United States
Environmental Protection
1=1 m m Agency
EPA/690/R-08/013F
Final
8-27-2008
Provisional Peer Reviewed Toxicity Values for
3,3' -Dimethylbenzidine
(CASRN 119-93-7)
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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Acronyms and Abbreviations
bw	body weight
cc	cubic centimeters
CD	Caesarean Delivered
CERCLA	Comprehensive Environmental Response, Compensation and
Liability Act of 1980
CNS	central nervous system
cu.m	cubic meter
DWEL	Drinking Water Equivalent Level
FEL	frank-effect level
FIFRA	Federal Insecticide, Fungicide, and Rodenticide Act
g	grams
GI	gastrointestinal
HEC	human equivalent concentration
Hgb	hemoglobin
i.m.	intramuscular
i.p.	intraperitoneal
IRIS	Integrated Risk Information System
IUR	inhalation unit risk
i.v.	intravenous
kg	kilogram
L	liter
LEL	lowest-effect level
LOAEL	lowest-observed-adverse-effect level
LOAEL(ADJ)	LOAEL adjusted to continuous exposure duration
LOAEL(HEC)	LOAEL adjusted for dosimetric differences across species to a human
m	meter
MCL	maximum contaminant level
MCLG	maximum contaminant level goal
MF	modifying factor
mg	milligram
mg/kg	milligrams per kilogram
mg/L	milligrams per liter
MRL	minimal risk level
MTD	maximum tolerated dose
MTL	median threshold limit
NAAQS	National Ambient Air Quality Standards
NOAEL	no-ob served-adverse-effect level
NOAEL(ADJ)	NOAEL adjusted to continuous exposure duration
NOAEL(HEC)	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
p-OSF	provisional oral slope factor
p-RfC	provisional inhalation reference concentration
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p-RfD
provisional oral reference dose
PBPK
physiologically based pharmacokinetic
ppb
parts per billion
ppm
parts per million
PPRTV
Provisional Peer Reviewed Toxicity Value
RBC
red blood cell(s)
RCRA
Resource Conservation and Recovery Act
RDDR
Regional deposited dose ratio (for the indicated lung region)
REL
relative exposure level
RfC
inhalation reference concentration
RfD
oral reference dose
RGDR
Regional gas dose ratio (for the indicated lung region)
s.c.
subcutaneous
SCE
sister chromatid exchange
SDWA
Safe Drinking Water Act
sq.cm.
square centimeters
TSCA
Toxic Substances Control Act
UF
uncertainty factor
l^g
microgram
[j,mol
micromoles
voc
volatile organic compound
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PROVISIONAL PEER REVIEWED TOXICITY VALUES FOR
3,3'-DIMETHYLBENZIDINE (CASRN 119-93-7)
Background
On December 5, 2003, the U.S. Environmental Protection Agency's (EPA's) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1.	EPA's Integrated Risk Information System (IRIS).
2.	Provisional Peer-Reviewed Toxicity Values (PPRTV) used in EPA's Superfund
Program.
3.	Other (peer-reviewed) toxicity values, including:
~	Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR),
~	California Environmental Protection Agency (CalEPA) values, and
~	EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in EPA's Integrated Risk Information System (IRIS). PPRTVs are
developed according to a Standard Operating Procedure (SOP) and are derived after a review of
the relevant scientific literature using the same methods, sources of data and Agency guidance
for value derivation generally used by the EPA IRIS Program. All provisional toxicity values
receive internal review by two EPA scientists and external peer review by three independently
selected scientific experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the
multi-program consensus review provided for IRIS values. This is because IRIS values are
generally intended to be used in all EPA programs, while PPRTVs are developed specifically for
the Superfund Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a five-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV manuscripts conclude
that a PPRTV cannot be derived based on inadequate data.
Disclaimers
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and RCRA program offices are advised to carefully review the information provided
in this document to ensure that the PPRTVs used are appropriate for the types of exposures and
circumstances at the Superfund site or RCRA facility in question. PPRTVs are periodically
updated; therefore, users should ensure that the values contained in the PPRTV are current at the
time of use.
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It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
users are strongly encouraged to read the entire PPRTV manuscript and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center for OSRTI. Other EPA programs or external parties who may
choose of their own initiative to use these PPRTVs are advised that Superfund resources will not
generally be used to respond to challenges of PPRTVs used in a context outside of the Superfund
Program.
Questions Regarding PPRTVs
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) may 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), or OSRTI.
INTRODUCTION
No chronic RfD or RfC for 3,3'-dimethylbenzidine (o-tolidine) is available on IRIS (U.S.
EPA, 2007), in the Health Effects Assessment Summary Table (HEAST) (U.S. EPA, 1997) or in
the Drinking Water Standards and Health Advisories list (U.S. EPA, 2006). Although a cancer
assessment for 3,3'-dimethylbenzidine is not available on IRIS (U.S. EPA, 2007) or the Drinking
Water list (U.S. EPA, 2006), the HEAST (U.S. EPA, 1997) lists a cancer weight-of-evidence
classification of B2 and an oral slope factor of 9.2 per (mg/kg-day) for 3,3'-dimethylbenzidine,
based on increased incidence of mammary tumors in rats orally treated with the chemical for 30
days (Griswold et al., 1968). The source document for this cancer assessment was a Health and
Environmental Effects Profile (HEEP) for 3,3'-Dimethylbenzidine (U.S. EPA, 1987). The HEEP
and a subsequent Cancer Reportable Quantity document (U.S. EPA, 1988a) that derived an oral
slope factor of 27.4 per (mg/kg-day) from the same data are the only relevant documents
included on the Chemical Assessments and Related Activities (CARA) lists (U.S. EPA, 1991,
1994).
The International Agency for Research on Cancer (IARC, 1972a, 1987) has classified
3,3'-dimethylbenzidine in category Group 2B, possible human carcinogen, based on sufficient
evidence in animals and no data in humans. The National Toxicology Program (NTP, 2007)
Eleventh Report on Carcinogens lists 3,3'-dimethylbenzidine as reasonably anticipated to be a
human carcinogen based on sufficient evidence in animals. NTP (1991a) has studied the
carcinogenic potential of 3,3'-dimethylbenzidine as part of the Benzidine Dye Initiative (NTP,
1982), an intensive research program designed to evaluate the toxic and carcinogenic effects of
benzidine congeners and related dyes.
The American Conference of Governmental Industrial Hygienists (ACGIH, 2001, 2006)
categorized 3,3'-dimethylbenzidine in group A3, as a confirmed animal carcinogen with
unknown relevance to humans, but did not set a Threshold Limit Value (TLV) due to an absence
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of inhalation data. Based on its potential carcinogenicity, the National Institute for Occupational
Safety and Health (NIOSH, 2007) established a REL of 0.02 mg/m3 as a 60-minute ceiling for
3,3'-dimethylbenzidine. The Occupational Safety and Health Administration (OSHA, 2007)
does not have a PEL for 3,3'-dimethylbenzidine, but jointly published with NIOSH (1981) a
health hazard alert that concluded that this chemical may present a cancer risk to workers and
recommended that worker exposure be reduced to the lowest feasible level. CalEPA (2001) has
not derived a REL or cancer potency factor for 3,3'-dimethylbenzidine.
No Agency for Toxic Substances and Disease Registry (ATSDR, 2007) Toxicological
Profile or World Health Organization (WHO, 2007) Environmental Health Criteria document are
available for 3,3'-dimethylbenzidine. Literature searches were conducted from the 1960's
through December, 2006 for studies relevant to the derivation of provisional toxicity values for
3,3'-dimethylbenzidine. Databases searched included: TOXLINE (Special, including NTIS
subfile), MEDLINE (including PubMed cancer subset), BIOSIS, TSCATS/TSCATS2, CCRIS,
DART/ETIC, GENETOX, HSDB, RTECS and Current Contents.
REVIEW OF PERTINENT DATA
Human Studies
No information was located regarding effects of 3,3'-dimethylbenzidine in humans,
except as part of a mixture. Most occupational exposures to 3,3'-dimethylbenzidine involve
mixed exposure with benzidine and/or other biphenyl amine compounds (ACGIH, 2001).
Elevated risks for cancer of the urinary tract have been found in workers exposed to
combinations of 3,3'-dimethylbenzidine and benzidine (ACGIH, 2001). However, because
benzidine is a known human bladder carcinogen (IARC, 1972b, 1982, 1987), these data are not
helpful in assessing the carcinogenic potential of 3,3'-dimethylbenzidineper se (ACGIH, 2001).
Although 3,3'-dimethylbenzidine is structurally similar to (is a congener of) benzidine, there is
no indication that it is metabolized to benzidine (IARC, 1972a, NTP, 1991a).
As indicated above, most studies involving occupational exposure to 3,3'-
dimethylbenzidine included confounding exposure to benzidine. Workers in a study by Quellet-
Hellstrom and Rench (1996) were not exposed to benzidine, although they were still exposed to
other benzidine congeners at higher levels than to 3,3'-dimethylbenzidine. This study included a
cohort of 704 workers (585 men and 119 women) employed between mid-1965 and 1989 in an
arylamine production plant in Connecticut. The chemicals produced at the facility during this
time period (in order of production volume) were 3,3'-dichlorobenzidine, o-dianisidine and 3,3'-
dimethylbenzidine. Benzidine had been produced in the plant prior to mid-1965, but only
workers hired after June 15, 1965 and never exposed to benzidine were included in the cohort.
Cancer incidence data were collected by matching the cohort roster from the company medical
department with the following sources: cancer cases registered at the Connecticut Tumor
Registry (CTR), death certificates of deceased workers indicating cancer associated with death
and cancer cases reported by the employees via mail survey confirmed by attending physician.
There were 27 total cases of cancer identified, 23 among male workers (including 3 cases of non-
melanoma skin cancer that were not considered cases for this study) and 4 among female
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workers. Among male workers, there were statistically significant increases in standardized
incidence ratio (SIR) for bladder cancer (SIR = 8.3; 95% CI: 3.3 to 17.1) based on 7 observed
cases and testicular cancer (SIR=11.4; 95% CI: 1.4 to 41.1) based on 2 observed cases. The
male workers diagnosed with testicular cancer had no reported exposure to arylamines and one
had worked only 15 days, so these cancers were not considered to be exposure-related. The only
elevated SIR among female workers was for breast cancer, but this was not statistically
significant (3 cases; SIR=1.9; CI 95%: 0.4 to 5.6).
Workers were scored for cumulative exposure to arylamines and sorted into no-, low- and
high-exposure groups (Quellet-Hellstrom and Rench, 1996). Workers were also stratified
according to length of follow-up (<5 yrs or 5+ yrs). No bladder cancer was seen in workers with
less than 5 years of follow-up. Among those with greater than 5 years of follow-up, the risk of
bladder cancer increased with increasing exposure. The SIR increased from 0 in the no-exposure
group (based on 0 cases) to 6.4 (95% CI: 0.8-23.1) in the low-exposure group based on 2 cases
and 17.3 (95% CI: 5.6-40.5) in the high-exposure group based on 5 cases. The high-exposure
group in which the bladder cancer cases were concentrated comprised chemical operators who
worked with the arylamines over a long period of time and mechanics who came into close
contact with the chemicals when repairing equipment (short, intense exposures). The authors
noted that the average age of 52 years among those diagnosed with bladder cancer in this cohort
was relatively young for this disease (compared with 68 years for Connecticut men overall). All
of the bladder cancer cases were current or ex-smokers. Smoking is a known risk factor for
bladder cancer and was considered by the authors to probably have contributed to the bladder
cancer risk observed in this study. Due to the mixed chemical exposures and confounding effect
of smoking, it is not clear to what extent 3,3'-dimethylbenzidine may have contributed to the
observed bladder cancer risk in the exposed workers.
Animal Studies
Oral Exposure
NTP (1991a) performed short-term, subchronic and chronic oral studies in rats. In the
short-term study, groups of 5 male and 5 female 48-day old F344/N rats were exposed to 0, 600,
1250, 2500, 5000 or 7500 ppm of 3,3'-dimethylbenzidine dihydrochloride in drinking water for
14 days. Based on reported water consumption and body weight data and the molecular weight
of 3,3'-dimethylbenzidine dihydrochloride, doses of 3,3'-dimethylbenzidine can be estimated as
0, 40, 90, 111, 127 and 142 mg/kg-day in males and 0, 47, 75, 139, 150 and 189 mg/kg-day in
females. Animals were observed twice daily. Feed and water consumption were recorded by
cage (5 animals/cage) weekly and twice weekly, respectively. Body weight was recorded
initially and weekly thereafter. All animals were necropsied and the following organ weights
were measured: brain, heart, right kidney, liver, lung, right testicle and thymus. Complete
histopathological examinations were performed on all control animals, males exposed to 5000
ppm and females exposed to 7500 ppm. Based on the findings in these groups, selected tissues
were examined in the lower-dose groups.
All 5 males and 1/5 females in the 7500 ppm group died, as did 1/5 males at 5000 ppm
(NTP, 1991a). All deaths occurred by day 13 of the study. A specific cause of death was not
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reported, but deaths were probably related to marked decreases in water consumption and body
weight in the higher-dose animals. At 2500 ppm and above, the animals actually lost weight
during the study, leading to marked terminal body weight deficits of 37-61% compared to
controls. At 1250 ppm, the animals gained weight, but more slowly than controls, leading to
terminal body weights that were 11-14% lower than controls. There were corresponding dose-
related decreases in water consumption compared to controls in both males and females. The
deficit in water consumption was 25-30%) at 600 ppm and increased to 83-88%) at 7500 ppm.
Feed consumption data were not reported. Clinical observations included thinness and kyphosis
(hunched-back) in all treated groups, along with urine stains, skin cold to touch, rough hair coat,
ataxia and reddish discharge at eyes and nares in the 7500 ppm groups. Gross necropsy revealed
absence of body fat in the 5000 and 7500 ppm groups, small thymus gland in the 2500 ppm and
5000 ppm groups, and small seminal vesicles in the 7500 ppm males. Organ weight changes
were observed in the 2500, 5000 and 7500 ppm groups, but were considered by the researchers
to be secondary to the marked body weight changes in these groups. Histopathological
examination showed liver lesions in males at >2500 ppm and females at >5000 ppm, including
hepatocyte necrosis and brown pigmentation of the cells lining the hepatic sinusoids (incidence
data not reported). Other effects at >2500 ppm were increased severity of nephropathy and bone
marrow hypocellularity. Lymphocytic atrophy of the thymus, spleen and mandibular and
mesenteric lymph nodes, necrosis and vacuolation of adrenal cortical cells, focal acinar cell
hypertrophy of the pancreas and, in males; increased immature sperm forms in the testis and
epididymis were also reported, although dose levels for these effects were not specified. The
low dose of 600 ppm (40-47 mg/kg-day) is a LOAEL for clinical signs and a marked decrease in
water consumption in rats exposed for 14 days.
In the NTP (1991a) subchronic study, groups of 10 male and 10 female 55-day old
F344/N rats were exposed to 0, 300, 500, 1000, 2000 or 4000 ppm of 3,3'-dimethylbenzidine
dihydrochloride in drinking water for 13 weeks. The average amount of 3,3'-dimethylbenzidine
consumed per day can be estimated as 0, 16, 22, 44, 86 or 144 mg/kg-day in males and 0, 18, 27,
50, 100 or 266 mg/kg-day in females, calculated as described above for the 14-day study.
Animals were observed twice daily. Feed and water consumption were recorded by cage (5
animals/cage) weekly and twice weekly, respectively. Body weight was recorded initially and
weekly thereafter. At week 13, blood samples were collected from all surviving animals for
hematology and clinical chemistry analyses that included liver enzymes and thyroid hormones.
At necropsy, weights of the following organ weights were recorded: brain, heart, liver, lung,
right kidney, right testis and thymus. Complete histopathological examinations were performed
on all controls, the two high-dose groups (2000 and 4000 ppm), and all animals that died or were
killed moribund. Based on the findings in these groups, selected organs were examined in the
lower-dose groups.
All rats receiving 4000 ppm and 4/10 males and 3/10 females receiving 2000 ppm, died
before study termination (NTP, 1991a). Deaths occurred on weeks 2 through 4 in the 4000 ppm
group and weeks 6 through 13 in the 2000 ppm group. Deaths appear to have been related to
marked decreases in water consumption and body weight in the high-dose groups. Final mean
body weight of treated rats relative to controls was decreased 9-12%> in males and females at 300
ppm, with the deficit increasing to 42-48% at 2000 ppm. Water consumption relative to controls
was decreased 20% in males and 43% in females at 300 ppm, with the deficit increasing to 54-
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64% at 2000 ppm (based on data for weeks 7 and 13). Feed consumption data were not reported.
Clinical observations included red nasal exudate, thinness, stains on fur and urine stains in all
treatment groups, appearing as early as week 1 or 2 in the 2000 and 4000 ppm groups.
Hematology and clinical chemistry results are shown in Table 1. Hematological findings
included significant dose-related decreases in hematocrit and red blood cell count in males at
>1000 ppm and females at all dose levels. Hemoglobin was reduced only in females at 2000
ppm. Leukocytes were significantly increased in males and females at 1000 ppm. Clinical
chemistry changes included significant increases in serum sorbitol dehydrogenase (SDH) in all
male and female groups, alanine aminotransferase (ALT) in males at 1000 ppm and females at
2000 ppm, lactate dehydrogenase (LDH) in males at 1000 ppm and blood urea nitrogen (BUN)
in males at 2000 ppm. There were significant decreases in serum thyroxin (T4) levels in all male
and female treatment groups and significant decreases in serum triiodothyronine (T3) values in
all treated females. Serum levels of thyroid stimulating hormone (TSH) were not different from
controls.
Table 1. Significant Hematology and Clinical Chemistry Findings in Rats Treated with
3,3'-Dimethylbenzidine Dihydrochloride in Drinking Water for 13 Weeks (NTP, 1991a)
Males
0 mg/kg-day
16 mg/kg-day
22 mg/kg-day
44 mg/kg-day
86 mg/kg-day
Parameter
Hematocrit (%)
45.2  0.54a
48.2 0.76
44.8 0.74
40.3 0.79
40.4 0.57
Erythrocytes (10'/|iL)
8.78  0.094
9.15 0.132
8.59  1.151
7.9 0.146
7.9 0.105
Leukocytes (10 7|iL)
5.4 0.146
5.0 0.264
5.4 0.197
6.7 0.363
7.0  0.732 b
Lymphocytes (107|iL)
3.96 0.256
3.82 0.219
4.12 0.200
5.02  0.240b
5.57  0.510b
Monocytes (10 7|iL)
0.13 0.023
0.08 0.022
0.12 0.018
0.15 0.030
0.30  0.064b
BUN (mg/dL)
17.9 0.62
18.5 0.81
18.8 0.80
20.4 0.99
25.0  3.08b
LDH (IU/L)
590  49.47
762  59.90b
663  52.99
1018 36.62
623 53.88
SDH (IU/L)
8.7 0.616
13.8  0.854
26.5 4.145
32.7 2.511
14.3  1.498
ALT (mg/dL)
40  2.46
33  1.42
47 5.99
54  3.89b
43 5.85
T4 (ng/dL)
4.73 0.178
3.06  0.158
2.98 0.181
3.06 0.136
2.80 0.163






