United States
Environmental Protection
1=1 m m Agency
EPA/690/R-13/008F
Final
6-12-2013
Provisional Peer-Reviewed Toxicity Values for
3,3 '-Dimethoxybenzidine
(CASRN 119-90-4)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Jason C. Lambert, PhD, DABT
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
ICF International
9300 Lee Highway
Fairfax, VA 22031
PRIMARY INTERNAL REVIEWERS
Anuradha Mudipalli, MSc, PhD
National Center for Environmental Assessment, Research Triangle Park, NC
Dan D. Petersen, PhD, DABT
National Center for Environmental Assessment, Cincinnati, OH
This document was externally peer reviewed under contract to
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
Questions regarding the contents of this document may be directed to the U.S. EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center (513-569-7300).
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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS iii
BACKGROUND 1
DISCLAIMERS 1
QUESTIONS REGARDING PPRTVS 1
INTRODUCTION 2
REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER) 4
HUMAN STUDIES 9
Oral and Inhalation Exposures 9
ANIMAL STUDIES 10
Oral Exposure 10
Short-term Study 10
Sub chronic-duration Studies 11
Chronic-duration Studies 12
Developmental and Reproduction Studies 15
Inhalation Exposure 16
Sub chronic-duration Studies 16
Chronic-duration Studies 16
Developmental and Reproduction Studies 16
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 16
DERIVATION 01 PROVISIONAL VALUES 23
FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC ORAL
REFERENCE DOSES 23
FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION REFERENCE CONCENTRATIONS 24
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 24
MODE-OF-ACTION (MOA) DISCUSSION 26
Mutagenic Mode of Action (MOA) 26
Key Events 26
Strength, Consistency, Specificity of Association 27
Dose-Response Concordance 27
Temporal Relationships 27
Biological Plausibility and Coherence 27
Early-Life Susceptibility 28
Conclusions 28
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES 28
Derivation of Provisional Oral Slope Factor (p-OSF) 28
Derivation of Provisional Inhalation Unit Risk (p-IUR) 32
APPENDIX A. PROVISIONAL SCREENING VALUES 33
APPENDIX B. DATA TABLES 36
APPENDIX C. BMD MODELING OUTPUTS FOR 3,3'-DIMETHOXYBENZIDINE 47
APPENDIX D. REFERENCES 50
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COMMONLY USED ABBREVIATIONS
BMC
benchmark concentration
BMCL
benchmark concentration lower bound 95% confidence interval
BMD
benchmark dose
BMDL
benchmark dose lower bound 95% confidence interval
HEC
human equivalent concentration
HED
human equivalent dose
IUR
inhalation unit risk
LOAEL
lowest-observed-adverse-effect level
LOAELadj
LOAEL adjusted to continuous exposure duration
LOAELhec
LOAEL adjusted for dosimetric differences across species to a human
NOAEL
no-ob served-adverse-effect level
NOAELadj
NOAEL adjusted to continuous exposure duration
NOAELhec
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
POD
point of departure
p-OSF
provisional oral slope factor
p-RfC
provisional reference concentration (inhalation)
p-RfD
provisional reference dose (oral)
RfC
reference concentration (inhalation)
RfD
reference dose (oral)
UF
uncertainty factor
UFa
animal-to-human uncertainty factor
UFC
composite uncertainty factor
UFd
incomplete-to-complete database uncertainty factor
UFh
interhuman uncertainty factor
UFl
LOAEL-to-NOAEL uncertainty factor
UFS
subchronic-to-chronic uncertainty factor
WOE
weight of evidence
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
3,3'-DIMETHOXYBENZIDINE (CASRN 119-90-4)
BACKGROUND
A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value
derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant
scientific literature using established Agency guidance on human health toxicity value
derivations. All PPRTV assessments receive internal review by a standing panel of National
Center for Environment Assessment (NCEA) scientists and an independent external peer review
by three scientific experts.
The purpose of this document is to provide support for the hazard and dose-response
assessment pertaining to chronic and subchronic exposures to substances of concern, to present
the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to
characterize the overall confidence in these conclusions and toxicity values. It is not intended to
be a comprehensive treatise on the chemical or toxicological nature of this substance.
The PPRTV review process provides needed toxicity values in a quick turnaround
timeframe while maintaining scientific quality. PPRTV assessments are updated approximately
on a 5-year cycle for new data or methodologies that might impact the toxicity values or
characterization of potential for adverse human health effects and are revised as appropriate. It is
important to utilize the PPRTV database (http://hhpprtv.ornl.gov) to obtain the current
information available. When a final Integrated Risk Information System (IRIS) assessment is
made publicly available on the Internet (www.epa.gov/iris). the respective PPRTVs are removed
from the database.
DISCLAIMERS
The PPRTV document provides toxicity values and information about the adverse effects
of the chemical and the evidence on which the value is based, including the strengths and
limitations of the data. All users are advised to review the information provided in this
document to ensure that the PPRTV used is appropriate for the types of exposures and
circumstances at the site in question and the risk management decision that would be supported
by the risk assessment.
Other U.S. Environmental Protection Agency (EPA) programs or external parties who
may choose to use PPRTVs are advised that Superfund resources will not generally be used to
respond to challenges, if any, of PPRTVs used in a context outside of the Superfund program.
QUESTIONS REGARDING PPRTVS
Questions regarding the contents and appropriate use of this PPRTV assessment should
be directed to the EPA Office of Research and Development's National Center for
Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300).
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INTRODUCTION
3,3'-Dimethoxybenzidine is an intermediate in the production of bisazobiphenyl dyes
used for coloring textiles, paper, plastic, rubber, and leather and in the production of
o-dianisidine diisocyanate for use in isocyanate-based adhesives and polyurethane elastomers
(NTP, 1990). The empirical formula for 3,3'-dimethoxybenzidine is C14H16N2O2 (see Figure 1).
A table of physicochemical properties is provided below (see Table 1). In this document,
"statistically significant" denotes ap-value <0.05.
Figure 1. 3,3'-Dimethoxybenzidine Structure
Table 1. Physicochemical Properties of 3,3'-Dimethoxybenzidinea
Property (unit)
Value
Boiling point (°C)
356
Melting point (°C)
137.5
Density (g/cm3)
Not available
Vapor pressure (Pa at 20 °C)
Negligible
pH (unitless)
Not available
Solubility in water (g/100 mL at 18.5 °C)
0.006
Relative vapor density (air =1)
Not available
Molecular weight (g/mol)
244.3
Flash point (°C)
206
Octanol/water partition coefficient (unitless)
1.81
'Values from Into://www.ede.gov/niosh/ipesneng/neng1582.html except for boiling point which was retrieved from
http://chem.sis.itilm.mh.gov/chemidpliis/.
The EPA's Integrated Risk Information System (IRIS) (U.S. EPA, 2011) does not list a
chronic oral reference dose (RfD), a chronic inhalation reference concentration (RfC), or a
cancer assessment for 3,3'-dimethoxybenzidine. Subchronic or chronic RfDs or RfCs for
3,3'-dimethoxybenzidine are not listed in the HEAST (U.S. EPA, 2010) or the Drinking Water
Standards and Health Advisories list (U.S. EPA, 2006). HEAST (U.S. EPA, 2010) reports a
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cancer weight-of evidence (WOE) classification of Group B2 (Probable Human Carcinogen), an
oral slope factor (OSF) of 1.4 x 10 2 (mg/kg-day) and an oral unit risk factor of
_n _ i
4.0 x 10 ((J-g/L) based on increased incidence of forestomach papillomas in hamsters
(Sellakumar et al., 1969). The 1994 CARA list (U.S. EPA, 1994) includes a Health and
Environmental Effects Profile (HEEP) for 3,3'-dimethoxybenzidine reporting a human
carcinogen potency factor (ql*) of 1.41 x 10 2 (mg/kg-day) 1 for oral exposure but does not
include any noncancer toxicity values. No occupational exposure limits for
3,3'-dimethoxybenzidine have been derived by the American Conference of Governmental
Industrial Hygienists (ACGIH, 2009), the National Institute of Occupational Safety and Health
(NIOSH, 2010), or the Occupational Safety and Health Administration (OSHA, 2006). The
International Agency for Research on Cancer (IARC, 2000) has reviewed the carcinogenic
potential of 3,3'-dimethoxybenzidine and placed it in Group 2B, "Possibly carcinogenic to
humans." The toxicity of 3,3'-dimethoxybenzidine has not been reviewed by the Agency for
Toxic Substances and Disease Registry (ATSDR, 2010) or the World Health Organization
(WHO, 2010). 3,3'-Dimethoxybenzidine is classified as "Reasonably Anticipated to be a Human
Carcinogen" based on sufficient data from animal studies in the 12th Report on Carcinogens
(NTP, 2011). No noncancer toxicity values for exposure to 3,3'-dimethoxybenzidine have been
derived by the California Environmental Protection Agency (CalEPA, 2008, 2009). CalEPA
(2009) has prepared a quantitative estimate of carcinogenic potential for
3,3'-dimethoxybenzidine and reports a No Significant Risk Level (NSRL) of 0.15 (J,g/day.
Literature searches were conducted from 1900 through August 2011 for studies relevant
to the derivation of provisional toxicity values for 3,3'-dimethoxybenzidine, CAS No. 119-90-4.
Searches were conducted using EPA's Health and Environmental Research Online (HERO)
database of scientific literature. HERO searches the following databases: AGRICOLA;
American Chemical Society; BioOne; Cochrane Library; DOE: Energy Information
Administration, Information Bridge, and Energy Citations Database; EBSCO: Academic Search
Complete; GeoRef Preview; GPO: Government Printing Office; Informaworld; IngentaConnect;
J-STAGE: Japan Science & Technology; JSTOR: Mathematics & Statistics and Life Sciences;
NSCEP/NEPIS (EPA publications available through the National Service Center for
Environmental Publications [NSCEP] and National Environmental Publications Internet Site
[NEPIS] database); PubMed: MEDLINE and CANCERLIT databases; SAGE; Science Direct;
Scirus; Scitopia; SpringerLink; TOXNET (Toxicology Data Network): ANEUPL, CCRIS,
ChemlDplus, CIS, CRISP, DART, EMIC, EPIDEM, ETICBACK, FEDRIP, GENE-TOX,
HAPAB, HEEP, HMTC, HSDB, IRIS, ITER, LactMed, Multi-Database Search, NIOSH, NTIS,
PESTAB, PPBIB, RISKLINE, TRI; and TSCATS; Virtual Health Library; Web of Science
(searches Current Content database among others); World Health Organization; and Worldwide
Science. The following databases outside of HERO were searched for toxicity values: ACGIH,
ATSDR, CalEPA, EPA IRIS, EPA HEAST, EPA HEEP, EPA OW, EPA TSCATS/TSCATS2,
NIOSH, NTP, OSHA, and RTECS.
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REVIEW OF POTENTIALLY RELEVANT DATA
(CANCER AND NONCANCER)
Table 2 provides information for all of the potentially relevant studies relating to
3,3'-dimethoxybenzidine toxicity. Entries for the principal studies are bolded.
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Table 2. Summary of Potentially Relevant Data for 3,3'-Dimethoxybenzidine (CASRN 119-90-4)
Category
Number of
Male/Female,
Strain, Species,
Study Type, and
Duration
Dosimetryb
Critical Effects
NOAELb'c
LOAELbc
Reference (Comments)
Notes3
Human
1. Oral (mg/kg-day)b
None
2. Inhalation (mg/m3)b
Subchronic
None
Chronic
None
Developmental
None
Reproductive
None
Carcinogenic
438 (sex not
reported),
occupational,
duration not reported
Not reported
A total of 88 cases of
uroepithelial cancer consisting of
67 in bladder; 5 in upper urinary
tract; 16 in bladder and upper
urinary tract
None
Not
reported
Hamasaki et al. (1996);
(abstract) (subjects were
exposed to a mixture of
compounds that included
3,3' -dimethoxybenzidine)
400/0, occupational,
duration not reported
Not reported
A total of 6 workers with bladder
cancer
None
Not
reported
Fruminetal. (1990) (subjects
were exposed to a mixture of
compounds that included
3,3' -dimethoxybenzidine)
585/119,
occupational,
8624 person-years
Not reported
Bladder cancer
None
Not
reported
Ouellet-Hellstrom and Rench
(1996); (subjects were
exposed to a mixture of
compounds that included
3,3' -dimethoxybenzidine)
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Table 2. Summary of Potentially Relevant Data for 3,3'-Dimethoxybenzidine (CASRN 119-90-4)
Category
Number of
Male/Female,
Strain, Species,
Study Type, and
Duration
Dosimetryb
Critical Effects
NOAELb'c
LOAELbc
Reference (Comments)
Notes3
Animal
1. Oral (mg/kg-day)b
Subchronic
(Screening Value)
10/sex, F344N, rat,
drinking water, ad
libitum, 91 days
0,13, 22,39, 70,
120 (male);
0,24, 49,60,103,
187 (female)
Increased relative kidney and
liver weights in males and
females; decreased thymus
weights in males
None
13
NTP (1990); Morgan et al.
(1989)
PS
(noncancer)
Chronic/
Carcinogenicity
3/3 per dose, 14/15
(10 mg/day dose),
F344, rats, oral by
gavage, 52 weeks
0,0.2,0.6, 1.9,
5.6, 18.8, 56.4
(male)
0,0.3,0.9,3.1,
9.4,31.2, 93.6
(female)
Decreased survival time and
body weight; tumors in lower
intestinal tract, skin, ear, and
forestomach (incidence not
statistically significant compared
to control)
None
None
Hadidian (1968) (animals
were followed for 6-months
after exposure concluded)
42, sex and strain
unreported, rat,
orally by gavage,
14 months
0, 33 (first
3 weeks of
study), 16 (over
subsequent
13 months of
study)
Decreased survival time
None
None
Pliss (1963, 1965), as cited
by NTP (1990) (animals
initially received 30 mg
gavage doses 3x/week for the
first 3 weeks of study but due
to poor survival was reduced
to 15 mg for an additional
13 months)
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Table 2. Summary of Potentially Relevant Data for 3,3'-Dimethoxybenzidine (CASRN 119-90-4)
Category
Number of
Male/Female,
Strain, Species,
Study Type, and
Duration
Dosimetryb
Critical Effects
NOAELb'c
LOAELbc
Reference (Comments)
Notes3
Chronic/
Carcinogenicity
45-75/45-75 per
dose, F344N, rat,
drinking water, ad
libitum, 21 months
0,6,12,21
(male);
0,7,14,23
(female)
Increased liver lesions;
hematopoietic cell proliferation
in the spleen; thrombi in the
atrium; histiocytic cellular
infiltration in the lung; Increased
tumors in multiple organs
including: Zymbal gland,
preputial gland, clitoral gland,
skin basal cells, skin squamous
cells, small intestines, large
intestines, oral cavity, liver,
mammary gland; increased
mortality due to tumors
None
None
NTP (1990); Morgan et al.
(1990) (high mortality rate at
all doses tested)
PS (cancer)
10 rats/sex from
control and high
dose group, F344N,
rat, drinking water,
ad libitum, 9 months
0, 21 (male),
23 (female)
Increased kidney, liver weight;
decreased hemoglobin,
erythrocytes, hematocrit, mean
corpuscular hemoglobin
None
21
NTP (1990); Morgan et al.
