html. 
 
9.  U.S. Environmental Protection Agency, 1991, Integrated Risk Information System 
(IRIS): Ethyl Chloride (CAS# 75-00-3), available at 
https://cfpub.epa.gov/ncea/iris2/chemicalLanding.cfm?substance_nmbr=523. 
 
10. National Toxicology Program, 1989, NTP Technical Report on the Toxicology and 
Carcinogenesis Studies of Ethyl Bromide (TR 363), Research Triangle Park, NC: 
National Institutes of Health, available at 
http://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr363.pdf. 
94  Contains Nonbinding Recommendations 
FORMALDEHYDE (CAS# 50-00-0) 
 
Potential for Human Exposure 
 
Formaldehyde exposure occurs in air, water, and food, and it is a common endogenous 
component of biological materials and is a naturally occurring component of many foods 
such as meat, dairy products, fruit, and vegetables. Levels of daily exposure to formaldehyde 
via the dietary route have been estimated in the range of 1.5 to 119 milligrams (mg) per day 
(Ref. 1,2). Formaldehyde is also a product of normal human metabolism and is essential for 
the biosynthesis of certain amino acids. The human body produces and uses 53 to 92 grams 
(g) (878 to 1210 mg/kilogram (kg) body weight/day for a 60 to 70 kg person) of 
formaldehyde per day, which is rapidly metabolized and cleared from blood plasma (Ref. 2). 
Formaldehyde is used in the synthesis and formulation of pharmaceuticals. In some cases, 
formaldehyde can function as the active ingredient in a drug (Ref. 3). Formaldehyde is also 
found as a component of some consumer products and can be produced during cooking or 
smoking (Ref. 1). 
 
Mutagenicity/Genotoxicity 
 
Formaldehyde is a mutagenic compound (Ref. 4,5). Formaldehyde induced mutations in the 
Ames test with and without S9 activation. It induced deletions, point mutations, insertions, 
and cell transformations in mammalian cells (Ref. 4-5). Formaldehyde is also clastogenic 
causing chromosomal aberrations, micronuclei, and sister chromatid exchanges in rodent and 
human primary cell lines. In vivo studies have also detected genotoxic effects primarily at the 
site of contact (Ref. 4).  
 
Carcinogenicity 
 
The International Agency for Research on Cancer (IARC) considers formaldehyde to be a 
Group 1 carcinogen, that is carcinogenic in humans based on cancer of the nasopharynx and 
leukemia (Ref. 6). The European Committee for Risk Assessment has assigned formaldehyde 
to Category 1B (may cause cancer) based on sufficient evidence from animal studies, but 
only limited evidence of carcinogenicity in humans (Ref. 7). There are several oral and 
inhalation animal studies conducted with formaldehyde (summarized in table below). The 
carcinogenicity of formaldehyde is specific to inhalation, whereas the weight of evidence 
indicates formaldehyde is not carcinogenic via the oral route (Ref. 6, 8, 9, 10). Formaldehyde 
was negative in oral carcinogenicity studies in rodents. In carcinogenicity studies conducted 
by the inhalation route, tumors in the nasal cavity have been observed in rodents.  
 
The nasal tumors observed following inhalation of formaldehyde have been attributed to 
continuous cycles of tissue degeneration and regeneration (cytolethality/regenerative cellular 
proliferation (CRCP)) rather than to a direct genotoxic effect (Ref. 11). Formation of DNA-
protein crosslinks (DPX) is probably involved in the cytolethality. Predicted additional cancer 
risks for an 80-year continuous environmental exposure to formaldehyde was modeled, with 
the risk predictions obtained from what Conolly et al. (Ref. 11) expected to be significant 
overestimates of real-world exposures to formaldehyde.    
 
In agreement with IARC (Ref. 6) the U.S. Environmental Protection Agency (EPA) (Ref. 12) 
and the National Toxicology Program (NTP) 15th Report on Carcinogens conclude that 
nasopharyngeal cancer and myeloid leukemia (ML) in humans can be attributed to 
95  Contains Nonbinding Recommendations 
formaldehyde exposure (Ref. 13). The conclusion that formaldehyde causes cancer has been 
peer reviewed by the National Academy of Science (Ref. 14). The reviews acknowledged that 
hazard identification for formaldehyde was not straightforward, especially with respect to 
possible leukemogenicity, in part due to its endogenous production and high reactivity. The 
most useful studies on the risk of formaldehyde causing ML are the large cohort studies of 
chemical workers and embalmers (Ref. 15, 16), which conclude that there is a causal 
association between formaldehyde exposure and mortality from ML (Ref. 15, 16).  In contrast 
the European Committee for Risk Assessment concluded that formaldehyde is not a human 
systemic carcinogen (Ref. 7). Albertini and Kaden (Ref. 17) concluded that overall, the 
available literature on genetic changes following formaldehyde exposure did not provide 
convincing evidence that exogenous exposure, and specifically exposure by inhalation, 
induces mutations as a direct DNA-reactive effect at sites distant from the portal-of-entry 
tissue. This would include proposed mode of actions that involve a stem cell effect at the port 
of entry with circulation back to the bone marrow. Mutations in the bone marrow or in any 
other tissues beyond the point of contact have not been observed.  
 
Since 2010, two short-term carcinogenicity studies have been conducted and published by the 
NTP in strains of genetically predisposed mice (male C3B6129F1-Trp53tm1Brdp53 haplo-
insufficient mice and male B6.129- Trp53tm1Brd) (Ref. 18). These carcinogenicity studies 
were conducted to test the hypothesis that formaldehyde inhalation would result in an 
increased incidence and/or shortened latency to nasal and lymphohematopoietic tumors and 
to investigate hypotheses that formaldehyde may induce leukemia by a mechanism not 
involving DNA adduct formation. This proposed mechanism assumes that inhaled 
formaldehyde could cause significant genetic damage to stem cells in the nasal epithelium or 
circulating in local blood vessels. These damaged stem cells could reach the systemic 
circulation, undergo lodgment, and become leukemic stem cells. The animals were exposed 
to 7.5 or 15 parts per million (ppm) formaldehyde 6 hours/day, 5 days/week, for 8 weeks and 
mice were monitored for approximately 32 weeks. At the highest concentrations, significant 
cell proliferation and squamous metaplasia of the nasal epithelium were observed; however, 
no nasal tumors were observed. No cases of leukemia were seen in either strain, and a low 
incidence of lymphoma in exposed mice was not considered related to exposure. In addition, 
no significant changes in hematological parameters were noted. Under the conditions of these 
studies, the authors concluded that formaldehyde inhalation did not cause leukemia in these 
strains of genetically predisposed mice (Ref. 18). 
 
Multiple studies in rats (Ref. 19-21) and monkeys (Ref. 21, 22) conducted with sensitive 
analytical methods that can measure endogenous versus exogenous formaldehyde DNA or 
protein adducts have demonstrated that inhaled exogenous formaldehyde is not systemically 
absorbed or reaches sites distant from the point of initial contact. In addition to these studies, 
the available data on the toxicokinetics of formaldehyde suggest that no significant amount of 
free formaldehyde would be transported beyond the portal of entry. 
 
96  Contains Nonbinding Recommendations 
Formaldehyde  Details of Carcinogenicity Studies 
 
Animals/  Most Sensitive 
Duration/  TD  
50
Study  Dose  Controls  Doses  Tumor 
Exposure  (mg/kg/d) 
Group  Site/Type/Sex 
Ref. 23  42-60/  35- or 64- 59  3:  No tumors  NC 
   group  weeks,  50, 100, 200 
C3H  inhalation   mg/m3  
Mouse  
R  ef. 24  40-54/sex  2 years,  50-62  3:  Nasal  43.9b 
/ group  inhalation  2, 5.6, 14.3  turbinates/ 
B6C3F1  ppm   Squamous cell 
Mouse  M: 0.644,  carcinoma/ 
1.93, 4.83  Male 
F: 0.686, 
2.06, 5.15 
mg/kg/da 
Ref. 24  73- 2 years,  79  3:  Nasal  0.798b  
   80/sex/  inhalation  2, 5.6, 14.3  turbinates/ 
group  ppm   Squamous cell 
F344 Rat  M: 0.129,  carcinoma/ 
0.386, 0.965  Male 
F: 0.184, 
0.552, 1.38 
mg/kg/da 
Ref. 25  100/  Lifetime,  99  1:  Nasal mucosa /  1.82b 
   group   i nhalation   14.8 ppm   Squamous cell 
Male  0.952  carcinoma/ 
Sprague  mg/kg/da  Male 
Dawley 
Rat 
Ref. 26  45/group   4, 8, or  134  2:  Nasal cavity /  NCc 
   Male  13 weeks,  10, 20 ppm   Male 
Wistar  i nhalation 
Rat  
Ref. 27  30/group  3 or  30  3:  No tumors for  NC 
   (Undama 28months,  0.1, 1.0; 10  undamaged 
ged)  inhalation   ppm   animalsd 
Male 
Wistar 
Rat 
Ref. 28  15-16/  24 months,  16  1:  Nasal cavity/  NC 
  group  inhalation   12.4 ppm  One squamous 
  Female  cell carcinoma 
Sprague 
Dawley 
Rat  
97  Contains Nonbinding Recommendations 
Animals/  Most Sensitive 
Duration/  TD  
50
Study  Dose  Controls  Doses  Tumor 
Exposure  (mg/kg/d) 
Group  Site/Type/Sex 
Ref. 29  47-97/  24 months,  46  5:  Nasal cavity  0.48b 
  group   inhalation   0.7, 2, 6, 10,  /Squamous cell 
  Male  15 ppm   carcinoma/ 
F344 Rat   0.045, 0.129,  Male 
0.386, 0.643, 
0.965 
mg/kg/da 
Ref. 30  20-22/  28 months,  22  3:  Nasal cavity  0.98b 
  group   inhalation   0.3, 2, 15  /Mixed tumor 
  Male  ppm  type/ Male 
F344 Rat   0.0193, 
0.129, 0.965 
mg/kg/da 
Ref. 31  88/ group   Lifetime,  132  1:  No tumors  NC 
  Male  inhalation   10 ppm  
  Syrian 
Golden 
Hamster  
Ref. 32  70/sex/  2 years,  70  3:  No tumors  NC 
  group  drinking  M: 1.2, 15, 
  Wistar  water  82  
Rat   F: 1.8, 21, 
109 
mg/kg/day 
Ref. 33  50/sex/  Lifetime,  50  7:  Lymphoblastic  424b  
   group  drinking  10, 50, 100,  leukemia-
Sprague  water   500, 1000,  lymphosarcom
Dawley  1500,  a / Malee 
Rat   2500 ppm  
M: 0.359, 
1.79, 3.59, 
17.9, 35.9, 
53.8 
F: 0.410, 
2.05, 4.10, 
20.5, 41.0, 
61.5 
mg/kg/daya 
R  ef. 34  20/sex/  24 months,  20  3:  No tumors  NC 
group  drinking  10, 50, 300 
Wistar  water   mg/kg/d 
Rat  
a The milligrams per kilogram per day doses are taken from the Carcinogenic Potency Database (CPDB). 
b TD  taken from the CPDB (Ref. 35). 
50
c Not calculated given the limited duration of dosing. 
d After 28 months of exposure, animals damaged by electrocoagulation experienced an increase in nasal cavity 
tumors. 
98  Contains Nonbinding Recommendations 
e There were concerns about study design (pooling of lymphomas and leukemias diagnosed, lack of reporting of 
nonneoplastic lesions and historical control data, discrepancies of data between this study and Sofritti (Ref. 36) 
(second report of this study), and lack of statistical analysis) (Ref. 4, 6, 10). 
mg = milligram; kg = kilogram; d = day; m = meter; NC = not calculated; M = male; F = female; ppm = parts 
per million. 
 
Mode of Action for Carcinogenicity 
 
Formaldehyde was only clearly carcinogenic in studies conducted by the inhalation route in 
rodents. Tumors in the nasal cavity have been observed and are considered a site of contact 
effect in rodents. The nasal tumors observed following inhalation of formaldehyde were 
attributed to continuous cycles of tissue degeneration and regeneration (CRCP) rather than to 
a direct genotoxic effect (Ref. 11). Formation of DPX is probably involved in the 
cytolethality of formaldehyde but not considered as the driving mechanism to 
carcinogenicity. In recent reviews of the mode of action of formaldehyde and relevance of rat 
nasal tumors to humans, the role of cytotoxicity and regenerative cell proliferation was 
reaffirmed. The reviews indicate that although DPX are a good biomarker of exposure, they 
may not meaningfully contribute to cancer via genotoxic effects except at concentrations that 
result in tissue levels well above endogenous levels (Ref. 37, 38).  
 
Regulatory and/or Published Limits 
 
For oral exposure to the general population, the Agency for Toxic Substances & Disease 
Registry, Health Canada, International Programme on Chemical Safety (IPCS), and U.S. EPA 
limit for formaldehyde is 0.2 mg/kg/day or 10 mg/day for a 50 kg person, which is based on a 
noncancer endpoint (reduced weight gain and histological changes to the gastrointestinal tract 
and kidney) (Ref. 10, 12, 39, 40). No oral carcinogenicity risk estimates exist for 
formaldehyde because carcinogenicity is specific to the inhalation route of exposure. 
 
Occupational limits have been set for air at workplaces by the National Institute for 
Occupational Safety and Health (recommended exposure limit time-weighted average (TWA) 
0.016 ppm), American Conference of Governmental Industrial Hygienists (TWA 0.1 ppm), 
DFG MAKs (TWA 0.3 ppm), European Union (binding occupational exposure limit 0.3 
ppm) and Occupational Safety and Health Administration (permissible exposure limit TWA 
0.75 ppm). 
 
For inhalation exposure to the general population, the U.S. EPA, IPCS, and Health Canada 
have developed inhalation cancer risk values (Ref. 12, 10, 40). The U.S. EPA limit is based 
on a linear cancer model, and Health Canada/IPCS developed nonlinear and linear cancer 
models.  Using the linear method from all three agencies, a daily inhaled dose of 16 to 32 
micrograms (g)/day would result in a 1 in 105 excess risk of cancer. However, more recent 
scientific analysis supports the use of the Health Canada/IPCS nonlinear model, which 
incorporates mechanistic data (Ref. 11, 41-43). Conolly et al. (Ref. 11, 41) developed a 
nonlinear/linear mechanistic-based model using empirical rodent and human data for the two 
modes of action with formaldehyde tumorigenicity: CRCP and DPX.  
 