Females
0 mg/kg-day
18 mg/kg-day
27 mg/kg-day
50 mg/kg-day
100 mg/kg-day
Parameter
Hematocrit (%)
48.3 0.68
46.1 0.69
44.6  0.72
41.4 0.95
37.6 0.78
Hemoglobin (g/dL)
16.3 0.16
16.0 0.13
15.8 0.19
15.8 0.21
15.4  0.42b
Erythrocytes (10'/|iL)
8.86 0.098
8.06  0.262
8.13 0.131
7.58 0.175
7.07 0.173
Leukocytes (10 7|iL)
4.3 0.152
4.2 0.292
4.9 0.376
5.7 0.283
5.9  0.658b
Lymphocytes (107|iL)
3.25 0.116
3.64 0.284
4.11 0.334
4.50 0.186
4.86  0.409
SDH (IU/L)
5.5 0.654
9.4 0.581
16.0 2.708
14.8  1.504
13.0  1.612
ALT (mg/dL)
30  1.4
27  1.28
30 2.08
37 2.74
51 7.86
T3 (ng/dL
102.42 3.11
72.58 4.48
69.58 5.26
55.24 3.50
47.43  5.60
T4 (ug/dL)
2.50 0.091
1.87 0.092
1.77 0.131
1.69 0.087
1.95 0.198
a mean  standard error for groups of 10 animals.



significantly different (p<0.05) from control group by Dunn's or Shirley's test
/?<0.01