(1990) (the 9-month time
point was a scheduled interim
sacrifice in the 21-month
study; low- and mid-dose
animals not examined at
interim sacrifice)
120/120 per dose,
BALBc, mouse,
drinking water,
ad libitum,
112 weeks
0, 6, 12, 23, 46,
91, 182 (male);
0, 6, 13,26, 52,
102, 204 (female)
Decreased body weight gain; no
carcinogenic effects
91
182
Schieferstein et al. (1990)
30/30, Syrian
golden, hamster,
feed, ad libitum,
lifetime
0, 57 (male);
0, 54 (female)
No carcinogenic effects
None
None
Saffiotti et al. (1967)
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Table 2. Summary of Potentially Relevant Data for 3,3'-Dimethoxybenzidine (CASRN 119-90-4)
Category
Number of
Male/Female,
Strain, Species,
Study Type, and
Duration
Dosimetryb
Critical Effects
NOAELb'c
LOAELbc
Reference (Comments)
Notes3
Chronic/
Carcinogenicity
Number, sex, and
strain not reported,
hamster, study type
and duration not
reported
171, 571 (male);
161, 536 (female)
Forestomach papillomas
None
None
Sellakumar et al. (1969)
2. Inhalation (mg/m3)b
Subchronic
None
Chronic
None
Developmental
None
Reproductive
None
Carcinogenic
None
aNotes: IRIS = Utilized by IRIS, date of last update; PS = Principal study; NPR = Not peer reviewed.
bDosimetry: Animal doses presented. For all discontinuous exposures, NOAEL and LOAEL values are converted to a continuous (daily) exposure.
°Not reported by the study authors; determined from available data for this document.
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HUMAN STUDIES
Oral and Inhalation Exposures
No information is available regarding oral exposure of humans to
3,3'-dimethoxybenzidine. No studies investigating the effects of subchronic inhalation exposure
to 3,3'-dimethoxybenzidine in humans have been identified. Chronic inhalation exposure to
3,3'-dimethoxybenzidine in humans has been evaluated in occupational studies involving the
production or usage of benzidine and benzidine congeners (Frumin et al., 1990; Hamasaki et al.,
1996; Ouellet-Hellstrom and Rench, 1996). No human studies involving exposure to
3,3'-dimethoxybenzidine alone were identified.
Hamasaki et al. (1996) evaluated a cohort of 438 workers (sex not reported) employed in
a plant producing and using aromatic amines including benzidine sulfate, beta-naphthylamine,
alpha-naphthylamine, and 3,3'-dimethoxybenzidine. The results presented here are as reported
in the abstract because the original publication was only available in Japanese. Among the
438 workers, a total of 88 cases of uroepithelial cancer occurred from 1949 to 1995, resulting in
an incidence rate of 20.1%. The average exposure time of individuals with cancer was
7.40 years. The average latency period was 26.79 years, and the average age of onset was
52.59 years. The duration of exposure of all workers evaluated was not provided. Of the
88 cases, 67 reported tumor sites in the bladder only and another 16 reported tumor sites in the
bladder and upper urinary tract. A total of 28 of the workers with cancer died of uroepithelial
cancer (31.8%). The authors reported survival rates of 87.9%, 74.0%>, 65.9%>, and 56.3%> for
5, 10, 15, and 20 years, respectively.
Frumin et al. (1990) investigated the occurrence of bladder cancer in textile dyeing and
printing workers. A total of 400 male workers were evaluated over a 4-year period using urine
cytology, during which time, 2 workers were diagnosed with bladder cancer. The authors
presented case reports of these two workers along with three other workers that self-reported
bladder cancers and one worker that was not diagnosed with bladder cancer until 2 years after the
screening process. All of the workers evaluated mixed dyes and pigments and applied them to
cloth. As a result, these workers were exposed to a large number of dyes including
3,3'-dimethoxybenzidine, 3,3'-dimethylbenzidine, and benzidine. The duration of exposure of
all workers evaluated was not provided. The average latency period was 23.3 years and ranged
from 16 to 32 years. A total of six workers were diagnosed with bladder cancer. These
6 workers had a mean age of 56.5 years at time of cancer detection; according to the study
authors, this age is 9-14 years less than the mean age at detection of nonoccupational bladder
cancer in men. The study authors concluded that occupational exposure to benzidine dyes and
dyes made from benzidine congeners cause an increased risk of bladder cancer. However, the
authors noted that their screening method and low number of cases did not allow for a statistical
analysis of their results.
Ouellet-Hellstrom and Rench (1996) evaluated a cohort of 704 workers (585 men,
119 women) employed in a plant that produced 3,3'-dichlorobenzidine, 3,3'-dimethylbenzidine,
and 3,3'-dimethoxybenzidine. Plant records were used to identify workers employed at the plant
between June 15, 1965, and December 31, 1989. Workers who may have been exposed to
benzidine were excluded from the study. Plant records were cross-referenced with the
company's medical records, death certificates, and the Connecticut Tumor Registry to identify
cancer cases. In addition, a survey was sent out by mail to all members of the cohort for which a
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current address was available. Additional mailings and telephone calls were used as follow-up
measures for nonrespondents. A total of 8624 person-years of observation were collected from
the cohort. A total of 24 malignant cancer cases were identified, including cancer of the buccal
cavity, bladder cancer, kidney cancer, brain cancer, breast cancer, and testicular cancer. Only
bladder cancer and testicular cancer were statistically significant. Of the three workers
diagnosed with testicular cancer, two were never exposed to the dyes, and the third was only
exposed for 15 days. An eight-fold increase in the risk of bladder cancer was observed in
individuals exposed to the dyes. All of the workers diagnosed with bladder cancer were current
or ex-smokers. The authors concluded that an association exists between exposure to the dyes
and bladder cancer, and that while smoking is known to be related to bladder cancer, it would not
by itself explain such a large increase in bladder cancer incidence. Because workers were likely
exposed to multiple dyes, this study was not able to determine cancer risks from one specific
dye.
ANIMAL STUDIES
Oral Exposure
The effects of oral exposure of animals to 3,3'-dimethoxybenzidine have been evaluated
in short-term (NTP, 1990), subchronic- (Morgan et al., 1989; NTP, 1990) and chronic-duration
(Hadidian et al., 1968; NTP, 1990; Schieferstein et al., 1990; Pliss, 1963, 1965; Saffiotti et al.,
1967; Sellakumar et al., 1969) studies.
Short-term Study
NTP (1990) sponsored a 14-day drinking water study with 3,3'-dimethoxybenzidine
dihydrochloride (purity 98%) in F344N rats. Groups of five male and five female rats were
exposed to 0, 200, 350, 750, 1500, or 4500 ppm for two consecutive weeks. Based on
body-weight data and water consumption data reported in the study, daily doses of
3,3'-dimethoxybenzidine dihydrochloride are estimated as 0, 18, 29, 57, 101, and 127 mg/kg-day
in males and 0, 19, 32, 61, 141, and 214 mg/kg-day in females, respectively. Water and feed
were provided ad libitum. Animals were observed for mortality and clinical signs twice daily.
The study authors recorded body weights before treatment and on Treatment Days 7 (males) or
4 (females) and also on Treatment Day 14. The rats were necropsied, and relative organ weights
for the brain, lungs, heart, liver, kidney, right testis, and thymus were recorded. The study
authors performed comprehensive histopathology on several tissues (including gross lesions,
tissue masses, associated lymph nodes, and 33 organs) on all rats in the 4500 ppm group. In
addition, the spleen, bone marrow (sternum), and thymus were examined histologically in male
rats in the 1500 ppm group, and bone marrow (sternum) was examined histologically in
1500 ppm female rats. This study was peer reviewed and performed in accordance with Good
Laboratory Practice (GLP) regulations.
All rats lived until the end of the study (NTP, 1990). Organ weight results are presented
in Table B.l. Final mean body weights of animals dosed with 4500 ppm were decreased when
compared to initial body weights. Water consumption was decreased in a dose-dependent
manner. Relative liver weights were increased at 200 ppm and at doses of 750 ppm and greater
in males and 1500 ppm and greater in females. No effects on relative liver weights were
observed in males treated with 350 ppm. Relative kidney weights were increased at doses of
350 ppm and greater in males and 1500 ppm and greater in females. The study authors noted
that no microscopic changes were observed in these organs. However, detailed results of the
histopathological examinations were not provided. Increases in relative brain, lung, heart, and
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right testis weights were measured in males treated with 4500 ppm. Relative brain and thymus
weights were increased in females treated with 4500 ppm. Lymphoid depletion was observed in
the spleen of males and females and in the thymus of males at 4500 ppm. Animals dosed with
4500 ppm that lost weight also had bone marrow hypocellularity. Based on increased relative
kidney weights in males, a LOAEL of 350 ppm (average daily dose of 29 mg/kg-day) and a
NOAEL of 200 ppm (average daily dose of 18 mg/kg-day) are established for 2-week oral
exposure to 3,3'-dimethoxybenzidine in rats.
Subchronic-duration Studies
The study by NTP (1990) is selected as the principal study for deriving the screening
subchronic p-RfD. NTP (1990) reported a 13-week oral study in which groups of 10 male and
10 female Fischer 344N rats were administered 0, 170, 330, 630, 1250, or 2500 ppm
3,3'-dimethoxybenzidine dihydrochloride (purity 98%) in drinking water. Results of this study
were also reported by Morgan et al. (1989). Respective corresponding daily doses were
estimated as 0, 13, 22, 39, 70, and 120 mg/kg-day for males and 0, 24, 49, 60, 103, and
187 mg/kg-day for females, respectively (Morgan et al., 1989). Animals were obtained from
Frederick Cancer Research Facility at 4 weeks of age and acclimated to laboratory conditions for
at least 2 weeks prior to study initiation. Food was provided ad libitum, and fresh water was
supplied twice weekly. Animals were observed daily for mortality and clinical signs of toxicity.
Body weights and food consumption were measured once per week. Water consumption was
measured twice per week. At study termination, blood samples were collected from the
retro-orbital sinus of all animals for hematology. At the end of the treatment period, all
surviving animals were sacrificed and necropsied. Selected organs were weighed, and complete
histopathological examinations were performed. This study was peer reviewed and performed in
accordance with GLP guidelines.
All animals survived until the end of the study (Morgan et al., 1989; NTP, 1990). No
signs of clinical toxicity were reported. Water consumption decreased in a dose-dependent
manner (see Table B.2). At 1250 ppm, water consumption was decreased by approximately 33%
in males and 56% in females after 13 weeks of exposure when compared to controls. At
2500 ppm, a 43% decrease in water consumption was observed in males and a 60% decrease in
females when compared to controls. Decreased body weights were noted in males (see
Table B.3) at 1250 (10%) and 2500 ppm (19%) and in females (see Table B.4) at 2500 ppm
(8%>). The study authors reported significant treatment-related increases in relative organ
weights for the liver and kidney in males of all exposure groups (see Table B.3) and the liver at
>630 ppm and the kidney at >330 ppm in females (see Table B.4). Significantly decreased
relative thymus weights were seen in males at all doses; however, this effect was not observed in
females at any dose (data not shown). Statistically significant changes were reported for
leukocyte, lymphocyte, and neutrophil counts in males and neutrophil and erythrocyte counts and
hematocrit values in females. However, the study authors concluded that none of these changes
were reliable based on nonoptimal experimental sampling procedure (e.g., mechanical stress of
harvested cells). Decreased creatinine was seen in all males and females treated with
3,3'-dimethoxybenzidine dihydrochloride (see Table B.5). The study authors concluded that
these changes could be due to loss of muscle mass or the result of assay interference from
bilirubin or hemoglobin. Mean serum triiodothyronine (T3) was decreased in females at
>330 ppm; no significant effects were seen in males. Decreases in mean serum thyroxine (T4)
were seen in all treated males and in females at >330 ppm (see Table B.5). The authors noted
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that because thyrotropin (TSH) remained unchanged, these changes are not a direct effect on the
thyroid gland and are most likely due to competition for the carrier protein for these hormones.
No other treatment-related effects in clinical chemistry or hematology parameters were reported.
Chronic nephropathy and foci of regenerative tubular epithelium were reported in
females at 2500 ppm after 90 days of treatment. Chronic nephropathy was seen in all control and
treated male rats and high-dose (2500 ppm) female rats (Morgan et al., 1989). However,
increased severity of the lesions was seen in 2500 ppm males (1.8) compared to control (1.0) and
1250 ppm males (1.0). Increased pigment (lipofuscin) in the cytoplasm of thyroid follicular cells
was observed in all males and females at 1250 (severity 1.6 and 1.5, respectively) and 2500 ppm
(severity =1.5 and 3.0, respectively) with no effects seen in the thyroid of any control animals
(Morgan et al., 1989). For this study, a LOAEL of 170 ppm (13 mg/kg-day) is determined based
on increased relative organ-weight changes in the liver and kidney, and decreased relative
thymus weight in male rats; a NOAEL is not established.
Chronic-duration Studies
Hadidian et al. (1968) reported the effects of 3,3'-dimethoxybenzidine administered
orally by gavage to male and female F344 rats. Three rats per sex per dose were administered 0,
0.1, 0.3, 1, 3, or 30 mg/day 3,3'-dimethoxybenzidine 5 days per week for 52 weeks. A total of
14 males and 15 females were administered 10 mg/day 3,3'-dimethoxybenzidine by the same
route and procedure. The study authors noted that 10 mg/day was one-third the maximum
tolerated dose of 3,3'-dimethoxybenzidine and felt that using a larger number of animals at this
dose would best reveal the carcinogenic effects of the compound. Based on reference average
body weights for this strain of rat (U.S. EPA, 1988), the estimated duration-adjusted (5/7 days
per week exposure) daily doses are 0, 0.2, 0.6, 1.9, 5.6, 18.8, and 56.4 mg/kg-day for males and
0, 0.3, 0.9, 3.1, 9.4, 31.2, and 93.6 mg/kg-day for females, respectively. The test material was
dissolved in a vehicle consisting of NaCl, sodium carboxymethylcellulose, polysorbate 80, and
benzyl alcohol. The purity of the 3,3'-dimethoxybenzidine used was not reported. Following
administration of the test substance for 52 weeks, the animals were observed for an additional
6 months. Animals were examined for signs of clinical toxicity five times per week during the
treatment. Body weight was measured every other week. Following the 6-month observation
period, all animals were sacrificed. Organ weights for the liver, spleen, kidneys, adrenal glands,
and the pituitary were obtained. Gross necropsies were performed on the liver, spleen, kidneys,
adrenal glands, pituitary, lungs, esophagus, stomach, intestines, bladder, gonads, thyroids, and
mammary glands. Tissues appearing abnormal during gross examination were examined for
histopathology.
Average survival time decreased in male and female rats of all treatment groups
>0.3 mg/day (see Table B.6) (Hadidian et al., 1968). However, statistical analysis was not
reported and could not be conducted because the average survival time of control animals was
not reported. Body weights also decreased in a dose-dependent fashion in female rats (see
Table B.6). No effects on liver weight were seen. Organ weights for other tissues were not
reported. The study authors noted that organ weights for the spleen, adrenal glands, and the
pituitary were rarely affected by treatment. No dose-related trends in nonneoplastic lesions were
seen. For neoplastic lesions, the study authors concluded that treatment with
3,3'-dimethoxybenzidine resulted in intestinal tract adenocarcinomas, skin carcinomas, ear duct
carcinomas, and a tumor of the forestomach (see Table B.7). However, statistical analysis of
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these results indicates that none of the tumors are statistically increased compared to controls.
The small number of animals evaluated precludes the determination of a NOAEL or LOAEL.
Pliss (1963, 1965) administered 3,3'-dimethoxybenzidine (purity unknown) via gavage to
42 rats (sex, strain unknown). Pliss (1963) is a review of benzidine and its derivatives and only
briefly mentions tumors found following exposure to 3,3'-dimethoxybenzidine. Pliss (1965) is
written in a foreign language. Therefore, the summary of these studies is based on information
provided by NTP (1990). Animals were administered 30 mg 3,3'-dimethoxybenzidine via
gavage 3 times per week. Due to poor survival, the dose was reduced to 15 mg after 3 weeks and
administered for 13 months. No further information was available for study protocol, and study
results were limited to survival and positive tumor results seen at terminal sacrifice. Based on
reference body weight of rats of unknown sex and strain (U.S. EPA, 1988), estimated daily doses
are 0, 33 (first 3 weeks of study), and 16 mg/kg-day (over subsequent 13 months). Of the 42 rats
treated with 3,3'-dimethoxybenzidine, only 18 survived until the end of the study. No data
regarding time of mortality were reported. The survival rate of the control animals was not
provided. Of the 18 animals that survived until the end of the study, Zymbal gland tumors were
reported in 2 animals (sex unknown), and 1 animal had an ovarian tumor. The study authors
noted that none of the 50 control animals developed tumors at these sites. Due to the high rate of
mortality seen in treated animals and poor study design/reporting, no LOAEL or NOAEL can be
determined from this study.
