United States
Environmental Protection
1=1 m m Agency
EPA/690/R-12/032F
Final
12-03-2012
Provisional Peer-Reviewed Toxicity Values for
oToluidine
(CASRN 95-53-4)
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
Chris Cubbison, PhD
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
ICF International
9300 Lee Highway
Fairfax, VA 22031
PRIMARY INTERNAL REVIEWERS
Audrey Galizia, Dr PH
National Center for Environmental Assessment, Washington, DC
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	10
Oral Exposures	10
Inhalation Exposures	10
Subchronic-duration Studies	10
Chronic-duration Studies	10
Developmental Studies	10
Reproductive Studies	10
Carcinogenicity Studies	10
ANIMAL STUDIES	11
Oral Exposures	11
Subchronic-duration Studies	12
Chronic-duration/Carcinogenic Studies	13
Developmental and Reproduction Studies	16
Inhalation Exposures	16
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)	16
Short-term Studies	16
Other Studies	17
DERIVATION OI PROVISIONAL VALUES	22
DERIVATION OI ORAL REFERENCE DOSE	22
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)	22
Derivation of Chronic Provisional RfD (Chronic p-RfD)	25
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS	25
CANCER WEIGHT-OF-EVIDENCE (WOE) DESCRIPTOR	25
MODE-OF-ACTION DISCI SSION	26
Summary of Mutagenicity and Genetic Toxicology Studies	26
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES	26
Derivation of Provisional Oral Slope Factor (p-OSF)	26
Derivation of Provisional Inhalation Unit Risk (p-IUR)	29
APPENDIX A. PROVISIONAL SCREENING VALUES	30
APPENDIX B. DATA TABLES	32
APPENDIX C. BMD OUTPUTS	38
APPENDIX D. REFERENCES	43
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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
NOAELrec
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
o-TOLUIDINE (95-53-4)
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 (http://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
0/7/70-Toluidine (o-toluidine; also referred to as 2-methylaniline or 2-aminotoluene) is a
synthetic aromatic amine with amine and methyl groups in an ortho (1,2) configuration.
o-Toluidine is used as an intermediate in the production of dyes, pigments, and rubbers (IARC,
2000). The empirical formula for o-toluidine is C7H9N (see Figure 1). A table of
physicochemical properties is provided below (see Table 1).
CH.
Figure 1. o-Toluidine Structure
Table 1. Selected Physicochemical Properties Table
(o-Toluidine)a
Property (unit)
Value
Boiling point (°C)
200.3
Melting point (°C)
-16.3
Density (g/cirf)
0.9984
Vapor pressure (kPa at 20 °C)
0.013
Solubility in water (mg/L at 25°C)
15
Relative vapor density (air = 1)
3.72
Molecular weight (g/mol)
107.16
Flash point (°C)
85
Octanol/water partition coefficient (unitless)
1.32
aValues obtained from IARC (2000).
No RfD, RfC, or cancer assessment for o-toluidine is included in the EPA's IRIS
database (U.S. EPA, 2012a) or on the Drinking Water Standards and Health Advisories List
(U.S. EPA, 2006). No RfD or RfC values have been reported in the Health Effects Assessment
Summary Tables (HEAST; U.S. EPA, 2012b). The Chemical Assessments and Related
Activities (CARA) list (U.S. EPA, 1994a) includes a listing for a Health and Environmental
Effects Profile (HEEP) for o-toluidine. The toxicity of o-toluidine has not been reviewed by the
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ATSDR (2008). The World Health Organization (WHO, 2011) has not produced an
Environmental Health Criteria Document for o-toluidine but did produce a Concise International
Chemical Assessment Document (WHO, 1998). CalEPA (2008) has not derived acute toxicity
values for o-toluidine. The American Conference of Governmental Industrial Hygienists
(ACGIH) has derived an 8-hour time-weighted average (TWA) exposure limit of 2 ppm
(8.8 mg/m3) along with a skin notation for o-toluidine (ACGIH, 2008). The National Institute of
Occupational Safety and Health (NIOSH, 2010) has not established a Recommended Exposure
Limit (REL) but considers o-toluidine to be a potential occupational carcinogen that may be
absorbed through skin. NIOSH set an immediately dangerous to life or health (IDLH) value for
o-toluidine at 50 ppm (220 mg/m3). The Occupational Safety and Health Administration
(OSHA, 2010) has derived a TWA Permissible Exposure Level (PEL) of 5 ppm (22 mg/m3) for
o-toluidine.
The HEAST (U.S. EPA, 201 lb) does not include a cancer assessment for o-toluidine, but
HEEP (U.S. EPA, 1984) provides a potency factor of 0.24 (mg/kg-day) 1 for oral exposure. The
International Agency for Research on Cancer (IARC, 2010) has classified o-toluidine as a
Group 1 agent (Carcinogenic to Humans) based on sufficient evidence in humans and animals.
o-Toluidine is included in the 12th Report on Carcinogens (NTP, 2011) and is listed as
Reasonably Anticipated to be a Human Carcinogen. CalEPA (2008) has derived an inhalation
slope factor of 1.8 x 10 1 per mg/kg-day, an inhalation unit risk of 5.1 x 10 5 per (J,g/m3, and an
oral slope factor of 1.8 x 10 1 per mg/kg-day for o-toluidine.
Literature searches were conducted on sources published from 1950s through
November 2011 for studies relevant to the derivation of provisional toxicity values for
o-toluidine, CASRN 95-53-4. 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 also searched for health-
related information: 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 an overview of the database for o-toluidine and includes all potentially
relevant repeated subchronic- and chronic-duration studies. Entries for the principal studies are
bolded. Unless otherwise qualified, the phrase "significant" means "statistical significance" with
ap-wdXut of less than 0.05).
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Table 2. Summary of Potentially Relevant Data for o-Toluidine (CASRN 95-53-4)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAEL'
BMDL/
BMCLa
LOAELab
Reference
(Comments)
Notes0
Human
1. Oral (mg/kg-d)a
Subchronic
ND
Chronic
ND
Developmental
ND
Reproductive
ND
Carcinogenicity
ND
2. Inhalation (mg/m3)a'd
Subchronic
ND
Chronic
ND
Developmental
ND
Reproductive
ND
Carcinogenicity
1643 male and
106 female (total 1749),
retrospective cohort,
from <5-yr to over 20-yr
exposure to o-toluidine
with or without aniline
Categories were
"definitely
exposed," "possibly
exposed," and
"probably not
exposed"
Bladder cancer
standardized incidence ratio [SIR]
(90% confidence interval [CI])
Definitely exposed:
6.48 (3.04-12.2)
Duration of employment
<5 yr = 0
5 to <10 yr= 8.8 (0.45-41.7)
>10 yr = 27.2 (11.8-53.7)
p < 0,001 for linear trend
NA
Not run
NA
Ward et al.
(1991)