99  Contains Nonbinding Recommendations 
Acceptable Intake (AI) for Inhalation Exposure 
 
Rationale for selection of study for AI calculation 
 
The AI for inhalation is based on the carcinogenicity model developed by Conolly et al. (Ref. 
11). Figure 1 represents the dose-response hockey stick-shaped model developed by Conolly 
et al., for a mixed population of smokers and nonsmokers. The rat dose response for 
CRCP/DPX was used by Connolly for the human model in absence of an alternative model. 
Since the exposure related tumor risk predicted by clonal growth models was extremely 
sensitive to cell kinetics, Conolly decided to evaluate human cancer risk associated with 
formaldehyde exposure using both the raw J-shaped dose response and a hockey stickshaped 
transformation of the rat data. This model incorporates the nonlinear-based mechanism at the 
high dose region (CRCP) and the linear mechanism at the low dose region (DPX). As noted 
above, the translation of DPX into mutations and an assumed linear dose response emerging 
from such mutations is not established experimentally. Moreover, experimental results 
suggest that DPX are not leading to mutations in a linear fashion. Thus, the linear dose-
response model at low doses reflects a conservative and practical approach and is not dictated 
by experimental data. 
 
 
Figure 1. Dose-response model hockey stick-shaped model from (Ref. 11) representing mixed smokers and 
nonsmokers. The dose (milligrams per day) was based on converting air concentration (parts per million) to 
daily dose using the International Council for Harmonisation guidance for industry Q3C(R8) Impurities: 
Guidance for Residual Solvents (December 2021) assumptions for human breathing volume (28,800 liters/day).1 
 
 
1 We update guidances periodically.  To make sure you have the most recent version of a guidance, check the 
FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents.  
100  Contains Nonbinding Recommendations 
Calculation of Inhalation AI 
 
The linear low-dose region of Figure 1 was used to determine the dose at a 1 in 100,000 
excess cancer risk. Linear regression at the low-dose region, which is less than or equal to 
24.74 mg/day (converted from 0.7 ppm), results in an equation of y = 1.62E-06x  3.27E-06.  
The dose of 24.74 mg/day was the point at which there is a predicted upward inflection of 
additional risk. Solving for a 1 in 100,000 excess cancer risk in the regression line (y) results 
in an AI of 8.2 mg/day (see Figure 1 dose equivalent to the 1:100,000 risk). 
 
Risk (y) = 1.62E-06x(dose)  3.27E-06 
0.00001 = 1.62E-06x  3.27E-06 
x = (0.00001 + 3.27E-06) / 1.62E-06 
Dose (x) = 8.2 mg/day 
 
Lifetime AI (inhalation) = 8 mg/day or 215 parts per billion (ppb), whichever is lower 
 
Rationale for the Concentration Limit 
 
Formaldehyde is considered a mutagenic carcinogen by the inhalation route of exposure.  The 
acceptable intake of 8 mg/day represents an upper limit over a 24-hour time period, which is 
considered acceptable as it limits excess cancer risk to 1 in 100,000. As described in the 
introduction section of Appendix 3 of the International Council for Harmonisation guidance 
for industry M7(R2) Assessment and Control of DNA Reactive (Mutagenic) Impurities in 
Pharmaceuticals to Limit Potential Carcinogenic Risk, other considerations may affect 
final product specifications. Formaldehyde is known to cause local irritation and sensitization 
effects. Therefore, the World Health Organization recommends a limit of 81.4 ppb in air as a 
30-minute average (Ref. 44) and Health Canada recommends a short-term exposure limit of 
100 ppb as a 1-hour average (Ref. 45). These recommended values provide at least a 10-fold 
margin of exposure to the lowest level at which symptoms have been observed. To protect 
patients from the potential for local irritation and sensitization effects of formaldehyde by the 
inhalation route of exposure, a concentration-based limit of 215 ppb is recommended (8 
mg/day over 24 hours of exposure is equal to a concentration limit of 215 ppb). That is: 
 
(0.008 g/day/28.8 cubic meters (m3)/day) * 1 / 1293 g/m3) = 215 ppb 
 
  Human breathing volume/day = 28.8 m3 /day 
  Air mass/m3 at standard conditions = 1,293 g 
 
The limit of 215 ppb could either be interpreted as the concentration of formaldehyde in air 
(which is the basis of the exposure limit) or the concentration of formaldehyde relative to 
drug substance.  See Note 3 for examples to clarify how the 215 ppb limit in air relates to a 
limit in active pharmaceutical ingredient (API) or drug product. 
 
Permissible Daily Exposure (PDE) for All Other Routes 
 
See section IV (4) of the introduction to this addendum that addresses formaldehyde exposure 
from the environment. 
 
PDE (all other routes) = 10 mg/day 
101  Contains Nonbinding Recommendations 
REFERENCES (FOR FORMALDEHYDE) 
 
1.  Organization for Economic Co-operation and Development (OECD), 2002, Screening 
Information Dataset (SIDS) Initial Assessment Report. Formaldehyde.  
 
2.  European Food Safety Authority, 2014, Scientific Report. Endogenous Formaldehyde 
Turnover in Humans Compared With Exogenous Contribution From Food Sources, 
EFSA Journal, 12(2):3550, available at 
https://www.efsa.europa.eu/de/efsajournal/pub/3550. 
 
3.  Bingham E, Cohrssen B, and Powell CH, 2001, Patty's Toxicology Volumes 19 5th ed., 
New York, N.Y: John Wiley & Sons, 981. 
 
4.  International Agency for Research on Cancer (IARC), 2006, Formaldehyde, 2-
Butoxyethanol and 1-Tertbutoxypropan-2-ol. In: IARC Monographs on the Evaluation of 
Carcinogenic Risks to Humans: Volume 88, 1478. PMID:17366697.  
 
5.  Ma TH and Harris MM, 1988, Review of the Genotoxicity of Formaldehyde, Mutat Res, 
196:3759.  
 
6.  IARC, 2012, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: 
Chemical Agents and Related Occupations: Volume 100 F.  
 
7.  Committee for Risk Assessment, RAC Opinion Proposing Harmonised Classification and 
Labelling at EU Level of Formaldehyde. 
https://echa.europa.eu/documents/10162/b8dfa022-9544-72e8-dcaa-7491dff3c0. 
 
8.  World Health Organization (WHO), 2005, Background Document for Development of 
WHO Guidelines for Drinking-Water Quality, WHO/SDE/WSH/05.08/48.  
 
9.  Scheuplein RJ, 1985, Formaldehyde: The Food and Drug Administrations Perspective.  
Chapter 16. In: Turoski V, editor, Formaldehyde: Analytical Chemistry and Toxicology, 
Washington, DC: American Chemical Society, 210:237245.   
 
10. International Programme on Chemical Safety (IPCS), 2002, IPCS Concise International 
Chemical Assessment Document 40. Formaldehyde.  
 
11. Conolly RB, Kimbell JS, et al., 2004, Human Respiratory Tract Cancer Risks of Inhaled 
Formaldehyde: Dose-Response Predictions Derived From Biologically-Motivated 
Computational Modeling of a Combined Rodent and Human Dataset, Toxicol Sci, 
82:279296. 
 
12. U.S. Environmental Protection Agency, 1990, Integrated Risk Information System 
(IRIS): Chemical Assessment Summary: Formaldehyde (CASRN 50-00-0).  
 
13. National Toxicology Program, 2021, Formaldehyde CAS No. 50-00-0. Report on 
Carcinogens, 15th Edition, Research Triangle Park, NC: U.S. Department of Health and 
Human Services, Public Health Service. 
 
102  Contains Nonbinding Recommendations 
14. National Research Council, 2011, Review of the Environmental Protection Agencys 
Draft IRIS Assessment of Formaldehyde, Washington, DC: The National Academies 
Press. 
 
15. Hauptmann M, Lubin JH, Stewart PA, Hayes RB, and Blair A, Mortality From 
Lymphohematopoietic Malignancies Among Workers in Formaldehyde Industries, J Natl 
Cancer Inst, 95:16151623. 
 
16. Hauptmann M, Stewart PA, Lubin JH, Freeman LEB, Hornung RW, Herrick RF, Hoover 
RN, Fraumeni JF, Blair A, and Hayes, RB, 2009, Mortality From Lymphohematopoietic 
Malignancies and Brain Cancer Among Embalmers Exposed to Formaldehyde, J Natl 
Cancer Inst, 101:16961708. 
 
17. Albertini RJ and Kaden DA, 2017, Do Chromosome Changes in Blood Cells Implicate 
Formaldehyde as a Leukemogen?, Crit Rev Toxicol, 47:145184. 
 
18. Morgan DL, Dixon D, King DH, Travlos GS, Herbert RA, French JE, Tokar EJ, Waalkes 
MP, and Jokinen MP, 2017, NTP Research Report on Absence of Formaldehyde-Induced 
Neoplasia in Trp53 Haploinsufficient Mice Exposed by Inhalation: Research Report 3, 
Durham, NC: National Toxicology Program, PMID: 30016014. 
 
19. Lu K, Collins LB, Ru H, Bermudez E, and Swenberg JA, 2010, Distribution of DNA 
Adducts Caused by Inhaled Formaldehyde Is Consistent With Induction of Nasal 
Carcinoma But Not Leukemia, Toxicol Sci, 116:441451.   
 
20. Edrissi B, Taghizadeh K, Moeller BC, Kracko D, Doyle-Eisele M, Swenberg JA, and 
Dedon PC, 2013, Dosimetry of N6-formyllysine Adducts Following [13C2H2]-
Formaldehyde Exposures in Rats, Chem Res Toxicol, 26:14211423. 
 
21. Yu R, Lai Y, Hartwell HJ, Moeller BC, Doyle-Eisele M, Kracko D, Bodnar WM, Starr 
TB, and Swenberg JA, 2015, Formation, Accumulation, and Hydrolysis of Endogenous 
and Exogenous Formaldehyde-Induced DNA Damage, Toxicol Sci, 146:170182.  
 
22. Moeller BC, Lu K, Doyle-Eisele M, McDonald J, Gigliotti A, and Swenberg JA, 2011, 
Determination of N2-hydroxymethyl-dG Adducts in the Nasal Epithelium and Bone 
Marrow of Non-human Primates Following 13CD2-Formaldehyde Inhalation Exposure, 
Chem Res Toxicol, 24:162164.  
 
23. Horton AW, Tye R, and Stemmer KL, 1963, Experimental Carcinogenesis of the Lung. 
Inhalation of Gaseous Formaldehyde or an Aerosol of Coal Tar by C3H Mice, J Natl 
Cancer Inst, 30:3143.  
 
24. Kerns WD, Pavkov KL, Donofrio DJ, Gralla EJ, and Swenberg JA, 1983, Carcinogenicity 
of Formaldehyde in Rats and Mice After Long-Term Inhalation Exposure, Cancer Res, 
43:43824392. 
 
25. Sellakumar AR, Snyder CA, Solomon JJ, and Albert RE, 1985, Carcinogenicity of 
Formaldehyde and Hydrogen Chloride in Rats, Toxicol Appl Pharmacol, 81:401406. 
 
103  Contains Nonbinding Recommendations 
26. Feron VJ, Bruyntjes JP, Woutersen RA, Immel HR, and Appelman LM, 1988, Nasal 
Tumours in Rats After Short-Term Exposure to a Cytotoxic Concentration of 
Formaldehyde, Cancer Lett, 39:101111. 
 
27. Woutersen RA, van Garderen-Hoetmer A, Bruijntjes JP, Zwart A, and Feron V J, 1989, 
Nasal Tumours in Rats After Severe Injury to the Nasal Mucosa and Prolonged Exposure 
to 10 ppm Formaldehyde, J Appl Toxicol, 9:3946.  
 
28. Holmstrom M, Wilhelmsson B, and Hellquist H, 1989, Histological Changes in the Nasal 
Mucosa in Rats After Long-Term Exposure to Formaldehyde and Wood Dust, Acta Oto-
Laryngologica, 108:274283.  
 
29. Monticello TM, Swenberg JA, Gross EA; Leininger JR, Kimbell JS, Seilkop S, Starr TB, 
Gibson JE, and Morgan KT, 1996, Correlation of Regional and Nonlinear Formaldehyde-
Induced Nasal Cancer With Proliferating Populations of Cells, Cancer Res, 56:1012
1022. 
 
30. Kamata E, Nakadate M, Uchida O, Ogawa Y, Suzuki S, Kaneko T, Saito M, and 
Kurokawa Y, 1997, Results of a 28-Month Chronic Inhalation Toxicity Study of 
Formaldehyde in Male Fisher-344 Rats, J Toxicol Sci, 22:239254. 
 
31. Dalbey WE, 1982, Formaldehyde and Tumors in Hamster Respiratory Tract, Toxicology, 
24:914. 
 
32. Til HP, Woutersen R, Feron VJ, Hollanders VHM, and Falke HE, 1989, Two-Year 
Drinking-Water Study of Formaldehyde in Rats, Fd Chem Toxic, 27:7787. 
 
33. Soffritti M, Maltoni C, Maffei F, and Biagi R, 1989, Formaldehyde: An Experimental 
Multipotential Carcinogen, Toxicol Ind Health, 5:699730. 
 
34. Tobe M, Naito K, and Kurokawa Y, 1989, Chronic Toxicity Study on Formaldehyde 
Administered Orally to Rats, Toxicology, 56:7986. 
 
35. Carcinogenic Potency Database, website available at 
https://files.toxplanet.com/cpdb/index.html.  
 
36. Soffritti M, Belpoggi F, et al., 2002, Results of Long-Term Experimental Studies on the 
Carcinogenicity of Formaldehyde and Acetaldehyde in Rats, Ann N Y Acad Sci, 982:87
105. 
 
37. Thompson CM, Gentry R, Fitch S, Lu K, and Clewell HJ, 2020, An Updated Mode of 
Action and Human Relevance Framework Evaluation for Formaldehyde-Related Nasal 
Tumors, Critical Reviews in Toxicology, 50:919952. 
 
38. Scientific Committee on Occupational Exposure Limits, 2016, SCOEL/REC/125 
Formaldehyde Recommendation from the Scientific Committee on Occupational 
Exposure Limits, available at https://op.europa.eu/en/publication-detail/-
/publication/7a7ae0c9-c03d-11e6-a6db-01aa75ed71a1/language-en. 
 
104  Contains Nonbinding Recommendations 
39. Agency for Toxic Substances and Disease Registry, 1999, Toxicological Profile for 
Formaldehyde, U.S. Department of Health and Human Services, Public Health Service, 
available at http://www.atsdr.cdc.gov/toxprofiles/tp.asp?id=220&tid=39.  
 