NTP (1990) evaluated the effects of 3,3'-dimethoxybenzidine in a 21-month chronic
study in rats. Results of this study are also reported by Morgan et al. (1990). Fischer 344N rats
were obtained from Simonsen Laboratories at 4 weeks of age and acclimated to laboratory
conditions for 14-21 days. Groups of 45-75 male and female Fischer 344N rats were exposed
to 0, 80, 170, or 330 ppm 3,3'-dimethoxybenzidine dihydrochloride (purity 98%) in drinking
water. Corresponding estimated daily doses were 0, 6, 12, and 21 mg/kg-day for males and 0, 7,
14, and 23 mg/kg-day for females, respectively (Morgan et al., 1990). Animals were observed
twice daily for signs of clinical toxicity and weighed once a week for the first 15 weeks and once
a month afterwards. A total of 10 animals per sex from the control and 330 ppm groups only
were selected for interim sacrifice following 9 months of exposure. Hematology, serum
chemistry, and urine analyses were performed during the 9-month interim sacrifice only. Gross
necropsy and histologic examinations were performed on all animals. Organ weights were
obtained during necropsy. This study was peer reviewed and conducted according to GLP
guidelines.
At 9 months, significant treatment-related increases in relative kidney and liver weights
were observed in both male and female rats of the 330 ppm groups (NTP, 1990) (see Table B.8).
In males, decreased hemoglobin, erythrocyte counts, hematocrit, and mean corpuscular
hemoglobin were observed and were indicative of mild anemia. No evidence of renal damage
was seen from the urinalysis. The study authors also reported basophilic and/or eosinophilic foci
of altered cells in the liver (8/10 males and 5/10 females). However, results for control rats were
not reported. Carcinomas of the preputial gland (1/10 rats) and Zymbal gland (2/10 rats) were
observed in males. In one female rat, a carcinoma of the clitoral gland was observed (NTP,
1990). None of these lesions were observed in the control animals during this interim sacrifice.
Based on increased relative organ weights in the liver and kidney, and hematology effects seen in
males, a LOAEL of 330 ppm (21 mg/kg-day) is determined for 9 months of exposure in rats.
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Because animals from the mid- and low-dose groups were not evaluated during the 9-month
interim sacrifice, a NOAEL cannot be established.
Dose-dependent decreases in body weight and water consumption were observed in both
male and female rats after 21 months of exposure (NTP, 1990) (see Table B.9). Clinical signs
reported by the study authors included increased incidence of tissue masses on the head, over the
dorsum, and in the genital area of dosed groups. Significant increased mortality due to tumors
was reported in both males and females at all doses (see Table B.10), resulting in the shortening
of the study from 24 to 21 months. The study authors noted that the decreased survival was
mostly due to the formation of neoplasms of the skin, Zymbal gland, preputial gland (in males),
and clitoral and mammary glands (in females). Tumors first appeared following 32 weeks of
exposure in the Zymbal gland and clitoral gland (in females). Treatment-related increases in
nonneoplastic lesions were observed in the lung, liver, heart, and spleen (see Table B. 11).
Neoplastic lesions were reported in multiple tissues including the Zymbal gland, preputial gland,
clitoral gland, skin basal cells, skin squamous cells, small intestines, large intestines, oral cavity,
liver, and mammary gland (see Table B. 12). NTP (1990) concluded that there was clear
evidence of carcinogenic activity of 3,3'-dimethoxybenzidine in both male and female rats. Due
to the increased rate of mortality seen in all dose groups treated with 3,3'-dimethoxybenzidine,
determination of a NOAEL or a LOAEL is not feasible.
Schieferstein et al. (1990) conducted a 2-year chronic toxicity and carcinogenicity study
in mice. BALBc mice (up to 24/sex/dose group) were given 0, 20, 40, 80, 160, 315, or 630 ppm
3,3'-dimethoxybenzidine dihydrochloride (>99.5% pure) in their drinking water for 112 weeks.
Based on recommended water consumption and reference body weight values (U.S. EPA, 1988),
corresponding daily doses are estimated here at 0, 6, 12, 23, 46, 91, and 182 mg/kg-day in males
and 0, 6, 13, 26, 52, 102, and 204 mg/kg-day in females. Methods of measuring water
consumption, food consumption, or body weight were not reported in the study. Animals were
sacrificed and necropsied on Weeks 13, 26, 39, 52, 78, and 112. Mice that died during study
were also necropsied. Complete histopathological examinations were recorded for all animals.
No treatment-related changes in mortality were observed at any dose (Schieferstein et al.,
1990). Histopathological analysis also revealed no treatment-related effects. Decreased water
consumption was reported in high-dose male and female mice (data not provided), and the study
authors reported that this may have been due to an unpleasant taste and not necessarily reflective
of 3,3'-dimethoxybenzidine toxicity. No data on organ weights were provided in the study.
Decreases in weight gain were noted in high-dose males (10.7%) and females (13.3%). The
study authors noted that the decrease in weight gain may be related to the decreased water
consumption and may not be reflective of 3,3'-dimethoxybenzidine toxicity. However, the
authors also noted that a 10% or greater decrease in body-weight gain can alter normal lifespan
by mechanisms not related to tumor induction. Therefore, a LOAEL of 630 ppm
(182 mg/kg-day) and a NOAEL of 315 ppm (91 mg/kg-day) are identified based on decreased
body-weight gain in male mice.
Saffiotti et al. (1967) investigated the effects of aromatic amines on bladder cancer in
hamsters. Groups of 30 male and 30 female Syrian golden hamsters were administered 0 or
0.1%) (w/w) 3,3'-dimethoxybenzidine (purity unspecified) in the diet, ad libitum, from 8 weeks of
age through the remainder of the lifespan. No further information on testing duration was given;
however, the average lifespan of a hamster is 2.5 years (U.S. EPA, 1988). Based on the
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estimated average chemical intake of 60 mg/week provided by the study authors and average
reference body weights for mature Syrian golden hamsters (U.S. EPA, 1988), daily doses are
estimated at 0 and 57 mg/kg-day for males, and 0 and 54 mg/kg-day for females. Animals were
weighed and evaluated for clinical toxicity every 2 weeks during the study. It is unknown if
clinical chemistry parameters were evaluated by the study authors as this is not discussed in the
study. At the end of the study, histopathological examinations were conducted on all bladders
and most kidneys, livers, and adrenal glands. In addition, any other organs where gross lesions
were observed were also examined microscopically. Because this study is presented as a book
chapter, it is not clear whether or not it is peer reviewed. GLP compliance is also unknown.
Survival rates are not reported (Saffiotti et al., 1967). Data concerning body weights,
clinical toxicity, clinical chemistry, and organ weights are not provided. The study authors
reported that no treatment-related tumors were seen in any organs evaluated except for the
bladder where a small transitional cell carcinoma was seen in a single male that died during
Week 144 of treatment. Histopathological data for groups or individuals were not provided.
Because this study provides limited data and appears to focus mainly on induction of bladder
cancer, determination of a LOAEL or NOAEL is not feasible.
Sellakumar et al. (1969) evaluated the effects of 3,3'-dimethoxybenzidine in hamsters.
No information on strain, sex, husbandry, test duration, or compound purity is given. The
authors of this document noted that the study groups were similar to those reported by
Saffiotti et al. (1967). However, no further explanation of study groups is given. Animals were
treated with 0.3% or 1% (w/w) 3,3'-dimethoxybenzidine in the diet. Assuming the protocol for
this study is similar to the previous study on 3,3'-dimethoxybenzidine conducted by
Saffiotti et al. (1967), daily doses are estimated as 171 and 571 mg/kg-day for males, and 161
and 536 mg/kg-day for females (based on estimated weekly chemical intakes of 180 mg at 0.3%
and 600 mg at 1.0%, and reference body weights for mature Syrian golden hamsters [U.S. EPA,
1988]). Discussion on examination protocol is limited to findings in the bladder, liver, bile duct,
and forestomach. This study is presented as an abstract for a conference proceeding, and no
further study details were found. Therefore, it is unknown if the information is peer reviewed.
GLP compliance is also unknown.
Survival data were not reported (Sellakumar et al., 1969). The study authors reported the
induction of 4 transitional cell bladder carcinomas, liver cell and cholangiomatous tumors
(number not reported), and diffuse chronic intrahepatic obstructing cholangitis (63%) in the
0.3%) group. Results for controls were not provided. Therefore, evaluation of these effects for
significance is not feasible. At 1.0%, 3,3'-dimethoxybenzidine had no effect on the formation of
bladder or liver tumors but caused a 37% increase in forestomach papillomas, compared to 2% in
controls. No information was provided to determine if these effects were seen in males, females,
or both. Due to the limited amount of information provided for this study, it is not feasible to
determine a NOAEL or LOAEL.
Developmental and Reproduction Studies
Gray and Ostby (1993) published a developmental study using two
dimethoxybenzidine-based dyes, Chicago Sky Blue (CSB) and Azoic Diazo Component 48
(ADC) (purities unreported). CSB is a tetrasodium salt of a naphthalene, dimethoxybenzidine
and disulphonate conjugate, whereas ADC's chemical structure is virtually identical to a
dimethoxybenzidine. Female CD-I mice (number not reported) were administered 0 or
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1000 mg/kg-day CSB or ADC orally by gavage in a vehicle of 0.2 mL water on Gestation Days
(GDs) 8-12. Dams were weighed both before and after exposure on GD 7 and GD 13,
respectively. Litters were randomly reduced to seven pups each. Female pups were discarded,
and male pups were weaned on Day 30 and necropsied on Days 46-47 and 187-190. Body,
right testis, cauda epididymis, and seminal vesicle weights were reported. Cauda epididymal
sperm counts and testicular sperm head counts were measured. Histopathological examinations
were conducted on the testes. Treatment with ADC resulted in a 4.0 to 1.2 g (p < 0.05) decrease
in maternal-weight gain during the treatment period. Treatment with CSB yielded no change in
maternal parameters. No significant treatment-related effects on the development of male mice
were observed after treatment with either CSB or ADC. Based on the decrease in
maternal-weight gain, a maternal LOAEL of 1000 mg/kg-day is established; no maternal
NOAEL is determined. No developmental LOAEL is established. The developmental NOAEL
for this study is 1000 mg/kg-day.
Inhalation Exposure
Subchronic-duration Studies
No studies could be located regarding the effects of subchronic inhalation exposure of
animals to 3,3'-dimethoxybenzidine.
Chronic-duration Studies
No studies could be located regarding the effects of chronic inhalation exposure of
animals to 3,3'-dimethoxybenzidine.
Developmental and Reproduction Studies
No studies could be located regarding the effects of inhaled 3,3'-dimethoxybenzidine on
reproduction or fetal development.
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)
Little information on the toxicokinetics of 3,3'-dimethoxybenzidine is available.
3,3'-Dimethoxybenzidine has been detected in the urine of workers following occupational
exposure (IARC, 1974). Rodgers et al. (1983) reported that 3,3'-dimethoxybenzidine was
rapidly metabolized by rats after intravenous administration; specifically, 30 minutes after an
intravenous injection of 14C-3,3'-dimethoxybenzidine, <2% of the bolus dose was recovered as
parent compound from the exposed animal. Three days after oral exposure, approximately 85%
of the administered 14C-3,3'-dimethoxybenzidine dose was excreted in the feces or urine with
greater than 90% of the excreted radiolabel in the form of metabolites (Rodgers et al., 1983).
Although a full metabolic profile has not been established for 3,3'-dimethoxybenzidine, GC/MS
analyses indicated a multitude of different phase II conjugates including/V-acetylated,
O-demethylated, hydroxylated, and glucuronidated species (Rodgers et al., 1983).
Table 3 summarizes the studies examining genotoxicity (e.g., clastogenicity,
mutagenicity) of 3,3'-dimethoxybenzidine. Anderson and Styles (1978), Chung et al. (2000),
Haworth et al. (1983), Krishna et al. (1986), Martin and Kennelly (1981), Probst et al. (1981),
and Messerly et al. (1987) indicate the mutagenicity of 3,3'-dimethoxybenzidine in Salmonella
typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 when metabolically
activated. Makena and Chung (2007) also found dimethoxybenzidine to be mutagenic in
Salmonella strain TA102 with metabolic activation. De France et al. (1986), Gregory et al.
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(1981), and Prival et al. (1984) investigated the mutagenic effects of certain
dimethoxybenzidine-based dyes. De France et al. (1986) found no significant results while
Gregory et al. (1981) and Prival et al. (1984) concluded that these dyes are mutagenic when
chemically-reduced to release benzidine. E. coli strains W3110 and P3478 were exposed to
3,3'-dimethoxybenzidine by Fluck et al. (1976), but the study yielded inconclusive results.
Martelli et al. (2000) conducted a study examining the effects of 3,3'-dimethoxybenzidine
on rat and human hepatocytes, as well as human urinary bladder cells in vitro and rat urinary
bladder cells in vivo. This study revealed dose-dependent DNA fragmentation and increased
frequencies of micronucleated cells in both rat and human hepatocytes, as well as increased
DNA damage to urinary bladder cells in vitro and in vivo. Galloway et al. (1987, 1985) provided
evidence of sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary
cells with and without metabolic activation after exposure to 3,3'-dimethoxybenzidine
dihydrochloride. In a study involving Drosophila melanogaster conducted by Yoon et al.
(1985), no sex-linked mutagenic effects were observed after exposure to
3,3'-dimethoxybenzidine. In an NIH 3T3 transfection assay, Reynolds et al. (1990) analyzed
several benign and malignant tumors from control and treated rats from the carcinogenic study
conducted by NTP (1990). Reynolds et al. (1990) reported a high percentage of
dimethoxybenzidine-induced tumors containing activated H-ras and N-ras oncogenes, compared
to low percentages of spontaneously occurring tumors in control rats, indicating that the
increased tumor incidence of treated rats was directly related to the mutagenicity of the chemical.
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Table 3. Other Studies
Test
Materials & Methods
Results
Conclusions
References
Genotoxicity
Salmonella strains TA98, TA100, TA1535
and TA1538 were exposed to 4, 20, 100, 500,
or 2500 ng/plate 3,3'-dimethoxybenzidine in
an Ames assay. Cultures were evaluated for
mutagenic activity with metabolic activation
by Aroclor 1254-induced rat liver S9.
Authors reported positive results for all
strains.
These results indicate that
3,3'-dimethoxybenzidine is
mutagenic in Salmonella strains
TA98, TA100, TA1535, and
TA1538 with metabolic
activation.
Anderson and Styles
(1978)
Genotoxicity
Salmonella typhimurium strains TA98 and
TA100 were exposed to 3, 10, 30, 100, 300,
or 1000 ng/plate 3,3'-dimethoxybenzidine in
an Ames assay. Resulting cultures were
examined for mutagenicity with and without
Aroclor 1254-induced rat liver S9.
Authors reported positive results in both
strains with metabolic activation.
These results suggest
mutagenicity of
3,3'-dimethoxybenzidine in
strains TA98 and TA100 with
metabolic activation.
Chung et al. (2000)
Genotoxicity
Salmonella strains TA98, TA100, TA 1535,
and TA1537 were exposed to
0-1000 ng/plate (concentrations vary over 3
laboratory test locations)
3,3'-dimethoxybenzidine in an Ames assay as
part of an evaluation of 250 chemicals.
Cultures were evaluated for mutagenic
activity without metabolic activation, and
with Aroclor 1254-induced rat and hamster
liver S9 activation.
Authors reported positive results at all
three testing facilities but did not report
findings regarding specific strains, dose
levels, state of metabolic activation, nor
did they present statistical analysis.