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Table 2. Summary of Potentially Relevant Data for o-Toluidine (CASRN 95-53-4)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry"
Critical Effects
NOAEL'
BMDL/
BMCLa
LOAELab
Reference
(Comments)
Notes0
Carcinogenicity
Not reported but
apparently the same
cohort as listed above
was used
Not reported
19 additional cases of bladder cancer in
the same cohort from 1973 to 1988
NA
Not run
NA
Markowitz and
Levin (2004)


1643 male and
106 female (total 1749),
retrospective cohort,
from <5 yr to over 20 yr;
bladder cancer incidence
was determined from
December 31, 1988 to
December 31, 1994 (re-
analysis of Ward et al.,
1991)
Categories were
"definitely
exposed," "possibly
exposed," and
"probably not
exposed"
Bladder cancer
SIR (95% CI)
Definitely exposed:
5.84 (2.91-10.45)*
Duration of employment
<5 yr= 1.25 (0.03-6.97)
5-<10 yr= 3.67 (0.09-20.44)
>10 yr = 11.09 (5.07-21.05)*
*p < 0.001
NA
Not run
NA
Carreon et al.
(2010)


2160 males, cohort, at
least 6 mo of
employment from
1955-1984
Not reported
Bladder cancer; for subgroup exposed to
0-toluidine,	standardized mortality ratio
[SMR] = 1589
Relative risk (95% CI) for years of
employment:
1-4	yr = 6.73 (1.59-28.41)*
>5 yr = 7.65 (1.03-56.87)*
*p < 0.05
test for trendp = 0.002
NA
Not run
NA
Sorahan et al.
(2000)

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Table 2. Summary of Potentially Relevant Data for o-Toluidine (CASRN 95-53-4)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry"
Critical Effects
NOAEL'
BMDL/
BMCLa
LOAELab
Reference
(Comments)
Notes0
Carcinogenicity
2160 males, cohort, at
least 6 mo of
employment from
1955-1984 (update of
study listed above)
Not reported
Bladder cancer; for subgroup exposed to
0-toluidine,	SMR =
1116 (95% CI: 230-3261)
Relative risk (95% CI) for years of
employment:
1-4	yr= 4.68 (1.66-13.2)*
>5 yr = 6.99 (1.69-28.9)*
*p < 0.05
test for trendp < 0.001
NA
Not run
NA
Sorahan (2008)

Animal
1. Oral (mg/kg-day)a
Subchronic
10 rat (sex and strain not
specified), diet, duration
not specified but at least
91 d
Not specified;
authors added
o-toluidine (final
concentration of 7.5
to 12 mg per d) in
rice flour diet
Three animals exhibited metaplasia and
early epithelial proliferation in bladder
mucous membrane
ND
Not run
ND
Ekman and
Strombeck
(1947)


20/0 F344/N rat, diet,
7 d/wk, 13 wk
0 or 301
(Adjusted)6''
Decreased body weight; increased
relative weight of the liver, kidney,
spleen, and testes; and increased
incidence of lesions in the liver,
kidneys, bladder, and spleen
None
Not run
301
NTP (1996a)
PS, PR

20/0 F344/N rat, diet,
7 d/wk, 26 wk
0 or 285
(Adjusted)e,f
Decreased body weight; increased
relative weight of the liver, kidney,
spleen, and testes; and increased
incidence of lesions in the liver, kidneys,
bladder, and spleen
None
Not run
285
NTP (1996b)

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Table 2. Summary of Potentially Relevant Data for o-Toluidine (CASRN 95-53-4)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry"
Critical Effects
NOAEL'
BMDL/
BMCLa
LOAELab
Reference
(Comments)
Notes0
Subchronic
20/0 F344/N rat, diet,
7 d/wk, 13 wk with
13 wk recovery
0 or 304
(Adjusted)e'f
Decreased body weight after treatment
and recovery; increased relative spleen
weight; and increased incidence of
lesions in the kidney, liver, and spleen
after recovery
None
Not run
304
NTP (1996c)

Chronic
50/50 F344 rat, diet,
7 d/wk, 101-104 wk
0, 231 and 525 in
males; 0, 264, and
600 in females
(Adjusted)6'8
Decreased survival
None
Not run
None; 231
is a frank
effect level
(PEL)
NCI (1979a)

Developmental
ND
Reproductive
ND
Carcinogenicity
50/50 F344 rat, diet,
7 d/wk, 101-104 wk
0, 63.0, and 138.8
in males; 0, 63.7
and 140.4 in
females6*1
Significant increase in several types of
cancer, including skin (fibromas), spleen,
and bladder cancer; BMDL based on
subcutaneous fibroma in males
NA
10.4
NA
NCI (1979a)


25/0 CD rat, diet,
7 d/wk, 18 mo followed
by 6 mo on the control
diet
0, 72.1, and
144.1eh
Significant increase in the incidence of
dermal subcutaneous fibroma and
fibrosarcoma
NA
4.5
NA
Weisburger et
al. (1978a)
PS, PR