Gross necropsy revealed paucity of body fat and reddening of the glandular mucosa of the
stomach of treated rats (dose groups not specified) (NTP, 1991a). Histopathological examination
showed lesions in the liver (hepatocyte necrosis and brown pigmentation within sinusoidal lining
cells) and kidneys (nephropathy, karyomegaly of renal tubule epithelial cells); atrophy of the
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thymus, spleen, mandibular and mesenteric lymph nodes and bone marrow; pancreatic acinar
degeneration; and, in males, immature sperm forms in the testis and epididymis. The incidences
and/or severity (severity scores provided for renal lesions only) of all of these lesions were dose-
related (see Table 2). Although most of these effects were seen only in the 2000 and 4000 ppm
dose groups, liver and kidney lesions were seen in the lower-dose groups as well. The most
sensitive effect, pigmentation of liver sinusoid lining cells, was significantly increased in females
of all dose groups. The low dose of 300 ppm (16 mg/kg-day in males and 18 mg/kg-day in
females) in this 13-week rat study is a LOAEL for liver histopathology (pigmentation), serum
chemistry changes (increased SDH, decreased T3 and T4), hematological effects (decreased
hematocrit and RBC count), decreases in water intake and body weight, and clinical signs.
In the NTP (1991a) chronic study, 6-week old male and female F344/N rats were
exposed to 3,3'-dimethylbenzidine dihydrochloride in drinking water at concentrations of 0, 30,
70 or 150 ppm (NTP, 1991a). Although originally intended to be a 2-year study with interim
sacrifices at 9 and 14 months, the study was instead terminated at 14 months due to high
mortality among the treated rats. The study included a total of 70 rats/sex in the control group,
45/sex at 30 ppm, 75/sex at 70 ppm and 70/sex at 150 ppm. Of these, 10/sex in the control and
150 ppm groups were used for the scheduled sacrifice at 9 months. Time weighted average
(TWA) doses of 3,3'-dimethylbenzidine are estimated as 0, 1.3, 3.0 and 8.3 mg/kg-day in males
and 0, 2.2, 5.1 and 9.6 mg/kg-day in females, in the control, low-, mid- and high-dose groups,
respectively (dose estimates reported by NTP were adjusted for differences in molecular weight
between 3,3'-dimethylbenzidine and 3,3'-dimethylbenzidine dihydrochloride). The animals were
observed twice daily. Body weights were recorded initially, once weekly for 14 weeks, at week
17 and once per month thereafter. Feed consumption was measured one week/month, and water
consumption was measured one week per month in 3-day and 4-day segments. Clinical
observations were made at body weight determinations. Necropsy and complete histopathology
examination were performed on all animals. Organ weights (liver, kidney, brain), hematology,
clinical chemistry (including liver enzymes and thyroid hormones) and urinalysis were assessed
only in rats sacrificed at 9 months.
Only rats from the control and high-dose (150 ppm) groups were examined in the
scheduled sacrifice at 9 months (NTP, 1991a). In high-dose males and females sacrificed at 9
months, body weight was decreased 17-20% compared to controls. Both absolute and relative
liver weights were significantly increased about 2-fold in the high-dose rats of both sexes.
Smaller, but statistically significant, increases were seen in absolute and relative kidney weight
in both sexes. Hematology results showed significant decreases in hematocrit, hemoglobin and
red blood cell count in high-dose males and females (Table 3). Serum chemistry findings
suggested effects on the liver (significant increases in SDH and ALT in high-dose males and
females), kidney (significant increases in BUN and creatinine in high-dose males) and thyroid
(significant decreases in T4 and increases in TSH in high-dose males and females). Urine
volume was significantly and markedly decreased in high-dose groups of both sexes, with
corresponding increases in urine osmolality, specific gravity, protein concentration and
creatinine concentration.
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Table 2. Histological Lesions in Rats in 13-Week Drinking Water Studies of 3,3'-Dimethylbenzidine Dihydrochloride (NTP, 1991a)

Male

Female
Dose (mg/kg-day)
0
16
22
44
86
144

0
18
27
50
100
266
LIVER
hepatocyte necrosis
pigment
0/10
0/10
0/10
1/10
0/10
0/10
0/10
0/10
7/10g
10/10g
3/10
9/10

0/10
0/10
1/10
10/10
6/10
10/10
4/10f
10/10
7/10
9/10
7/9
8/9
KIDNEY
nephropathy
10/10
(l.l)a
b
10/10
(1.0)
10/10
(1.6)
10/10
(2.6)
10/10
(2.6)

2/10
(1.0)
5/10
(1.0)
10/10
(1.0)
10/10
(1.0)
10/10
(2.2)
7/9
(2.1)
karyomegalyc







0/10
0/10
0/10
7/10
9/10
0/9
THYMUS
atrophy
0/10


0/10
5/6
9/9

0/10


2/10
7/8
5/5
SPLEEN
atrophy
0/10


0/10
5/108
10/10

0/10


0/10
4/10f
9/9
MANDIBULAR LYMPH NODE
atrophy
0/10


0/10
7/10g
10/10

1/10


0/10
5/10
7/7
MESENTERIC LYMPH NODE
atrophy
0/10


0/10
1/10
2/9

0/10


0/10
4/10f
6/7
BONE MARROW
hypocellularity (atrophy)
0/10


0/10
8/108
10/10

0/10


0/10
10/10
9/9
PANCREAS
degeneration"1
0/10


0/10
4/10f
10/10

0/10



2/10
8/9
TESTES
immature sperm
0/10


1/10
3/10
7/10

NAe
NA
NA
NA
NA
NA
a values in parentheses are average severity grades for affected animals; l=minimal, 2=slight, 3=moderate
b organ not examined in animals at this dose level
c terminology preferred by Pathology Working Group for the lesion diagnosed as megalocytosis by the laboratory pathologist
d terminology preferred by Pathology Working Group for the lesion diagnosed as acinar hypertrophy by the laboratory pathologist
e not applicable
f significantly different (p<0.05) from the control group by Fisher exact test
/K0.01
8

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Table 3. Significant Hematology, Clinical Chemistry and Urinalysis Findings in
Rats Treated with 3,3'-Dimethylbenzidine Dihydrochloride in Drinking
Water for 9 Months (NTP, 1991a)


Males
Females
Parameter
0 mg/kg-day
8.3 mg/kg-day
0 mg/kg-day
9.6 mg/kg-day
Hematology




Hematocrit (%)
43.9  0.71a
35.5  1.06d
45.6 0.72
34.5  0.51d
Hemoglobin (g/dL)
16.6 0.16
15.1 0.34d
16.1 0.12
14.7  0.33d
Erythrocytes (10'/|iL)
8.61 0.13
6.90  0.22d
8.26 0.11
6.10  0.08d
Leukocytes (107|iL)
8.0 0.21
10.0  0.64d
4.4 0.39
7.2  0.72d
MCH (pg)b
19.3 0.20
22.0  0.40d
19.5 0.27
24.2  0.39d
MCHC (%)
37.6 0.42
42.7  0.73d
35.4 0.55
42.8  0.79d
MCV (u3)
51.0  0.18
51.5 0.32
55.1 0.23
56.4  0.19d
Serum Chemistry




BUN (mg/dL)
18.7  1.17
28.6  5.33
16.9 0.43
14.9  1.2ld
Creatinine (mg/dL)
0.69 0.05
1.00  0.13
0.64  0.02
0.65 0.02
Serum glucose (mg/dL)
147 5.6
150 5.8
120 3.2
190  18. ld
ALT (mg/dL)
73.3 9.87
85.1 4.78c
30.2 3.70
98.6  42.5d
SDH (IU/L)
14.8  1.90
31.1 3.86d
7.1  1.43
71.4  40.24d
T3 (ng/dL)
81.7 8.45
114.0  8.16
104.8 3.88
94.7  4.67
T4 (ug/dL)
3.5 0.26
2.2  0.30d
3.44 0.15
2.44  0.20d
TSH (ng/mL)
337.6 25.3
501.3 54.5C
321.1 23.4
486.2 73.6C
Urinalysis




Urine osmolality (mOSM/kg)
1350 271
2730  303
1568  246
2247 164
Osmolality ratio (urine/serum)
4.21 0.86
8.44  0.92
4.96 0.77
7.03  0.53
Urine creatinine (mg/dL)
163.7 32.1
271.2 26.0C
151.3 21.3
245.4  14.1d
Urine volume (mL/16 h)
7.3  1.24
2.5  0.53d
3.1 0.46
0.9  0.14d
Urine specific gravity
1.03 0.00
1.06  0.00d
1.04 0.00
1.06  0.00d
Urine protein (mg/dL)
51.00  10.69
300.00  0.00d
45.00  12.58
300.00  0.00d
Creatinine excretion rate
8.91 0.33
5.57  0.49d
3.71 0.32
2.07  0.32d
(mg/16 h)




a mean  standard error for groups of 10 animals.



rank transformed data analyzed.




0 significantly different (><0.05) from control group by Wilcoxon's test
d/?<0.01


Histopathological examination of high-dose rats at 9 months (NTP, 1991a) revealed
nonneoplastic (hepatocellular hypertrophy, fatty change, cystic degeneration) and neoplastic
(neoplastic nodules, hepatocellular carcinomas) effects in the liver, nonneoplastic effects in the
spleen (atrophy in males and females) and kidney (increased incidence in females and severity in
both sexes of nephropathy) and preneoplastic and neoplastic (malignant and benign) lesions in
the lung, skin, oral cavity, preputial/clitoral gland, small intestine, large intestine and Zymbal's
gland in both males and females (see Table 4). NTP commented that the short latency of
neoplastic effects was unusual and shows the carcinogenic potency of 3,3'-dimethylbenzidine.
Based on the non-neoplastic effects observed after 9 months of exposure, including
histopathology in the liver, spleen and kidneys, and hematology, serum chemisty, and urinalysis
changes, a subchronic LOAEL of 150 ppm (8.3-9.6 mg/kg-day) was identified for rats. A
NOAEL was not identified because lower-dose groups were not evaluated at 9 months.
9

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Table 4. Treatment-Related Lesions in Rats in the 9 Month Exposure Evaluation (NTP, 1991a)

Male
Female
Dose (mg/kg-day)
0
8.3
0
9.6
No. animals examined
10
10
10
10
LIVER




hepatocellular carcinoma
0
2
0
0
neoplastic nodule3
0
5e
0
1
hepatocyte hypertrophy
0
10f
0
10f
basophilic focus
0
10f
0
0
fatty changeb
1
10f
0
10f
cystic degeneration
0
7f
0
0
LUNG




alveolar/bronchiolar carcinoma
0
1
0
0
alveolar/bronchiolar adenoma
0
0
0
1
alveolar epithelium hyperplasia
0
7f
0
1
SKIN




basal cell carcinoma
0
1
0
0
sebaceous gland adenoma
0
1
0
0
squamous papilloma
0
0
0
1
ORAL CAVITY




squamous cell carcinoma
0
0
0
1
PREPUTIAL/CLITORAL GLAND




adenoma
0
1
0
2
carcinoma
0
2
0
3
SMALL INTESTINE




mucinous adenocarcinoma
0
2
0
0
LARGE INTESTINE




adenomatous polyp
0
3
0
0
ZYMBAL'S GLAND




carcinoma
0
2
0
3
adenoma
0
1
0
2
squamous papilloma
0
3
0
1
squamous hyperplasia
0
3
0
1
focal hyperplasia
0
1
0
0
KIDNEY




nephropathy0
10 (1.0)
10 (3.4)
3 (1.0)
10 (3.0)
SPLEEN




lymphoid atrophyd
0
10f
0
7f
aterm previously used for lesions currently classified as hepatocellular adenoma


b diagnosed as cytoplasmic vacuolization by the study pathologist


c values in parentheses are average severity grades; 1= minimal, 2= mild, 3= moderate, 4=marked