These results suggest
mutagenicity of
3,3'-dimethoxybenzidine to
various strains of Salmonella
typhimurium.
Haworth et al. (1983)
Genotoxicity
Salmonella strains SV50 and TA98 were
exposed to 0.03, 0.10, 0.30, or 1 mg/plate
3,3'-dimethoxybenzidine in an Ames assay
and a complete azo dye protocol experiment.
Cultures were evaluated for mutagenicity
without metabolic activation, and with
activation by hamster liver S9 and Aroclor
1254-induced rat liver S9 fraction.
Authors reported a positive result in
strain TA98 with rat liver S9 fraction
and negative results with and without
metabolic activation in SV50 for the
Ames assay. Statistically significant
positive results were reported in strain
SV50 for the azo dye protocol
experiment, but the number of
revertants/plate did not double.
These results suggest
mutagenicity of
3,3'-dimethoxybenzidine in strain
TA98 with metabolic activation.
Krishna et al. (1986)
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Table 3. Other Studies
Test
Materials & Methods
Results
Conclusions
References
Genotoxicity
Salmonella strains TA98 and TA1538 were
exposed to 20, 100, 500, or 2500 ng/plate
3,3'-dimethoxybenzidine in an Ames assay
examining mutagenicity of azo dyes.
Cultures were evaluated for mutagenicity with
sodium phenobarbitone-induced rat liver S9.
Authors reported statistically significant
positive results in both strains with
metabolic activation by rat liver S9.
These results suggest
mutagenicity of
3,3'-dimethoxybenzidine in
strains TA98 and TA1538 with
metabolic activation.
Martin and Kennelly
(1981)
Genotoxicity
Salmonella strain TA98 was exposed to
1.0 |imo 1/plate while strain TA100 was
exposed to 0.5 |imo 1/p late
3,3'-dimethoxybenzidine. Cultures were
evaluated for mutagenicity with and without
Aroclor 1254-induced male Sprague-Dawley
rat liver S9 fraction.
Authors reported negative results in both
strains without metabolic activation, and
positive results in both strains with
activation.
These results indicate that
3,3'-dimethoxybenzidine is
mutagenic with exogenous
metabolic activation in strains
TA98 and TA100.
Messerly et al. (1987)
Genotoxicity
Salmonella strains TA98 and TA100 were
exposed to dihydrochloride salt of
3,3'-dimethoxybenzidine in an Ames assay
examining the mutagenic effects of
benzidine-based dyes.
Results were positive in both strains
when dyes were first reduced with
sodium dithionate.
3,3' -dimethoxybenzidine
dihydrochloride is mutagenic in
strains TA98 and TA100 when
reduced to release benzidine.
Gregory et al. (1981), as
cited by NTP (1990)
Genotoxicity
Salmonella strain TA98 was exposed to
0-1000 nmol/plate (exact concentrations
unreported) 3,3'-dimethoxybenzidine and an
unreported number of hydrazone dyes in
which it is incorporated. Cultures were
evaluated for mutagenic activity with
metabolic activation by hamster liver S9.
Authors reported no significant
mutagenic effects of
3,3'-dimethoxybenzidine or its
corresponding hydrazone dyes.
Dyes of the hydrazone class
containing
3,3'-dimethoxybenzidine are not
considered mutagenic because of
their resistance to enzymatic
reduction.
De France et al. (1986)
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Table 3. Other Studies
Test
Materials & Methods
Results
Conclusions
References
Genotoxicity
Salmonella strain TA98 was exposed to 0,
0.1, 0.3, or 1.0 nmol/plate
3,3'-dimethoxybenzidine dihydrochloride and
3 monoazo dyes incorporating it in an Ames
assay modified to include preincubation with
flavin mononucleotide (FMN). Cultures were
evaluated for mutagenicity with hamster liver
S9 fraction.
Authors reported positive results for 2 of
the 3 o-dianisidine dyes containing
3,3'-dimethoxybenzidine in the presence
of FMN.
Any soluble compound that can
be reduced to release free
3,3' -dimethoxybenzidine
considered mutagenic under the
conditions of this assay.
Prival et al. (1984)
Genotoxicity
Salmonella strain TA102 was exposed to 5,
10, 50, or 100 ng/plate
3,3'-dimethoxybenzidine in a preincubation
Ames assay and evaluated for mutagenicity
with and without activation by Aroclor
1254-induced rat liver S9 fraction.
Authors reported strongly positive
results with rat liver S9, and negative
results without rat liver S9.
These results suggest
mutagenicity of
3,3'-dimethoxybenzidine in
Salmonella strain TA102 with
metabolic activation.
Makena and Chung (2007)
Genotoxicity
Salmonella strains C3076, D3052, G46,
TA98, TA100, TA1535, TA1537, and
TA1538, and it. coli strains WP1 and WP2
were exposed to unreported concentrations of
3,3'-dimethoxybenzidine in an Ames assay.
Cultures were evaluated for mutagenic
activity with and without Aroclor
1254-induced liver S9 fraction. Authors also
conducted an autoradiographic assay for
unscheduled DNA synthesis (UDS).
For the Ames assay, authors reported
positive results in strains TA100, TA98,
and TA1538 with metabolic activation,
and negative results in these strains
without activation and in all other
strains. Authors also reported positive
results for the hepatocyte UDS test.
These results suggest
mutagenicity of
3,3'-dimethoxybenzidine in
strains TA100, TA98, and
TA1538 with metabolic
activation, which can also be
detected by a test for UDS.
Probst et al. (1981)
Genotoxicity
E. coli strains W3110 and P3478 were
exposed to 500 ng/plate
3,3'-dimethoxybenzidine using a rapid
screening technique. Cultures were evaluated
for mutagenic effects without metabolic
activation.
Test results were negative for both
strains.
Insolubility of the test substance
could have prevented it from
diffusing through the agar and
reaching the indicator organism,
resulting in inconclusive results.
Fluck et al. (1976)
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Table 3. Other Studies
Test
Materials & Methods
Results
Conclusions
References
Genotoxicity
Male Sprague-Dawley rat hepatocytes were
exposed to 0, 56, 100, or 180 (imol/plat
3,3'-dimethoxybenzidine for 20 hours in a
DNA damage/alkaline elution assay.
Authors reported a dose-dependent
increase in frequency of DNA
single-strand breaks and/or alkali-labile
sites (DNA elution rate).
Exposure of rat hepatocytes to
3,3'-dimethoxybenzidine causes
DNA fragmentation in rat
hepatocytes in a dose-dependent
manner.
Martelli et al. (2000)
Genotoxicity
Human hepatocytes taken from 2 donors
(1 male, 1 female) were exposed to 0, 56
(male hepatocytes only), 100, or
180 (imol/plate 3,3'-dimethoxybenzidine for
20 hours in a DNA damage assay.
Authors reported a dose-dependent
increase in DNA elution rate in primary
cultures.
Exposure of human hepatocytes
to 3,3'-dimethoxybenzidine
causes DNA fragmentation in a
dose-dependent manner.
Martelli et al. (2000)
Genotoxicity
Human urinary bladder cells taken from
5 donors (4 male, 1 female) were exposed to
0, 100 (3 of 5 donors), or 180 (imol/plate
3,3'-dimethoxybenzidine for 20 hours and
evaluated in a Comet assay.
Authors reported increased nuclear
DNA damage in all bladder cells
exposed to 3,3'-dimethoxybenzidine.
3,3'-dimethoxybenzidine causes
increased damage to nuclear
DNA in human bladder cells.
Martelli et al. (2000)
Genotoxicity
Male Sprague-Dawley rat hepatocytes were
exposed to 56, 100, or 180 (imol/plate
3,3'-dimethoxybenzidine for 48 hours in a
micronucleus assay.
Authors reported increased frequencies
of micronucleated cells at the highest
dose level in 1 of 3 experiments. Pooled
data showed a dose-dependent increase
in micronucleated cell frequency with
significance reported at 100 and
180 (imol.
Exposure of rat hepatocytes to
3,3'-dimethoxybenzidine may
cause increased frequencies of
micronucleated cells.
Martelli et al. (2000)
Genotoxicity
Male Sprague-Dawley rats were administered
960 mg/kg of 3,3'-dimethoxybenzidine in a
single treatment by gastric intubation.
Distilled water (0.01 mg/g body weight) was
used as the vehicle with 0.5%
carboxymethylcellulose as a suspending
agent. Rats were sacrificed 4 hours after
exposure. Liver and urinary bladder cells
were removed and evaluated for DNA
damage.
Authors reported no clinical signs of
toxicity in any of the rats. DNA damage
was observed in urinary bladder cells
collected from all animals in the form of
migration of the DNA from the urinary
bladder mucosa. Authors reported no
DNA damage in liver cells.
Exposure of rats to
3,3'-dimethoxybenzidine results
in increased DNA damage in the
urinary bladder mucosa but not in
the liver.
Martelli et al. (2000)
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Table 3. Other Studies
Test
Materials & Methods
Results
Conclusions
References
Genotoxicity
Chinese hamster ovary cells were exposed to
various unreported doses of
3,3'-dimethoxybenzidine dihydrochloride and
evaluated for sister chromatid exchanges, and
chromosomal aberrations.
In 1985, authors reported positive
evidence of sister chromatid exchanges
with and without metabolic activation,
and negative results for chromosomal
aberrations with and without activation.
A reanalysis of the chromosomal
aberration data in 1987 revealed a
weakly positive result without metabolic
activation, and a positive result with
activation.
These results indicate induction
of chromosomal aberrations and
sister chromatid exchanges by
3,3'-dimethoxybenzidine in
Chinese hamster ovary cells both
with and without metabolic
activation.
Galloway et al. (1985),
Galloway et al. (1987)
Genotoxicity
Adult male Drosophila melanogaster were
exposed to 3,3'-dimethoxybenzidine by
feeding (100 ppm) or injection (200 ppm) and
evaluated for the induction of sex-linked
recessive lethal.
Negative for sex-linked mutations
induced by injection or feeding.
Results indicate that
3,3'-dimethoxybenzidine does
not cause sex-linked mutations in
adult male Drosophila
melanogaster.
Yoonetal. (1985)
Genotoxicity
Benign and malignant tumors were obtained
from control and treated rats in the NTP
(1990) study and evaluated for the presence of
activated oncogenes in a NIH 3T3 DNA
transfection assay.
Tumors of rats treated with
3,3'-dimethoxybenzidine contained a
higher percentage of activated H-ras and
N-ras oncogenes than the single
spontaneous tumor from a control rat.
Results suggest that
3,3'-dimethoxybenzidine and
other benzidine derived
compounds cause point mutations
in the ras gene family in rats.
Reynolds et al. (1990)
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DERIVATION OF PROVISIONAL VALUES
Table 4 presents a summary of noncancer reference values. Table 5 presents a summary
of cancer values. The cancer toxicity value was converted to human equivalent dose (HED)
units, and the conversion process is presented in the section on derivation of provisional cancer
potency values. IRIS data are indicated in the table if available.
Table 4. Summary of Reference Values for 3,3'-Dimethoxybenzidine (CASRN 119-90-4)
Toxicity Type
(Units)
Species/
Sex
Critical Effect
/>-Reference
Value
POD
Method
POD
UFC
Principal Study
Subchronic p-RFD
(mg/kg-day)
Rat/M
Increased relative
liver weight
1 x 1(T3
LOAEL
13
10,000
NTP (1990)
Chronic p-RfD
(mg/kg-day)
None
Subchronic p-RfC
(mg/m3)
None
Chronic p-RfC
(mg/m3)
None
"Oral Screening value provided in Appendix A.
Table 5. Summary of Cancer Values for 3,3'-Dimethoxybenzidine (CASRN 119-90-4)
Toxicity Type
Species/
Sex
Tumor Type
Cancer Value
Principal
Study
p-OSF
Rat/M
Combined tumor types
1.6 (mg/kg-day) 1
NTP (1990)
p-IUR
None
None
None
None
FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC ORAL
REFERENCE DOSES
It is inappropriate to derive a provisional subchronic or chronic p-RfD for
3,3'-dimethoxybenzidine. No quantitative human studies examining the effects of subchronic or
chronic oral exposure to 3,3'-dimethoxybenzidine alone have been identified. The available
human studies involve occupational exposure to a mixture of compounds including
3,3'-dimethoxybenzidine. In animals, useful dose-response data for nonneoplastic effects
following subchronic or chronic exposure are limited to the NTP (1990) study of
3,3'-dimethoxybenzidine dihydrochloride in rats following 13 weeks or 21 months of exposure,
respectively. After 13-weeks of 3,3'-dimethoxybenzidine exposure, male and female rats
exhibited significant changes in relative organ weights and hematological/serum chemistry
parameters, as well as chronic nephropathy and accumulation of pigment in follicular cells of the
thyroid. However, as a function of dose, changes in relative organ weight were the most
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sensitive effects observed. Compared to control, significantly increased relative liver weight and
decreased relative thyroid weight were observed in male rats at the lowest
3,3'-dimethoxybenzidine exposure dose. A subchronic p-RfD cannot be confidently derived here
due to the high level of uncertainty associated with the lack of reliable study data; however a
"screening level" value for subchronic oral exposure is provided in Appendix A.
As previously discussed, the NTP (1990) chronic study was terminated at 21 months
because of significantly decreased survival at all doses tested, primarily due to extensive
neoplastic formation. Treatment-related increases in tumors were seen in the liver, small
intestines, large intestines, Zymbal gland, preputial gland, oral cavity, and skin. For
nonneoplastic lesions, dose-dependent increases were seen in the liver, spleen, heart, and lungs.
Because of the exceedingly high rates of mortality and neoplastic effects at all doses, a NOAEL
for nonneoplastic effects is not identified suggesting that a threshold for nonneoplastic effects
may occur at a dose much lower than those tested. As such, a chronic p-RfD cannot be derived.
In addition, a "screening level" value for chronic oral exposure cannot be supported.
FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION REFERENCE CONCENTRATIONS
Due to a complete lack of exposure-response data for the inhalation route in any species,
it is inappropriate to derive a subchronic or chronic p-RfC for 3,3'-dimethoxybenzidine.
Derivation of "screening values" is also precluded.
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR
Table 6 identifies the cancer WOE descriptor for 3,3'-dimethoxybenzidine.
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Table 6. Cancer WOE Descriptor for 3,3'-Dimethoxybenzidine
Possible WOE Descriptor
Designation
Route of Entry (Oral,
Inhalation, or Both)
Comments
"Carcinogenic to Humans "
Not selected
N/A
No human cancer studies involving exposure to 3,3'-dimethoxybenzidine alone are
available.
"Likely to Be Carcinogenic to
Humans"
Selected
Oral administration by
drinking water
Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), the available
evidence of carcinogenicity in rats orally exposed to 3,3'-dimethoxybenzidine supports
the "Likely to Be Carcinogenic to Humans" descriptor. There are limited available
human data; what human data are available comes from studies or reports of human
exposure to mixtures that included 3,3'-dimethoxybenzidine. NTP (1990) reported
treatment-related increases in a number of tumor types located in multiple tissues
including the Zymbal gland, preputial gland, clitoral gland, skin basal cells, skin
squamous cells, small intestines, large intestines, oral cavity, liver, and mammary
gland in rats exposed to 3,3'-dimethoxybenzidine orally by drinking water for
21 months (see Table B.12). Tumors were also reported at the 9-month interim
sacrifice. The observation of tumors in multiple animal tissues following a short
latency period, and significant evidence of mutagenicity and clastogenicity in several
experimental cell systems, including human, is suggestive of a mutagenic carcinogen.