30/0 F344 rat, diet,
7 d/wk, 72 wk followed
by 21-wk recovery
0 or 44.2e'h
Significant increases in fibromas of the
skin, spleen, and mammary tissue, and in
peritoneal tumors; nonsignificant
increases in neoplasms of the bladder,
liver, and mammary carcinomas;
decreased survival (significance not
reported)
NA
Not run
NA
Hecht et al.
(1982)

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Table 2. Summary of Potentially Relevant Data for o-Toluidine (CASRN 95-53-4)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry"
Critical Effects
NOAEL3
BMDL/
BMCLa
LOAELab
Reference
(Comments)
Notes0
Carcinogenicity
50/50 B6C3FJ mouse,
diet, 7 d/wk, 102-103 wk
0, 23.0, and 73.8 in
males; 0, 26.5, and
91.9 in females6'11
Significant increase in hepatocellular
carcinomas, adenomas, and
hemangiosarcomas; BMDL based on
hepatocellular adenomas and carcinomas
in females
NA
22.2
NA
NCI (1979b)

25/25 CD-I mouse, diet,
7 d/wk, 18 mo followed
by 3 mo on the control
diet
0,210.5, and 420.9
in males; 0, 211.8,
and 423.7 in
femalese,h
Significant increase in vascular tumors
NA
23
NA
Weisburger et
al. (1978b)