diagnosed as lymphoid depletion by the study pathologist



e significantly different (p<0.05) from the control group by Fisher exact test


'/KO.Ol




10

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8-27-2008
In the main (14-month) part of the study, dose-related increases in mortality were seen in
both sexes at all dose levels starting soon after the 9-month (36-week) mark (NTP, 1991a). All
high-dose males were dead by week 55, at which time fewer than 25% of high-dose females
remained alive. Survival to the end of the study at 14 months was significantly reduced in mid-
and high-dose males and females and marginally reduced even in low-dose males (p=0.06) and
females (p=0.05). The early deaths resulted from tumor formation. Body weight gain was
reduced throughout the study in high-dose males and mid- and high-dose females. At study
termination, body weights were reduced by about 30% in high-dose males and 20-25% in mid-
and high-dose females, compared with controls. Water intake compared to controls was reduced
20-30% in high-dose males for the first 13 weeks of the study and in high-dose females for the
first year of the study. In both groups, water consumption was higher than controls over the last
few weeks of the study (increased 135% in males and 40% in females). Data on food intake
were not reported.
Nonneoplastic lesions occurred in the liver in males and females from all treated groups
(NTP, 1991a). As summarized in Table 5, significant increases were found for cystic
degeneration and fatty change, as well as foci of cellular alteration (basophilic, eosinophilic and
mixed-cell foci; possible precursors to neoplasms) and hematopoietic cell proliferation
(presumably secondary to inflammation associated with neoplasms). The hepatotoxicity was
considered by NTP to be of mild severity. The other noteworthy nonneoplastic effect was a
dose-related increase in severity of nephropathy in males (incidence at or near 100% in all
groups, minimal-to-mild severity in controls and low- and mid-dose males and moderate-to-
marked in high-dose males) and in incidence and severity of nephropathy in females (incidence
78%) in controls and close to 100% in all treated groups, severity minimal-to-mild in control and
low-dose females and moderate in mid- and high-dose females). Based mainly on cystic
degeneration and foci of cellular alteration in the liver, this study identified a LOAEL of 30 ppm
(1.3 mg/kg-day in males and 2.2 mg/kg-day in females) for chronic toxicity in rats.
Table 5. Incidence of Nonneoplastic Liver Lesions in Rats in 14-month Exposure Evaluation
(NTP, 1991a)

Male
Female
Dose (mg/kg-day)
0
1.3
3.0
8.3
0
2.2
5.1
9.6
cystic degeneration
0/60
24/45a
67/75a
5 l/60a
0/60
3/45
12/74a
1 l/60a
focal or multifocal necrosis
3/60
4/45
10/75
5/60
0/60
3/45
7/74a
2/60
fatty change
1/60
2/45
1/75
7/60a
0/60
0/45
4/74
2/60
basophilic focus
1/60
3 l/45a
54/75a
27/60a
0/60
13/45a
ll/74a
3/60
eosinophilic focus
0/60
0/45
57/75a
53/60a
0/60
7/45a
57/74a
38/60a
mixed cell focus
0/60
37/45a
54/75a
30/60a
0/60
34/45a
49/74a
32/60a
hematopoietic cell proliferation
0/60
2/45
27/75a
15/60a
0/60
7/45a
19/74a
8/60a
" /?<().05 by Fisher exact test, based on effective rates
11

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Tissue masses were observed on the head, back and ventral posterior area of the rats after
as few as 24 weeks (NTP, 1991a). These masses represented primarily Zymbal's gland tumors,
epithelial skin tumors and preputial/clitoral gland tumors, respectively, all of which were found
at significantly increased incidence in treated rats (see Table 6). Significant increases in tumor
incidence also occurred in the liver, oral cavity, small and large intestine, mammary gland, lung,
brain and mesothelium. While the tumor increases occurred primarily in the mid- and high-dose
groups, the most sensitive tumors (basal cells in males, Zymbal's and clitoral gland tumors in
females) were increased in the low-dose groups as well. NTP concluded there was clear
evidence of carcinogenic activity of 3,3'-dimethylbenzidine in male and female F344/N rats in
this study.
A previous cancer study in rats included a group of twenty 45-day-old Sprague-Dawley
females that was administered 3,3'-dimethylbenzidine by gavage in sesame oil, in 10 doses of 50
mg/rat (total dose 500 mg/rat) at 3 day intervals over 30 days, followed by a 9-month
observation period (Griswold et al., 1968). Using the reference body weight for female Sprague-
Dawley rats of 0.204 kg (U.S. EPA, 1988b), it can be estimated that this dosing regimen
provided an average dose of approximately 81 mg/kg-day of 3,3'-dimethylbenzidine over the 30
day dosing period. A negative control group (pooled from several experiments) comprised 140
young female Sprague-Dawley rats given the sesame oil vehicle alone. Endpoints evaluated
included morbidity/mortality checks twice daily, body weight and inspection for abnormal tissue
masses weekly, gross necropsy and histological examination of mammary tissue, intestinal tract,
pituitary, liver, ovaries, adrenals and any gross lesions. Of the 20 rats exposed to 3,3'-
dimethylbenzidine, 16 survived the 9 month observation period and were necropsied; 3 of these
16 rats (19%) had mammary lesions. The lesions identified by microscopic examination in these
3 rats were 4 carcinomas and 1 hyperplasia. In the control group, 132/140 rats survived to
necropsy. Of these, 5 (4%) had mammary lesions (3 carcinomas, 1 fibroadenoma and 1
hyperplasia). The difference in the incidences of treated and control rats with mammary lesions
is statistically significant (p=0.04; Fisher exact test conducted for this review). Identification of
a NOAEL or LOAEL for systemic toxicity is precluded by inadequate reporting of non-
neoplastic effects.
A cancer bioassay in mice was conducted by Schieferstein et al. (1989). There were 7
groups of 120 male and 120 female BALB/c mice that were exposed to 0, 5, 9, 18, 35, 70 or 140
ppm of 3,3'-dimethylbenzidine dihydrochloride in drinking water. Average doses of 3,3'-
dimethylbenzidine (calculated from dose estimates for 3,3'-dimethylbenzidine dihydrochloride at
specific time periods in the paper, and adjusted for differences in molecular weight between 3,3'-
dimethylbenzidine and 3,3'-dimethylbenzidine dihydrochloride) were 0, 0.4, 0.8, 1.5, 2.8, 7.4 and
11 mg/kg-day in males and 0, 0.4, 0.7, 1.4, 2.6, 5.4 and 11 mg/kg-day in females. Sacrifices
were scheduled for weeks 13, 26, 39, 52, 78 and 116 (24/sex/dose at each time, except
8/sex/dose at week 39 and 16/sex/dose at week 52). Endpoints evaluated included body weight
and water consumption (averaged over three 4-week periods), survival and histopathological
examination of 40 selected tissues. The probable cause of death or morbidity was determined for
each dead or moribund animal. Body weight was similar to controls in males and females of all
dose groups throughout the study. Water consumption was also similar to controls in all groups,
except high-dose males, which had 10-20% lower water intake than controls throughout the
study. There was no dose-related effect on mortality in male or female mice. The number of
12

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Table 6. Treatment-Related Neoplastic Lesions in Rats in the 14-month Exposure Evaluation (NTP,
1991a)a

Male
Female
Dose (mg/kg-day)
0
1.3
3.0
8.3
0
2.2
5.1
9.6
SKIN
keratoacanthoma
sebaceous gland adenoma
basal cell adenoma
basal cell carcinoma
basal cell adenoma or carcinoma
squamous cell papilloma
squamous cell carcinoma
squamous cell papilloma or carcinoma
1/60
0/60
0/60
0/60
0/60
0/60
0/60
0/60
1/44
0/44
10/44"
1/44
11/44"
0/45
2/45
2/45
8/67"
7/72"
52/72"
4/68
54/72"
8/72"
10/74"
17/74"
5/27"
5/49"
29/45"
2/43
30/45"
15/55"
13/59"
27/59"
0/60c
0/60c
0/60
0/60
0/60
0/60
0/60
0/60
0/45
0/45
3/45
0/45
3/45
1/45
2/45
3/45
0/75
1/75
5/64"
5/69"
10/69"
6/72"
4/64
9/72"
1/60
1/60
5/41"
4/46"
9/46"
5/55"
7/41"
12/55"
ZYMBAL'S GLAND
adenoma
carcinoma
adenoma or carcinoma
1/60
0/60
1/60
1/44
2/45
3/45
13/72"
21/74"
32/74"
16/54"
23/60"
36/60"
0/60
0/60
0/60
4/45"
2/45
6/45"
11/72"
22/74"
32/74"
12/57"
35/59"
42/59"
PREPUTIAL GLAND (tf) or CLITORAL
GLAND (?)
adenoma
carcinoma
adenoma or carcinoma
2/60
0/60c
2/60
4/44
0/45
4/44
4/72
2/75
6/72
8/49"
1/60
9/49"
0/60
0/60
0/60
9/45"
5/45"
14/45"
32/73"
11/72"
42/73"
17/58"
18/55"
32/58"
LIVER
neoplastic nodule
hepatocellular carcinoma
neoplastic nodule or hepatocellular carcinoma
0/60
0/60
0/60
0/44
0/45
0/45
29/72"
12/72"
35/72"
26/49"
12/55"
33/55"
0/60
0/60c
0/60
0/45
0/45
0/45
7/58"
1/74
7/58"
3/36"
1/60
4/36"
ORAL CAVITY
squamous cell papilloma
squamous cell carcinoma
squamous cell papilloma or carcinoma
0/60c
0/60c
0/60
0/45
0/45
0/44
3/75
1/75
4/67
2/60
3/60
5/32"
0/60
0/60
0/60
3/45
1/45
3/45
7/73"
2/64
9/73"
9/59"
4/41"
13/59"
SMALL INTESTINE
adenomatous polyp
adenocarcinoma
adenomatous polyp or adenocarcinoma
0/60c
0/60
0/60
0/45
0/45
0/45
1/75
3/74
4/74
1/60
8/59"
8/59"
0/60c
0/60
0/60
1/45
0/45
1/45
1/75
2/72
3/72
0/60
5/57"
5/57"
LARGE INTESTINE
adenomatous polyp
adenocarcinoma
adenomatous polyp or adenocarcinoma
0/60
0/60
0/60
0/44
0/45
0/45
6/67"
0/67
6/67"
9/38"
7/36"
15/38"
0/60
0/60c
0/60
1/45
0/45
1/45
6/70"
1/75
7/70"
4/46"
1/60
4/46"
MAMMARY GLAND
Adenocarcinoma
--
--
--
--
0/60
1/45
3/71
6/51"
LUNG
alveolar/bronchiolar adenoma
alveolar/bronchiolar carcinoma
alveolar/bronchiolar adenoma or carcinoma
1/60
0/60c
1/60
0/45
0/45
0/45
7/73
1/75
8/73"
6/57"
0/60
6/57"
1/60
0/60c
1/60
1/45
0/45
1/45
3/63
0/74
3/63
3/41
1/60
4/41
MESOTHELIUM (ALL ORGANS)
mesothelioma (benign/malignant)
0/60
0/45
3/67
4/38"
--
--
--
--
" incidence based on effective rates (denominator is number of animals alive at first occurrence of tumor type in any dose group), except as noted
b /?	0.05 by Fisher exact test
c only overall rates reported by NTP and reproduced here
13