In addition, 3,3'-dimethoxybenzidine has been classified as '•'•Reasonably Accepted to be
a Human Carcinogen" by the 12th Report on Carcinogens (NTP, 2011). Studies
evaluating the carcinogenic potential of inhaled 3.3'-dimethoxybenzidine in animals
were not identified. Occupational studies indicate increased incidences of bladder
cancer in workers exposed to 3,3'-dimethoxybenzidine (Frumin et al., 1990; Hamasaki
et al., 1996; Ouellet-Hellstrom and Rench, 1996). However, these workers were
employed in textile dyeing and printing facilities and were exposed to other
compounds as well. No studies involving exposure to 3,3'-dimethoxybenzidine alone
were identified.
"Suggestive Evidence of
Carcinogenic Potential"
Not selected
N/A
The evidence from human and animal data is more than suggestive of carcinogenicity,
which raises a concern for carcinogenic effects but is judged sufficient for a stronger
conclusion.
"Inadequate Information to Assess
Carcinogenic Potential"
Not selected
N/A
Available adequate information exists to assess carcinogenic potential.
"Not Likely to Be Carcinogenic to
Humans "
Not selected
N/A
No strong evidence of noncarcinogenicity in humans or animals is available.
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MODE-OF-ACTION (MOA) DISCUSSION
The Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) define mode of
action (MOA) as "a sequence of key events and processes, starting with interaction of an agent
with a cell, proceeding through operational and anatomical changes, and resulting in cancer
formation". Examples of possible modes of carcinogenic action for any given chemical include
"mutagenicity, mitogenensis, inhibition of cell death, cytotoxicity with reparative cell
proliferation, and immunologic suppression".
Human studies involving exposure to 3,3'-dimethoxybenzidine alone are not available.
However, occupational studies involving the exposure of 3,3'-dimethoxybenzidine
simultaneously with benzidine or benzidine congeners indicate that 3,3'-dimethoxybenzidine
may cause tumors in the bladder. In animals, the available evidence suggests that tumors
observed following oral exposure to 3,3'-dimethoxybenzidine arise from genetic mechanisms
(e.g., oncogene activation; see "Key Events" section below). In rats, tumors have been reported
in a number of tissues including the Zymbal gland, preputial gland, clitoral gland, skin, small
intestines, large intestines, oral cavity, liver, and mammary gland. Based on the weight of the
evidence, it is determined that 3,3'-dimethoxybenzidine is carcinogenic by a mutagenic MOA.
Mutagenic Mode of Action (MOA)
Key Events
For 3,3'-dimethoxybenzidine, the proposed MOA involves the occurrence of a number of
key events. These include (1) metabolic activation of parent compound to reactive intermediates
that bind covalently to DNA, (2) genetic alteration of oncogenes including Ras, and (3) tumor
formation following proliferation of initiated cells. Reynolds et al. (1990) provided data to
support this MOA. The authors evaluated a wide range of neoplasms formed in the rat following
exposure to 3,3'-dimethoxybenzidine and found codon-specific mutations in the H-ras and N-ras
oncogenes with the large majority found in H-ras. Additionally, an evaluation of malignant and
benign tumors from rats treated with 3,3'-dimethoxybenzidine showed that 62% contained
activated H-ras or N-ras genes compared with detection of the activated oncogenes in
1/38 spontaneous tumors from control rats (Reynolds et al., 1990). The increased incidence of
activated Ras oncogenes coupled with mutational specificity at codons 13 and 61 of H-ras
suggest that the increased incidence of both benign and malignant tumors observed in rats
exposed to 3,3'-dimethoxybenzidine is related to its mutagenic effects.
Support for a mutagenic MOA is also provided by in vitro tests as described in Table 3.
In a number of Salmonella strains, 3,3'-dimethoxybenzidine was shown to cause mutagenicity
following metabolic activation. Positive results for clastogenicity/mutagenicity (sister chromatid
exchanges, micronucleated cells, DNA damage, and chromosomal aberrations) were seen in
mammalian cells both with and without metabolic activation. In vivo data in rats indicated an
increase in DNA damage to urinary bladder cells.
Evidence also exists for a mutagenic MOA for the structurally similar compound
benzidine and a number of its metabolites (Morgan et al., 1994). Morgan et al. (1994) also
indicated that a number of metabolites of benzidine are more mutagenic than the parent
compound. Mono- and diacetylated metabolites were indicated to be about 10 times as
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mutagenic as benzidine while A-hydroxy-A',A"-di acetyl benzidine glucoronide was about
100 times as mutagenic as benzidine, following incubation with P-glucoronidase to release the
hydroxylated diacetylamine.
Strength, Consistency, Specificity of Association
Reynolds et al. (1990) evaluated a large number of tumor types from animals treated with
3,3'-dimethoxybenzidine (NTP, 1990) or its derivative dye, C.I. Direct Blue 15 (NTP, 1992), and
found that the majority contained codon-specific mutations in the ras oncogene. The role of the
ras oncogene in the carcinogenic effects of 3,3'-dimethoxybenzidine is supported by the high
incidence of the codon-specific mutations (19/21 tumors with an activated Ras oncogene) and
the high incidence of ras gene activation (21/34) in tumors from treated animals as compared to
the low incidence of oncogene activation in spontaneous tumors obtained from control animals
(1/38). Reynolds et al. (1990) reported similar findings in tumors obtained from rats treated with
3,3'-dimethylbenzidine. In tumors from treated animals, Ras gene activation was seen in
13/16 tumors and codon-specific mutations in 12/13 tumors with activated Ras oncogene.
Dose-Response Concordance
Data to evaluate the dose-response concordance between mutagenesis and tumor
formation following exposure to 3,3'-dimethoxybenzidine are unavailable. No data indicating
the dose distribution of mutations or activated oncogenes in the tumors evaluated by
Reynolds et al. (1990) were provided.
Temporal Relationships
For 3,3'-dimethoxybenzidine, the temporal relationship between mutagenesis and tumor
formation cannot be assessed at this time. Reynolds et al. (1990) evaluated tumors collected
from rats exposed to 3,3'-dimethoxybenzidine for types of mutations. However, data on the
incidence or types of mutations formed prior to the generation of neoplasms in these tissues are
not available.
Biological Plausibility and Coherence
Reynolds et al. (1990) provides data supporting the biological plausibility of a mutagenic
MO A for 3,3 '-dimethoxybenzi dine. In tumors from rats treated with 3,3'-dimethoxybenzidine,
point mutations were detected at codons 12, 13, and 61 and shown to lead to activation of Ras
oncogenes. Similar findings were seen in the structurally similar compound
3,3'-dimethylbenzidine (Reynolds et al., 1990). Combined with a lack of oncogene activation in
tumors taken from control animals, and the large body of in vitro evidence indicating
clastogenicity/mutagenicity, these data suggest a mutagenic MO A for 3,3'-dimethoxybenzi dine.
An evaluation of the results from chronic studies in rats exposed to
3,3'-dimethoxybenzidine provides additional evidence of an association between mutagenesis
and tumor formation (NTP, 1990). After only 9 months of exposure, carcinomas of the
preputial, clitoral, and Zymbal glands were seen in treated rats but not in controls, although the
statistical significance of these neoplasias is not clear. Following 21 months of exposure to
3,3'-dimethoxybenzidine, a number of rare tumors were reported in rats including those found in
the intestinal tract, Zymbal gland, skin, and oral cavity (NTP, 1990). These data are supportive
of a mutagenic MO A, because most mutagenic compounds are associated with multiple, unusual
tumor sites and a short latency period for tumorigenesis (NTP, 1990).
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Early-Life Susceptibility
An increased early-life susceptibility is assumed in individuals exposed to carcinogens
with a mutagenic MOA (U.S. EPA, 2005b). For 3,3'-dimethoxybenzidine, sufficient data are not
available to develop separate risk estimates for childhood exposure because no information
evaluating tumor formation during early life after exposure to 3,3'-dimethoxybenzidine has been
reported.
Conclusions
The weight of evidence (WOE) for 3,3'-dimethoxybenzidine tumorigenicity supports a
mutagenic MOA. Data from a battery of in vitro studies in both bacteria and eukaryotic cells
show that exposure to 3,3'-dimethoxybenzidine causes clastogenic/mutagenic effects (see
Table 3). Formation of codon-specific mutations in Ras oncogenes in tumors taken from rats
exposed to 3,3'-dimethoxybenzidine was also seen while only one tumor from control rats
contained an activated Ras oncogene (Reynolds et al., 1990). Lastly, the reporting of rare tumors
at multiple sites, along with the short latency period before tumor formation provide further
support for a mutagenic MOA for 3,3'-dimethoxybenzidine (NTP, 1990). Because a mutagenic
MOA for the carcinogenic effects of 3,3'-dimethoxybenzidine is proposed, a linear approach is
used to extrapolate from the POD to determine the p-OSF (U.S. EPA, 2005b). No data are
available to develop estimates of risk from early-life exposure to 3,3'-dimethoxybenzidine.
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
Derivation of Provisional Oral Slope Factor (p-OSF)
The 21-month study by NTP (1990) is selected as the principal study. The cancer
endpoint is the incidence of combined primary tumor types in male rats; combined tumor data in
female rats were also evaluated. This study is generally well conducted, and the data from this
study support a quantitative cancer dose-response assessment. This study is a peer-reviewed
technical report from the NTP, has been performed according to GLP principles, and otherwise
meets the standards of study design and performance with numbers of animals, examination of
potential toxicity endpoints, and presentation of information. Details are provided in the
"Review of Potentially Relevant Data" section. The NTP (1990) chronic study represents the
only study in the database with useful data for deriving the p-OSF. As previously discussed in
the "Review of Potentially Relevant Data," other chronic studies for 3,3'-dimethoxybenzidine
suffer from a number of deficiencies including small sample size, poor reporting of data, and low
rates of survival.
NTP (1990) reported treatment-related tumor types in a number of tissues in both male
and female rats after exposure to 3,3'-dimethoxybenzidine in drinking water for 21 months.
Tissues with observed tumors include the Zymbal gland, preputial gland, clitoral gland, skin
basal cells, skin squamous cells, small intestines, large intestines, oral cavity, liver, mesothelium,
and mammary gland. Cancer-dose-response modeling was performed for all of these tumor
types; it should be noted that some tumor types were not included in the dose-response modeling
analyses due to an irregular dose response (e.g., incidence decreases with increasing dose such as
mammary fibroadenomas in female rats). Incidence data used for dose-response modeling were
based on effective rates (the number of animals alive during the first occurrence of the tumor
being modeled) as reported by NTP (1990). The effective rates for combined tumor types were
extracted from the individual animal data provided by NTP (1990).
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The significant increases in tumor incidence seen in multiple tissues are characteristic of
a mutagenic MOA and indicate that the overall risk of tumor formation is spread throughout the
body. Because all of these tumors contribute to the overall cancer risk, an underestimation of
risk of cancer development would result from a dose-response assessment based on any one type.
Therefore, the overall cancer risk for 3,3'-dimethoxybenzidine based on combined tumor
incidence was evaluated in both male and female rats. For carcinogens that produce tumors at
multiple sites, combining incidence is an appropriate way to estimate cancer risk (U.S. EPA,
2005a).
The following dosimetric adjustments were made for oral drinking water treatment in
adjusting doses for cancer analysis (p-OSF). The low-dose conversion is shown below for
convenience:
(DOSEhEd)nTP, 1990
Body-weight adjustment
where,
BWh
BWa
Body-weight adjustment
(DOSEhed)nTP, 1990
(DOSEhed) NTP, 1990
= (Dose) ntp, 1990 x body-weight adjustment
1/4
= (BWa - BWh)
= 70 kg (human reference body) (U.S. EPA, 1997)
= 0.363 kg (average body weight for male F344 rats)
(Morgan et al., 1989)
= (0.363 -70)1/4 = 0.26835
= (Dose)„ x 0.26835
= 6 mg/kg-day x 0.26835
= 1.61 mg/kg-day
Table 7 presents BMD input data for incidence of combined primary tumors in male rats
exposed to 3,3'-dimethoxybenzidine by drinking water for 21 months (see Table B. 12 for
incidences of individual tumor types). Combined tumor incidence in female rats was also
evaluated by BMD analysis; however, the male rat data provided a slightly lower BMDiohed and
BMDLiohed-
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Table 7. BMD Input for Combined Primary Tumors in Male F344/N Rats
Exposed to 3,3'-Dimethoxybenzidine Dihydrochloride for 21 Months"
(Dose)„ (mg/kg-day)
(DOSEhed),, (mg/kg-day)
Number of Rats
Response (Combined
Tumor Incidence)
0
0
59
22b
6
1.61
45
41
12
3.22
75
70
21
5.64
60
60
aNTP (1990).
b16/59 control male rats had preputial adenoma or carcinoma; the incidence of other tumor types did not exceed
2/59.
Table 8 presents the BMD modeling results. Adequate model fit is obtained for
incidence of combined primary tumors using the multistage-cancer model, and the BMD
modeling results yields a BMDiohed of 0.122 mg/kg-day and a BMDLiohed of 0.095 mg/kg-day.
The OSF calculated from adult exposure is derived from the BMDLiohed, the 95% lower bound
on the human equivalent exposure associated with a 10% extra cancer risk (represented by the
0.1 BMR in the calculation of a p-OSF below). It is representative of an upper bound risk
estimate for continuous lifetime exposure. As discussed in the MOA section,
3,3'-dimethoxybenzidine is a mutagenic carcinogen. However, because no data on early-life
susceptibility are available, the BMDLiohed is representative of an upper bound risk estimate for
continuous lifetime exposure without consideration of increased susceptibility during childhood.
Because a linear, mutagenic MOA has been determined for neoplasms caused by
3,3'-dimethoxybenzidine, a linear extrapolation to low dose was calculated as the ratio
0.1/BMDLiohed, as shown below.
Table 8. Model Predictions for Combined Malignant Tumors in the Male Rat
Exposed to 3,3'-Dimethoxybenzidine Dihydrochloride in Drinking Water for 21 Months"
Model
Goodness of
Fit />-Valucb
AICC for
Fitted Model
BMD10hedc
(mg/kg-day)
BMDL10hedc
(mg/kg-day)
Conclusions
Multistage Cancer
0.18
149.289
0.122
0.095
Selected as lowest BMDL for
POD
aNTP (1990).
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
°AIC = Akaike Information Criteria; BMD = benchmark dose; BMDL lower confidence limit (95%) on the
benchmark dose.
p-OSF
(unadjusted)
0.1 BMDLiohed
= 0.1 0.095 mg/kg-day
1.1 (mg/kg-day)"1
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An adjustment was performed for a shorter-than-lifetime observation period (U.S. EPA,
1980). The NTP (1990) rat bioassay was terminated after 21 months (compared to the
experimental rat life span of 24 months), due to increased mortality from the formation of
tumors. Therefore, it is unclear if a sufficient period of time had elapsed to fully evaluate the
carcinogenicity of 3,3'-dimethoxybenzidine at the lowest dose. Because of the truncated
experimental protocol seen in the NTP (1990) study, it is unknown how a full 2-year exposure to
3,3'-dimethoxybenzidine may have influenced the tumor incidence in low-dose rats. As a result,
"3
an adjustment factor of (L/Le) is applied to the unadjusted p-OSF, where L = the lifetime of the
animal (in this case, the experimental lifetime) and Le = the duration of experimental dosing.
Using this adjustment, a p-OSF of 1.6 (mg/kg-day)-1 is derived as follows:
p-OSF — p-OSF (unadjusted) x (L ~ Le)
= 1.1 (mg/kg-day )_1 x (24 months 21 months)3
= 1.6 (mg/kg-day)"1
The p-OSF for 3,3'-dimethoxybenzidine should not be used with exposures exceeding the
point of departure (BMDLiohed = 0.095 mg/kg-day), because above this level the fitted
dose-response model better characterizes what is known about the carcinogenicity of
3,3'-dimethoxybenzidine.
The human equivalent dose was also calculated for the central estimate (BMDio)
associated with the selected point of departure (combined tumors in male rats); A BMDio hed of
0.122 mg/kg-day was calculated. The unadjusted slope of the linear extrapolation from the
central estimate (0.122 mg/kg-day) is 0.8 (mg/kg-day )_1 and the adjusted slope is
1.2 (mg/kg-day)_1.