40. Health Canada, 2001, Priority Substances List Assessment Report: Formaldehyde, 
Ottawa: Ministry of Public Works and Government Services, available at http://www.hc-
sc.gc.ca/ewh-semt/pubs/contaminants/psl2-lsp2/index_e.html.  
 
41. Conolly RB, Kimbell JS, Janszen D, Schlosser, PM, Kalisak D, Preston J, and Miller FJ, 
2003, Biologically Motivated Computational Modeling of Formaldehyde Carcinogenicity 
in the F344 Rat, Toxicol Sci, 75:432447. 
 
42. Starr TB and Swenberg JA, 2013, A Novel Bottom-Up Approach to Bounding Low-Dose 
Human Cancer Risks From Chemical Exposures, Regul Toxicol Pharmacol, 65:311315. 
 
43. Swenberg JA, Moeller BC, Lu K, Rager JE, Fry RC, and Starr TB, 2013, Formaldehyde 
Carcinogenicity Research: 30 Years and Counting for Mode of Action, Epidemiology, 
and Cancer Risk Assessment, Toxicol Pathol, 41:181189. 
 
44. World Health Organization (WHO), 2010, WHO Guidelines for Indoor Air Quality: 
Selected Pollutants. 3 Formaldehyde. 
 
45. Health Canada, Formaldehyde in your home, available at 
https://www.canada.ca/en/health-canada/services/environmental-workplace-
health/reports-publications/air-quality/formaldehyde-indoor-air-environment-workplace-
health.html.  
105  Contains Nonbinding Recommendations 
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 to 80 micrograms (g)/day (Ref. 1). 
 
Mutagenicity/Genotoxicity 
 
Glycidol is mutagenic/genotoxic in vitro and in vivo. 
 
The International Agency for Research on Cancer (IARC) (Ref. 2) and Chemical 
Carcinogenesis Research Information System (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 National Toxicology Program (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 milligrams (mg)/kilogram (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 
106  Contains Nonbinding Recommendations 
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 2-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). 
 
Glycidol  Details of Carcinogenicity Studies 
 
Most 
Animals/ 
Duration/  Sensitive  TD50 
Study  Dose  Controls  Doses 
Exposure  Tumor  (mg/kg/d) 
Group 
Site/Sex 
  50/sex/  2 years  50  2:  Mammary  4.15 
group  5 days/wk,  26.5; 53.8  gland/Female 
Ref. 5a 
F344/N  oral gavage  mg/kg/d 
rats 
  50/sex/  2 years  50  2:  Harderian gland  32.9 
group  5 days/wk,  17.7; 35.4  /Female 
Ref. 5 
B6C3F1  oral gavage  mg/kg/d 
mice 
  12-20/  60  weeks  Yes  1:  Spleen/Female  56.1c 
  sex/group  Twice/  M: 15.8 
Ref. 8  Syrian  wk,  F:  17.9 
Golden  gavage  mg/kg/d 
hamsters 
  20  520 days  Yes  1:  No tumors  NAc 
Ref. 9  ICR/Ha  3 times/wk,  5% 
(bCited  Swiss  skin painting 
in Ref. 2)  mice 
Studies listed are in the Carcinogenic Potency Database (CPDB) (Ref. 10) unless otherwise noted. 
a Carcinogenicity study selected for acceptable intake calculation. 
b Not in CPDB. 
c Not a standard carcinogenicity design. Only one dose, intermittent dosing, and small sample size (Ref.7). 
mg = milligram; kg = kilogram; d = day; wk = week; M = male; F = female; NA = not applicable. 
 
Mode of Action of Carcinogenicity 
 
Glycidol is a mutagenic carcinogen, and the acceptable intake (AI) is calculated by linear 
extrapolation from the TD . 
50
 
Regulatory and/or Published Limits 
 
No regulatory limits have been published, for example by the U.S. Environmental Protection 
Agency, the World Health Organization, or the Agency for Toxic Substances and Disease 
Registry. 
 
107  Contains Nonbinding Recommendations 
Acceptable Intake 
 
Rationale for selection of study for AI calculation 
 
The most suitable carcinogenicity data for human cancer potency assessment come from the 
2-year oral studies conducted in F344/N rats and B6C3F1 mice by the NTP (Ref. 5). The 
most sensitive organ site was female mammary glands with a TD  of 4.15 mg/kg/day. 
50
 
Calculation of AI 
 
Lifetime AI = TD /50,000 x 50 kg 
50
 
Lifetime AI = 4.15 (mg/kg/day)/50,000 x 50 kg 
 
Lifetime AI = 4 g/day 
108  Contains Nonbinding Recommendations 
REFERENCES (FOR GLYCIDOL) 
 
1.  Bakhiya N, Abraham K, Gurtler R, Appel KE, and Lampen A, 2011, Toxicological 
Assessment of 3-Chloropropane-1,2-diol and Glycidol Fatty Acid Esters in Food. Mol 
Nutr Food Res, 55:509521. 
 
2.  International Agency for Research on Cancer (IARC), 2000, Monographs on the 
Evaluation of the Carcinogenic Risks to Humans: Volume 77, Geneva: World Health 
Organization,), 469, available at https://monographs.iarc.fr/index.php. 
 
3.  National Cancer Institute/Chemical Carcinogenesis Research Information System, 2013, 
National Library of Medicine, available at 
https://www.ncbi.nlm.nih.gov/pcsubstance?term=%22Chemical%20Carcinogenesis%20R
esearch%20Information%20System%20(CCRIS)%22%5BSourceName%5D%20AND%
20hasnohold%5Bfilt%5D and search on CAS number. 
 
4.  Irwin RD, Eustis SL, Stefanski S, and Haseman JK, 1996, Carcinogenicity of Glycidol in 
F344 Rats and B6C3F1 Mice, J Appl Toxicol, 16(3):201209. 
 
5.  National Toxicology Program (NTP), 1990, 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) (NTP TR 374), Research Triangle Park, NC: National Institutes of 
Health. 
 
6.  NTP, 2007, 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, 13:181. 
 
7.  California Environmental Protection Agency, 2010, No Significant Risk Level (NSRL) 
for the Proposition 65 carcinogen Glycidol, available at 
https://oehha.ca.gov/media/downloads/proposition-65/chemicals/glycidolnsrl073010.pdf.  
 
8.  Lijinsky W and Kovatch RM, 1992, A Study of the Carcinogenicity of Glycidol in Syrian 
Hamsters, Toxicol Ind Health, 8(5):267271. 
 
9.  Van Duuren BL, Langseth L, Goldschmidt BM, and Orris L, 1967, Carcinogenicity of 
Epoxides, Lactones, and Peroxy Compounds. VI. Structure and Carcinogenic Activity, J 
Natl Cancer Inst, 39:12171228. 
 
10. Carcinogenic Potency Database, website available at 
https://files.toxplanet.com/cpdb/index.html. 
109  Contains Nonbinding Recommendations 
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. 
 
The International Agency for Research on Cancer (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 the U.S. Environmental Protection Agency (EPA) as Group B2 or a probable human 
carcinogen (Ref. 7). 
 
There are seven hydrazine carcinogenicity studies cited in the Carcinogenic Potency Database 
(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  is in Ref. 11. The most 
50
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 less than 50 animals per group (and a single dose level in one case), and the 
calculated TD  values were higher (less potent) than those for the drinking water study of 
50
110  Contains Nonbinding Recommendations 
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 noninhalation acceptable intake (AI) calculation for hydrazine. 
 
Hydrazine  Details of Carcinogenicity Studies 
 
Most 
Animals/  Sensitive 
Duration/  TD50 
Study  Dose  Controls  Doses  Tumor 
Exposure  (mg/kg/d) 
Group  Site/Type/S
ex 
  50/sex/ group  Lifetime,  50  3:  Liver/Female  41.6 
  Wistar rats  drinking  M: 0.1; 1.5, 
water  2.5. 
Ref. 9 
F: 0.11, 
0.57, 2.86 
mg/kg/d 
  100/sex/  1 year with  150  4:  Nasal  0.194 
  group  18-month  M:1.37,  adenamatous 
F344 rats  observation,  6.87, 27.5,  polyps/Male 
 
inhalation  137 
Ref. 11a 
F: 1.96, 
9.81, 39.3, 
196 
g/kg/d 
  50/sex/ group  2 years,  50  3:  Negative  NA, 
  Bor:NMRI,  drinking  M: 0.33,  negative 
SPF-bred  water  1.67, 8.33.  study 
Ref. 12 
NMRI mice  F: 0.4, 2.0, 
10.0 
mg/kg/d 
  200  1 year with  Yes  3:  Nasal  4.16 
male Golden  12-month  0.02, 0.08,  adenomatous 
Ref. 11 
Syrian  observation,  0.41  polyps/Male 
hamsters  inhalation  mg/kg/d 
  400 female  1 year with  Yes  1:  Negative  NA 
C57BL/6  15-month  0.18 
Ref. 11 
mice  observation,  mg/kg/d 
inhalation 
  50/sex/ group  Lifetime,  Not  1:  Lung/Male  2.20c 
Ref. 13  Swiss mice  drinking  concurre ~1.7-2 
water  nt  mg/kg/d 
  25  40 weeks  85  1:  Lung/Female  5.67d 
Ref. 14  female Swiss  5d/wk,  Untreated  ~5 mg/kg/d 
mice  gavage 
111  Contains Nonbinding Recommendations 
Most 
Animals/  Sensitive 
Duration/  TD50 
Study  Dose  Controls  Doses  Tumor 
Exposure  (mg/kg/d) 
Group  Site/Type/S
ex 
  50/sex/  Lifetime,  Yes  3:  Liver/Female  38.7 
  F344/DuCrj  drinking  M: 0.97, 
rats  water  1.84, 3.86 
Ref. 10be 
F:1.28, 2.50, 
5.35 
mg/kg/d 
  50/sex  Lifetime,    3:  Liver/Female  52.4 
Crj:BDF1  drinking  M: 1.44, 
Ref. 10e 
Mice  water  2.65, 4.93 
F: 3.54, 
6.80, 11.45 
mg/kg/d 
Studies listed are in the Carcinogenic Potency Database (CPDB) (Ref. 8). 
a Carcinogenicity study selected for inhalation acceptable intake (AI) calculation. 
b Carcinogenicity study selected for noninhalation TD  (see Note 4) and AI calculations. 
50
c Excluded by the U.S. Environmental Protection Agency (Ref. 7); no concurrent controls. Liver negative. 
d Animal survival affected. Liver negative. 
e Not in CPDB 
mg = milligram; kg = kilogram; d = day; wk = week; M = male; F = female; NA = not applicable. 
 
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. 
 
Regulatory and/or Published Limits 
 
The U.S. 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 times 10-5 per microgram (g)/liter (L). At the 1 in 100,000 risk level, 
this equates to a concentration of 0.1 g of hydrazine/L of water or about 0.2 g/day for a 50 
kg human. This limit is a linearized multistage extrapolation based on the observation of 
hepatomas in a multidose 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 reevaluated by the U.S. EPA. 
 
The U.S. EPA (Ref. 7) has also published an inhalation slope factor of 17 per milligrams 
(mg)/kilogram (kg)/day and an inhalation unit risk of 4.9x10-3 per g/cubic meter (m3). At the 
1 in 100,000 risk level, this equates to an air concentration of 2 x 10-3 g/m3 of hydrazine or 
0.04 g/day assuming a person breathes 20 m3/day. This limit is a linearized multistage 
extrapolation based on the observation of nasal cavity adenoma or adenocarcinoma in male 
rats in a multidose 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 
112  Contains Nonbinding Recommendations 
U.S. 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 
 
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 four mouse studies and two 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 U.S. 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 U.S. EPA (Ref. 7) was 
proprietary but is likely the same one described in Vernot et al. (Ref. 11). Given that the 
threshold of toxicological concern was derived via linear extrapolation from TD  values for 
50
hundreds of carcinogens, that same approach was used in the derivation of a compound-
specific AI for hydrazine. The methodology used by the U.S. 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 1 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  value of 0.194 mg/kg/day, after being adjusted, as standard 
50
practice, to account for 1 versus 2 years of exposure. 
 
Calculation of AI 
 
Lifetime AI = TD /50,000 x 50 kg 
50
 
Lifetime AI = 38.7 (mg/kg/day)/50,000 x 50 kg 
 
Lifetime AI = 39 g/day 
 
Calculation of Inhalation AI 
 
Lifetime AI = TD /50,000 x 50 kg 
50
 
Lifetime AI = 0.194 (mg/kg/day)/50,000 x 50 kg 
 
Lifetime inhalation AI = 0.2 g/day 
113  Contains Nonbinding Recommendations 
REFERENCES (FOR HYDRAZINE) 
 
1.  Choudary G and Hansen H, 1998, Human Health Perspective on Environmental Exposure 
to Hydrazines: A Review, Chemosphere, 37:801843. 
 
2.  Von Burg R and Stout T, 1991, Hydrazine, J Appl Toxicol, 11:447450. 
 
3.  Toth B, 2000, A Review of the Natural Occurrence, Synthetic Production and Use of 
Carcinogenic Hydrazines and Related Chemicals, In Vivo, 14(2):299319. 
 
4.  Hazardous Substance Database: Hydrazine (302-01-2), 2005 June 24 (cited 2013 
February 27), available at https://pubchem.ncbi.nlm.nih.gov/compound/9321.  
 
5.  Liu YY, Schmeltz I, and Hoffman D, 1974, Chemical Studies on Tobacco Smoke. 
Quantitative Analysis of Hydrazine in Tobacco and Cigarette Smoke, Anal Chem, 46: 
885889. 
 
6.  International Agency for Research on Cancer, 1999, Monographs on the Evaluation of the 
Carcinogenic Risks to Humans: Volume 71, Lyon, France: World Health Organization, 
1006, (Online) 1972-PRESENT (Multivolume work), . 
 
7.  U.S. Environmental Protection Agency, 1991, Integrated Risk Information System 
(IRIS): Chemical Assessment Summary: Hydrazine/Hydrazine Sulfate (302-01-2), 
available at 
https://cfpub.epa.gov/ncea/iris/iris_documents/documents/subst/0352_summary.pdf. 
 
8.  Carcinogenic Potency Database, website available at 
https://files.toxplanet.com/cpdb/index.html. 
 
9.  Steinhoff D and Mohr U, 1988, The Question of Carcinogenic Effects of Hydrazine, Exp 
Pathol, 33:133140. 
 
10. Matsumoto M, Kano H, Suzuki M, Katagiri T, Umeda Y, and Fukushima S, 2016, 
Carcinogenicity and Chronic Toxicity of Hydrazine Monohydrate in Rats and Mice by 
Two-Year Drinking Water Treatment, Regul Toxicol Pharmacol, 76:6373. 
 