2. Inhalation (mg/m3)a
Subchronic
ND
Chronic
ND
Developmental
ND
Reproductive
ND
Carcinogenicity
ND
""Dosimetry: NOAEL, BMDL/BMCL, and LOAEL values are converted to an adjusted daily dose (ADD in mg/kg-d) for oral noncancer effects and to a human equivalent
dose (HED in mg/kg-d) for oral carcinogenic effects. All long-term exposure values (4 wk and longer) are converted from a discontinuous to a continuous (weekly)
exposure. Values from animal developmental studies are not adjusted to a continuous exposure.
bNot reported by the study author, but determined from data.
°PS = Principal study, PR = Peer reviewed, NPR = Not peer reviewed, NA = Not applicable, ND = No data.
dThese studies were occupational studies where exposure is presumably via inhalation, but dermal and oral exposures are also possible.
"Compound administered as toluidine hydrochloride.
fAverage daily doses were provided in the study report in mg/kg-day presumably based on the concentrations in the diet (also provided in the study report), body weights,
and food consumption (both of which were routinely measured).
8Doses were converted from ppm (as presented in the study report) by using the following equation ppm x average daily food consumption x (1 body weight) x (days
dosed ^ total days). Average food consumption was obtained from EPA (1988) and average body weights were obtained from the study report (NCI, 1979).
hThese doses are HEDs that were calculated as follows: average daily dose in mg/kg-day x (average animal body weight average human body weight)0'25.
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HUMAN STUDIES
Oral Exposures
No human oral exposure studies on o-toluidine were identified
Inhalation Exposures
The effects of inhalation exposure of humans to o-toluidine have been evaluated in five
carcinogenicity studies (i.e., Ward et al., 1991; Markowitz and Levin, 2004; Carreon et al., 2010;
Sorahan et al., 2000; Sorahan, 2008).
Subchronic-duration Studies
No subchronic-duration studies on o-toluidine were identified.
Chronic-duration Studies
No chronic-duration studies on o-toluidine were identified.
Developmental Studies
No developmental toxicity studies on o-toluidine were identified.
Reproductive Studies
No reproductive toxicity studies on o-toluidine were identified.
Carcinogenicity Studies
Human studies have been limited to those examining cancer outcomes, specifically
bladder cancer, in subjects presumed to have occupational exposure to azo dyes. Although for
the purpose of this document, the exposures are considered via inhalation because there are no
specifics in the study reports. Occupational exposures to compounds, such as aromatic amines,
might have an appreciable dermal component, likely due to the relatively low vapor pressure of
these compounds (Baan et al., 2008). The majority of the studies examined exposures to
mixtures of several chemicals, including o-toluidine, and, in some cases, other suspected or
known carcinogens (i.e., Ward et al., 1991; Markowitz and Levin, 2004; Carreon et al., 2010;
Sorahan et al., 2000; Sorahan, 2008).
Ward et al. (1991) used a retrospective cohort design to investigate the incidence of
bladder cancer in 1749 workers (1643 males, 106 females) at a New York chemical plant with
exposures to o-toluidine and aniline. The workers were assigned to three groups: (1) "definitely
exposed" (n = 708; those who worked in the o-toluidine department), (2) "possibly exposed"
(n = 288; maintenance, janitors, yard workers, and shipping), (3) and "probably not exposed"
(n = 753; all others). The observed (O) incidence rate of bladder cancer in the chemical plant
was compared to that of the expected (E) general population of the State of New York (excluding
New York City). Workers in the "definitely exposed" group had a standardized incidence ratio
(SIR) for bladder cancer of 6.48 (90% confidence interval [CI]: 3.04-12.2; O/E = 7/1.08). SIRs
for <5 years, 5 to <10 years, or 10 years or more of employment were 0 (O/E = 0/0.75),
8.8 (O/E = 1/0.11), and 27.2 (O/E = 6/0.22) respectively. Using <5 years as a reference group,
the standard rate ratios for these categories were 1.0, 3.31, and 16.0, which were significant for
trend (p < 0.001). Ward et al. (1996) reported urinary levels of o-toluidine in both exposed
(98.7 ± 119.4 |ig/L) and unexposed (2.8 ±1.4 |ig/L) workers. They further looked at the
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hemoglobin adducts showing higher levels of o-toluidine than aniline, thereby suggesting that
exposure to o-toluidine exceeded that of aniline.
Markowitz and Levin (2004) conducted a follow-up study of the Ward et al. (1991)
cohort and found an additional 19 cases of bladder cancer based on data from attorneys who
represented civil litigation and from a bladder cancer screening program sponsored by the
chemical plant, which employed the workers. Their analysis indicated that further follow-up
should be conducted to characterize the occupational cohort.
Another reanalysis by Carreon et al. (2010) changed the group assignments of the
workers based on a walk-through and observations of the workers in the chemical plant.
Although the categories remained the same, the number of individuals in the categories changed,
with more being placed in the definitely exposed category (i.e., definitely exposed, n = 962;
possibly exposed, n= 187; and probably not exposed, n = 600). The bladder cancer incidence
was determined for the period of December 31, 1988 to December 31, 1994. The calculated SIR
for the updated "definitely exposed" cohort was reported as 5.84 (95% CI: 2.91-10.45), which
was significantly higher than the "probably not exposed" group. Duration of exposure was also
reevaluated with an SIR of 11.09 (95% CI: 5.07-21.05) for those employed for 10 years or more.
These results support the initial Ward et al. (1991) study, which also found a significant excess
risk of bladder cancer among the cohort of workers in the chemical plant.
Sorahan et al. (2000) examined the cancer mortality and incidence in 2160 males working
in a factory manufacturing chemicals for the rubber industry for at least 6 months from 1955 to
1984 in northern Wales. Exposure assessments were done by using job histories for 300
different jobs from 1930 to 1984. However, a specific exposure-versus-time matrix for
o-toluidine was not possible, and the subjects were evaluated by duration of employment in the
o-toluidine department. There were 1131 observed deaths between 1955 and 1996, which were
quite close to the expected deaths of 1114.5. Of the workers in the o-toluidine department
(n = 53), there were three deaths due to bladder cancer, compared to the expected rate of 0.2, the
standard mortality ratio (SMR) was reported to be 1589 (no CI provided). Relative risks (RRs)
of bladder cancer mortality due to working in the o-toluidine department for 1-4 years or
>5 years, using untagged employment history, were both elevated, at 4.44 (95% CI: 0.76-25.79)
and 5.48 (95% CI: 0.51-59.14), respectively. However, RRs of total bladder cancer incidence
for those working in the o-toluidine department for 1-4 years or >5 years were 6.73 (95% CI:
1.59-28.41) and 7.65 (95% CI: 1.03-56.87), respectively, which were significantly increased
and followed a significant trend.