Based on a WOE evaluation, 3,3'-dimethoxybenzidine is carcinogenic by a mutagenic
MOA. Carcinogens with a mutagenic MOA are assumed to be associated with an increased
early-life susceptibility (U.S. EPA, 2005b). However, no sufficient data are available to develop
separate risk estimates for childhood exposure to 3,3'-dimethoxybenzidine. Therefore, the
p-OSF of 1.6 (mg/kg-day) 1 calculated from adult exposure data is not reflective of the presumed
early-life susceptibility for this compound, and age-dependent adjustment factors (ADAFs)
should be applied to this parameter when assessing cancer risks. Example evaluations of cancer
risks based on age at exposure and ADAFs for three specific age groups have been established as
indicated in Section 6 of the Supplemental guidance for assessing susceptibility from early-life
exposure to carcinogens (U.S. EPA, 2005b). Currently, the ADAFs and their age groups are 10
for <2 years, 3 for 2 to <16 years, and 1 for >16. When estimating cancer risk from early life
(<16 years of age) exposure to 3,3'-dimethoxybenzidine, the 10-fold and 3-fold adjustments in
slope factor should be combined with age-specific exposure estimates. These ADAFs and their
age groups may be revised over time, and the most current guidance on assessing susceptibility
from childhood exposure to carcinogens can be found at www.epa.gov/cancerguidelines/. When
estimating risk for exposure to 3,3'-dimethoxybenzidine, it is recommended that age-specific
values for both exposure and cancer potency be used and that age-specific values for cancer
potency are determined using the appropriate ADAFs. For each age group, a cancer risk is
derived with these values summed across age groups to obtain the total risk for the exposure
period of interest.
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Derivation of Provisional Inhalation Unit Risk (p-IUR)
No human or animal studies examining the carcinogenicity of 3,3'-dimethoxybenzidine
following inhalation exposure have been identified. Therefore, derivation of an IUR is
precluded.
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APPENDIX A. PROVISIONAL SCREENING VALUES
DERIVATION OF A SCREENING SUBCHRONIC p-RFD VALUE
For reasons noted in the main PPRTV document, it is inappropriate to derive provisional
toxicity values for 3,3'-dimethoxybenzidine. However, information is available for this chemical
which, although insufficient to support derivation of a provisional toxicity value, under current
guidelines, may be of limited use to risk assessors. In such cases, the Superfund Health Risk
Technical Support Center summarizes available information in an Appendix and develops a
"screening value." Appendices receive the same level of internal and external scientific peer
review as the PPRTV documents to ensure their appropriateness within the limitations detailed in
the document. Users of screening toxicity values in an appendix to a PPRTV assessment should
understand that there is considerably more uncertainty associated with the derivation of an
appendix screening toxicity value than for a value presented in the body of the assessment.
Questions or concerns about the appropriate use of screening values should be directed to the
Superfund Health Risk Technical Support Center.
The 13-week rat study by NTP (1990) is selected as the principal study for the
derivation of a screening subchronic p-RfD. This study is a range-finding study in a
peer-reviewed report conducted for the NTP, National Institutes of Health and has been
performed according to GLP principles, and otherwise meets the standards of study design and
performance with regards to the numbers of animals and the examination of potential toxicity.
This study and its results were also published as a peer-reviewed article (Morgan et al., 1989).
Details are provided in the "Review of Potentially Relevant Data" section and are summarized in
Table A.l below. After 13 weeks of 3,3'-dimethoxybenzidine exposure, male and female rats
experienced significant changes in relative organ weights and hematological/serum chemistry
parameters, as well as chronic nephropathy and accumulation of pigment in follicular cells of the
thyroid. However, the original study authors ascribed the observed hematological/serum
chemistry changes in part to mechanical hemolysis during sample processing. Furthermore,
while the study authors noted reduced T3 and T4 levels in male and female rats, the changes
were not significant compared to controls, and thyrotropin (TSH, which is responsive to T3 and
T4 levels) concentrations in these animals were not different from controls. Therefore, relative
organ-weight changes, pigmentation of thyroid follicular cells, and chronic nephropathy were
considered in the selection of a critical effect for subchronic exposure. While nephropathy and
thyroid follicular cell pigmentation occurred primarily at higher 3,3'-dimethoxybenzidine
exposure doses, significant organ-weight changes occurred at doses lower than all other effects
considered (see Table A. 1). Relative liver and kidney weights were statistically significantly
increased as a function of increasing dose in both male and female rats; males appeared to be
more sensitive than females to the liver-weight changes, while the kidney-weight changes were
comparable in males and females. Thymus weights were statistically significantly reduced in
male rats at all doses tested; however, compared to controls, thymus weights in females were
unchanged even at the highest 3,3'-dimethoxybenzidine dose. As such, decreased relative
thymus weights were not further considered. In male rats, the magnitude of change in relative
liver weight at the lowest exposure dose was greater than in the kidney, and greater than that
observed in liver or kidney of female rats at the lowest dose (see Tables B.3 and B.4). Therefore,
increased relative liver weight in male rats is chosen as the critical effect for derivation of a
subchronic oral screening value.
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Table A.l. Summary of Relevant Subchronic Oral Systemic Toxicity Studies
for 3,3'-Dimethoxybenzidine
References
#/Sex
(M/F),
Species
Exposure
(mg/kg-day)
Frequency/
Duration
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Endpoint
NTP (1990)
10/10,
rat
Male: 0, 13,
22, 39, 70, or
120
Female: 0,
24, 49, 60,
103, or 187
Ad libitum in
drinking water
for 13 weeks
Male: 22
Female: 24
Male: 39
Female: 49
Increased relative
kidney weight
NTP (1990)
10/10,
rat
Male: 0, 13,
22, 39, 70, or
120
Female: 0,
24, 49, 60,
103, or 187
Ad libitum in
drinking water
for 13 weeks
Male: none
Female: 49
Male: 13
Female: 60
Increased relative
liver weight
NTP (1990)
10/10,
rat
Male: 0, 13,
22, 39, 70, or
120
Female: 0,
24, 49, 60,
103, or 187
Ad libitum in
drinking water
for 13 weeks
Male: none
Female: 187
Male: 13
Female: none
Decreased relative
thymus weight in
males
NTP (1990);
Morgan et al.
(1989)
10/10,
rat
Male: 0, 70,
or 120
Female: 0,
103, or 187
Ad libitum in
drinking water
for 13 weeks
Male: none
Female: none
Male: 70
Female: 103
Thyroid pigment in
follicular cells
NTP (1990);
Morgan et al.
(1989)
10/10,
rat
Male: 0, 70,
or 120
Female: 0,
103, or 187
Ad libitum in
drinking water
for 13 weeks
Female: none
Female: 103
Kidney multifocal
chronic nephropathy
BMD modeling was conducted with the EPA's BMD software (BMDS version 1.4.1).
For continuous data such as the male rat relative liver weight (see Table B.3.), the data were
modeled with all the continuous models available within the software. An adequate fit was
judged based on the goodness-of-fit-p-value, scaled residue at the range of benchmark response
(BMR), and visual inspection of the model fit. Among all the models attempted, none provided
adequate fit to the liver-weight data; therefore, the LOAEL of 13 mg/kg-day is used as the POD
for derivation of a subchronic oral screening value as follows:
Screening Subchronic p-RfD = LOAEL UFc
= 13 mg/kg-day ^ 10,000
= 1 x 10~3 mg/kg-day
Table A.2 summarizes the uncertainty factors for the subchronic oral screening value for
3,3'-dimethoxybenzidine.
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Table A.2. Uncertainty Factors for Screening Subchronic p-RfD for
3,3'-Dimethoxybenzidine (NTP, 1990)
UF
Value
Justification
ufa
10
A UFa of 10 is applied for interspecies extrapolation to account for potential toxicokinetic and
toxicodynamic differences between rats and humans. There are no data to determine whether
humans are more or less sensitive than rats to liver effects of 3,3'-dimethoxybenzidine.
ufd
10
A UFd of 10 is applied because there are no acceptable two-generation reproduction studies or
developmental studies.
UFh
10
A UFh of 10 is applied for intraspecies differences to account for potentially susceptible
individuals in the absence of information on the variability of response in humans.
ufl
10
A UFl of 10 is applied because the POD was developed using a LOAEL.
UFS
1
A UFS of 1 is applied because a subchronic study (NTP, 1990) was utilized as the principal
study.
UFC
10,000
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APPENDIX B. DATA TABLES
Table B.l. Selected Organ Weight to Body-Weight Ratios in F344N Rats
Exposed to Oral 3,3'-Dimethoxybenzidine for 14 Days"
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
Male rat
0 ppm
200 ppm
(18)
350 ppm
(29)
750 ppm
(57)
1500 ppm
(101)
4500 ppm
(127)
Final body weight (g)
235 ± 1.2
241 ±6.2 (+3)
235 ± 4.0 (0)
232 ±7.2
(-1)
225 ±9.9
(-4)
141 ±4.2**
(-40)
Relative organ weights (mg/g)
Brain
7.3 ±0.11
7.6 ±0.22
(+4)
7.6 ±0.08
(+4)
7.5 ±0.12
(+3)
7.8 ±0.27
(+7)
11.9 ±0.43**
(+63)
Lungs
4.0 ±0.09
4.3 ±0.16
(+8)
4.2 ±0.10
(+5)
4.2 ±0.09
(+5)
4.1 ±0.09
(+3)
5.5 ±0.29**
(+38)
Heart
2.8 ±0.08
3.1 ±0.23
(+11)
2.9± 0.08 (+4)
3.0 ±0.15
(+7)
3.0 ±0.03
(+7)
3.3 ±0.07**
(+18)
Liver
43.4 ±0.74
46.7 ±0.41*
(+8)
45.0 ±0.70
(+4)
48.2 ±0.45**
(+11)
51.5 ± 0.41**
(+19)
47.8 ±3.60**
(+10)
Kidney
3.5 ±0.08
3.9 ±0.27
(+11)
3.9 ± 0.15*
(+11)
3.8 ±0.10*
(+09)
4.0 ±0.09**
(+14)
5.1 ±0.25**
(+46)
Right testis
5.3 ± 0.15
5.4 ±0.24
(+02)
5.3 ±0.08
(0)
5.6 ±0.14
(+6)
5.6 ±0.13
(+6)
7.7 ±0.26**
(+45)
Female rat
0 ppm
200 ppm
(19)
350 ppm
(32)
750 ppm
(61)
1500 ppm
(141)
4500 ppm
(214)
Final body weight (g)
163 ±4.2
163 ±4.1
(0)
160 ± 1.9
("2)
156 ±2.9
(-4)
157 ±4.2
(-4)
135 ±3.3**
(-17)
Relative organ weights (mg/g)
Brain
10.2 ±0.34
10.4 ±0.26
(+2)
11.0 ±0.40
(+8)
10.6 ±0.21
(+4)
10.4 ±0.26
(+2)
11.9 ±0.49*
(+17)
Liver
37.0 ±0.95
39.2 ±0.96
(+6)
37.9 ± 16 (+2)
39.3 ±0.46
(+6)
41 ±0.57**
(+11)
45.6 ± 1.50**
(+23)
Kidney
3.7 ±0.15
3.7 ±0.23
(0)
3.7 ±0.08
(0)
3.9 ±0.08
(+5)
4.1 ± 0.13*
(+11)
4.6 ±0.23**
(+24)
Thymus
2.2 ±0.10
2.3 ±0.10
(+5)
2.4 ±0.24
(+9)
2.1 ±0.08
("5)
2.0 ±0.07
(-9)
1.7 ±0.10**
(-23)
aNTP (1990).
Notes: Means ± SE (% change relative to controls); n = 5 for each group; p values are vs. the controls by Dunn's or
Shirley's test.
*p<0.05; **p<0.01.
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Table B.2 Water Consumption of Male and Female F344N Rats
Exposed to Oral 3,3'-Dimethoxybenzidine for 13 Weeks"
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
Male rat
0 ppm
170 ppm
(13)
330 ppm
(22)
630 ppm
(39)
1250 ppm
(70)
2500 ppm
(120)
Water consumption (ml/animal/day)
Week 7b
21
21(0)
17 (-19)
16 (-24)
13 (-38)
12 (-43)
Week 13b
21
22 (+5)
20 (-5)
17 (-19)
14 (-33)
12 (-43)
Female rat
0 ppm
170 ppm
(24)
330 ppm
(49)
630 ppm
(60)
1250 ppm
(103)
2500 ppm
(187)
Water consumption (ml/animal/day)
Week 7b
27
23 (-15)
29 (+7)
16 (-41)
13 (-52)
10 (-63)
Week 13b
25
21 (-16)
29 (+16)
14 (-44)
11 (-56)
10 (-60)
aNTP (1990).
bValue (% change relative to controls).
Table B.3. Selected Organ Weight to Body-Weight Ratios in the F344N Male Rat
Exposed to Oral 3,3'-Dimethoxybenzidine for 13 Weeks"
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
0 ppm
170 ppm
(13)
330 ppm
(22)
630 ppm
(39)
1250 ppm
(70)
2500 ppm
(120)
Sample size
10
10
10
10
10
10
Final body weight (g)
326 ±6.18
319 ± 5.58
("2)
325 ±4.54
(0)
318 ±5.69
("2)
295 ±5.51**
(-10)
265 ±5.45**
(-19)
Relative organ weights (mg/g)
Liver
25.1 ±0.20
27.7 ±0.19**
(+10)
27.9 ±0.21**
(+11)
29.3 ±0.30**
(+17)
31.3 ±0.35**
(+25)
32.8 ±0.58**
(+31)
Right kidney
3.0 ±0.04
3.1 ±0.04*
(+3)
3.2 ±0.04**
(+7)
3.4 ±0.04**
(+13)
3.5 ±0.06**
(+17)
4.0 ±0.06**
(+33)
Thymus
1 ± 0.03
0.9 ±0.02*
(-18)
0.9 ±0.06**
(-18)
0.9 ±0.04**
(-18)
0.8 ±0.06**
(-27)
0.8 ±0.01**
(-27)
aNTP (1990).
Notes: Means ± SE (% change relative to controls); p values are vs. the controls by Dunn's or Shirley's test.
*p<0.05; **p<0.01.
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Table B.4. Selected Organ Weight to Body-Weight Ratios in F344N Female Rats
Exposed to Oral 3,3'-Dimethoxybenzidine for 13 Weeks"
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
0 ppm
170 ppm (24)
330 ppm (49)
630 ppm (60)
1250 ppm (103)
2500 ppm (187)
Sample size
10
10
10
10
10
10
Final body weight (g)
179 ±2.20
176 ± 2.22
("2)
178 ± 1.65
(-1)
175 ± 1.46
("2)
174 ±3.44
("3)
164 ±2.63*
("8)
Relative organ weights (mg/g)
Liver
25.9 ±0.40
26.2 ±0.36
(+1)
27.0 ±0.39
(+4)
28.4 ±0.97**
(+10)
28.3 ±0.24**
(+9)
30.2 ±0.46**
(+17)
Right kidney
3.2 ±0.05
3.3 ±0.05
(+3)
3.5 ±0.05**
(+9)
3.9 ±0.06**
(+22)
4.0 ±0.09**
(+25)
4.2 ±0.05**
(+31)
aNTP (1990).
Notes: Means ± SE (% change relative to controls); p values are vs. the controls by Dunn's or Shirley's test.
*p<0.05; **p<0.01.