11. Vernot EH, MacEwen JD, Bruner RH, Haun CC, Kinkead ER, Prentice DE, et al., 1985, 
Long-Term Inhalation Toxicity of Hydrazine, Fundam Appl Toxicol, 5:l0501064. 
 
12. Steinhoff D, Mohr U, and Schmidt WM, 19990, On the Question of the Carcinogenic 
Action of Hydrazine  Evaluation on the Basis of New Experimental Results, Exp 
Pathol, 39:19. 
 
13. Toth B, 1972, Hydrazine, Methylhydrazine and Methylhydrazine Sulfate Carcinogenesis 
in Swiss Mice. Failure of Ammonium Hydroxide to Interfere in the Development of 
Tumors, Int J Cancer, 9:109118. 
 
14. Roe FJC, Grant GA, and Millican DM, 1967, Carcinogenicity of Hydrazine and 1,1- 
Dimethylhydrazine for Mouse Lung, Nature, 16:375376. 
 
114  Contains Nonbinding Recommendations 
15. Becker RA, Barrows LR, and Shank RC, 1981, Methylation of Liver DNA Guanine in 
Hydrazine Hepatotoxicity: Dose-Response and Kinetic Characteristics of 7-
Methylguanine and O6-Methylguanine Formation and Persistence in Rats, 
Carcinogenesis, 2:11811188. 
 
16. Bosan WS and Shank RC, 1983, Methylation of Liver DNA Guanine in Hamsters Given 
Hydrazine, Toxicol Appl Pharmacol, 70:324334. 
 
17. Bosan WS and Shank RC, MacEwen JD, Gaworski CL, and Newberne PM, 1987, 
Methylation of DNA Guanine During the Course of Induction of Liver Cancer in 
Hamsters by Hydrazine or Dimethylnitrosamine, Carcinogenesis, 8:439444. 
 
18. Saffhill R, Fida S, Bromley M, and OConnor PJ, 1988, Promutagenic Alkyl Lesions Are 
Induced in the Tissue DNA of Animals Treated With Isoniazid, Human Toxicol, 7:311
317. 
 
19. Leakakos T and Shank RC, 1994, Hydrazine Genotoxicity in the Neonatal Rat, Toxicol 
Appl Pharmacol, 126:295300. 
 
20. Mathison B, Murphy SE, and Shank RC, 1994, Hydralazine and Other Hydrazine 
Derivatives and the Formation of DNA Adducts, Toxicol Appl Pharmacol, 127:9198. 
 
21. Biancifiori C, 1970, Hepatomas in CBA/Cb/Se Mice and Liver Lesions in Golden 
Hamsters Induced by Hydrazine Sulfate, J Natl Cancer Inst, 44:943. 
 
22. FitzGerald BE and Shank RC, 1996, Methylation Status of DNA Cytosine During the 
Course of Induction of Liver Cancer in Hamsters by Hydrazine Sulphate, Carcinogenesis, 
17:27032709. 
115  Contains Nonbinding Recommendations 
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 grams (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 percent hydrogen peroxide (Ref. 2). 
 
Mutagenicity/Genotoxicity 
 
Hydrogen peroxide is mutagenic and genotoxic in vitro but not in vivo. 
 
The International Agency for Research on Cancer (IARC) (Ref. 3) and European 
Commissions 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 milligrams (mg)/kilogram (kg), or to catalase-deficient 
C57BL/6NCr1BR mice in drinking water at 200; 1,000; 3,000; and 6,000 parts per million 
for 2 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 Carcinogenic Potency Database 
(CPDB) (Ref. 6), in which mice were treated with hydrogen peroxide in drinking water at 0.1 
or 0.4 percent 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 
less than 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 percent hydrogen peroxide administered in drinking water was 
defined as the lowest observed adverse effect level, equivalent to an average daily dose-rate 
per kilogram body weight per day of 167 mg/kg/day. 
 
Studies of 6-month duration or longer are summarized in the following table (adapted from 
Ref. 2); they are limited in the numbers of animals and used a single dose level. Most studies 
116  Contains Nonbinding Recommendations 
did not meet the criteria for inclusion with a TD  calculation in the CPDB. DeSesso et al. 
50
(Ref. 2) noted that, out of 14 carcinogenicity studies (two subcutaneous studies in mice, two 
dermal studies in mice, six drinking water studies (two in rats and four in mice), one oral 
intubation study in hamsters, and three buccal pouch studies), only three 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 of the U.S. Food and Drug Administration (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 (SCCP) 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 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, 
esophagus, 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 U.S. 
FDA conclusion above (Ref. 10). 
 
Hydrogen Peroxide  Details of Oral Carcinogenicity Studies 
 
Study  Animals/  Duration/  Controls  Doses  Notes 
Dose  Exposure 
Group 
  48-51/sex/ group  100  Yes  2:  TD50 7.54 g/kg/d 
C57BL/6J mice  weeks,  0.1;  0.4%  for female duodenal 
Ref. 5a  drinking  M: 167;  carcinoma. 
water  667 
F: 200; 800 
  29 mice  700  No  1:    No tumors reported. 
  C57BL/6J  days,  0.4%  Time-dependent 
  total male  Drinking  induction of erosions 
  and female  water  and nodules in stomach 
  (additional groups  and nodules and plaques 
  sampled at  in duodenum. 
Ref. 7  intervals from 7  After a recovery period 
to 630 days of  following 140 days of 
treatment; or 10  H O treatment, by 10 to 
2 2 
to 30 days after  30 days without 
cessation of  treatment there were 
treatment at 140  fewer mice with lesions. 
days) 
117  Contains Nonbinding Recommendations 
Study  Animals/  Duration/  Controls  Doses  Notes 
Dose  Exposure 
Group 
  18 C3H/HeN  6  No  1:  2 mice with duodenal 
mice  months,  0.4%  tumors (11.1%). 
Ref. 8 
total male  Drinking 
and female  water 
  22  6  No  1:  7 mice with duodenal 
B6C3F1 mice  months,  0.4%  tumors (31.8%). 
Ref. 8 
total male  drinking 
and female  water 
  21 C57BL/6N   7  No  1:  21 mice with duodenal 
Ref. 8  mice  months,  0.4%  tumors (100%). 
total male and  drinking 
female  water 
  24 C3HCb/s   6  No  0.4% only  22 mice with duodenal 
mice  months,  tumors (91.7%). 
Ref. 8 
total male  drinking 
and female  water 
  21 female  6  11  1:  2 mice with duodenal 
Ref. 9  C3H/HeN mice  months,  0.4%  tumors (9.5%). 
drinking  None in controls. 
water 
  22 female  6  12  1:  7 mice with duodenal 
Ref. 9  B6C3F1  months,  0.4%  tumors (31.8%). 
Mice  drinking  None in controls. 
water 
  24 female  6  28  1:  22 mice with duodenal 
Ref. 9  C3HCb/s  mice  months,  0.4%  tumors (91.7%). 
drinking  None in controls. 
water 
  3 male rats  21  3  1:  No tumorigenic effect 
Ref. 12  weeks,  1.5%  observed. 
drinking 
water 
  Male and female  2 years,  Yes  2:  No tumorigenic effect 
Ref. 13  rats  drinking  0.3%  observed. 
(50/sex/group)  water  0.6% 
  Hamsters, sex not  15 weeks  Yes  1:  No tumorigenic effect 
  reported  and 6  70 mg/kg/d  observed. 
Ref. 14  (20/group)  months, 
oral 
gavage 
(5 d/wk) 
*a Their studies are not in the Carcinogenic Potency Database but are summarized in Ref. 2 
Catalase deficient. 
M = male; F = female; g = grams; kg = kilogram; d = day; wk = week; H O  = hydrogen peroxide;  
2 2
 
118  Contains Nonbinding Recommendations 
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 bodys 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 nonlinear, threshold effect. 
 
Regulatory and/or Published Limits 
 
Annex III of the European Union 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 percent. Higher levels up to 6 percent are also permitted providing products are 
prescribed by dental practitioners to persons older than 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 
percent hydrogen peroxide in the product, the amount of hydrogen 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). 
119  Contains Nonbinding Recommendations 
 
U.S. FDA  hydrogen peroxide is generally recognized as safe up to 3 percent for long-term 
over-the-counter-use as an antigingivitis/antiplaque 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 percent of the estimated 
endogenous production of 6.8 g/day, that is, 68 mg/day (or 68,000 micrograms (g)/day) 
would not significantly add to background exposure, but would usually exceed limits based 
on quality, in a pharmaceutical. The International Council for Harmonisation guidance for 
industry M7(R2) Assessment and Control of DNA Reactive (Mutagenic) Impurities in 
Pharmaceuticals to Limit Potential Carcinogenic Risk notes that when calculating acceptable 
intakes from compound-specific risk assessments, an upper limit would be determined by a 
quality limit of 0.5 percent, or, for example, 500 g in a drug with a maximum daily dose of 
100 mg.1 
 
1 We update guidances periodically.  To make sure you have the most recent version of a guidance, check the 
FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents.  
120  Contains Nonbinding Recommendations 
REFERENCES (FOR HYDROGEN PEROXIDE) 
 
1.  Halliwell B, Clement MV, and Long LH, 2000, Hydrogen Peroxide in the Human Body, 
FEBS Lett, 486:1013. 
 
2.  DeSesso JM, Lavin AL, Hsia SM, and Mavis RD, 2000, Assessment of the 
Carcinogenicity Associated With Oral Exposures to Hydrogen Peroxide, Food and Chem 
Toxicol, 38:102141. 
 
3.  International Agency for Research on Cancer, 1999, Monographs on the Evaluation of the 
Carcinogenic Risks to Humans: Re-Evaluation of Some Organic Chemicals, Hydrazine 
and Hydrogen Peroxide: Volume 71, Lyon, France: World Health Organization. 
 
4.  European Commissions Joint Research Center, 2003, European Union Risk Assessment 
Report: Hydrogen Peroxide (CASRN 7722-84-1), 38, available at 
https://echa.europa.eu/documents/10162/a6f76a0e-fe32-4121-9d9d-b06d9d5f6852. 
 
5.  Ito A, Watanabe H, Naito M, and Naito Y, 1981, Induction of Duodenal Tumors in Mice 
by Oral Administration of Hydrogen Peroxide, Jpn J Cancer Res, 72: 174175. 
 
6.  Carcinogenic Potency Database, website available at 
https://files.toxplanet.com/cpdb/index.html. 
 
7.  Ito A, Naito M, Naito Y, and Watanabe H, 1982, Induction and Characterization of 
Gastro-Duodenal Lesions in Mice Given Continuous Oral Administration of Hydrogen 
Peroxide, Jpn J Cancer Res, 73: 315322. 
 
8.  Ito A, Watanabe H, Naito M, Naito Y, and Kawashima K, 1984, Correlation Between 
Induction of Duodenal Tumor by Hydrogen Peroxide and Catalase Activity in Mice, Jpn J 
Cancer Res, 75:1721. 
 
9.  Ito A, Watanabe H, Aoyama H, Nakagawa Y, and Mori M, 1986, Effect of 1,2-
Dimethylhydrazine and Hydrogen Peroxide for the Duodenal Tumorigenesis in Relation 
to Blood Catalase Activity in Mice, Hiroshima J Med Sci,35:197200. 
 
10. U.S. Food and Drug Administration, final rule, Irradiation in the Production, Processing, 
and Handling of Food (53 FR 53176 at 53198-53199, December 30, 1988). 
 
11. Scientific Committee on Consumer Products, 2007, European Commissions 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, available at 
https://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/sccp_o_122.pdf. 
 
12. Hiroto N and Yokoyama T, 1981, Enhancing Effect of Hydrogen Peroxide Upon 
Duodenal and Upper Jejunal Carcinogenesis in Rats, Gann, 72: 811812. 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, Lyon, France, International 
Agency for Cancer Research, 11:86. (Cited in Ref. 2) Li Y, Noblitt T, Zhang A, Origel A, 
121  Contains Nonbinding Recommendations 
Kafrawy A, and Stookey G, 1993, Effect of Long-Term Exposure to a Tooth Whitener 
(Abstract), J Dent Res, 72:1162. (Cited in Ref. 2). 
 
14. Tredwin CJ, Naik S, Lewis NJ, and Scully C, 2006, Hydrogen Peroxide Tooth-Whitening 
(Bleaching) Products: Review of Adverse Effects and Safety Issues, Br Dent J, 200:371
376. 
 
15. De Bont R and Larebeke N, 2004, Endogenous DNA Damage in Humans: A Review of 
Quantitative Data, Mutagenesis, 19:169185. 
 
16. Finkel T and Holbrook NJ, 2000, Oxidants, Oxidative Stress and the Biology of Ageing, 
Nature, 408:23947. 
 
17. Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 
November 2009 on cosmetic products. 
 
18. U.S. Food and Drug Administration, advanced notice of proposed rulemaking, Oral 
Health Care Drug Products for Over-the-Counter Human Use; Antigingivitis/Antiplaque 
Drug Products; Establishment of a Monograph (68 FR 32231 at 3223232286, May 29, 
2003). 
122  Contains Nonbinding Recommendations 
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. 
 
The World Health Organization (WHO) (Ref. 1) reports that the methyl chloride 
concentration in the air in rural sites is in general below 2.1 micrograms (g)/cubic meter 
(m3) (1.0 parts per billion), and in urban cities it is equal to 0.27 to 35 g/m3 (0.13 to 17 ppb), 
corresponding to approximately 20 to 700 g daily intake (human respiratory volume of 20 
m3 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 g/liter in a well 
sample (Ref. 1). 
 
Mutagenicity/Genotoxicity 
 
Methyl chloride is mutagenic and genotoxic in vitro but equivocal in vivo. WHO (Ref. 1) and 
U.S. Environmental Protection Agency (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 the International Agency for Research on Cancer (IARC) as 
Group 3, inadequate evidence for the carcinogenicity to humans (Ref. 3), and by the U.S. 
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 parts per million (ppm)). Although not of 
statistical significance, cortical adenoma was also seen at 464 milligrams (mg)/m3 (225 ppm), 
and development of renal cortical microcysts in mice was seen in the 103 mg/m3 (50 ppm) 
dose group and to some extent in the 464 mg/m3 (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. 
123  Contains Nonbinding Recommendations 
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 P450 (CYP) isozyme 
believed to be responsible, CYP2E1, is present in male mouse kidney and is androgen-
dependent; female mice had CYP2E1 levels only 20 to 25 percent of those in males. 
Generation of formaldehyde has been demonstrated in renal microsomes 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 P450 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), and it 
was not 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 P450 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 U.S. 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 
to 1310 mg/kilogram (kg)/day; Ref. 5). In addition, based on the limitations of human 
relevance, the U.S. EPA classified methyl chloride as a group D compound, that is Not 
Classifiable as to Human Carcinogenicity. 
 