Sorahan (2008) provided an update, with bladder cancer incidence and deaths examined
through December 2005. The SMR for workers in the o-toluidine department was 1116
(95%) CI: 230-3261). Relative risk of bladder cancer due to working in the o-toluidine
department for 1-4 years or >5 years remained elevated and significant, consistent with the 2000
analysis at 4.68 (95% CI: 1.66-13.2) and 6.99 (95% CI: 1.69-28.9), respectively.
ANIMAL STUDIES
Oral Exposures
In animal studies, o-toluidine is generally administered as o-toluidine HC1. The effects of
oral exposure of animals to o-toluidine HC1 have been evaluated in subchronic-duration (Ekman
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and Strombeck, 1947; NTP, 1996a,b,c), chronic-duration (NCI, 1979a,b), and cancer (NCI,
1979a,b; Weisburger et al., 1978a,b; Hecht et al., 1982) studies. The NTP rat studies examined
13-week (designated as NTP, 1996a), 26-week (designated as NTP, 1996b), and 13-week with a
13-week recovery period (designated as NTP, 1996c) exposures. The NCI (1979) and
Weisburger et al. (1978) studies were conducted in rats and mice. Rat studies were designated as
NCI (1979a) and Weisburger et al. (1978a), while mouse studies were designated NCI (1979b)
and Weisburger et al. (1978b) as indicated in Table 2. When the complete study was discussed
throughout the report, the letter designations were not employed. Two subacute (14-day) studies
were also conducted and are reported under the section of "Other Data" below.
Subchronic-duration Studies
In a study by Ekman and Strombeck (1947), 10 rats (sex and strain not specified) were
given a diet of rice flour with o-toluidine that was supplemented with a slice of carrot every other
day (duration not specified). The methods of the study are not well reported, and the dose of the
experiment was lowered soon after study initiation. The study authors reported the dose in the
latter part of the study as approximately 7.5 to 12 mg of o-toluidine per day, with levels about
twice as high during the beginning of the study. Study investigators reported the average
lifespan of the animals to be 91 days. No other methods or details of the experiment were
provided. Three of the rats were stated to have changes in the mucous membrane of the bladder,
with metaplasia and early epithelial proliferation.
NTP (1996)
The 13-week component of the peer-reviewed rat study by NTP (1996a) is selected
as the principal study for derivation of the screening subchronic p-RfD. NTP (1996a,b,c)
conducted a study performed under Good Laboratory Practices (GLP) that compared the toxicity
and carcinogenicity of o-nitrotoluene and o-toluidine HC1 administered in feed at one dose level.
Sixty male F344/N rats (20/group) were administered 5000 ppm of o-toluidine HC1 (100% pure
as tested by the study laboratory) in the diet and sacrificed after continuous exposure for
13 weeks (Group 1) or 26 weeks (Group 2). A third group was administered 5000-ppm
o-toluidine HC1 for 13 weeks, followed with a 13-week recovery period before sacrifice. The
study authors reported the daily doses received by these groups as 301 mg/kg-day for the
13-week continuous exposure, 285 mg/kg-day for the 26-week continuous exposure, and
304 mg/kg-day for the 13-week exposure with 13-week recovery group. Homogeneity and
stability tests were conducted. Results demonstrated that 9% of the test compound was lost due
to preparation, but the dose formulations were homogeneous and stable for 28 days when stored
at -20°C to 5°C. Samples stored at room temperature lost 10% of the test material. There were
a total of 40 unexposed control rats. However, 20 were used to examine the effects of altered
gastrointestinal flora (which were used as part of the testing for o-nitrotoluene). Clinical signs,
body weight, and food consumption were recorded throughout the study. At study termination,
animals were necropsied, and the following organs were weighed: epididymis, right kidney,
liver, spleen, and right testis. Histopathology was only routinely performed on gross lesions,
epididymides, liver, kidneys, testes, spleen, and urinary bladder. Continuous data with a normal
distribution were analyzed with a Dunnett's test. A Fischer's Exact test was used to evaluate
histopathological lesions, and a Wilcoxon rank sum test was used for the placental glutathione
»Y-transferase-positive data.
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No mortalities were observed among treated or control animals (NTP, 1996a,b,c).
Although statistical analyses were not performed, there was a 11% decrease in body-weight,
relative to concurrent controls after 13 weeks of treatment. The lowered relative body-weight
persisted after the 13-week recovery period (11% lower than the concurrent control). After
26 weeks of treatment, o-toluidine-treated animals had a body-weight 18% lower than the
concurrent controls. This was accompanied by lower food consumption. There was a significant
increase in the relative weight of the right kidney, liver, spleen, and right testis, but not in the
epididymides, after both 13 (see Table B. 1) and 26 weeks (see Table B.2) of treatment. After
13 weeks of treatment followed by 13 weeks of recovery, only the relative weight of the spleen
remained significantly elevated (155% of the control weight) (see Table B.2). However, the
spleen weight, after the recovery period, was not as elevated as after 13 weeks (350% of the
controls) or 26 weeks (433% of the controls) of continuous exposure. In the liver, accumulation
of hemosiderin pigment of the Kupffer cell cytoplasm was significantly increased in all groups
(20/20 after 13 weeks and 26 weeks, 11/20 after a 13-week recovery period compared to 0/20 in
the controls after 13 weeks and 0/10 after 26 weeks). Placental isozyme of glutathione
^'-transferase (PGST)-positive foci (number and size) were also found to be significantly
increased at 26 weeks (not measured in the other two treatment groups). All treated animals
experienced a significant increase in hemosiderin pigment accumulation in the tubule epithelium
of the kidney. There was a significant increase in transitional cell hyperplasia in the urinary
bladder after 13 (10/20) and 26 (17/20) weeks of treatment but not after the 13-week recovery
period (0/20). At necropsy, the spleens were stated to be grossly enlarged, with granular plaques
on the capsular surface, with some plaques containing fluid-filled cysts at 13 weeks. At 13 and
26 weeks, all the treated animals had the following histopathological lesions in the spleen:
congestion, hematopoietic cell proliferation, hemosiderin pigmentation, and fibrosis capsules.
These were significantly increased compared with the incidence in the controls, and with the
exception of hematopoietic cell proliferation, were still significantly increased after a 13-week
recovery period. There was thrombosis in the spleen in a few animals after 13 (3/20) and
26 (2/20) weeks of treatment that were not observed in either the controls or the 13-week
recovery animals. The only end points that seemed to progress in severity from 13 weeks to
26 weeks were the increase in spleen weight and the increase in the severity of fibrosis capsules
in the spleen. After a 13-week recovery period, effects were still observed in the spleen, with