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8-27-2008
dead/moribund mice ranged from 17-25 in male groups and 15-21 in female groups (including
controls). The primary cause of death was neoplasms in both males and females. In males, there
was a statistically significant trend for increased death due to neoplasms with increased dose.
Histopathological examination revealed that among male mice found dead or moribund there
was a dose-related increase in the incidence of lung alveolar cell adenomas or adenocarcinomas
(see Table 7). Pairwise comparisons showed a significant increase in the high-dose group and
marginally significant increases in the 35- and 70-ppm groups (0.05 
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8-27-2008
Table 7. Lung Alveolar Cell Adenomas or Adenocarcinomas Observed in BALB/c Mice Exposed
to 3,3'-Dimethylbenzidine Dihydrochloride in Drinking Water (Schieferstein et al., 1989)
Dose (mg/kg-day)
Males
0
0.4
0.8
1.5
2.8
7.4
11
13 week sacrifice
0/24a
0/24
0/24
0/24
0/24
0/24
0/24
26 week sacrifice
0/23
1/24
1/24
0/23
0/23
0/24
0/24
39 week sacrifice
0/8
0/8
1/8
0/8
2/8
0/8
0/8
52 week sacrifice
1/15
3/16
1/14
5/14
2/15
4/16 (1)
2/16
78 week sacrifice
11/23
4/20
8/18
8/23 (2)
5/18
7/21
8/20
112 week sacrifice
3/10 (1)
5/10 (3)
0/4
6/10
3/8 (1)
4/7 (1)
4/7 (1)
Found dead or moribund
5/16 (2)
7/16 (2)
5/25 (2)
5/18 (2)
7/24 (6)
11/20 (5)
13/20 (10)b

Females
0
0.4
0.7
1.4
2.6
5.4
11
13 week sacrifice
0/24
0/24
0/24
1/24
0/24
0/24
0/24
26 week sacrifice
0/24
0/24
0/24
0/24
0/24
1/24
0/24
39 week sacrifice
0/8
0/8
1/8
0/8
0/8
1/8
0/8
52 week sacrifice
0/16
1/15
2/16
1/13
3/16
0/16
4/16 (1)
78 week sacrifice
4/21
1/23
8/20 (1)
5/21 (1)
4/20
2/21
5/18 (2)
112 week sacrifice
1/7
2/8
4/9
4/5 (2)
5/11 (3)
5/10
3/11 (1)
Found dead or moribund
7/19 (5)
4/17 (3)
3/19 (3)
4/20 (2)
5/17 (2)
4/15 (2)
4/18 (2)
a incidence reported for adenomas or adenocarcinomas combined; number of mice with adenocarcinomas shown in ()
bp< 0.05
Other Studies
Carcinogenicity Studies (Parenteral Exposure)
As summarized by U.S. EPA (1987) and NTP (1991a), early cancer studies found that
3,3'-dimethylbenzidine was carcinogenic in rats when administered by chronic weekly
subcutaneous injections or subcutaneous implantation (Pliss, 1963; Pliss and Zabezhinsky, 1970;
Spitz et al., 1950). These studies were generally limited by small numbers of animals, lack of
concurrent controls and use of toxic doses (NTP, 1991a). Tumors were induced in various body
tissues distant from the site of administration, including Zymbal gland, mammary gland,
preputial gland, forestomach, skin, liver, lungs, external auditory canal and/or hematopoietic
system. Increased incidences of mammary and lung tumors also occurred in the offspring of
mice injected subcutaneously during gestation (Golub et al., 1974).
15