124  Contains Nonbinding Recommendations 
Methyl Chloride  Details of Carcinogenicity Studies (Only Inhalation Studies 
Available) 
 
Most 
Animals/ 
Duration/  Sensitive  TD50 
Study  Dose  Controls  Doses 
Exposure  Tumor  (mg/kg/d) 
Group 
Site/Sex 
  120/sex/  24  Yes  3:  Kidney tumors  1,360.7b 
Ref. 4  group  months    103; 464;  in males only. 
(summarized  B6C3F1  6 h/d,       2064  No finding in 
in Ref. 1 and  mice  5 d/wk,  mg/m3 (50;  females. 
Ref. 2)a  inhalation  225; 1000 
ppm) 
  120/sex/  24  Yes  3:  No findings in  NA 
  group  months   103; 464;  males and 
Ref. 4  Fisher  6 h/d,   2064  females. 
(summarized  344 rats  5 d/wk.  mg/m3 (50; 
in Ref. 1 and  inhalation  225; 1000 
Ref. 2)  ppm) 
Note: Studies not listed in the Carcinogenic Potency Database. 
a Carcinogenicity study selected for acceptable intake calculation. 
b TD  calculated based on carcinogenicity data (see Note 5).  
50
mg = milligram; kg = kilogram; d = day; h = hour; wk = week; m3 = cubic meter; ppm = parts per million; NA = 
not applicable. 
 
Regulatory and/or Published Limits 
 
The WHO (Ref. 1) developed a guideline value for the general population of 0.018 mg/m3, 
and the U.S. EPA (Ref. 2) developed a reference concentration of 0.09 mg/m3. Both were 
based on the potential for adverse central nervous system 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 /50,000 x 50 kg 
50
 
Lifetime AI = 1,360.7 mg/kg/day /50,000 x 50 kg 
 
Lifetime AI = 1,361 g/day 
125  Contains Nonbinding Recommendations 
REFERENCES (FOR METHYL CHLORIDE) 
 
1.  World Health Organization, 2000, Concise International Chemical Assessment Document 
(CICAD) 28: Methyl chloride, available at 
http://www.inchem.org/documents/cicads/cicads/cicad28.htm. 
 
2.  U.S. Environmental Protection Agency, 2001, Toxicological Review of Methyl Chloride 
(CAS No. 74-87-3): In Support of Summary Information on the Integrated Risk 
Information System (IRIS), available at 
https://cfpub.epa.gov/ncea/iris/iris_documents/documents/toxreviews/1003tr.pdf. 
 
3.  International Agency for Research on Cancer, 1999, Methyl Chloride. In: Monographs on 
the Evaluation of the Carcinogenic Risks to Humans: Volume 71, Lyon, France: World 
Health Organization. 
 
4.  Chemical Industry Institute of Toxicology (CIIT), 1981, 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. EPA/OTS Doc #878212061, 
NTIS/OTS0205952. 
 
5.  European Food Safety Authority (EFSA), 2014, Endogenous Formaldehyde Turnover in 
Humans Compared With Exogenous Contribution From Food Sources, EFSA Journal, 12 
Suppl 2:3550. 
126  Contains Nonbinding Recommendations 
STYRENE (CAS# 100-42-5) 
 
Potential for Human Exposure 
 
Styrene exposure to the general population occurs via environmental contamination and 
dietary exposure (Ref. 1). In the general population, indoor and outdoor air account for the 
largest exposures. However, smoking one pack of cigarettes would likely lead to the 
inhalation of milligram quantities of styrene (Ref. 2). Styrene has been detected as a natural 
constituent of a variety of foods and beverages, the highest levels occurring in cinnamon. 
Polystyrene and its copolymers are widely used as food-packaging materials and monomers 
such as styrene can migrate to food at low levels. The daily intake of styrene from dietary 
sources has been estimated to be 1 to 4 micrograms (g) in the United Kingdom, 2 to 12 g in 
Germany, and 9 g in the United States (Ref. 3, 4). Styrene is used in the synthesis of active 
pharmaceutical ingredients. 
 
Mutagenicity/Genotoxicity 
 
Styrene has produced contradictory findings in the in vitro Ames test, and it is predominantly 
inactive in the in vivo chromosome aberration, micronucleus and unscheduled DNA synthesis 
assays when conducted according to Organization for Economic Co-operation and 
Development (OECD) guidelines. Inconsistent results in the Ames test were attributed to 
styrene volatility, poor solubility, and different metabolic systems (Ref. 5). Styrene was 
positive for mutagenicity in the Ames test only with metabolic activation (Ref. 5), where it is 
converted to electrophilic intermediates (e.g., styrene-7,8-oxide) to enable formation of 
covalent adducts with DNA. Most of the genetic damage associated with styrene exposure is 
thought to be due to styrene-7,8-oxide, the main metabolite of styrene, which is further 
detoxified to styrene glycol. Styrene exposure elevated DNA adducts (N7-guanine, O6-
guanine, and N1-adenine) and sister chromatid exchanges in both animal models and in 
humans, and DNA strand breaks in humans (Ref. 5, 6). In a critical review of styrene 
genotoxicity based on the requirements outlined in the current OECD guidelines, Moore et al. 
(Ref. 7) concluded that it is unclear whether unmetabolized styrene is mutagenic in the Ames 
test, while the styrene-7,8-oxide metabolite is clearly mutagenic. The authors also noted that 
most styrene-7,8-oxide Ames positive data was collected without using exogenous metabolic 
activation, meaning that styrene-7,8-oxide was not further metabolized to styrene glycol.  
 
Styrene was mutagenic in glycophorin A variant frequencies in erythrocytes from 28 workers 
exposed via inhalation to greater than or equal to 85 milligrams (mg)/cubic meters styrene 
(Ref. 8). Lymphocytes from styrene-exposed workers had increased mutation frequencies 
(MFs) at the hypoxanthine-guanine phosphoribosyl transferase HPRT locus (Ref. 9).  
 
Two in vitro mammalian gene mutation studies were identified. In the HPRT assay, styrene 
induced only small increases in HPRT MFs in V79 cells (Ref. 10). Similarly, in V79 cells, 
styrene induced increases in mutations at the HPRT locus with exogenous metabolic 
activation system (Ref. 11). No rodent in vivo mutation studies evaluating styrene or styrene-
7,8-oxide were identified. 
 
Based on standard regulatory tests, there is no convincing evidence that styrene possesses 
significant genotoxic potential in vivo from the available data in experimental animals. 
However, genotoxicity associated with styrene exposure (related to formation of styrene-7,8-
127  Contains Nonbinding Recommendations 
oxide) has been proposed as a possible mode of action for styrene-induced carcinogenicity in 
experimental animals and humans (Ref. 1).  
 
Carcinogenicity 
 
The International Agency for Research on Cancer (IARC) classified styrene and the 
metabolite styrene-7,8-oxide in Group 2A, as probably carcinogenic to humans based on 
limited evidence in humans and sufficient evidence in experimental animals (Ref. 5). 
Styrene is also reasonably anticipated to be a human carcinogen by the National Institutes of 
Health (NIH) (Ref. 1). Possible modes of action for styrene-induced carcinogenicity involve 
genotoxic and cytotoxic effects together with immunosuppression (Ref. 1). The U.S. National 
Toxicology Program (NTP) listed styrene as reasonably anticipated to be a human 
carcinogen in its 12th and 14th Reports on Carcinogens (Ref. 12, 13). The National 
Research Council concluded that reasonably anticipated to be a human carcinogen was an 
appropriate carcinogenicity classification for styrene, due to limited carcinogenicity evidence 
in humans, sufficient evidence in animal studies, and other mechanistic data supporting 
carcinogenicity (Ref. 6). 
 
A recent systematic review of epidemiologic studies of exposure to styrene concluded that 
besides some limitations of available research as lack of quantitative estimates of styrene, the 
risk of specific cancers found no strong and consistent evidence of a causal association 
between styrene and non-Hodgkins lymphoma and its subtypes, all leukemia, subtypes of 
leukemia, or cancers of the esophagus, pancreas, lung, kidney, or other sites (Ref. 14).  
 
In the Carcinogenic Potency Database, styrene is reported to be carcinogenic in mice via the 
oral and inhalation routes and rats via the inhalation route (Ref. 15). The NIH Report on 
Carcinogens (Ref. 1) considered the most robust studies to be the 2-year studies via (1) oral 
exposure in B6C3F1 mice and (2) inhalation exposure in CD-1 mice. In male B6C3F1 mice, 
oral exposure to styrene increased the combined incidence of alveolar and bronchiolar 
adenomas and carcinomas (Ref. 16). In the inhalation study, in male and female CD-1 mice, 
there was an increase in the incidence of pulmonary adenomas and an increase in pulmonary 
carcinomas in females in the high-dose group (Ref. 17). 
 
The IARC evaluated nine studies each (with various routes of application) in mice and rats 
for styrene and three each in mice and rats for styrene-7,8-oxide. For styrene, one study with 
transplacental exposure followed by gavage using O20 mice demonstrated an increase of lung 
carcinoma and adenoma in pups whereas a second study in C57BL mice was negative (Ref. 
18). Two out of five studies with inhalation in CD-1 mice reported an increase in lung 
bronchoalveolar tumors  (Ref. 16, 19); whereas the other three (in C57BL/6 mice) were 
negative (Ref. 19). One study in mice with oral application found increased lung tumors and 
a positive trend for hepatocellular carcinoma (Ref. 16). Another study with intraperitoneal 
(i.p.) application gave negative results (Ref. 20). In two studies in CD (SD derived) or SD 
rats with whole body inhalation exposure, styrene produced negative results in one (Ref. 21) 
but increased mammary gland tumors in the other (Ref. 22); whereas four oral studies, three 
with gavage (Ref. 17, 22) and one via drinking water (Ref. 23), were negative. The observed 
increase in mammary gland tumors was not dose-dependent and was not considered reliable 
evidence of carcinogenicity by NIH Report on Carcinogens (Ref. 1); the IARC (Ref. 5) also 
noted short treatment duration and incomplete reporting of the study. Other studies in rats 
with transplacental exposure followed by gavage (Ref. 17), i.p. application, or subcutaneous 
application (Ref. 22) were also negative.  
128  Contains Nonbinding Recommendations 
 
Styrene-7,8-oxide was tested in three studies in mice, one by gavage (Ref. 24) and two by 
skin application (Ref. 25, 26). In the oral gavage study styrene-7,8-oxide increased squamous 
cell tumors in forestomach in males and females and hepatocellular tumors in males. The 
studies by skin application were inadequate for evaluation due to the limited reporting of 
study details and the lack of controls. In rats, styrene-7,8-oxide was tested in two studies with 
oral exposure by gavage (Ref. 22, 24) and one by transplacental exposure followed by gavage 
(Ref. 27). In both studies by gavage, squamous cell tumors of the forestomach were increased 
and in one of the studies mammary gland tumors where also increased in males. In the study 
by transplacental exposure followed by gavage, forestomach tumors were increased. The 
IARC concluded that there is sufficient evidence for carcinogenicity of styrene and styrene-
7,8-oxide in experimental animals (Ref. 5).   
 
The NTP concluded that the evidence from studies in rats was insufficient for reaching a 
conclusion concerning the carcinogenicity of styrene (Ref. 1). An evaluation of the available 
data from eight oncogenicity studies by Cruzan et al. (Ref. 21) concluded that there was clear 
evidence that styrene did not induce cancer in rats. It has been proposed that the reason for 
lung tumor induction in mice, but not rats, may involve differential metabolism of styrene in 
the two species (Ref. 1). 
 
Styrene  Details of the Most Relevant Carcinogenicity Studies 
 
Most 
Animals/  Sensitive 
Duration/  TD
50 
Study  Dose  Controls  Doses  Tumor 
Exposure  mg/kg/d)a 
(
Group  Site/Type/Se
x 
Ref. 16  50/sex/  78 weeks,   20  2:  Lung/ Male b  360 
   group  o ral gavage  150, 300 
M&F  mg/kg/d 
B6C3F1 
m  ouse  
Ref. 17  70/sex/  98-104  70  4:  Lung/ Male  154c 
   group  weeks,  20, 40, 80, 
CD1  i nhalation  160 ppm, 
m  ouse   22.3, 44.6, 
89.3, 179 
mg/kg/d 
Ref. 16  70/sex/  78 -107  40  3:  No Tumors  NC 
   group  weeks,   500, 1,000, 
Fischer  o ral gavage  2,000 
3 44 rats  mg/kg/d 
Ref. 21  70/sex/  104 weeks,  70  4:   No Tumors  NC 
   group   i nhalation  50, 200, 500, 
CD rats  1,000 ppm 
129  Contains Nonbinding Recommendations 
Most 
Animals/  Sensitive 
Duration/  TD
50 
Study  Dose  Controls  Doses  Tumor 
Exposure  mg/kg/d)a 
(
Group  Site/Type/Se
x 
Ref. 22  30/sex/  52 weeks,  60  5:  Mammary  23.3 
   group   i nhalation  25, 50, 100,  tissue/ 
S  D rats  200, 300  Female d 
ppm 
Ref. 22  40/sex/  52 weeks,  40  2:  No Tumors  NC 
  group   gavage   50, 250 
  SD Rats   mg/kg/day 
Ref. 22  40/sex/  s.c. once,  40  1:  No Tumorse  NC 
  group   then  50 mg (s.c.), 
  SD Rats   i.p. 4 times  50 mg (i.p.) 
at 2-month 
intervals 
a The TD  values are taken from the Carcinogenic Potency Database (CPDB) (Ref. 15) 
50
b Despite having a statistically significant dose-trend per CPDB, the author concluded that there was no 
convincing evidence of carcinogenicity in mice 
c Carcinogenicity study selected for the acceptable intake calculation, milligrams per kilogram per day dose 
conversion taken from CPDB  
d Author opinion: Styrene, was found to cause an increase in total (benign and malignant) and malignant 
mammary tumors. Cruzan et al. (Ref. 21) noted no obvious dose response in the data. Furthermore, the study 
findings were not considered reliable evidence of carcinogenicity by the National Institutes of Health Report on 
Carcinogens (Ref. 1) and the International Agency for Research on Cancer (Ref. 5) noted short treatment 
duration and incomplete reporting of the study. 
e Study limited to acute exposures and a nonstandard study design 
mg = milligram; kg = kilogram; d = day; ppm = parts per million; SD = Sprague-Dawley; NC = not calculated; 
s.c. = subcutaneous injection; i.p. = intraperitoneal injection. 
 