relative weight still increased and numerous lesions still observed. The LOAEL is 5000 ppm
(285 to 304 mg/kg-day), the only dose tested, based on the numerous effects observed among the
three groups.
Chronic-duration/Carcinogenic Studies
NCI (1979a)
NCI (1979a) sponsored a chronic-duration/carcinogenic feeding study of o-toluidine HC1
(purity >99% as measured by the study laboratory) to male and female F344 rats
(20 controls/sex, 50/dose/sex), obtained from the NCI Frederick Cancer Research Center animal
farm. The rats received concentrations of 0, 3000, or 6000 ppm in the diet for a period of 101 to
104 weeks (equivalent to 231 and 525 mg/kg-day in males and 264 and 600 mg/kg-day in
females based on average body weight from the study and daily food consumption [U.S. EPA,
1988]). The human equivalent doses (HEDs) for the carcinogenicity study are equivalent to 63.0
and 138.8 mg/kg-day in males and 63.7 and 140.4 mg/kg-day in females. Doses were selected
based on a 7-week study that was conducted to determine a maximum tolerated dose. Diets were
freshly prepared every 1 to 1.5 weeks and stored at 5°C, but there is no indication that the diets
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were tested for concentration, stability, or homogeneity. Animals were checked twice daily for
morbidity and mortality. Clinical observations, palpations, and body-weight measurements were
conducted once per month. Animals were necropsied and evaluated for gross signs of toxicity,
and tissues were examined microscopically. The statistical tests conducted were numerous and
typical of those conducted in NCI studies. Animals that died from other than natural causes were
statistically censored at the time of death; animals dying from natural causes were not
statistically censored. A time-adjusted analysis was applied when numerous early deaths
resulted from causes that were not associated with the formation of tumors.
There was a significant decrease in survival relative to controls, at both doses, in both
male and female rats; there was approximately 90%, 27%, and 0% (males) and 80%, 55%, and
21% (females) survival at the end of the study for control, low, and high doses, respectively
(estimated from Figure 2 of NCI, 1979a). The majority of mortalities in the high-dose group
occurred late in the exposure, after Week 60. Decreases in mean body weights of surviving rats
relative to concurrent controls were also noted beginning at around 40 weeks of treatment for the
low-dose group and at 10 weeks of treatment for the high-dose group. Proliferative lesions in the
spleen, abdomen, scrotum, urinary bladder epithelium, and renal pelvis of dosed rats were
observed. There was an increase in the incidence of liver necrosis in high-dose females and both
low- and high-dose male groups and bladder epithelial hyperplasia in both low- and high-dose
groups in males and females (see Table B.3). o-Toluidine also induced subcutaneous fibromas
and mammary fibroadenomas (see Table B.4). Statistical analyses conducted by the authors
showed a significant, dose-related increase in subcutaneous fibroma in male (but not female)
rats, with the incidence at both dose levels (28/50 low, 27/49 high) significantly higher than
matched controls (0/20) in male rats. Sarcomas in multiple organs and fibrosarcomas in multiple
organs were found to be significantly elevated in male rats as compared to controls. In female
rats, sarcomas (fibrosarcomas, angiosarcomas, and osteosarcomas) of multiple organs were
found to be significantly higher than controls. The study authors also reported that o-toluidine
significantly induced urinary bladder transitional cell carcinomas in female rats,
fibroadenomas/adenomas of the mammary gland in female rats, and mesotheliomas in multiple
organs or tunica vaginalis in male rats. A NOAEL for noncancer effects was not reported by the
study authors. No NOAEL can be derived from the data. There is no LOAEL because the
lowest dose (231 mg/kg-day) is a frank effect level (FEL) based on decreased survival.
Weisburger et al. (1978a)
The peer-reviewed rat study by Weisburger et al. (1978a) is selected as the principal
study for derivation of the cancer p-OSF. Weisburger et al. (1978a) administered o-toluidine
HC1 (purity 97-99%) to groups of 25 male Sprague-Dawley-derived CD rats from Charles River
in a 24-month study beginning at concentrations of 8000 or 16,000 ppm in the diet for 3 months,
then reduced to 4000 or 8000 ppm in the diet for 15 additional months, after which they were fed
a control diet for 6 additional months before scheduled sacrifice. Several chemicals were tested
and reported in this report. For all compounds, the higher dose was selected to be the maximum
tolerated dose, but if high mortality occurred or body weight became 10% lower than the
concurrent control, the doses were reduced. Body weight was routinely measured to keep track
of the differences. Each compound was run in conjunction with a control, which is considered
the concurrent control. The pooled control combines the control groups for each compound
(start times were stratified over an 8-month period). The time-weighted human equivalent doses
are 72.1 and 144.1 mg/kg-day calculated based on the 3-month exposure to 8000 or 16,000 ppm,
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the 15-month exposure to 4000 or 8000 ppm, and the 6 months on control diet (using average
body weight [U.S. EPA, 1994b] and daily food consumption [U.S. EPA, 1988]), and the ratio of
animal-to-human body-weight adjustment raised to the Vi power. Necropsies were performed
only on animals that died or were sacrificed after 6 months of exposure. The following tissues
were processed for histopathology: gross lesions and tumor masses, lung, liver, spleen, kidney,
adrenals, intestines, stomach, heart, bladder, reproductive organs, and pituitary gland. A
Fischer's Exact test was used to determine the differences between treatment groups and the
control groups. There was a significant increase in subcutaneous fibromas and fibrosarcomas in
both the low- (18/23) and high- (21/24) dose groups compared to the control group (concurrent:
0/16; pooled: 18/111) (see Table B.5). There was a nonsignificant increase in bladder tumors in
both the low- (3/23) and high- (4/24) dose groups compared to the control (concurrent: 0/16;
pooled: 5/111). Multiple tumors (this is assumed to be more than one tumor per rat, although the
study authors did not specify) were significantly increased above the pooled controls in the
high-dose group (8/24) but not the low-dose group (6/23) compared to the control groups
(concurrent: 3/16; pooled: 14/111).
Hecht et al. (1982)
Hecht et al. (1982) administered o-toluidine HC1 (purity not reported) to 30 male F344
rats at a concentration of 0.028 mol/kg diet for 72 weeks. Study authors calculated a mean daily
dose of o-toluidine HC1 to be 0.062 g/rat based on measured mean daily food consumption. This
is equivalent to 163 mg/kg-day based on a standard average body weight of 0.380 kg in F344 rats
during a chronic-duration study (U.S. EPA, 1994b). The human equivalent dose based on an
animal-to-human body-weight ratio raised to the Vi power is 44.2 mg/kg-day. After 72 weeks of