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Toxicity and Carcinogenicity of Related Compounds
There is in vivo evidence in humans, rats and dogs indicating that some 3,3'-
dimethylbenzidine-based azo dyes are metabolized to 3,3'-dimethylbenzidine (ACGIH, 2001;
Boeniger, 1978; NIOSH, 1981; NTP, 1991a). Rats treated with one of these dyes, C.I. Acid Red
114, in the drinking water for up to two years developed a similar array of tumors as found for
3,3'-dimethylbenzidine (NTP, 1991b). Trypan blue and Evans blue, two 3,3'-
dimethylbenzidine-based dyes, were carcinogenic in rats when injected subcutaneously or
intraperitoneally (IARC 1975a,b, 1987). 3,3'-Dimethylbenzidine-based dyes (trypan blue, Evans
blue, benzopurpurin 4B) had no effect on testicular development in male offspring of pregnant
mice treated with 1000 mg/kg-day doses by gavage on days 8-12 of gestation (Gray and Ostby,
1993). The offspring were examined 44-50 days after birth (post-puberty) and/or 86-87 days
after birth (young adult).
Genotoxicity
The genotoxicity of 3,3'-dimethylbenzidine has been evaluated in a variety of test
systems. Based on results of studies summarized by U.S. EPA (1987), NTP (1991a), and Chung
et al. (2006), as well as more recent studies (Claxton et al., 2001; Oda, 2004), 3,3'-
dimethylbenzidine is genotoxic in both bacteria and eukaryotes. Extensive testing in Salmonella
typhimurium has shown that 3,3'-dimethylbenzidine produces reverse mutations in frameshift-
sensitive strains (e.g., TA98 and TA1538) with metabolic activation; response was weak or
negative in the absence of metabolic activation or in tester strains designed to detect base-pair
substitutions (e.g., TA100 and TA1535; Reid et al., 1984) or that are sensitive to reactive oxygen
species (e.g., TA102; Makena and Chung, 2007). Also in bacteria, 3,3'-dimethylbenzidine
produced positive results in a growth differential test using repair-deficient and proficient strains
of Escherichia coli. In eukaryotes, 3,3'-dimethylbenzidine produced positive results with or
without metabolic activation in tests for forward mutation in mouse L5178Y lymphoma cells,
DNA repair in isolated rat and hamster hepatocytes, unscheduled DNA synthesis in HeLa cells
and sister chromatid exchange and chromosomal aberrations in cultured Chinese hamster ovary
cells. 3,3'-Dimethylbenzidine also produced positive results in a cell transformation assay using
Fischer rat embryo cells. In vivo assays in mice for micronucleus formation in bone marrow and
inhibition of DNA synthesis in the testis also were positive. In vivo studies with Drosophila
found that 3,3'-dimethylbenzidine induced sex-linked recessive lethal mutations when
administered by feeding or injection, but did not induce reciprocal translocations.
Using the NIH 3T3 DNA transfection assay, Reynolds et al. (1990) analyzed tumors
(both benign and malignant) from several tissues of control and treated rats from the NTP
carcinogenicity bioassays for 3,3'-dimethylbenzadine dihydrochloride (NTP, 1991a) for the
presence of activated ras oncogene (specifically H-ras or N-ra.v), While spontaneous tumors in
control animals had a very low frequency of oncogene activation (1/38), 81% (13/16) of the
tumors from 3,3'-dimethylbenzidine-treated animals1 contained activated oncogenes. Southern
blot analysis was used to identify the activated oncogenes as primarily H-ras oncogenes (12/13
1 The researchers do not distinguish between tumors from animals treated with 3,3'-dimethylbenzadine
dihydrochloride and those treated with C.I. Acid Red 114. They appear to have considered animals from both
groups together as 3,3'-dimethylbenzidine exposed.
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tumors) in the tumors from animals treated with 3,3'-dimethylbenzidine. Mutations at codons
12, 13 and 61 were identified in the altered H-ras oncogenes from these tumors.
Oda (2004) showed, using the SOS/umu assay system, that the human acetyltransferase
encoded by the gene NAT1 was primarily responsible for activating 3,3'-dimethylbenzidine to a
genotoxic intermediate. Strains of S. typhimurium overexpressing NAT1 (strain NM6001) and
NAT2 (strain NM6002) were tested for induction of umuC gene expression, expressed as an
increase in P-galactosidase activity in culture. The 3,3'-dimethylbenzidine treatment did not
cause gene induction in the parent strain (NM6000), which lacked acetyltransferase activity; in
contrast, dose-dependent induction was observed in the NM6001 strain. Gene induction in the
NM6002 strain was much lower, indicating that the NAT1 enzyme was more important than
NAT2 in the production of DNA damage.
Role of Metabolism in Carcinogenicity
Metabolism is believed to play an important role in the carcinogenic action of benzidines,
many of which are known to be metabolized to DNA-reactive metabolites (Morgan et al. 1994).
However, little information is available on the metabolism of 3,3'-dimethylbenzidine. Dieteren
(1966) analyzed the urine of workers engaged in the manufacture of 3,3'-dimethylbenzidine (o-
tolidine) and reported detection of diacetylated and hydroxylated metabolites, as well as the
parent compound. None of the compounds detected in urine were quantified. Mongrel dogs
given intraperitoneal doses of 70 mg/kg 3,3'-dimethylbenzidine excreted about 40% of the dose
in the urine, with about 4% excreted as parent compound; the balance was reported to consist of
"the conjugated form" without further identification (Sciarini and Meigs, 1961). Rats treated
with 3,3'-dimethylbenzidine excreted N-acetyl 3,3'-dimethylbenzidine, N,N'-diacetyl-
dimethylbenzidine and parent compound in the urine (Tanaka et al. 1982, as cited in NTP,
1991a).
Metabolism of 3,3'-dimethylbenzidine may be similar to that of the related compound
benzidine. ATSDR (2001) and Morgan et al. (1994) reviewed the available data on metabolism
of benzidine, and ATSDR (2001) provides a thorough discussion of the role of metabolism in the
carcinogenicity of benzidine. Although the metabolism of benzidine is quite complex, the three
major reactions are N-acetylation, N-oxidation and N-glucuronidation. Each of these pathways
leads to reactive metabolites that interact with DNA (ATSDR, 2001; Morgan et al., 1994). A
number of known metabolites of benzidine are mutagenic, more so than the parent compound;
mono- and diacetylated metabolites have been shown to be about 10 times as mutagenic as
benzidine, while N-hydroxy-N,N'-diacetylbenzidine glucuronide is about 100 times as
mutagenic (after release of the glucuronide moiety) (Morgan et al., 1994). N-acetylated
benzidine DNA adducts have been observed in rodents and in humans (ATSDR, 2001).
Species differences in the metabolism of benzidine have been observed both in vivo and
in vitro (ATSDR, 2001). For example, studies using liver slices showed that dog liver does not
acetylate benzidine, in contrast to both rat and human liver. Rat liver produced more N,N'-
diacetylbenzidine, while human liver produced more N-acetylbenzidine. Both in vivo and in
vitro data indicate that dogs produce glucuronidated metabolites of benzidine (ATSDR, 2001).
Species differences in metabolic pathways are believed to contribute to tumor site specificity that
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differs between species (e.g., benzidine is associated with liver tumors in rats but with bladder
tumors in dogs and humans) (ATSDR, 2001). It is not known whether these differences in
metabolism and tumor site specificity also extend to 3,3'-dimethylbenzidine, as data on its
metabolism is sparse and there are no data with which to clearly identify human tissues
susceptible to 3,3'-dimethylbenzidine carcinogenesis.
FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC
ORAL RfD VALUES FOR 3,3'-DIMETHYLBENZIDINE
No data were located regarding nonneoplastic health effects of 3,3'-dimethylbenzidine in
humans. Pertinent data on nonneoplastic effects in animals are available only from the NTP
(1991a) subchronic and chronic studies of 3,3'-dimethylbenzidine dihydrochloride in rats.
Subchronic RfD
In the subchronic NTP (1991a) study, rats were exposed to 0, 300, 500, 1000, 2000 or
4000 ppm in drinking water for 13 weeks. The low dose of 300 ppm (16 mg/kg-day in males
and 18 mg/kg-day in females) was a LOAEL for effects on the liver indicated by histopathology
(brown pigmentation in sinusoidal lining cells) and serum chemistry changes (increased SDH),
thyroid (indicated by decreases in serum T3 and T4) and blood (decreases in red blood cell count
and hematocrit). General toxicity at this dose was also indicated by decreases in water intake
(20-43%) and body weight (9-12% lower than controls) and the presence of clinical signs of
toxicity (thinness, urine stains, red nasal exudate), although the extent of occurrence of clinical
signs at this dose was not reported. At higher doses, there were also effects on the kidneys
(nephropathy), spleen (atrophy), bone marrow (atrophy), thymus (atrophy), lymph nodes
(atrophy), pancreas (degeneration) and testis (immature sperm). Early deaths occurred at 2000
ppm (86-100 mg/kg-day) and above.
The chronic NTP (1991a) study included interim sacrifices in rats exposed to 0 or 150
ppm in drinking water (8.3 mg/kg-day in males and 9.6 mg/kg-day in females) for 9 months,
which represents a subchronic duration. Effects were similar to those observed in the 13-week
study: hepatotoxicity indicated by histopathology (hepatocellular hypertrophy, fatty change,
cystic degeneration) and serum chemistry (increased SDH and ALT), nephrotoxicity indicated by
histopathology (nephropathy) and serum chemistry (increased BUN and creatinine), hypothyroid
indicated by changes in serum hormone levels (decreased T4 and increased TSH), anemia
(decreased red blood cell count, hemoglobin and hematocrit), splenic atrophy and reduced body
weight (17-20%) lower than controls). In addition, neoplastic effects were found in both males
and females, including malignant and benign tumors in the liver, lung, skin, oral cavity,
preputial/clitoral gland, intestines and Zymbal's gland.
The NTP (1991a) studies of 3,3'-dimethylbenzidine do not provide a suitable basis for
deriving a subchronic p-RfD, because sufficiently low doses were not employed. The low dose
in the 13-week study (16-18 mg/kg-day) was a LOAEL that produced a variety of toxic effects,
including overt generalized effects on body weight and clinical signs of toxicity. The 9-month
sacrifice in the chronic study found many of the same toxic effects as the 13-week study,
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including a large reduction in body weight, at half the dose of the 13-week study (8.3-9.6 mg/kg-
day). These data suggest that a threshold for nonneoplastic effects of 3,3'-dimethylbenzidine
could occur at far lower doses than were tested for subchronic exposure. Therefore, it would not
be appropriate to base a risk assessment value for non-carcinogenic end points on these data.
Chronic RfD
In the chronic NTP (1991a) study, rats were exposed to 0, 30, 70 or 150 ppm in drinking
water for 14 months. This study was terminated at 14 months due to high mortality in all treated
groups associated with tumor formation. Significant increases in tumor incidence were found for
skin, Zymbal's gland, preputial/clitoral gland, liver, oral cavity, small and large intestine,
mammary gland, lung and mesothelium, primarily in the 70 and 150 ppm groups, but also in the
30 ppm males (basal cell adenoma) and females (Zymbal's gland adenoma, clitoral gland
adenoma and carcinoma). Nonneoplastic effects unrelated to tumor formation occurred in the
liver (cystic degeneration) and kidney (nephropathy). The low dose of 30 ppm (1.3 mg/kg-day
in males and 2.2 mg/kg-day in females) was a LOAEL for both effects.
The 1.3-2.2 mg/kg-day chronic LOAEL did not clearly approach a threshold for
nonneoplastic effects due to a high incidence of liver cystic degeneration in males (53%
compared to 0% in controls) at the lowest tested dose in the study. Additionally, tumors and
related mortality occurred at this dose. Therefore, because the available chronic data do not
identify a NOAEL and suggest that a threshold for nonneoplastic effects could occur at far lower
doses than were tested, it is not appropriate to derive a chronic p-RfD for 3,3'-
dimethylbenzidine.
FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION RfC VALUES FOR 3,3'-DIMETHYLBENZIDINE
Derivation of RfC values for 3,3 '-dimethylbenzidine is precluded by a lack of inhalation
toxicity data.
PROVISIONAL CARCINOGENICITY ASSESSMENT
FOR 3,3'-DIMETHYLBENZIDINE
Weight-of-Evidence Descriptor
Information on the carcinogenicity of 3,3'-dimethylbenzidine in humans mainly consists
of studies of occupational exposure that included confounding exposure to benzidine. Risks for
urinary tract cancer were increased in workers with mixed exposure to 3,3'-dimethylbenzidine
and benzidine (ACGIH, 2001), but because benzidine is a known human bladder carcinogen
(IARC, 1972b, 1982, 1987), the data are insufficient for determining whether 3,3'-
dimethylbenzidine alone is carcinogenic. In arylamine production workers exposed to 3,3'-
dimethylbenzidine and other benzidine congeners, but not to benzidine, Quellet-Hellstrom and
Rench (1996) found a significant exposure-related increase in bladder cancer. No conclusions
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regarding the carcinogenicity of 3,3'-dimethylbenzidine alone can be drawn from this study due
to the mixed benzidine congener exposure and because all bladder cancer cases were current or
ex-smokers (smoking is a known risk factor for bladder cancer that probably contributed to the
observed cancer risk).
There is no indication that 3,3'-dimethylbenzidine is metabolized to benzidine (IARC,
1972a; NTP, 1991a), but concern for the carcinogenicity of 3,3'-dimethylbenzidine in humans is
raised by its structural similarity to benzidine, as well as human and animal data indicating that
some carcinogenic 3,3'-dimethylbenzidine-based azo dyes are metabolized to 3,3'-
dimethylbenzidine (ACGIH, 2001; Boeniger, 1978; IARC, 1975a,b; NIOSH, 1981; NTP, 1991a,
1991b).
There is sufficient evidence of 3,3'-dimethylbenzidine carcinogenicity in experimental
animals based on results of oral bioassays in rats and mice (Griswold et al., 1968; NTP, 1991a;
Schieferstein et al., 1989). In the NTP (1991a) study, rats that were exposed to 3,3'-
dimethylbenzidine dihydrochloride in drinking water showed development of malignant and
benign tumors in the liver, lung, skin, oral cavity, preputial/clitoral gland, intestines and
Zymbal's gland after only 9 months, with no tumors of any type in controls at 9 months. This
was a planned 24-month study that was terminated at 14 months due to high tumor-related
mortality. At 14 months, significant increases in tumor incidence were found for skin, Zymbal's
gland, preputial/clitoral gland, liver, oral cavity, small and large intestine, mammary gland, lung,
and mesothelium. NTP (1991a) concluded that this bioassay presented clear evidence of
carcinogenic activity in male and female F344/N rats. Rats (Sprague-Dawley) that were exposed
to 3,3'-dimethylbenzidine by gavage on 3 days/week for 30 days and subsequently observed for 9
months, had a significantly increased incidence of mammary tumors (Griswold et al., 1968). A
two-year drinking water study of 3,3'-dimethylbenzidine dihydrochloride in mice found an
increased incidence of lung alveolar cell adenomas or adenocarcinomas in males found dead or
moribund during the study, although not in males killed at scheduled times or in females
(Schieferstein et al., 1989). Lifetime dietary studies of 3,3'-dimethylbenzidine in hamsters
reported no evidence of carcinogenicity, but were limited by small group sizes, use of single
dose levels, examination of only a few tissues and insufficient reporting (Saffiotti et al., 1967;
Sellakumar et al., 1969). Supporting evidence for the carcinogencity of 3,3'-dimethylbenzidine
in animals is provided by limited subcutaneous studies in rats and mice that found systemic
tumor induction in a wide variety of target tissues (Golub et al., 1974; Pliss, 1963; Pliss and
Zabezhinsky, 1970; Spitz et al., 1950) and oral studies of a 3,3'-dimethylbenzidine-derived dye
that found similar results (NTP, 1991b).
The weight of the evidence is adequate to demonstrate carcinogenic potential to humans.
3,3'-Dimethylbenzidine has tested positive for carcinogenicity by the oral route in animal studies
in more than one species, sex, strain and site, and these findings are supported by results of
subcutaneous injection studies of 3,3'-dimethylbenzidine and oral studies of a 3,3'-
dimethylbenzidine-derived dye. Additionally, 3,3'-dimethylbenzidine is structurally similar to
(is a congener of) benzidine, a known human carcinogen. In accordance with the U.S. EPA
(2005a) cancer guidelines, the weight-of-evidence indicates that 3,3'-dimethylbenzidine is likely
to be carcinogenic to humans.
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Mode of Action Discussion
The U.S. EPA (2005a) Guidelines for Carcinogen Risk Assessment defines mode of
action as a sequence of key events and processes starting with the 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 include mutagenic, mitogenic, anti-apoptotic
(inhibition of programmed cell death), cytotoxic with reparative cell proliferation and
immunologic suppression.
There are no data on the carcinogenicity of 3,3'-dimethylbenzidine in humans. Available
evidence in laboratory animals indicates that oral exposure to 3,3'-dimethylbenzidine leads to
tumors in a wide variety of tissues including skin, Zymbal's gland, preputial/clitoral gland, liver,
oral cavity, small and large intestine, mammary gland, lung and mesothelium. A weight of