Mode of Action for Carcinogenicity 
 
A comprehensive review of the mechanisms that contribute to the carcinogenicity of styrene 
is presented in the IARC monograph (Ref. 5). Taking into consideration the available in vitro 
and in vivo genotoxicity data, the IARC concluded that there is strong evidence that styrene 
is genotoxic and that the mechanism is relevant to humans. Styrene is metabolically activated 
in animals and in humans to an electrophile, styrene-7,8-oxide, which interacts with 
nucleophilic macromolecules, such as proteins and DNA. DNA adducts are formed primarily 
by alkylation of N7-guanine.  Styrene-7,8-oxide DNA adducts have been observed in vitro, in 
rodents and in humans exposed to styrene. The IARC also indicated that there is strong 
evidence that both styrene and styrene-7,8-oxide alter cell proliferation and that styrene 
modulates receptor-mediated effects based on increased serum prolactin in humans exposed 
occupationally.   
 
The genotoxic potential and human relevance of the observed mouse tumors has been 
questioned (Ref. 28, 29, 30, 31). Other possible mechanisms contributing to the carcinogenic 
activity of styrene include oxidative stress, immunosuppression, and chronic inflammation.  
The mechanism suggested by Cruzan et al. (Ref. 28) as the main cause of mice lung tumors 
includes styrene metabolites inducing gene expression for metabolism of lipid, lipoprotein, 
cell cycle and mitotic M-M/G1 phases, and mild cytotoxicity and strong mitogenicity in mice 
130  Contains Nonbinding Recommendations 
lung cells, leading to excessive cell proliferation and hyperplasia. On the other hand, the 
authors assume that it would not be relevant in humans due to limited lung metabolism (by 
CYP2F2). The IARC concluded that the evidence for these mechanisms of action is moderate 
to weak. The various perspectives were evaluated in determining the overall conclusions 
regarding the genotoxic potential and human relevance of tumors associated with styrene 
administration. Ultimately, the IARC conclusions were used in supporting the derivation of 
the acceptable intake (AI) for styrene. 
 
Regulatory and/or Published Limits 
 
The World Health Organization (WHO) defined a tolerable daily intake (TDI) for styrene via 
the oral route of 7.7 g/kilogram (kg)/day (i.e., 0.385 mg per day based on 50 kg body 
weight) from which a drinking water guideline value of 20 g/L has been defined (i.e., 40 g 
per day based on consumption of 2 liters (L) of water per day) (Ref. 32). This WHO limit was 
based on reduced body weight gain in a 2-year rat drinking water study. The U.S. 
Environmental Protection Agency (EPA) oral reference dose (Ref. 33) for styrene is 
200 g/kg/day (i.e., 10 mg/day based on 50 kg body weight), based on noncancer endpoints. 
The associated U.S. EPA drinking water limit is 100 g/L (i.e., 200 g per day based on 
consumption of 2 L water per day). The Joint FAO/WHO Expert Committee on Food 
Additives maximum TDI for styrene (Ref. 34) from migration from food packaging is 0.04 
mg/kg/day (i.e., a maximum of 2 mg per day based on 50 kg body weight). A specific 
migration limit of 60 parts per million styrene into foods in polystyrene packaging (i.e., 60 
mg per day assuming the consumption of 1 kg food/day for adult humans) is considered 
permissible in the European Union (Ref. 4). 
 
Acceptable Intake 
 
Rationale for selection of study for AI calculation 
 
Since styrene is not considered to be a rat carcinogen, mouse lung tumor data were used to 
derive the AI. The most sensitive TD  was in the inhalation study of Cruzan et al. (Ref. 17). 
50
The AI derived from this inhalation study was considered suitable for all routes of 
administration as an increase in lung tumors were also seen in the carcinogenicity study in 
mice with gavage treatment (Ref. 16). The AI is expected to be a conservative limit as the 
mouse is known to have higher levels of CYP2F enzymes in comparison to humans, which is 
key to tumor formation (Ref. 28).   
 
Calculation of AI 
 
Lifetime AI = TD50/50,000 x 50 kg 
 
Lifetime AI =154 mg/kg/day/50,000 x 50 kg 
 
Lifetime AI = 154 g/day 
131  Contains Nonbinding Recommendations 
REFERENCES (FOR STYRENE) 
 
1.  National Institutes of Health, 2016, Report on Carcinogens, 14th edition, available at 
https://ntp.niehs.nih.gov/ntp/roc/content/profiles/styrene.pdf. 
 
2.  Capella KM, Roland K, et al., 2019, Ethylbenzene and Styrene Exposure in the United 
States Based on Urinary Mandelic Acid and Phenylglyoxylic Acid: NHANES 20052006 
and 20112012, Environ Res, 171:101110. 
 
3.  World Health Organization, 2000, Chapter 5.12 Styrene. Air Quality Guidelines: 2nd 
edition, available at 
http://www.euro.who.int/__data/assets/pdf_file/0018/123066/AQG2ndEd_5_12Styrene.p
df.  
 
4.  Gelbke HP, Banton M, et al., 2014, Derivation of Safe Health-Based Exposure Limits for 
Potential Consumer Exposure to Styrene Migrating Into Food From Food Containers, 
Food Chem Toxicol, 64:258269. 
 
5.  International Agency on Research of Cancer, 2019, IARC Monographs on the Evaluation 
of Carcinogenic Risks to Humans: Styrene, Stryrene-4,8-Oxide and Quinoline: Volume 
121, Lyon, France: World Health Organization. 
 
6.  National Research Council, 2014, Review of the Styrene Assessment in the National 
Toxicology Program 12th Report on Carcinogens, Washington, DC: The National 
Academies Press, available at https://doi.org/10.17226/18725. 
 
7.  Moore MM, Pottenger LH, and House-Knight T, 2019, Critical Review of Styrene 
Genotoxicity Focused on the Mutagenicity/Clastogenicity Literature and Using Current 
Organization of Economic Cooperation and Development Guidance, Environ Mol 
Mutagen, 60:624663. 
 
8.  Compton-Quintana PJ, Jensen RH, Bigbee WL, Grant SG, Langlois RG, Smith MT, and 
Rappaport SM, 1993, Use of the Glycophorin A Human Mutation Assay to Study 
Workers Exposed to Styrene, Environ Health Perspect, 99:297301. 
 
9.  Vodicka P, Soucek P, Tates AD, Dusinska M, Sarmanova J, Zamecnikova M, Vodickova 
L, Koskinen M, de Zwart FA, Natarajan AT, and Hemminki K, 2001, Association 
Between Genetic Polymorphisms and Biomarkers in Styrene-Exposed Workers, Mutat 
Res, 482:89103.  
 
10. Loprieno N, Abbondandolo A, Barale R, Baroncelli S, Bonatti S, Bronzetti G, 
Cammellini A, Corsi C, Corti G, Frezza D, Leporini C, Mazzaccaro A, Nieri R, Rosellini 
D, and Rossi AM, 1976, Mutagenicity of Industrial Compounds: Styrene and Its Possible 
Metabolite Styrene Oxide, Mutat Res, 40:317324.  
 
11. Beije B and Jenssen D, 1982, Investigation of Styrene in the Liver Perfusion/Cell Culture 
System. No Indication of Styrene-7,8-Oxide as the Principal Mutagenic Metabolite 
Produced by the Intact Rat Liver, Chem Biol Interact, 39:5776.  
 
132  Contains Nonbinding Recommendations 
12. National Toxicology Program, 2011, 12th Report on Carcinogens, Rep Carcinog, 12: iii
499.  
 
13. National Toxicology Program, 2016, 15th Report on Carcinogens, Research Triangle 
Park, NC: U.S. Department of Health and Human Services, Public Health Service, 
available at https://ntp.niehs.nih.gov/go/roc14.  
 
14. Collins JJ and Delzell E, 2018, A Systematic Review of Epidemiologic Studies of Styrene 
and Cancer, Crit Rev Toxicol, 48:443470. 
 
15. Carcinogenic Potency Database, website available at 
https://files.toxplanet.com/cpdb/index.html. 
 
16. National Cancer Institute, 1979, Technical Report Series No. 185: Bioassay of Styrene for 
Possible Carcinogenicity. NCI-CG-TR-185, 1979. 
 
17. Cruzan G, Cushman JR, et al., 2001, Chronic Toxicity/Oncogenicity Study of Styrene in 
CD-1 Mice by Inhalation Exposure for 104 Weeks, J Appl Toxicol, 21:185198. 
 
18. Ponomarkov V and Tomatis L, 1978, Effects of Long-Term Oral Administration of 
Styrene to Mice and Rats, Scand J Work Environ Health, 4:127135. 
 
19. Cruzan G, Bus JS, Banton MI, Sarang SS, Waites R, Layko DB, et al., 2017, Complete 
Attenuation of Mouse Lung Cell Proliferation and Tumorigenicity in CYP2F2 Knockout 
and CYP2F1 Humanized Mice Exposed to Inhaled Styrene for up to 2 Years Supports a 
Lack of Human Relevance, Toxicol Sci, 159:413421. 
 
20. Brunnemann KD, Rivenson A, Cheng SC, Saa V, and Hoffmann D, 1992, A Study of 
Tobacco Carcinogenesis. XLVII. Bioassays of Vinylpyridines for Genotoxicity and for 
Tumorigenicity in A/J Mice, Cancer Lett, 65:107113. 
 
21. Cruzan G, Cushman JR, et al., 1998, Chronic Toxicity/Oncogenicity Study of Styrene in 
CD Rats by Inhalation Exposure for 104 Weeks, Toxicol Sci, 46:266281. 
 
22. Conti B, Maltoni C, et al., 1988, Long-Term Carcinogenicity Bioassays on Styrene 
Administered by Inhalation, Ingestion and Injection and Styrene Oxide Administered by 
Ingestion in Sprague-Dawley Rats, and para-Methylstyrene Administered by Ingestion in 
Sprague-Dawley Rats and Swiss Mice, Ann N Y Acad Sci, 534:203234. 
 
23. Beliles RP, Butala JH, Stack CR, and Makris S, 1985, Chronic Toxicity and Three-
Generation Reproduction Study of Styrene Monomer in the Drinking Water of Rats, 
Fundam Appl Toxicol, 5:855868. 
 
24. Lijinsky W, 1986, Rat and Mouse Forestomach Tumors Induced by Chronic Oral 
Administration of Styrene Oxide, J Natl Cancer Inst, 77:471476. 
 
25. Weil CS, Condra N, Haun C, and Striegel JA, 1963, Experimental Carcinogenicity and 
Acute Toxicity of Representative Epoxides, Am Ind Hyg Assoc J, 24:305325. 
 
133  Contains Nonbinding Recommendations 
26. Van Duuren BL, Nelson N, Orris L, Palmes ED, and Schmitt FL, 1963, Carcinogenicity 
of Epoxides, Lactones, and Peroxy Compounds, J Natl Cancer Inst, 31:4155. 
 
27. Ponomarkov V, Cabral JRP, Wahrendorf J, and Galendo D, 1984, A Carcinogenicity 
Study of Styrene-7,8-Oxide in Rats, Cancer Lett, 24:95101. 
 
28. Cruzan G, Bus JS, et al., 2018, Based on an Analysis of Mode of Action, Styrene-Induced 
Mouse Lung Tumors Are Not a Human Cancer Concern, Reg Tox Pharm, 95:1728. 
 
29. European Food Safety Authority (EFSA), 2020, EFSA Panel on Food Contact Materials, 
Enzymes and Processing Aids (CEP) Assessment of the Impact of the IARC Monograph 
Vol. 121 on the Safety of the Substance Styrene (FCM No 193) for Its Use in Plastic 
Food Contact Materials, EFSA J, 18(10):6247. 
 
30. European Chemicals Authority, 2011, CLH Report for Styrene, Proposal for Harmonised 
Classification and Labelling, Based on Regulation (EC) No 1272/2008 (CLP Regulation), 
Annex VI, Part 2, Substance Name: Styrene, available at 
https://echa.europa.eu/documents/10162/fd574447-2888-e637-ae30-77df1b043a2f. 
 
31. European Chemicals Authority, 2021, Registry of CLH Intentions Until Outcome: 
Styrene, available  at https://echa.europa.eu/de/registry-of-clh-intentions-until-outcome/-
/dislist/details/0b0236e180a0fa4f. 
 
32. World Health Organization (WHO), 2003, Styrene in Drinking Water: Background 
Document for Development of WHO Guidelines for Drinking-water Quality 
WHO/SDE/WHS/03.04/27. 
 
33. U.S. Environmental Protection Agency, 1990, Integrated Risk Information System 
(IRIS): Styrene (CASRN 100-42-5), available at 
https://cfpub.epa.gov/ncea/iris/iris_documents/documents/subst/0104_summary.pdf. 
 
34. Joint Expert Committee on Food Additives, 1984, Styrene: WHO Food Additives Series 
19, available at http://www.inchem.org/documents/jecfa/jecmono/v19je15.htm. 
134  Contains Nonbinding Recommendations 
VINYL ACETATE (CAS# 108-05-4) 
 
Potential for Human Exposure 
 
Human exposure occurs primarily in the occupational setting with very little exposure to 
vinyl acetate in the general population (Ref. 1). Vinyl acetate is used in the synthesis of 
pharmaceuticals. 
 
Mutagenicity/Genotoxicity 
 
The mutagenicity and genotoxicity of vinyl acetate has been reviewed by Albertini (Ref. 2). 
Vinyl acetate is not mutagenic in the Ames test in multiple strains of Salmonella or in 
Escherichia coli and vinyl acetate mutagenicity in mammalian cells (at the tk locus human 
TK6 cells) appears to reflect mainly chromosome level or large mutational events, but normal 
growth mutants thought to reflect smaller gene mutations were also reported. Vinyl acetate 
also induced micronuclei and chromosome aberrations in vitro and chromosome aberrations 
in vivo and was positive in one out of five in vivo micronucleus studies. Small increases of 
micronuclei in mouse bone marrow were induced following intraperitoneal (i.p.) 
administration, but the genotoxicity was associated with elevated toxicity and mortality (Ref. 
3). 
 
There is extensive evidence that vinyl acetate genotoxicity is mediated by its metabolite 
acetaldehyde. Acetaldehyde is produced endogenously, and detoxification by aldehyde 
dehydrogenase is required to maintain intracellular homeostasis (Ref. 2). Given its response 
in mammalian cells, and rapid conversion to acetaldehyde, vinyl acetate is considered 
mutagenic.  See Mode of Action for Carcinogenicity information below for further details. 
 