dosing, animals were placed on control diet. Animals were sacrificed when found moribund or
after 93 weeks and were necropsied. It was stated that all major organs were processed for
histopathology, but a detailed list was not provided. There was a decrease in survival of dosed
rats beginning in the 18th month. Although the study authors stated that a chi-square test was
used, it appears that results were compared to the o-nitrosotoluene group and not the control.
Therefore, a Fisher's Exact test is used to analyze the data. There was a significant increase in
the following tumors in treated rats compared to the concurrent controls: skin fibromas (25/30
versus 1/27), spleen fibromas (10/30 versus 0/27), mammary fibroadenomas (11/30 versus 0/27),
and peritoneal tumors (14/30 versus 2/27). Smaller, nonsignificant, increases were observed for
liver hepatomas (2/30 treated; 0/27 controls), bladder papilloma (3/30 treated; 0/27 controls),
bladder carcinoma (1/30 treated; 0/27 controls), and mammary carcinomas (2/30 treated;
0/27 controls). The use of a single dose level precludes dose-response modeling.
NCI (1979b)
B6C3Fi mice (20 controls/sex, 50/dose/sex) were dosed in the diet at levels of 0, 1000, or
3000 ppm for a period of 102 to 103 weeks (NCI, 1979b). Study details were similar to those
discussed above in the rat study (NCI, 1979a). HEDs are 23.0 and 73.8 mg/kg-day in males and
26.5 and 91.9 mg/kg-day in females calculated based on average body weight from the study and
daily food consumption (U.S. EPA, 1988), and the ratio of animal-to-human body-weight
adjustment raised to the Vi power. Animals were checked twice daily for morbidity and
mortality. Clinical observations, palpations, and body-weight measurement were conducted
once per month. Animals were necropsied and evaluated for gross signs of toxicity, and tissues
were examined microscopically. There was a decrease in mean body weight in treated male and
female mice, but, in contrast to the rats, there was no significant dose-related decrease in
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survival. Statistical analyses conducted by the study authors indicate that incidence of
hemangiosarcomas of all sites in male mice is significantly increased (p < 0.05 trend test; see
Table B.6). In female mice, investigators reported a significant, dose-related increase in the
number of combined hepatocellular carcinomas and adenomas, which was not observed in male
mice.
Weisburger et al. (1978b)
Weisburger et al. (1978b) also administered o-toluidine HC1 (purity 97-99%) to groups
of 25/dose/sex CD-I mice in a 24-month study, beginning at concentrations of 16,000 or
32,000 ppm in the diet for 3 months, followed by 8000 or 16,000 ppm in the diet for 15
additional months, after which they were fed the control diet for 6 additional months before
scheduled sacrifice. Methodology mirrored that in the rat experiment, except the pituitary was
not routinely examined histologically. The time-weighted HEDs are 210.5 and 420.9 mg/kg-day
in male mice and 211.8 and 423.7 mg/kg-day in female mice. Necropsies were performed only
on animals that died after 6 months of exposure. Tissues were processed for histopathology.
There was a significant increase in vascular tumors in both the low- and high-dose groups
compared to the control in male and female groups (see Table B.7). Incidence of vascular
tumors in the simultaneous control, pooled control, low-dose group, and high-dose group were
0/14, 5/99, 5/14, and 9/11, respectively, in male mice and 0/15, 9/102, 5/18, and 9/21,
respectively, in female mice.
Developmental and Reproduction Studies
No studies could be located regarding the effects on development and reproduction
resulting from oral exposure of animals to o-toluidine. Hiles and Abdo (1990) report on two
dermal exposure studies that examined the developmental and reproductive effects of o-toluidine
that indicate chemical-related effects. The original sources of Malysheva and Zaitseva (1982)
and Malysheva et al. (1983) were unavailable for review at this time. The original study
publications were published in a foreign language. The brief summary of the two studies stated
that an unspecified number of animals (species not specified) were treated dermally (doses and
purity not reported) for 4 months with o-toluidine. The summary also stated treatment resulted
in changes in ovarian cycle, ovarian morphostructure, ability to reproduce in females, and
stimulation of spermatogenesis in males. It is further stated that progeny were also affected, but
no specific effects were reported.
Inhalation Exposures
There is no suitable information to provide in this regard.
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)
Short-term Studies
Haskell Laboratory (1994) conducted a study sponsored by Dupont & Co. that examined
urinary bladder toxicity in rats after a 14-day feeding exposure. o-Toluidine (purity 99.5%) was
added to the diet via an ethanol vehicle and administered to F344 rats (10-15 rats/sex/dose) for
14 days at dose levels of 0, 500, 3000, or 6000 ppm. The mean daily intake was estimated by the
study authors to be 40.4, 238, and 449 mg/kg-day in males and 43.5, 251, and 481 mg/kg-day in
females. It was reported that the test substance was not stable in the diet (30% decrease in
concentration after 7 days), and doses were not adjusted for test substance stability. Cage side
observations and checks for moribund animals were made twice daily, body weights were
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measured three times weekly, and urine was collected on Days 6-7 and Days 13-14. Average
daily food consumption was measured weekly. At sacrifice, blood was collected and analyzed
specifically for methemoglobin levels. Necropsies were performed at the end of the study
period, and bladder and duodenum were removed and processed for analysis of cell proliferation
(5 rats/sex/dose) and unscheduled DNA synthesis (5 rats/sex/dose).
None of the animals died during treatment (Haskell Laboratory, 1994). Clinical signs of
toxicity included wet and stained perineums in females of the high-dose group. There was a
significant decrease in body weight in high-dose males and females that was likely due to the
low weight gain during the first week of exposure. A significant decrease in food consumption
was noted in the mid- and high-dose males and females during the first week. Significant
increases in bladder epithelial cell proliferation were observed in mid- and high-dose females
and high-dose males. Histopathology revealed mild urothelial hyperplasia in females, slight
urothelial thickening in males, and unscheduled DNA synthesis (UDS) in both males and
females in the high-dose groups. Furthermore, the investigators observed a significant and
dose-related increase in methemoglobin levels in both sexes at all dose levels. The study authors
established a NOAEL of 500 ppm (equivalent to 40.4 and 43.5 mg/kg-day in males and females,
respectively) for urinary bladder toxicity (LOAEL is 238 and 251 mg/kg-day in male and
females, respectively). The study authors could not establish a NOAEL for methemoglobinemia;
therefore, the LOAEL is 40.4 and 43.5 mg/kg-day in males and females, respectively, based on
increased levels of methemoglobin.