evidence evaluation supports a determination that 3,3'-dimethylbenzidine is carcinogenic by a
mutagenic mode of action. Determination of the mode of action of carcinogens is addressed in
Section 5 of the 2005 Cancer Supplementary Guidance (U.S. EPA, 2005b) as follows:
"Determinations of chemicals that are operating by a mutagenic mode of action entails
evaluation of test results for genotoxic endpoints, metabolic profiles, physiochemical properties,
and structure-activity relationships." Evaluation of each of these elements is discussed below in
the mode of action discussion.
Mutagenic Mode of Action
Key Events  The proposed mode of action for 3,3'-dimethylbenzidine carcinogenicity
consists of the following key events: 1) metabolism to DNA-reactive metabolites, 2) binding to
DNA, 3) mutation of ras and possibly other oncogenes, and 4) proliferation of initiated cells.
Most of the support for this proposed mode of action is derived from the study published by
Reynolds et al. (1990), coupled with data from in vitro genotoxicity testing and from studies of
the related compounds, 3,3'-dimethoxybenzidine and benzidine. Reynolds et al. (1990) showed
codon-specific mutations, primarily in the H- ras oncogene, in a wide variety of rat tumors
induced by 3,3'-dimethylbenzidine2. In addition, a large majority of both benign and malignant
tumors (81%) from rats treated with 3,3'-dimethylbenzidine contained activated H- ras or N- ras
oncogenes, while only 1 of 38 spontaneous tumors from control rats contained activated
oncogenes. The much higher incidence of activated ras oncogenes and mutational specificity at
codons 12, 13 and 61 provide support for a genotoxic mode of action for the benign and
malignant tumors observed in rats treated with 3,3'-dimethylbenzidine.
Additional support for a mutagenic mode of action is provided by in vitro genotoxicity
tests, as described in more detail above in the Genotoxicity section. 3,3'-Dimethylbenzidine has
consistently given positive results in genotoxicity testing in bacteria and eukaryotes. In bacteria,
metabolic activation is required to obtain positive mutagenicity results, but in mammalian cells,
2 Tumors were from rats treated with 3,3'-dimethylbenzidine dihydrochloride or C.I. Acid Red 114, a dye derived
from 3,3'-dimethylbenzidine. Available data indicate that benzidine dyes are cleaved via azo reduction by intestinal
flora to release the parent benzidine molecule (in this case, 3,3 '-dimethylbenzidine), which is then taken up by the
gastrointestinal tract. Thus, exposure to the dye is believed to result in risks comparable to those associated with
exposure to the parent benzidine compound (Morgan et al., 1994).
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3,3'-dimethylbenzidine produced positive results with or without metabolic activation in tests for
gene mutation, DNA repair, unscheduled DNA synthesis, sister chromatid exchange and
chromosomal aberrations (U.S. EPA, 1987; NTP, 1991a). In vivo assays for micronucleus
formation and inhibition of DNA synthesis in mice and mutations in Drosophila were also
positive (U.S. EPA, 1987; NTP, 1991a).
There is also evidence for a mutagenic mode of action for the structurally similar
compound benzidine. A number of known metabolites of benzidine are mutagenic, more so than
the parent compound: mono- and diacetylated metabolites have been shown to be about 10 times
as mutagenic as benzidine, while N-hydroxy-N,N'-diacetylbenzidine glucuronide is about 100
times as mutagenic (after release of the glucuronide moiety) (Morgan et al., 1994). DNA
adducts with N-acetylated benzidine derivatives have been observed in both rodents and in
humans (ATSDR, 2001).
Strength, Consistency, Specificity of Association  Data from Reynolds et al. (1990)
show codon-specific mutations in the ras oncogene in a large majority of the tumors from
animals treated with 3,3'-dimethylbenzidine or its derivative dye C.I. Acid Red 114 in NTP
(1991a, 199b) cancer bioassays. The high incidence of these mutations (in 12/13 tumors with
activated ras oncogene) and the high incidence of ras gene activation (13/16 tumors) in tumors
from treated animals, in contrast with the very low incidence of oncogene activation in
spontaneous tumors from control animals (1/38) provides support for the role of mutation in the
ras oncogene as a precursor to tumor formation in rats treated with 3,3'-dimethylbenzidine. A
similar finding of ras oncogene activation (21/34 tumors) and mutations in these three codons
(19/21 tumors with activated ras oncogene) was observed in tumors from rats treated with 3,3'-
dimethoxybenzidine or its derivative dye C.I. Direct Blue 15 (Reynolds et al., 1990), providing
additional support for the importance of ras gene activation via mutation in the tumorigenicity of
these compounds.
Additional evidence for the association between mutagenesis and tumor formation results
from the observation that 3,3'-dimethylbenzidine exposure caused tumors in a wide variety of rat
tissues, including skin, Zymbal's gland, preputial/clitoral gland, liver, oral cavity, small and large
intestine, mammary gland, lung, and mesothelium, many of which are sites at which tumors are
infrequently found in F344 rats (NTP, 1991a) and also caused tumors in exposed mice
(Schieferstein et al., 1989). Induction of tumors at multiple sites and in different species is
characteristic of carcinogens acting via mutagenesis (U.S. EPA, 2005a). In addition, the short
latency-to-tumor formation (a high incidence was observed in rats after only 9 months, while no
tumors were observed in controls at this time; NTP, 1991a) is suggestive of a mutagenic effect.
Dose-Response Concordance  No data with which to evaluate the dose-response
concordance between mutagenesis and tumor formation after 3,3'-dimethylbenzidine exposure
are available. Reynolds et al. (1990) did not report the dose distribution of mutations or
activated oncogenes in the tumors evaluated. Furthermore, the high incidence of tumors in all
dose groups of the NTP (1991a) rat bioassay may have obscured a dose-response relationship for
mutation and/or oncogene activation.
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Temporal Relationships  In rats exposed to 3,3'-dimethylbenzidine, tumors were
observed in a significant fraction of the exposed animals after only 9 months of exposure (no
tumors were observed in controls at this time), and the bioassay was terminated at 14 months due
to high tumor-related mortality (NTP, 1991a). There are no data from any studies on the
incidence or types of mutations induced prior to tumor formation in tissues subsequently
developing tumors; thus, the temporal relationship between mutagenesis and the development of
tumors cannot be assessed.
Biological Plausibility and Coherence  The biological plausibility of a mutagenic
mode of action for 3,3'-dimethylbenzidine is supported by evidence of mutations leading to ras
oncogene activation in tumors from rats treated with 3,3'-dimethylbenzidine (Reynolds et al.,
1990). This study provides the critical link between in vitro evidence for mutagenicity and
tumor formation in a specific species. Similar findings with the related compound 3,3'-
dimethoxybenzidine and the lack of oncogene activation in spontaneous tumors from untreated
rats (Reynolds et al., 1990) augment the database supporting this particular mode of action for
benzidine congeners. Evidence for a variety of mutagenic metabolites observed both in vitro and
in vivo after exposure to the structurally similar carcinogen benzidine supports the plausibility of
this mode of action. In addition, in a study of known carcinogenic aryl amines that included
benzidine, the extent and persistence of measured DNA adducts in Beagles given oral doses of a
series of these compounds correlated with their potency to form bladder tumors (Beland et al.,
1983), providing further support for the relationship between mutagenesis and tumor formation
for aryl amines in general.
Early-Life Susceptibility  According to the Supplemental Guidance for Assessing
Susceptibility from Early-Life Exposure to Carcinogens (Supplemental Guidance) those exposed
to carcinogens with a mutagenic mode of action are assumed to have increased early-life
susceptibility. Data on 3,3 '-dimethylbenzidine are not sufficient to develop separate risk
estimates for childhood exposure. There are no data comparing the turnorigenicity of 3,3'-
dimethylbenzidine after exposure during early life with tumorigenicity after exposure during
adulthood. Exposure to 3,3'-dimethylbenzidine was commenced at about 6 weeks of age and
continued through adulthood in the chronic bioassays in rats (NTP, 1991a; Griswold et al.,
1968); the age at which exposure was commenced in the one bioassay in mice was not clearly
reported, but was apparently after 4 weeks of age (Scheiferstein et al., 1989). Limited
information from the structurally-related compound benzidine provides some toxicokinetic
plausibility for age-dependent differences in susceptibility to benzidine carcinogenesis. Human
expression of N-acetyl transferase 2 (NAT2) and glucuronosyl transferase (UGT), two enzymes
involved in benzidine metabolism, varies developmentally, with adult activity being reached at
1-3 years of age (NAT2) and 6-18 months (UGT). However, the importance of these two
enzymes in the metabolic pathways for 3,3'-dimethylbenzidine and in the formation of critical
DNA-reactive metabolites after exposure to this congener is not known. Oda (2004) found that
the NAT1 enzyme was more important than NAT2 in the production of DNA damage from 3,3'-
dimethylbenzidine in the SOS/umu assay system.
Conclusions  A weight of evidence evaluation supports a mutagenic mode of action
for 3,3'-dimethylbenzidine tumorigenicity. In vitro studies provide evidence that 3,3'-
dimethylbenzidine is capable of eliciting genotoxic effects in both bacteria and eukaryotic cells.
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More importantly, analysis of tumors from rats exposed to 3,3-dimethylbenzidine showed codon-
specific mutations in ras oncogenes in a large majority of tumors, while only one spontaneous
tumor from a control rat contained an activated ras oncogene (Reynolds et al., 1990). Finally,
the finding of tumors at multiple sites and in multiple species, as well as the brief latency to
tumor formation, provide additional support for a mutagenic mode of action for 3,3'-
dimethylbenzidine. Because a mutagenic mode of action for carcinogenicity is proposed for
3,3'-dimethylbenzidine, a linear approach would be appropriate to extrapolate from the point of
departure in the derivation of the oral slope factor (U.S. EPA, 2005a). There are no data with
which to develop separate estimates of risk from childhood exposure to 3,3'-dimethylbenzidine.
An important uncertainty in the quantitative cancer assessment for 3,3'-
dimethylbenzidine stems from the limited information on its metabolism. Although metabolic
activation may be a critical step in the cancer mode of action for 3,3 '-dimethylbenzidine, the
important DNA-reactive metabolites have not been established. Information on the closely
related compound benzidine suggests species variations in metabolism and DNA-reactive
metabolites that lead to species differences in tumor site (ATSDR, 2001) and possibly to species
differences in potency. Because there are few data on metabolites of 3,3'-dimethylbenzidine in
different species, and insufficient data on human tumors associated with 3,3'-dimethylbenzidine
exposure, it is not known whether these species variations also pertain to 3,3'-dimethylbenzidine.
Quantitative Estimates of Carcinogenic Risk
Oral Exposure
In the NTP (1991a) chronic study, 6-week old male and female F344/N rats were
exposed to 3,3'-dimethylbenzidine dihydrochloride in drinking water at concentrations of 0, 30,
70 or 150 ppm, which are equivalent to estimated time-weighted average doses of 0, 1.3, 3.0, and
8.3 mg/kg-day in males and 0, 2.2, 5.1, and 9.6 mg/kg-day in females, in the control, low-, mid-,
and high-dose groups respectively. Cancer dose-response modeling for 3,3'-dimethylbenzidine
was performed using the most prominent tumors: basal cell tumors (adenoma or carcinoma) in
the skin of male rats, squamous cell tumors (papilloma or carcinoma) in the skin of male rats,
liver tumors (neoplastic nodules or hepatocellular carcinoma) in male rats, Zymbal's gland
tumors (adenoma or carcinoma) in male and female rats, and clitoral gland tumors (adenoma or
carcinoma) in female rats. Incidences used for dose-response modeling were based on the
number of animals alive at first occurrence of the tumor being modeled in any dose group, as
reported by NTP (1991a).
In addition, dose-response modeling was performed for all significantly increased skin
tumors combined in male and female rats and for all significantly increased tumors (across sites)
combined in male and female rats. Combined skin tumors were modeled because significant
increases were found for six different tumor types in the skin (keratoacanthoma, sebaceous gland
adenoma, basal cell adenoma, basal cell carcinoma, squamous cell papilloma and squamous cell
carcinoma). Although distinct from each other in terms of the specific cell type/location affected
and the degree of development of the neoplasm, the fact that all of these tumor types were
increased suggests a nonspecific effect on the skin. Therefore, it appeared reasonable to estimate
overall skin cancer risk by modeling the combined skin tumor incidence data.
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Combined incidence of all significantly increased tumors across sites was modeled for a
similar reason. 3,3'-Dimethylbenzidine produced significant increases in tumor incidence in
many tissues, including skin, Zymbal's gland, preputial/clitoral gland, liver, oral cavity, small
intestine, large intestine, mammary gland, lung and mesothelium. The lack of tissue specificity
of the neoplastic response, which is characteristic of a mutagenic mode of action, means that the
overall risk of tumor formation is spread throughout the body. Because all of these tumors
contribute to the overall cancer risk, dose-response assessment based on any one type will
underestimate risk of developing cancer. Therefore, it is reasonable to estimate overall cancer
risk for this chemical based on combined tumor incidence. Combining incidence data for
significantly increased tumors across sites is an appropriate way to estimate risk for carcinogens
that produce tumors at multiple sites (U.S. EPA, 2005a).
The combined incidence data (effective rates) were extracted from the individual animal
data reported by NTP (1991a). Although this method of estimating overall tumor risk can
sometimes underestimate risk by inflating the control tumor incidence, that was not an issue for
3,3'-dimethylbenzidine, at least for females, as combined tumor incidence in female controls was
0/60 for skin tumors and 1/60 for all significantly increased tumors. All tumor incidence data
used for dose-response modeling are shown in Table 8.
In accordance with the U.S. EPA (2005a) cancer guidelines, the BMDLio (lower bound
on dose estimated to produce a 10% increase in tumor incidence over background) was estimated
using the U.S. EPA (2000) benchmark dose methodology. The incidence data were analyzed
using the multistage model available in the BMDS program (version 1.4.1) developed by U.S.
EPA. The polydegree was chosen as the lowest degree polynomial providing an adequate fit to
the data, as indicated by the chi-square goodness-of-fit test producing a/>value greater than or
equal to 0.1. The high-dose group was dropped where necessary to achieve an adequate fit. Risk
was calculated as extra risk. Confidence bounds were calculated by the BMDS software using a
maximum likelihood profile method.
Modeling results are shown in Table 8. An adequate fit was achieved for all tumor types
except combined skin tumors in males, in most cases after dropping the high-dose group. In both
males and females, the lowest BMDLio values were for combined tumors, with the value in
females being approximately half that in the males.
Human equivalent doses (BMDLio hed) were calculated for each animal BMDLio using
U.S. EPA's cross-species scaling factor of body weight raised to the 3/4 power (U.S. EPA,
2005a). Using this scaling factor, the straight dose (mg) in humans is obtained by multiplying
the straight animal dose (mg) by the ratio of human:animal body weight raised to the 3/4 power.
For doses expressed per unit body weight (mg/kg or mg/kg-day), the relationship is reciprocal
and the human dose (mg/kg) is obtained by multiplying the animal dose (mg/kg) by the ratio of
animal:human body weight raised to the 1/4 power. The BMDLio hed represents the chronic
daily dose (mg/kg-d) expected to result in 10% extra risk for tumor development extrapolated
from the animal bioassay data. The BMDLio hed values are shown in Table 8.
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Table 8. BMD Modeling of Cancer Incidence (Effective Rates) in Rats Treated with 3,3'-Dimethylbenzidine in Drinking Water for 14 Months (NTP,
1991a)
Tumor Type
Sex
Dose (mg/kg-day)
Poly-
degree
x2
/j-valuc
Benchmark Dose (mg/kg-day)
0
1.3
3
8.3
BMD10
BMDL10
BMDL10 iikd"
Skin
Basal cell adenoma or carcinoma
male
0/60
11/44
54/72
30/45
2b
1.00
0.74
0.36
0.098
Squamous cell papilloma or carcinoma
male
0/60
2/45
17/74
27/59
1
0.65
1.42
1.12
0.30
Combined skin tumors
male
1/60
13/44
61/74
41/59
NA
NA
NA
NA
NA
Liver
Neoplastic nodule or hepatocellular carcinoma
male
0/60
0/45
35/72
33/55
3b
0.29
1.66
1.44
0.39
Zymbal's gland
Adenoma or carcinoma
male
1/60
3/45
32/74
36/60
2b
0.35
1.35
0.94
0.26
Combined sites0
Combined tumors
male
5/60
19/45
73/74
58/60
2b
0.14
0.53
0.35
0.095