Carcinogenicity 
 
Vinyl acetate is classified as Group 2B, possibly carcinogenic to humans (Ref. 4). There are 
two oral carcinogenicity reports cited in the Carcinogenic Potency Database (CPDB) (Ref. 5). 
One mouse and one rat study, in which vinyl acetate was administered in drinking water, are 
limited as there are only two treatment groups and less than 50 animals per group. Uterine, 
esophageal and forestomach tumors were observed in Swiss mice; and liver, thyroid, and 
uterine tumors were observed in Fischer 344 rats. A number of nonsite-of-contact tumors 
(e.g., Zymbal gland, lung, liver, uterine, mammary gland) were observed in the oral 
carcinogenicity studies conducted by Maltoni et al. (Ref. 6) and Lijinsky et al. (Ref. 7). These 
tumors in Maltoni et al. (Ref. 6) all occurred with high background incidence. Therefore, 
without adjusting for age, these tumor data cannot be evaluated with certainty. Squamous cell 
carcinoma of the oral cavity, tongue, esophagus, and forestomach were all treatment related 
at 5000 parts per million (ppm). There were no tumors among mice administered 1,000 ppm 
(Ref. 8). In the oldest published oral carcinogenicity study, Lijinsky et al. (Ref. 7), there are a 
number of deficiencies in the study design, most notably that the drinking water solutions 
were prepared only once per week. The authors recognized a decomposition rate of 
approximately 8.5 percent per day. Therefore, by the end of the week the animals in the 2,500 
ppm group, for example, were exposed to approximately 1,300 ppm vinyl acetate and 
significant quantities of breakdown products, including acetaldehyde and acetic acid. The 
authors also did not purify the vinyl acetate prior to preparation of the drinking water 
solutions. Thus, the rats were also exposed to unspecified impurities. In addition, only 20 rats 
135  Contains Nonbinding Recommendations 
were in each group, so the statistical power for detecting true positive responses and for 
discriminating against false positive and false negative outcomes is compromised (Ref. 8). 
 
In addition to the CPDB, other carcinogenicity studies are available in the literature. An oral 
drinking water study was conducted by the Japan Bioassay Research Centre in accordance 
with the Organization for Economic Cooperation and Development guideline 453, including 
three treatment groups and 50 animals per group (Ref. 9, 10). Increases in tumors of the oral 
cavity, esophagus, and forestomach in Crj:BDF1 mice and statistically significant increases 
of tumors in the oral cavity of female F344:DuCrj rats at all doses were reported following 
drinking water administration of vinyl acetate. In another lifetime study, Minardi et al. (Ref. 
11) report increases in tumors in oral cavity and lips in 17-week-old and 12-day-old Sprague-
Dawley rats also administered vinyl acetate in the drinking water. Two treatments groups 
were included with more than 50 animals per group for the 12-day-old rats (offspring) but 
less than 50 per group for the 17-week-old animals (breeders). The 12-day-old rats were more 
sensitive with tumors in the oral cavity and lips; whereas an increase tumor response was not 
evident in the 17-week-old animals.  
 
Finally, Bogdanffy et al. (Ref. 12) administered vinyl acetate in drinking water for 10 weeks 
to male and female rats that were subsequently mated. The offspring were then culled into 
two groups of 60 for the main study and 30 for satellite groups and exposure in the drinking 
water continued to 104 weeks. The authors concluded that in the offspring there were no non-
neoplastic or neoplastic lesions observed that were compound related. Two squamous 
carcinomas were observed in the oral cavity of treated males, but the incidence of these 
tumors was within historical control ranges. Therefore, they were not considered related to 
vinyl acetate treatment. 
 
There are two inhalation carcinogenicity reports cited in the CPDB (Ref. 5). Vinyl acetate 
was not carcinogenic to CD-1 mice but induced nasal tumors in Sprague-Dawley rats (Ref. 
13). All but one of the 11 nasal tumors in rats (benign endo and exophytic papillomas and 
squamous-cell carcinomas) were observed at the terminal sacrifice at the high dose of 600 
ppm, indicating a late life dependency of tumor formation.  One benign tumor, of 
questionable relationship to exposure, was observed at the 200 ppm concentration (Ref. 13). 
In both species and both sexes, vinyl acetate induced morphological non-neoplastic lesions in 
the nasal cavity of the 200 and 600 ppm groups and in the trachea (mice only) and in the 
lungs of the 600 ppm groups.  
 
136  Contains Nonbinding Recommendations 
Vinyl Acetate  Details of Carcinogenicity Studies 
 
Animals/  Most Sensitive 
Duration/  TD  
50
Study  Dose  Controls  Doses  Tumor 
Exposure  (mg/kg/d) 
Group  Site/Type/Sex 
Ref. 6  37 F and  2 years,  37 F, 14  2:  Uterine, Female  3920b 
  13 M/  drinking  M  1,000 ppm 
  group  water  (103 mg/kg/d 
  Swiss  F and 96.3 
mice   mg/kg/d M), 
  5,000 ppm 
(578 mg/kg/d 
F and 546 
mg/kg/d M) 
Ref. 7  20/sex/  2 years,  20  2:  Liver, Male  132b 
  group  drinking  1,000 mg/L  
  F344 rat   water  (0.1 mg/kg/d 
    F and 0.062 
mg/kg/d M), 
2,500 mg/L 
(0.04 
mg/kg/d F 
and 0.025 
mg/kg/d M) 
Ref. 9  50/sex/  2 years,  50  3:  Oral cavity,  1854c 
  group  drinking  400 ppm (63  Male 
  Crj:BDF water  mg/kg F and 
1  
  mice   42 mg/kg/d 
M), 2,000 
ppm (301 
mg/kg/d F 
and 202 
mg/kg/d M), 
10,000 ppm 
(1,418 
mg/kg/d F 
and 989 
mg/kg/d M)  
137  Contains Nonbinding Recommendations 
Animals/  Most Sensitive 
Duration/  TD  
50
Study  Dose  Controls  Doses  Tumor 
Exposure  (mg/kg/d) 
Group  Site/Type/Sex 
Ref. 9  50/sex/  2 years,  50  3:  Oral cavity,  3057c 
  group  drinking  400 ppm (31  Male 
  F344/Du water  mg/kg/d F 
Crj rats  and 21 
  mg/kg/d M), 
2,000 ppm 
(146 mg/kg/d 
F and 98 
mg/kg/d M), 
10,000 ppm 
(575 mg/kg/d 
F and 442 
mg/kg/d M) 
Ref. 11  37F and  2 years,  Breeders  2:  Oral cavity and  983c 
  14M/  drinking  14M and  1,000 ppm  lips, Male 
group,  water  37F;  (70.6 
Breeders  Offspring  mg/kg/d), 
(17 wk  107M  5,000 ppm 
old);   and 99F  (353 
53 or  mg/kg/d)a 
83M and 
57 or 87F 
Sprague-
Dawley 
rat 
offspring 
(12 day 
old)  
Ref. 12  60/sex/  2 years,  60  3:  No tumors  NC 
  group  drinking  200 ppm (16 
  Crl:CD(S water  mg/kg/d F 
D)BR  and 10 
rats  mg/kg/d M), 
1,000 ppm 
(76 mg/kg/d 
F and 47 
mg/kg/d M), 
5,000 ppm 
(302 mg/kg/d 
F and 202 
mg/kg/d M) 
138  Contains Nonbinding Recommendations 
Animals/  Most Sensitive 
Duration/  TD  
50
Study  Dose  Controls  Doses  Tumor 
Exposure  (mg/kg/d) 
Group  Site/Type/Sex 
Ref. 13  60/sex/  2 years,  60  3:  No tumors  NC 
  group  inhalation   50 ppm (55.3 
  Charles  mg/kg/d F 
River  and 46.1 
CD1  mg/kg/d M), 
mice  200 ppm 
(221 mg/kg/d 
F and 184 
mg/kg/d M), 
600 ppm 
(664 mg/kg/d 
F and 554 
mg/kg/d M) 
Ref. 13  60/sex/  2 years,  20  3:  Nasal, Male  758b 
  group  inhalation  50 ppm (13.3 
  Charles  mg/kg/d F 
River CD  and 9.32 
(Sprague- mg/kg/d M), 
Dawley)  200 ppm 
Rats  (52.7 
mg/kg/d F 
and 36.9  
mg/kg/d M), 
600 ppm 
(158 mg/kg/d 
F and 111 
mg/kg/d M) 
a Calculated based on the International Council for Harmonisation guidance for industry Q3C(R8) Impurities: 
Guidance for Residual Solvents (December 2021) assumptions.1 
b Taken from the Carcinogenic Potency Database (CPDB) (Ref. 14). Carcinogenicity study selected for the 
acceptable intake calculation 
c Study not reported in CPDB, therefore TD  value calculated based on carcinogenicity data  
50
mg = milligram; kg = kilogram; d = day; M = male; F = female; ppm = parts per million; L = liter; NC = not 
calculated. 
 
Mode of action for carcinogenicity 
 
Vinyl acetate has been reviewed by the European Commissions Scientific Committee on 
Health and Environmental Risks (SCHER), which published a Risk Assessment Report in 
2008 (Ref. 1). Overall, SCHER supports the conclusion that the carcinogenic potential of 
vinyl acetate is expressed only when tissue exposure to acetaldehyde is high and when 
cellular proliferation is simultaneously elevated. This mode of action suggests that exposure 
levels, which do not increase intracellular concentrations of acetaldehyde, will not produce 
adverse cellular responses. As long as the physiological buffering systems are operative, no 
local carcinogenic effect by vinyl acetate should be expected at the no-observed adverse 
 
1 We update guidances periodically.  To make sure you have the most recent version of a guidance, check the 
FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents.  
139  Contains Nonbinding Recommendations 
effect level (NOAEL) for histological changes in respiratory rodent tissues. However, the 
SCHER indicated that local levels at or below the NOAEL are not free of carcinogenic risk, 
although the risk may be negligibly low. Hengstler et al. (Ref. 8) presented the case for vinyl 
acetate as a DNA-reactive carcinogen with a threshold dose response, which has also been 
described by Albertini (Ref. 2). Like acetaldehyde, vinyl acetate is not mutagenic in the 
standard bacterial reversion assay; evidence for DNA reactivity and site-of-contact 
carcinogenicity of vinyl acetate is that it occurs because of metabolic conversion to 
acetaldehyde. 
 
The genotoxicity profiles for acetaldehyde and vinyl acetate are almost identical, and vinyl 
acetate is not active as a clastogen without the addition of carboxylesterase (Ref. 8). 
Therefore, the clastogenic activity of vinyl acetate is attributed to metabolic formation of 
acetaldehyde. At high concentrations, enzyme activities are not able to oxidize all the 
generated acetaldehyde, and a low pH microenvironment is the result (Ref. 12). From 
consistent endogenous acetic acid exposure, tissues may sustain a reduction of 0.15 units in 
pH following vinyl acetate treatment without adverse effects (i.e., cytotoxicity and 
genotoxicity) (Ref. 15). However, when this practical threshold is exceeded, DNA damage, 
cytotoxicity, and regenerative cellular proliferation occur, resulting in tumor formation at the 
site of contact. 
 
There is clear evidence for the carcinogenicity of vinyl acetate in two animal species, in both 
sexes and for both inhalation and oral administration. Following both oral and inhalation 
administration, vinyl acetate is rapidly hydrolyzed at the site of contact by carboxylesterases, 
to acetic acid and acetaldehyde (Ref. 3, 16). Vinyl acetate exposure produces tumors at the 
site of first contact along the exposure routes. The dose response is thought to be nonlinear, 
with the observed tumor responses reflecting the target tissue-specific enzyme activities for 
activation and detoxification (Ref. 2). However, as noted in the acetaldehyde monograph, 
there are no published measurements, which would allow discrimination between the 
irritating effect and the potential mutagenic effect ion cancer development.   
 
Regulatory and/or Published Limits 
 
For vinyl acetate, the U.S. Environmental Protection Agency (EPA) integrated risk 
information system database calculated an inhalation reference concentration (RfC) for 
noncarcinogenic effects of 0.2 milligrams (mg)/cubic meter (m3), or 5.8 mg/day assuming a 
respiratory volume of 28.8 m3. The RfC was based on a human equivalent concentration of 
5 mg/m3 derived from Owen et al. 1988, which identified both a NOAEL and a lowest-
observed adverse effect level for histopathological effects of the nasal olfactory epithelia in 
rats and mice in a chronic 2-year study. A total adjustment factor of 30 was applied (Ref. 17). 
The U.S. EPA report did not include a carcinogenicity assessment for lifetime exposure to 
vinyl acetate. It is stated that RfCs can be derived for the noncarcinogenic health effects of 
substances that are carcinogens and to refer to other sources of information concerning the 
carcinogenic potential.   
 
Permissible Daily Exposure (PDE) for Oral Exposure 
 
Rationale for selection of study for PDE calculation 
 
Following oral administration, vinyl acetate is rapidly hydrolyzed at the site of contact by 
carboxylesterases, to acetic acid and acetaldehyde. Given the weight of evidence for a 
140  Contains Nonbinding Recommendations 
nonlinear dose response for the carcinogenicity of both vinyl acetate and acetaldehyde 
following oral administration and considering high background exposure to acetaldehyde 
from a wide variety of foods, the oral PDE recommended is based on that derived for 
acetaldehyde of 2 mg/day. 
 
PDE (oral) = 2 mg/day 
 
Acceptable Intake (AI) for all other routes 
 
Rationale for selection of study for AI calculation 
 
For routes of administration other than the oral route, the inhalation carcinogenicity study in 
rats (Ref. 13) was used for derivation of an AI. In this study, there were three treatment 
groups with 60 animals per sex per treatment group.  Animals were exposed 6 hours per day, 
5 days per week for 2 years to vinyl acetate. Carcinogenicity was observed in the nasal cavity 
of rats, with male being the more sensitive sex. The TD  for the nasal cavity in male rats is 
50
758 mg/kilogram (kg)/day, as reported in CPDB. The only other carcinogenicity study that is 
available with vinyl acetate administered via the inhalation route in mice is negative (Ref. 
13). Therefore, the rat inhalation study was selected for derivation of an AI. 
 
Although the dose-response relationship for carcinogenicity is thought to be nonlinear, as 
stated above, there are no published measurements which allow discrimination between a true 
threshold versus a nonlinear inflection point. Therefore, the AI was calculated using linear 
extrapolation. 
 