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Table B.5. Selected Serum Chemistry of Male and Female F344N Rats
Exposed to Oral 3,3'-Dimethoxybenzidine for 13 Weeksa'b
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
Male rat
0 ppm
170 ppm
(13)
330 ppm
(22)
630 ppm
(39)
1250 ppm
(70)
2500 ppm
(120)
Serum creatinine
(mg/dl)
0.67 ±0.015
0.58 ±0.013"
(-13)
0.57 ±0.015**
(-15)
0.50 ±0.030**
(-25)
0.61 ±0.028**
(-19)
0.56 ±0.034**
(-16)
Triiodothyronine (T3)
(ng/dl)
67.0 ±2.68
67.0 ±4.41
(0)
69.1 ±3.31
(+3)
65.9 ±2.46
("2)
65.5 ± 1.85
("2)
58.6 ±3.13
(-13)
Thyroxine (T4)
(Hg/dl)
4.0 ±0.14
3.4 ±0.22*
(-15)
3.6 ±0.16*
(-10)
2.9 ±0.14**
(-27)
3.4 ±0.16**
(-15)
2.8 ±0.19**
(-30)
Thyrotropin (TSH)
(ng/ml)
609 ± 55.3°
527 ± 39.2d
(-13)
639 ± 74.4e
(+5)
592 ± 27.0
("3)
668 ± 74.0d
(+10)
476 ± 52.3e
("22)
Female rat
0 ppm
170 ppm
(24)
330 ppm
(49)
630 ppm
(60)
1250 ppm
(103)
2500 ppm
(187)
Serum creatinine
(mg/dl)
0.71 ±0.031
0.62 ±0.025*
(-13)
0.61 ±0.038*
(-14)
0.54 ±0.029**
(-24)
0.62 ±0.025*
(-13)
0.57 ±0.021**
("20)
Triiodothyronine (T3)
(ng/dl)
98.4 ±2.16
97.7 ±4.54
(-1)
79.4 ±3.63**
(-19)
68.3 ±2.87**
(-31)
63.3 ±2.01**
(-36)
57.2 ±2.49**
(-42)
Thyroxine (T4)
(ng/dl)
3.9 ±0.17
3.4 ±0.17
(-13)
3.2 ±0.23*
(-18)
2.4 ±0.05**
(-38)
2.0 ±0.17**
(-49)
2.0 ±0.14**
(-49)
Thyrotropin (TSH)
(ng/ml)
461 ±21.7C
697 ± 62.9f
(+51)
730 ± 79.2e
(+58)
606 ± 47.8s
(+31)
962 ± 246. le
(+109)
605 ± 138.8d
(+31)
aNTP (1990).
''Mean ± SE for groups of 10 animals, unless otherwise specified (% change relative to controls).
°Five animals were examined.
dNine animals were examined.
eEight animals were examined.
fSix animals were examined.
8Seven animals were examined.
p < 0.05.
p < 0.01.
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Table B.6 Mean Survival Time and Mean Final Body Weights of Male and Female F344
Rats Following Oral Exposure to 3,3'-Dimethoxybenzidine for 52 Weeksa'b
Parameter
Exposure Group (Daily Average Dose mg/kg-day)
Male rat
0.1 mg/day
(0.2)
0.3 mg/day
(0.6)
1.0 mg/day
(1.9)
3.0 mg/day
(5.6)
10 mg/day
(18.8)c
30 mg/day
(56.4)
Survival time (days)d
568
568 (0)
503 (-11)
519 (-9)
506 (-11)
394 (-31)
Mean body weightd (g)
418
425 (+2)
359 (-14)
357 (-15)
393 (-6)
343 (-18)
Female rat
0.1 mg/day
(0.3)
0.3 mg/day
(0.9)
1.0 mg/day
(3.1)
3.0 mg/day
(9.4)
10 mg/day
(31.2)e
30 mg/day
(93.6)
Survival timed
548
548 (0)
517 (-6)
383 (-30)
447 (-18)
462 (-16)
Mean body weight (g)d
293
305 (+4)
249 (-15)
247 (-16)
232 (-21)
223 (-24)
aHadidian et al. (1968).
hn = 3 except where otherwise specified.
°n = 14.
Value (% change relative to lowest exposure dose group).
en = 15.
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Table B.7. Incidence of Neoplasms in Male and Female F344 Rats Following Oral
Exposure to 3,3'-Dimethoxybenzidine for 52 Weeksa'b
Parameter
Exposure Group (Daily Average Dose mg/kg-day)
Male Rat
0
mg/dayc
0.1
mg/day
(0.2)
0.3
mg/day
(0.6)
1.0
mg/day
(1.9)
3.0
mg/day
(5.6)
10
mg/day
(18.8)d
30
mg/day
(56.4)
Ear:
Squamous cell carcinoma
2
0
0
1
1
3
0
Skin:
Basal cell carcinoma
0
0
0
0
0
2
0
Squamous cell carcinoma
0
0
0
0
0
1
1
Stomach:
Papilloma
0
0
0
0
0
1
0
Pituitary:
Adenoma
2
0
0
0
0
1
0
Intestine:
Adenocarcinoma
0
0
0
0
0
2
0
Colon:
Adenocarcinoma
0
0
0
0
0
0
1
Testes:
Interstitial cell
123
2
2
2
2
2
0
Multiple
organs:
Metastasis
0
0
0
1
0
1
0
Female Rat
0
mg/dayc
0.1
mg/day
(0.3)
0.3
mg/day
(0.9)
1.0
mg/day
(3.1)
3.0
mg/day
(9.4)
10
mg/day
(31.2)e
30
mg/day
(93.6)
Ear:
Squamous cell carcinoma
0
0
0
0
0
2
1
Skin:
Basal cell carcinoma
0
0
0
0
0
0
1
Squamous cell carcinoma
0
0
0
0
0
2
0
Uterus:
Carcinoma
0
0
0
0
0
1
0
Endometrial carcinoma
0
0
0
0
0
1
0
Mammary
Adenocarcinoma
0
0
0
0
1
2
0
gland:
Fibroadenoma
10
0
0
0
0
1
1
Bladder:
Papilloma
1
0
0
0
1
0
1
Multiple
Metastasis
0
0
0
0
0
1
0
organs:
Lipoma
0
0
1
0
0
0
0
aHadidian et al. (1968).
bNumber of rats observed with lesion; n = 3 except where otherwise specified.
°n = 330 (240 vehicle controls and 90 untreated controls).
dn = 14.
en = 15.
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Table B.8. Selected Organ Weight to Body-Weight Ratios in F344N Rats
Exposed to Oral 3,3'-Dimethoxybenzidine for 9 Months"
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
Male rat
0 ppm
330 ppm (21)
Sample size
10
10
Final body weight (g)
390 ±7.7
373 ± 8.4 (-4)
Relative organ weights (mg/g)
Liver
25.5 ±0.40
28.7 ±0.67* (+13)
Kidney
6.1 ± 0.11
7.0 ±0.12* (+15)
Female rat
0 ppm
330 ppm (23)
Sample size
10
10
Final body weight (g)
232 ±3.9
223 ±3.3 (-4)
Relative organ weights (mg/g)
Liver
26.9 ±0.47
29.7 ±0.69* (+10)
Kidney
6.2 ±0.16
7.3 ±0.15* (+18)
aNTP (1990).
Notes: Means ± SE (% change relative to controls); p values are vs. the controls by Dunn's or Shirley's test.
*p < 0.05.
Table B.9. Water Consumption, Mean Body Weight, and Survival of Male and Female
F344N Rats Exposed to 3,3'-Dimethoxybenzidine in Drinking Water for 21 Months"
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
Male rat
0 ppm
80 ppm (6)
170 ppm (12)
330 ppm (21)
Water consumption13
26.6 ±3.3
25.0 ±5.1 (-6)
25.8 ±6.2 (-3)
22.0 ±4.9 (-17)
Mean body weight0
373
366 (-2)
359 (-4)
354 (-5)
Survival
44/60 (73%)
8/45 (18%)
0/75
0/60
Female rat
0 ppm
80 ppm (7)
170 ppm (14)
330 ppm (23)
Water consumption13
20.1 ±2.9
19.9 ±4.6 (-1)
19.4 ±3.1 (-3)
15.7 ±4.1 (-22)
Mean body weight0
251
244 (-3)
234 (-7)
231 (-8)
Survival
45/60 (75%)
15/45 (33%)
6/75 (8%)
0/60
aNTP (1990).
bGrams of water consumed per rat per day (% change relative to controls).
Estimated over the duration of the study (% change relative to controls).
dAnimals surviving until study termination (% alive at termination of study at 21 months).
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Table B.10. Survival Rates in F344N Rats Exposed to 3,3'-Dimethoxybenzidine
in Drinking Water for 21 Months"
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
Male rat
0 ppm
80 ppm (6)
170 ppm (12)
330 ppm (21)
Sample size
60
45
75
60
Natural deaths'3
9
9 (20)
25 (33)
14 (23)
Moribund
7
28 (62)
50 (67)
46 (77)
Animals surviving to end of study
44
8(18)
0(0)
0(0)
Survival ^-values0
<0.001
<0.001
<0.001
<0.001
Female rat
0 ppm
80 ppm (7)
170 ppm (14)
330 ppm (23)
Sample size
60
45
75
60
Natural deaths
5
3(7)
9(12)
9(15)
Moribund
10
27 (60)
60 (80)
51 (85)
Animals surviving until end of
study
45
15 (33)
6(8)
0(0)
Survival /?-valuesc
<0.001
<0.001
<0.001
<0.001
aNTP (1990).
dumber (% of exposure group).
°Life table pairwise comparisons.
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Table B.ll. Selected Nonneoplastic Lesions in F344N Rats Exposed to
3,3'-Dimethoxybenzidine in Drinking Water for 21 Months"
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
Male rat
0 ppm
80 ppm (6)
170 ppm (12)
330 ppm (21)
Number examined
60
45
74
60
Liver:
Cystic degeneration13
13 (22)
23** (51)
34** (46)
28** (47)
Centrilobular degeneration13
0(0)
4* (9)
9** (12)
10** (17)
Eosinophilic focusb
6(10)
15** (33)
35** (47)
38** (63)
Hematopoietic cell proliferation13
2(3)
15** (33)
39** (53)
41** (68)
Necrosis'3
4(7)
15** (33)
18** (24)
17** (28)
Regeneration13
5(8)
7(16)
22** (30)
18** (30)
Cytoplasmic vacuolization13
2(3)
2(4)
7(9)
10** (17)
Spleen:
Hematopoietic cell proliferation13
3(5)
13°* (31)
43d* (57)
38* (63)
Heart:
Atrium thrombi13
3(5)
15e* (34)
27* (36)
23* (38)
Lung:
Histiocytic cellular infiltration13
0(0)
3e (7)
10* (14)
6* (10)
Female rat
0 ppm
80 ppm (7)
170 ppm (14)
330 ppm (23)
Number examined
60
44
75
60
Liver:
Cystic degeneration13
1(2)
2(5)
1(1)
5(8)
Centrilobular degeneration13
1(2)
3(7)
8* (11)
5(8)
Eosinophilic focusb
5(8)
7(16)
20** (27)
28** (47)
Hematopoietic cell proliferation13
1(2)
18** (41)
43** (57)
41** (68)
Necrosis'3
1(2)
3(7)
13** (17)
18** (30)
Regeneration13
6(10)
3(7)
5(7)
4(7)
Cytoplasmic vacuolization13
3(5)
1(2)
4(5)
3(5)
Spleen:
Hematopoietic cell proliferation13
3(5)
22* (50)
50* (67)
47* (78)
Heart:
Atrium thrombi13
0(0)
lf (2)
0(0)
1(2)
Lung:
Histiocytic cellular infiltration13
0(0)
3f (7)
4(5)
18* (30)
aNTP (1990).
bNumber (% of exposure group examined).
°42 animals were examined.
d75 animals were examined.
e44 animals were examined.
f45 animals were examined.
Notes: *p < 0.05 vs. controls; **p < 0.01 vs. controls.
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Table B.12. Selected Analysis of Primary Tumors in Male and Female F344N Rats
Exposed to 3,3'-Dimethoxybenzidine Dihydrochloride in Drinking Water for 21 Monthsa'b
Parameter
Exposure Group (Daily Average Dose mg/kg-day)
Male Rat
0 ppm
80 ppm
(6)
170 ppm
(12)
330 ppm
(21)
Liver:
Neoplastic nodule
0/58
3/39
7/54**
6/35**
Neoplastic nodule or hepatocellular
carcinoma
1/58
4/39
7/54*
8/35**
Large intestine:
Adenomatous polyp
0/59
1/44
4/73
5/57*
Adenocarcinoma
0/59
0/42
4/67
3/50
Adenomatous polyp or adenocarcinoma
0/59
1/44
8/73**
8/57**
Small intestine:
Adenocarcinoma
0/59
4/44*
7/75*
5/60*
Zymbal gland:
Adenoma
0/58
4/44*
11/71**
9/53**
Carcinoma
0/58
7/45**
14/75**
21/60**
Adenoma or carcinoma
0/58
10/45**
25/75**
30/60**
Preputial gland:
Carcinoma
2/59
6/42
15/73**
19/59**
Adenoma or carcinoma
16/59
12/42
33/73*
29/59*
Oral cavity:
Squamous cell papilloma
1/59
7/44*
10/73*
9/57**
Squamous cell papilloma or carcinoma
1/59
8/44**
10/73*
11/57**
Skin:
Basal cell adenoma
1/59
31/42**
47/67**
35/50**
Basal cell carcinoma
1/59
4/44
18/71**
17/54**
Basal cell adenoma or carcinoma
2/59
32/44**
54/71**
40/54**
Squamous cell papilloma
0/58
5/42*
7/62**
5/41*
Squamous cell carcinoma
0/59
9/42**
24/65**
21/48**
Squamous cell papilloma or carcinoma
0/59
13/42**
28/65**
22/48**
All Tissues:
Mesothelioma
2/59
1/44
7/72*
6/56**
Combined
Sites0
Combined tumors
22/59
41/45**
70/75**
60/60**
Female Rat
0 ppm
80 ppm
(7)
170 ppm
(14)
330 ppm
(23)
Liver:
Neoplastic nodule or hepatocellular
carcinoma
0/59
1/44
0/47
3/38
Large intestine:
Adenomatous polyp or adenocarcinoma
0/59
1/44
1/48
3/35*
Zymbal gland:
Adenoma
0/59
3/44
4/48*
3/35*
Carcinoma
1/60
10/45**
17/74**
13/59**
Adenoma or carcinoma
1/60
12/45**
21/74**
16/59**
Skin:
Basal cell adenoma
0/59
3/44
3/48
2/35
Basal cell adenoma or carcinoma
0/59
4/44*
3/48
2/35
Clitoral gland:
Adenoma
5/58
15/44**
13/73
16/55**
Carcinoma
2/58
17/44**
41/74**
30/55**
Adenoma or Carcinoma
7/58
27/44**
48/74**
41/55**
Mammary
gland:
Adenocarcinoma
1/60
2/45
14/73**
20/57**
45
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Table B.12. Selected Analysis of Primary Tumors in Male and Female F344N Rats
Exposed to 3,3'-Dimethoxybenzidine Dihydrochloride in Drinking Water for 21 Monthsa'b
Parameter
Exposure Group (Daily Average Dose mg/kg-day)
Uterus:
Adenoma
0/56
3/34
1/19
2/8*
Adenoma or Carcinoma
0/59
4/44*
2/48
2/35
Combined
Sites'1
Combined tumors
5/60
38/45**
65/74**
53/59**
aNTP (1990).
bNumber of tumor-bearing animals/effective number of animals, i.e., number of animals living during first
occurrence of tumors in any dose group.
Includes sites with statistically increased tumor incidences (liver, small intestines, large intestine, Zymbal gland,
preputial gland, oral cavity, and skin).
Includes sites with statistically increased tumor incidences (liver, large intestine, Zymbal gland, skin, clitoral gland,
mammary gland, and uterus).
Notes: *p < 0.05 vs. controls; **p < 0.01 vs. controls.
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APPENDIX C. BMD MODELING OUTPUTS FOR 3,3'-DIMETHOXYBENZIDINE
Multistage Cancer Model with 0.95 Confidence Level
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2 EfrMDL BMP
0
17:58 06/15 2010
Multistage Cancer
Linear extrapolation
dose
Combined Significant Tumors in Males (NTP, 1990).