0
2.2
5.1
9.6

Skin
Combined skin tumors
female
0/60
6/45
19/72
21/54
1
0.94
1.87
1.48
0.35
Zymbal's gland
Adenoma or carcinoma
female
0/60
6/45
32/74
42/59
1
0.45
0.93
0.78
0.18
Clitoral gland
Adenoma or carcinoma
female
0/60
14/45
42/73
32/58
lb
1.00
0.63
0.50
0.12
Combined sites'1
Combined tumors
female
1/60
25/45
70/74
58/59
1
0.22
0.23
0.19
0.045
a human cancer equivalent dose of the BMDL10 calculated as: animal BMDL10 x (Wanjmai / Whlmlan)1/4 where Whuman = 70 kg (human reference body weight) and Wa^mai = 0.384 kg
for male rats and 0.218 kg for female rats (time weighted average body weights for the low-dose group in the study)
b high dose group dropped
c includes all sites with statistically increased tumor incidences (skin, zymbal's gland, preputial gland, liver, oral cavity, small intestine, large intestine, lung and mesothelium)
d includes all sites with statistically increased tumor incidences (skin, zymbal's gland, clitoral gland, liver, oral cavity, small intestine, large intestine, and mammary gland), as
well as the lung (marginal increase in females, but presumed treatment-related due to statistical increase in males)
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An oral slope factor calculated from adult exposure is derived from the BMDLio hed, the
95% lower bound on the human equivalent exposure associated with a 10% extra cancer risk, by
dividing the risk (as a fraction; in this case 0.1) by the BMDLio hed, and represents an upper
bound risk estimate for continuous lifetime exposure without consideration of increased early-
life susceptibility due to the 3,3'-dimethylbenzidine mutagenic mode of action (see below).
Since a linear, mutagenic mode of action has been implicated for 3,3'-dimethylbenzidine-induced
tumors, a linear extrapolation to low doses was performed. In order to linearly extrapolate
cancer risks from the BMDLio hed to the origin, a cancer oral slope factor was calculated as the
ratio 0.1/BMDLio hed- Taking the BMDLio hed of 0.045 mg/kg-day for combined tumors in
female rats as the point of departure, a provisional unadjusted oral slope factor of 2.2 (mg/kg-
day)"1 is calculated as follows:
p-OSF(unadjusted) 0.1/ BMDLio HED
= 0.1 / 0.045 mg/kg-day
= 2.2 (mg/kg-day)"1
An adjustment was used for shorter-than-lifetime observation period (U.S. EPA, 1980).
The NTP (1991a) bioassay was terminated after only 14 months (compared to the reference rat
life span of 24 months), due to early mortality associated with tumor formation. In the NTP
(1991a) study, a short duration of observation was imposed by the development of tumors
however, it was not clear that a sufficient period of time had elapsed to fully evaluate the
carcinogenicity of 3,3'-dimethylbenzidine in the low-dose treated rats. Specifically, as illustrated
in Table 8, the combined tumor incidence in male and female rats of the mid- and high-dose
treatment groups approached almost 100%, whereas there was an approximate 50% tumor
incidence in the low-dose treatment group. Due to the truncated experimental protocol in the
NTP (1991a) study it is not known how an increased duration (i.e. the full 2-year lifetime
exposure) may have influenced the tumor incidence in the low-dose treated rats. Therefore, an
adjustment factor of (L/Le)3 was applied to the unadjusted p-OSF, where L = the lifetime of the
animal and Le = the duration of experimental dosing. Using this adjustment, a provisional oral
slope factor of 11 (mg/kg-day)"1 is derived as follows:
P-OSF = P"0SF(unadjusted) x (L/Le)3
= 2.2 (mg/kg-day)"1 x (24 months/14 months)3
= 11 (mg/kg-day)"1
The oral slope factor for 3,3'-dimethylbenzidine should not be used with exposures
exceeding the point of departure (BMDLio hed = 0.045 mg/kg-day), because above this level the
fitted dose-response model better characterizes what is known about the carcinogenicity of 3,3'-
dimethylbenzidine. For exposures exceeding the point of departure, the uncertainty in risk
associated with the OSF may be significantly increased.
The human equivalent dose was also calculated for the central estimate associated with
the selected point of departure, the BMDiofor combined tumors in female rats (0.23 mg/kg-day).
A BMD io hed of 0.054 mg/kg-day was calculated. The	slope of the linear
extrapolation from the central estimate (0.054 mg/kg-day) is 1.8 (mg/kg-day)"1 and the adjusted
slope is 9 (mg/kg-day)"1.
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A weight of evidence evaluation has concluded that 3,3 '-dimethylbenzidine is
carcinogenic by a mutagenic mode of action. According to the Supplemental Guidance for
Assessing Susceptibility from Early-Life Exposure to Carcinogens (U.S. EPA, 2005b) those
exposed to carcinogens with a mutagenic mode of action are assumed to have increased early-life
susceptibility. Data on 3,3'-dimethylbenzidine are not sufficient to develop separate risk
estimates for childhood exposure. The oral slope factor of 11 (mg/kg-day)"1 calculated from data
from adult exposure does not reflect presumed early-life susceptibility for this chemical, 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 are given in Section
6 of the Supplemental Guidance (U.S. EPA, 2005b) which establishes ADAFs for three specific
age groups. The current ADAFs and their age groupings are 10 for <2 years, 3 for 2 to <16
years, and 1 for 16 years and above. The 10-fold and 3-fold adjustments in slope factor are to be
combined with age-specific exposure estimates when estimating cancer risks from early life (<16
years of age) exposure to 3,3'-dimethylbenzidine. These ADAFs and their age groups were
derived from the 2005 Supplemental Guidance (U.S. EPA, 2005b), and they may be revised over
time. The most current information on the application of ADAFs for cancer risk assessment can
be found at www.epa.gov/cancerguidelines/. In estimating risk, EPA recommends using age-
specific values for both exposure and cancer potency; for 3,3'-dimethylbenzidine, age-specific
values for cancer potency are calculated using the appropriate ADAFs. A cancer risk is derived
for each age group and these are summed across age groups to obtain the total risk for the
exposure period of interest.
Inhalation Exposure
There are no human or animal carcinogenicity data from which to derive an inhalation
unit risk for 3,3'-dimethylbenzidine.
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