Calculation of AI 
 
Lifetime AI = TD /50,000 x 50 kg 
50
 
Lifetime AI = 758 mg/kg/day x 50 kg 
 
Lifetime AI (all other routes) = 758 micrograms (g)/day 
141  Contains Nonbinding Recommendations 
REFERENCES (FOR VINYL ACETATE) 
 
1.  Scientific Committee on Health and Environmental Risks, 2008, Risk Assessment Report 
on Vinyl Acetate, available at 
https://ec.europa.eu/health/archive/ph_risk/committees/04_scher/docs/scher_o_108.pdf. 
 
2.  Albertini RJ, 2013, Vinyl Acetate Monomer (VAM) Genotoxicity Profile: Relevance for 
Carcinogenicity, Crit Rev Toxicol, 43:671706. 
 
3.  European Chemicals Agency, 2008, Summary Risk Assessment Report: Vinyl Acetate 
CASRN 108-05-4. 2008, available at https://echa.europa.eu/documents/10162/a3c24f78-
4c8d-44e9-a424-24ac30c9c8aa.   
 
4.  International Agency for Research on Cancer (IARC), 1995, Vinyl Acetate. In: IARC 
Monographs on the Evaluation of Carcinogenic Risks to Humans: Volume 63, Lyon, 
France: World Health Organization, 443, available at 
http://www.inchem.org/documents/iarc/vol63/vinyl-acetate.html. 
 
5.  Carcinogenic Potency Database, website available at 
https://files.toxplanet.com/cpdb/index.html. 
 
6.  Maltoni C, Ciliberti A, Lefemine G, and Soffritti M, 1997, Results of a Long-Term 
Experimental Study on the Carcinogenicity of Vinyl Acetate Monomer in Mice, Ann N Y 
Acad Sci, 837:20938. 
 
7.  Lijinsky W and Reuber MD, 1983, Chronic Toxicity Studies of Vinyl Acetate in Fischer 
Rats, Toxicol Appl Pharmacol, 68:4353. 
 
8.  Hengstler JG, Bogdanffy MS, et al., 2003, Challenging Dogma: Thresholds for Genotoxic 
Carcinogens? The Case of Vinyl Acetate, Annu Rev Pharmacol Toxicol, 43:485520. 
 
9.  Umeda Y, Matsumoto M, et al., 2004, Carcinogenicity and Chronic Toxicity in Mice and 
Rats Administered Vinyl Acetate Monomer in Drinking Water, J Occup Health, 46:87
99. 
 
10. Organization for Economic Co-operation and Development (OECD), 2009, OECD 453 
Guideline for the Testing of Chemicals: Combined Chronic Toxicity\Carcinogenicity 
Studies.  
 
11. Minardi F, Belpoggi B, et al., 2002, Results of Long-Term Carcinogenicity Bioassay on 
Vinyl Acetate Monomer in Sprague-Dawley Rats, Ann N Y Acad Sci, 982:106122. 
 
12. Bogdanffy MS, Tyler TR, et al., 1994, Chronic Toxicity and Oncogenicity Study with 
Vinyl Acetate in the Rat: In Utero Exposure in Drinking Water, Fundam Appl Toxicol, 
23:206214. 
 
13. Bogdanffy MS, Dreef-van der Meulen HC, Beems RB, Feron VJ, Cascieri TC, Tyler TR, 
Vinegar MB, and Rickard RW, 1994, Chronic Toxicity and Oncogenicity Inhalation 
Study With Vinyl Acetate in the Rat and Mouse, Fundam Appl Toxicol, 23:215229.  
 
142  Contains Nonbinding Recommendations 
14. Carcinogenic Potency Database, website available at 
https://files.toxplanet.com/cpdb/index.html. 
 
15. Nedergaard M, Goldman SA, et al., 1991, Acid-Induced Death in Neurons and Glia, J 
Neurosci, 11:248924897. 
 
16. Robinson DA, Bogdanffy MS, et al., 2022, Histochemical Localisation of 
Carboxylesterase Activity in Rat and Mouse Oral Cavity Mucosa, Toxicology, 180:209
220. 
 
17. U.S. Environmental Protection Agency, 1990, Integrated Risk Information System 
(IRIS): Vinyl Acetate (CASRN 108-05-4), available at 
http://cfpub.epa.gov/ncea/iris/iris_documents/documents/subst/0512_summary.pdf. 
 
143  Contains Nonbinding Recommendations 
NOTE 1 
 
The calculated TD  for 1-chloro-4-nitrobenzene is illustrated below because it was not listed 
50
in the Carcinogenic Potency Database. 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. 
 
Number of  Total 
Ppm  Dose (mg/kg/day)  Positive  Number of 
Animals  Animals 
0  0  3  50 
50  1.9  6  50 
225  9.8  4  50 
1,000  53.8  16  50 
ppm = parts per million; mg = milligram; kg = kilogram. 
 
The TD  is calculated from crude summary data of tumor incidence over background with 
50
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  is the proportion of animals with the specified tumor type for the 
0
control. Converting  and D into a simple linear equation results in the following: 
 
 
0
pp pp
 
ln 1 = D
0
Plotting the results and using the slope to1 reprppesent  results in the following graph for the 
dose response and  = 0.0059302912. 
 
 
 
144  Contains Nonbinding Recommendations 
The TD  can then be calculated as follows. 
50
 
Solving for TD  results in in the following equation. 
50
 
Therefore, the TD  = 0.693 / 0.0059302912 or 116.9 milligrams/kilogram/day. 
50
145  Contains Nonbinding Recommendations 
REFERENCES (FOR NOTE 1) 
 
1.  Matsumoto M, Aiso S, Senoh H, Yamazaki K, Arito H, Nagano K, et al., 2006, 
Carcinogenicity and Chronic Toxicity of para-Chloronitrobenzene in Rats and Mice by 
Two-Year Feeding, J. Environ Pathol Toxicol Oncol, 25:571584. 
 
2.  Gaylor DW and Gold LS, 1995, Quick Estimate of the Regulatory Virtually Safe Dose 
Based on the Maximum Tolerated Dose for Rodent Bioassays, Regul Toxicol Pharmacol, 
22:5763. 
 
3.  Sawyer C, Peto R, Bernstein L, and Pike MC, 1984, Calculation of Carcinogenic Potency 
From Long-Term Animal Carcinogenesis Experiments, Biometrics, 40:2740. 
146  Contains Nonbinding Recommendations 
NOTE 2 
 
The calculated TD  for ethyl bromide is illustrated below since it was decided to use the 
50
same study data but not the TD  calculated by the Carcinogenic Potency Database because 
50
the positive dose response was not statistically significant (see monograph for ethyl bromide).  
 
Number of  Total Number 
ppm  Dose (mg/kg/day)1 
Positive Animals  of Animals 
0  0  8  40 
100  22.9  23  45 
200  45.8  18  46 
400  91.7  21  46 
ppm = parts per million; mg = milligram; kg = kilogram. 
 
A TD  is calculated for each dose separately with the following equation (Ref. 1, 2):  
50
 
Where P is the proportion of animals with the specified tumor type observed at a certain dose 
(D in the equation) and P  is the proportion of animals with the specified tumor type for the 
0
control.  Converting  and D into a simple linear equation results in the following: 
 
 
0
pp pp
 
ln 1 =  D
0
Plotting the results and using the slope 1to repppresent  results in the following graphs for the 
dose response and  = 0.0215055234 for low dose, 0.0059671034 for mid dose, and 
0.0042161616 for the high dose. 
 
Low Dose 
 
 
147  Contains Nonbinding Recommendations 
Mid Dose 
 
 
 
High Dose 
 
 
 
The TD  can then be calculated as follows.  
50
 
Solving for TD  results in in the following equation.  
50
 
148  Contains Nonbinding Recommendations 
 
Therefore, the lowest TD  = 0.693 / 0.0215055234 or 32.2 milligrams/kilogram/day. 
50
149  Contains Nonbinding Recommendations 
REFERENCES (FOR NOTE 2) 
 
1.  Gaylor DW and Gold LS, 1995, Quick Estimate of the Regulatory Virtually Safe Dose 
Based on the Maximum Tolerated Dose for Rodent Bioassays, Regul Toxicol Pharmacol, 
22:5763. 
 
2.  Sawyer C, Peto R, Bernstein L, and Pike MC, Calculation of Carcinogenic Potency From 
Long-Term Animal Carcinogenesis Experiments, Biometrics, 40:2740. 
150  Contains Nonbinding Recommendations 
NOTE 3 
 
For formaldehyde the limit is 215 parts per billion (ppb) or 8 milligrams (mg)/day, whichever 
is lower. As written, this may be misunderstood. That is, the limit of 215 ppb could either be 
interpreted as the concentration of formaldehyde in air (which is the basis of the exposure 
limit), or the concentration of formaldehyde relative to drug substance. The following 
example could clarify how the limit in active pharmaceutical ingredient (API) or drug 
product would be derived: 
 
Example: Albuterol sulfate actuator with formaldehyde as an impurity in the API 
 
  Each actuator of albuterol delivers 90 micrograms (g) of API. 
 
  The API and any impurities will be diluted into air, which is inhaled with each 
actuator. The tidal volume of air is 500 milliliters (mL) for adult males and 400 mL 
for adult females (see Physiology, Tidal Volume, available at StatPearls - NCBI 
Bookshelf (NIH.gov), https://www.ncbi.nlm.nih.gov/books/NBK482502/). The more 
conservative dilution of formaldehyde into air would be for adult females with a 
lower tidal volume. 
 
  Convert the concentration limit of formaldehyde in air (215 ppb) to an absolute 
amount of formaldehyde based on the female tidal volume: 215 ppb formaldehyde = 
0.215 x 30 grams (g)/mole (molecular weight of formaldehyde)/24.45 = 0.263 
mg/cubic meter (m3).  0.263 mg/m3 x 1 m3/1,000 liter (L) x 0.4 L (tidal volume 
women) = 0.105 g formaldehyde. 
 
  Calculate the corresponding API limit: 0.105 g formaldehyde/90 g API = 0.12%. 
 
Example:  Albuterol sulfate actuator with formaldehyde as a drug product impurity 
 
  Each actuator of albuterol delivers 35 mg of drug product. 
 
  The absolute amount of formaldehyde associated with the 215 ppb in air limit will be 
the same, that is, 0.105 g formaldehyde. 
 
  Calculate the corresponding drug product limit: 0.105 g formaldehyde/35 mg drug 
product = 3 parts per million. 
151  Contains Nonbinding Recommendations 
NOTE 4 
 
The calculated TD  for hydrazine is illustrated below since it was not listed in the 
50
Carcinogenic Potency Database. 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 
 
Number of  Total 
ppm  Dose (mg/kg/day)  Positive  Number of 
Animals  Animals 
0  0  1  50 
20  1.28  0  50 
40  2.50  3  50 
80  5.35  6  50 
ppm = parts per million; mg = milligram; kg = kilogram. 
 
The TD  is calculated from crude summary data of tumor incidence over background with 
50
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  is the proportion of animals with the specified tumor type for the 
0
control. Converting  and D into a simple linear equation results in the following:  
 
 
0
pppp
 
ln 1 =  D
0
Plotting the results and using the slope t1o repppresent  results in the following graph for the 
dose response and  = 0.0179164668. 
 
 
 
152  Contains Nonbinding Recommendations 
The TD  can then be calculated as follows. 
50
 
Solving for TD  results in in the following equation. 
50
 
Therefore, the TD  = 0.693 / 0.0179164668 or 38.7 milligram/kilogram/day. 
50
153  Contains Nonbinding Recommendations 
REFERENCES (FOR NOTE 4) 
 
1.  Matsumoto M, Kano H, Suzuki M, Katagiri T, Umeda Y, and Fukushima S, 2016, 
Carcinogenicity and Chronic Toxicity of Hydrazine Monohydrate in Rats and Mice by 
Two-Year Drinking Water Treatment, Regul Toxicol Pharmacol, 76:6373. 
 
2.  Gaylor DW and Gold LS, 1995, Quick Estimate of the Regulatory Virtually Safe Dose 
Based on the Maximum Tolerated Dose for Rodent Bioassays, Regul Toxicol Pharmacol, 
22:5763. 
 
3.  Sawyer C, Peto R, Bernstein L, and Pike MC, 1984, Calculation of Carcinogenic Potency 
From Long-Term Animal Carcinogenesis Experiments, Biometrics, 40:2740. 
154  Contains Nonbinding Recommendations 
NOTE 5 
 
The calculated TD  for methyl chloride is illustrated below because it was not listed in the 
50
Carcinogenic Potency Database. Because the methyl chloride study (Ref. 1, 2) is based on 
inhalation, the inhaled parts per million concentrations need to be converted to dose. 
 
Number of  Total 
ppm  Dose (mg/kg/day)1  Positive  Number of 
Animals  Animals 
0  0  0  67 
50  28  0  61 
225  127  2  57 
1000  566  22  86 
ppm = parts per million; mg = milligram; kg = kilogram; mol = mole. 
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  is calculated from crude summary data of tumor incidence over background with 
50
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  is the proportion of animals with the specified tumor type for the 
0
control. Converting  and D into a simple linear equation results in the following:  
 
 
0
pppp
 
ln 1 =  D
0
Plotting the results and using the slope t1o repppresent  results in the following graph for the 
dose response and  = 0.0005092936.  
 
 
 
155  Contains Nonbinding Recommendations 
The TD  can then be calculated as follows. 
50
 
 
Solving for TD  results in in the following equation. 
50
 
Therefore, the TD50 = 0.693 / 0.0005092936 or 1360.7 milligram/kilogram/day.  
156  Contains Nonbinding Recommendations 
REFERENCES (FOR NOTE 5) 
 
1.  Chemical Industry Institute of Toxicology, 1981, 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. EPA/OTS Doc #878212061, 
NTIS/OTS0205952. 
 
2.  U.S. Environmental Protection Agency, 2001, Toxicological Review of Methyl Chloride 
(CAS No. 74-87-3): In Support of Summary Information on the Integrated Risk 
Information System (IRIS), available at 
https://cfpub.epa.gov/ncea/iris/iris_documents/documents/toxreviews/1003tr.pdf. 
 
3.  Gaylor DW and Gold LS, 1995, Quick Estimate of the Regulatory Virtually Safe Dose 
Based on the Maximum Tolerated Dose for Rodent Bioassays, Regul Toxicol Pharmacol, 
22:5763. 
 
4.  Sawyer C, Peto R, Bernstein L, and Pike MC, 1984, Calculation of Carcinogenic Potency 
From Long-Term Animal Carcinogenesis Experiments, Biometrics, 40:2740. 
 
157