Output for selected model: Multistage Cancer
NTP, 1990: Combined Significant Tumors in Males
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: C:\l\NTP_1990_SigTumor_M_MultiCanc_l.(d)
Gnuplot Plotting File: C:\l\NTP_1990_SigTumor_M_MultiCanc_l.pit
Tue Jun 15 17:58:58 2010
[add notes here]
The form of the probability function is:
P [response] = background + (l-background)*[l-EXP(
-betal*doseAl-beta2*doseA2-beta3*doseA3)]
The parameter betas are restricted to be positive
Dependent variable = DichPerc
Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
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Total number of parameters in model = 4
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0
Beta(l) = 0
Beta(2) = 0
Beta(3) = 5.80808e+017
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta(2) -Beta(3)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix)
Background Beta(l)
Background 1 -0.44
Beta(l) -0.44 1
Parameter Estimates
Variable
Background
Beta(l)
Beta(2)
Beta(3)
Estimate
0.38265
0.864045
0
0
95.0% Wald Confidence Interval
Std. Err. Lower Conf. Limit Upper Conf. Limit
¦ Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log(likelihood) #Param's Deviance Testd.f. P-value
Full model -70.8357 4
Fitted model -72.6447 2 3.618 2 0.1638
Reduced model -117.058 1 92.4447 3 <0001
AIC: 149.289
Goodness of Fit
Scaled
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Dose EstProb. Expected Observed Size Residual
0.0000
0.3826
22.576
22.000
59
-0.154
1.6100
0.8464
38.088
41.000
45
1.204
3.2200
0.9618
72.134
70.000
75
-1.285
5.6400
0.9953
59.717
60.000
60
0.534
ChiA2 = 3.41 d.f.
= 2 P-value = 0.1818
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 0.121939
BMDL = 0.095041
BMDU = 0.218288
Taken together, (0.095041, 0.218288) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 1.05218
49 3,3'-Dimethoxybenzidine
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APPENDIX D. REFERENCES
ACGIH (American Conference of Industrial Hygienists). (2009) 2009 TLVs and BEIs:
Threshold limit values for chemical substances and physical agents and biological exposure
indices. Cincinnati, OH: ACGIH. 594528
Anderson, D; Styles, JA. (1978) An evaluation of 6 short-term tests for detecting organic
chemical carcinogens: Appendix II- The bacterial mutation test. Br J Cancer 37(6):924-930.
594532
ATSDR (Agency for Toxic Substances and Disease Registry). (2010) Toxicological profile
information sheet. U.S. Department of Health and Human Services, Public Health Service,
Atlanta, GA. Available online at http://www.atsdr.cdc.gov/toxprofiles/index.asp. Accessed on
2/25/2010. 684152
CalEPA (California Environmental Protection Agency). (2008) All OEHHA acute, 8-hour and
chronic reference exposure levels (chRELs) as on December 18, 2008. Office of Environmental
Health Hazard Assessment, Sacramento, CA. Available online at
http://www.oehha.ca.gov/air/allrels.html. Accessed on 2/25/2010. 595416
CalEPA (California Environmental Protection Agency). (2009) OEHHA toxicity criteria
database. Office of Environmental Health Hazard Assessment, Sacramento, CA. Available
online at http://www.oehha.ca.gov/riskAChemicalDB/index.asp. Accessed on 2/25/2010.
595417
Chung, KT; Chen, SC; Wong, TY; et al. (2000) Mutagenicity studies of benzidine and its
analogs: Structure-activity relationships. ToxicolSci 56(2):351-356. 225330
De France, BF; Carter, MH; Josephy, PD. (1986) Comparative metabolism and mutagenicity of
azo and hydrazone dyes in the Ames test. Food Chem Toxicol 24(2): 165-169. 625777
Fluck, ER; Poirier, LA; Ruelius, HW. (1976) Evaluation of a DNA polymerase-deficient
mutant of E. coli for the rapid detection of carcinogens. Chem Biol Interact 15(3):219-231.
074993
Frumin, E; Velez, H; Bingham, E; et al. (1990) Occupational bladder cancer in textile dyeing
and printing workers: Six cases and their significance for screening programs. JOccup Med
32:887-890. 225334
Galloway, SM; Armstrong, MJ; Reuben, C; et al. (1987) Chromosome aberrations and sister
chromatid exchanges in Chinese hamster ovary cells: evaluations of 108 chemicals. Environ Mol
Mutagen, 10:1-175. 007768
Galloway, SM; Bloom, AD; Resnick, M; et al. (1985) Development of a standard protocol for
in vitro cytogenetic testing with Chinese hamster ovary cells: comparison of results for
22 compounds in two laboratories. Environ Mutagen 7:1-51. 625785
50
3,3'-Dimethoxybenzidine
-------
FINAL
6-12-2013
Gray, LE Jr; Ostby, JS. (1993) The effects of prenatal administration of azo dyes on testicular
development in the mouse: A structure activity profile of dyes derived from benzidine,
dimethylbenzidine, or dimethoxybenzidine. ToxicolSci 20(2): 177-183. 068031
Gregory, AR; Elliott, J; Kluge, P. (1981) Ames testing of Direct Black 38 parallels
carcinogenicity testing. JAppl Toxicol 1(6):308-313. 625786
Hadidian, Z; Fredrickson, TN; Weisburger, EK; et al. (1968) Tests for chemical carcinogens
Report on the activity of derivatives of aromatic amines, nitrosamines, quinolines, nitroalkanes,
amides, epoxides, aziridines, and purine antimetabolites. J Natl Cancer Inst 41(4):985-1036.
062691
Hamasaki, T; Aramaki, K; Hida, T; et al. (1996) [Clinical study of occupational uroepithelial
cancer], J UOEH 18:247-259. 229151
Haworth, S; Lawlor, T; Mortelmans, K; et al. (1983) Salmonella mutagenicity test results for
250 chemicals. Environ Mutagen 5(Suppl 1):3-142. 028947
IARC (International Agency for Research on Cancer). (1974) Some aromatic amines, hydrazine
and related substances, N-nitroso compounds and miscellaneous alkylating agents. IARC
monographs on the evaluation of carcinogenic risks to humans, vol 4. WHO. Available online
at http://monoeraphs.iarc.fr/ENG/Monoeraphs/vol4/volume4.pdf. 231090
IARC (International Agency for Research on Cancer). (2000) Some industrial chemicals.
IARC monographs on the evaluation of carcinogenic risks to humans, volume 77. Geneva:
WHO. Available online at http://monoeraphs.iarc.fr/ENG/Monoeraphs/vol77/index.php.
201837
Krishna, G; Xu, J; Nath, J. (1986) Comparative mutagenicity studies of azo dyes and their
reduction products in Salmonella typhimurium. J Toxicol Environ Health 18:111-119. 625787
Makena, P; Chung, KT. (2007) Evidence that 4-aminobiphenyl, benzidine, and benzidine
congeners produce genotoxicity through reactive oxygen species. Environ MolMutagen
48(5):404-413. 625788
Martelli, A; Robbiano, L; Carrozzino, R; et al. (2000) DNA damage induced by 3,3'
dimethoxybenzidine in liver and urinary bladder cells of rats and humans. Toxicol Sci 53:71-76.
233260
Martin, CN; Kennelly, JC. (1981) Rat liver microsomal azoreductase activity on four azo dyes
derived from benzidine, 3,3'-dimethylbenzidine or 3,3'-dimethoxybenzidine. Carcinogenesis
2(4):307-312. 625791
Messerly, EA; Fekete, JE; Wade, DR; et al. (1987) Structure-mutagenicity relationships of
benzidine analogues. Environ Mol Mutagen 10(3):263-274. 062707
Morgan, DL; Jameson, CW; Mennear, JH; et al. (1989) Thirteen-week toxicity studies of
3,3'-dimethoxybenzidine and CI direct blue 15 in the fisher 344 rat. Toxicology 59(3):297-309.
094786
51
3,3'-Dimethoxybenzidine
-------
FINAL
6-12-2013
Morgan, DL; Bucher, JR; Huff, JE; et al. (1990) Multi-organ carcinogenicity of
3,3'-dimethoxybenzidine dihydrochloride given in drinking water to F344/N rats. Int J Toxicol
9:79-91. 061197
Morgan, DL; Dunnick, JK; Goehl, T; et al. (1994) Summary of the National Toxicology
Program benzidine dye initiative. Environ Health Perspect 102(Suppl 2):63-78. 069233
NIOSH (National Institute for Occupational Safety and Health). (2010) NIOSH pocket guide to
chemical hazards. Index of chemical abstracts service registry numbers (CAS No.). Center for
Disease Control and Prevention, U.S. Department of Health, Education and Welfare,
Atlanta, GA. Available online at http://www.cdc.gov/niosh/npg/npgdcas.html. 625692
NTP (National Toxicology Program). (1990) Toxicology and carcinogenesis studies of
3,3'-dimethoxybenzidine dihydrochloride (CAS No. 20325-40-0) in F344/N rats (drinking water
studies). U.S. Department of Health and Human Services, Public Health Service, Research
Triangle Park, NC; TR 372. Available online at
http://ntp.niehs.nih.gov/ntp/htdocs/LT rptsZtr372.pdf.
NTP (National Toxicology Program). (1992) Toxicology and carcinogenesis studies of C.I.
direct blue 15 (CAS No. 2429-74-5) in F344 Rats (drinking water studies). National Toxicology
Program. Research Triangle Park, N.C. Available online at
http://ntp.ni ehs.nih.gov/?obi ectid=070952A0-F5Bl-AlFl-DB952886A955ACC A.
NTP (National Toxicology Program). (2011) 12th Report on carcinogens. U.S. Department of
Health and Human Services, Public Health Service, National Institutes of Health, Research
Triangle Park, NC. Available online at http://ntp.niehs.nih.gov/?obiectid=03C9AF75-E1BF-
FF40-DB A9EC0928DF8B15.
OSHA (Occupational Safety and Health Administration). (2006) Table Z-l limits for air
contaminants: occupational safety and health standards, subpart Z, toxic and hazardous
substances. U.S. Department of Labor, Washington, DC; OSHA Standard 1910.1000. Available
online at http://63.234.227.130/pls/oshaweb/owadisp.show document?p table STANDARDS#,
p id=9992. Accessed on 2/22/2010. 670067
Ouellet-Hellstrom, R; Rench, JD. (1996) Bladder cancer incidence in arylamine workers. J
Occup Environ Med 38(12): 1239-1247. 376069
Pliss, GB. (1963) On some regular relationships between carcinogenicity of aminodiphenyl
derivatives and the structure of substance. Acta Unio Internat Cont Cane 19:499-501. 376071
Pliss, GB. (1965) [On the carcinogenic properties of ortho-toluidine and dianisidine], Gigiena
Truda i Professional'nye Zabolevaniya 9(7): 18-22. 377970
Prival, MJ; Bell, SJ; Mitchell, VD; et al. (1984) Mutagenicity of benzidine and
benzidine-congener dyes and selected monoazo dyes in a modified Salmonella assay. Mutat Res
136:33-47. 625794
52
3,3'-Dimethoxybenzidine
-------
FINAL
6-12-2013
Probst, GS; McMahon, RE; Hill, LE; et al. (1981) Chemically-induced unscheduled DNA
synthesis in primary rat hepatocyte cultures: a comparison with bacterial mutagenicity using
218 compounds. Environ Mutagen 3:11-32. 018887
Reynolds, SH; Patterson, RM; Mennear, JH; et al. (1990) ras Gene activation in rat tumors
induced by benzidine congeners and derived dyes. Cancer Res 50(2):266-272. 062314
Rodgers, RM; Garvie-Gould, C; Scott, KF; et al. (1983) Metabolism, distribution, and excretion
of the carcinogenic aromatic amine, 3,3'-dimethoxybenzidine in the rat: formation of mutagenic
urinary and biliary metabolites. DrugMetab Dispos 11(4):293-300. 062715
Saffiotti, U; Cefis, F; Montesano, R; et al. (1967) Induction of bladder cancer in hamsters fed
aromatic amines. In: WB Deichman; KR Lampe; ed. Bladder cancer: A symposium.
Birmingham, AL: Aesculapius Publishing Company, pp. 129-135. 062317
Schieferstein, GJ; Sheldon, WG; Allen, RR; et al. (1990) Oncogenic evaluation of
3,3'-dimethoxybenzidine dihydrochloride in BALB/c mice. Int J Toxicol 9:71-77. 094675
Sellakumar, AR; Montesano, R; Saffiotti, U. (1969) Aromatic amines carcinogenicity in
hamsters: Abstract #309. Presented at American Association for Cancer Research, San
Francisco, CA. 376236
U.S. EPA (Environmental Protection Agency). (1980) Appendix C-Guidelines and
methodology used in the preparation of health effect assessment chapters of the consent decree
water criteria documents. Federal Register 45:79347-79357.
U.S. EPA (Environmental Protection Agency). (1988) Recommendations for and
documentation of biological values for use in risk assessment. Environmental Criteria and
Assessment Office, Cincinnati, OH; EPA/600/6-87/008. Available online at
http://cfpub.epa. gov/ncea/cfm/recordisplay.cfm?deid=34855#Download. 064560
U.S. EPA (Environmental Protection Agency). (1994) Chemical assessments and related
activities (CARA). Office of Health and Environmental Assessment, Washington, DC;
EPA/600/R-94/904. Available online at
http://nepis.epa.eov/Exe/ZyPURL.cgi?Dockev=6000lG8L.txt. Accessed on 2/25/2010. 596444
U.S. EPA (Environmental Protection Agency). (1997) Exposure factors handbook (final report)
1997. U.S. Environmental Protection Agency, Washington, DC; EPA/600/P-95/002Fa-c.
Available online at http://cfpub.epa.gov/ncea/cfm/recordisplav.cfm?deid=12464. 594981
U.S. EPA (Environmental Protection Agency). (2005a) Guidelines for carcinogen risk
assessment. Risk Assessment Forum, Washington, DC; EPA/630/P-03/001F. Federal Register
70(66): 17765-17817. Available online at http://www.epa.gov/raf/publications/pdfs/CANCER
GUIDELINES FINAL 3-25-05.PDF. Accessed on 6/12/2010. 086237
U.S. EPA (Environmental Protection Agency). (2005b) Supplemental guidance for assessing
susceptibility from early-life exposure to carcinogens. Risk Assessment Forum, Washington,
DC; EPA/630/R-03/003F. Available online at
http://www.epa.gov/ttn/atw/childrens supplement finat.pdf. Accessed on 6/12/2010. 088823
53
3,3'-Dimethoxybenzidine
-------
FINAL
6-12-2013
U.S. EPA (Environmental Protection Agency). (2006) 2006 Edition of the drinking water
standards and health advisories. Office of Water, Washington, DC; EPA/822/R-06/013.
Washington, DC. Available online at
http://www.epa.gov/waterscience/drinking/standards/dwstandards.pdf. Accessed on 2/25/2010.
091193
U.S. EPA (Environmental Protection Agency). (2010) Health effects assessment summary
tables (HEAST). Prepared by the Office of Research and Development, National Center for
Environmental Assessment, Cincinnati OH for the Office of Emergency and Remedial Response,
Washington, DC. Available online at http://epa-heast.ornl.eov/. Accessed on 2/25/2010.
595422
U.S. EPA (Environmental Protection Agency). (2010) Integrated risk information system
(IRIS). Office of Research and Development, National Center for Environmental Assessment,
Washington, DC. Available online at http://www.epa.gov/iris/. Accessed on 2/25/2010. 595423
WHO (World Health Organization). (2010) Online catalogs for the Environmental Health
Criteria series. Available online at http://www.who.int/topics/environmental health/en/.
Accessed on 2/25/2010. 595424
Yoon, JS; Mason, JM; Valencia, R; et al. (1985) Chemical mutagenesis testing in Drosophila.
IV. Results of 45 coded compounds tested for the National Toxicology Program. Environ Mol
Mutagen 7(3):349-367. 194373
54
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