Short et al. (1983) conducted a short-term toxicity study where they administered
o-toludine (purity 99.3%) to male F344 rats (10/time point) via daily gavage at a dose level of
225 mg/kg-day for 5, 10, or 20 days. Body weight, organ weight, and histopathology were
evaluated. Clinical signs reported by the study authors included transient cyanosis and rough
hair coat. Of the 30 rats treated, 10 of the rats died before the end of the 20 days of treatment.
Body weight was significantly lower after 5 and 10 days but was equal to the control by Day 20.
Spleen weight was significantly increased at all time points, but liver and kidney weights were
not affected. The effects on the spleen were confirmed with significant increases in spleen
congestion, hemosiderosis, and hematopoiesis in the survivors at all three time points.
Hypercellularity of bone marrow was significantly increased only at the 10-day time point. No
changes in histopathology were noted in the liver. The LOAEL is 225 mg/kg-day and was the
only dose tested.
Other Studies
Toxicokinetic data demonstrate that o-toluidine is readily absorbed via the
gastrointestinal tract and excreted within 24 hours in the urine (Cheever et al., 1980; Son et al.,
1980). A greater amount of o-toluidine is excreted unchanged (21%) compared to either of the
other toluidine isomers (2.5%). o-Toluidine is not mutagenic in Ames assays (see Table 3).
However, in a review or o-toluidine genotoxicity, Danford (1991) states that this is only the case
in standard Ames assays, and positive mutagenicity results have been obtained at high
concentrations using different types of metabolic activation systems. Additionally, Danford
(1991) states that most yeast studies have also been negative, but o-toluidine has been found to
cause deletion mutations in yeast at concentrations that reduced survival (Brennan and Schiestl,
1999). o-Toluidine was weakly positive for Trp+ revertants in modified yeast (Saccaromyces
cerevisiae C658-K42) (Morita et al., 1989). There have been mixed results with cytogenic
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assays. Danford (1991) concludes that it is a clastogen but only with prolonged exposure.
Morita et al. (1997) summarized results from six independent micronuclei assays in male
B6C3Fi mice receiving one to four intraperitoneal (ip) injections at doses up to 1000 mg/kg (the
LD50 was stated to be 800 mg/kg). There was a small—but significant—increase in
micronucleated polychromatic erythrocytes in the bone marrow only with the 800- or
1000-mg/kg dose. This was not considered biologically significant, due to the deaths in these
animals, and the tests were considered negative. Chromosomal aberrations have been found in
Chinese hamster ovary (CHO) cells in vitro (Gulati et al., 1985) but not after ip injection to
B6C3Fi mice in vivo (McFee et al., 1989). o-Toluidine has been found to cause sister chromatid
exchange (SCE) in both CHO cells in vitro (Gulati et al., 1985) and in B6C3Fi mice in vivo
(McFee et al., 1989) and to cause transformations in BALB/c-3T3 and Syrian hamster embryo
(SHE) transformation assays (Matthews et al., 1993; Kerckaert et al., 1998) (see Table 3).
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Table 3. Other Studies
Tests
Materials and Methods
Results
Conclusions
References
Toxicokinetic
Studied distribution of 14C following single
dose of either 50- or 400 mg/kg subcutaneous
dose of o-[methyl-14C]toluidine to male F344
rats (2/dose) after 24 and 48 h.
Urine was the major excretory pathway at
both dose levels, followed by fecal
excretion and breath. Tissue
concentration of radiolabel after 48 h was
highest in liver with approximately 0.12%
and 0.325% at the 50- and 400-mg/kg
dose, respectively.
NA
Sonet al. (1980)
Toxicokinetic
Studied excretion and metabolism of 14C
following single oral dose of 50 or 500 mg/kg
of o-toluidine to male Sprague-Dawley rats;
/Moluidinc and /w-toluidine were also tested.
92% of o-toluidine was excreted via the
urine within 24 h of dosing. The amount
of parent compound excreted was greater
for o-toluidine (21%) than either of the
other isomers (2.5%).
Aminomethylphenols were the metabolite
detected in the urine.
NA
Cheever et al.
(1980)
Genotoxicity
Tested for reverse mutation in Salmonella
typhimurium (Ames assay) TA1535, TA1537,
TA1538, TA98, and TA100 with and without
metabolic activation at a dose level of 1000 |ig
or 9.33 (imole.
Nonmutagenic in all strains tested.
o-Toluidine was not considered
to be mutagenic.
Simmon (1979)
Genotoxicity
Tested for reverse mutation in Salmonella
typhimurium (Ames assay) TA100 with
metabolic activation and for DNA damage in
Chinese hamster lung fibroblasts (V79) with
metabolic activation incubated for 2 h with
concentrations ranging from 0.3 to 10.0 mM
(32-1070 mg).
Nonmutagenic and did not cause DNA
breakage.
o-Toluidine was not considered
to be mutagenic or cause DNA
damage.
Zimmer et al. (1980)
Genotoxicity
Tested for sperm head abnormalities using ip
of 0.05, 0.1, 0.2, 0.25, 0.3, 0.4, or
0.5 mg/kg-day for 5 days in male (CBA x
BALB/c)Fi mice.
Negative.
o-Toluidine did not cause sperm
head abnormalities.
Topham (1980)
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Table 3. Other Studies
Tests
Materials and Methods
Results
Conclusions
References
Genotoxicity
Tested for reverse mutation in Salmonella
typhimurium (Ames assay) TA1535, TA1537,
TA1538, TA98, and TA100 with and without
metabolic activation at doses of 0.32, 1.0, 3.2,
or 10.0 ng/plate.
Nonmutagenic in all test strains.
o-Toluidine was not considered
to be mutagenic.
Baker and Bonin
(1981)
Genotoxicity
Tested in vivo mutagenicity in the bone
marrow micronucleus test (injected at 0 and 24
h, samples taken at 48, 72, and 96 h); dose
level was at 50, 80, and 100% of LD50 in
B6C3Fi mice (800 mg/kg).
Nonclastogenic.
o-Toluidine is not considered to
be clastogenic.
Salamone et al.
(1981)
Genotoxicity
Tested for chromosomal aberrations and sister
chromatid exchanges (SCEs) in Chinese
hamster ovary (CHO) cells with concentrations
up to 500 ng/ml in the absence of metabolic
activation and 5000 ng/ml in the presence of
metabolic activation.
The highest dose was generally cytotoxic.
However, in the one assay that was not
toxic at 500 |ig/plate without metabolic
activation, there was nearly twice as many
SCEs as in the vehicle control. There was
also an increase in chromosomal
aberrations, with a concentration of 500
Hg/plate or greater depending on the assay
conditions.
o-Toluidine was considered
positive for inducing SCE in the
presence and absence of
metabolic activation.
o-Toluidine was considered
positive for chromosomal
aberration only when fixation
was done at 6 h.
Gulati et al. (1985)
Genotoxicity
Tested for chromosomal aberrations and sister
chromatid exchanges (SCE) in vivo after a
single ip injection at doses of 150, 300, or 600
(maximum tolerated dose) mg/kg to male
B6C3Fi mice.
There was not an increase in chromosomal
aberrations or micronuclei, but there was
an increase in the frequency of SCE in two
separate trials.
o-Toluidine was positive for
inducing SCE but not
chromosomal aberrations or
micronuclei in vivo.
McFee et al. (1989)
Genotoxicity
Tested for Trp+ reversion in Saccaromyces
cerevisiae C658-K42 (strain with increased
permeability), with concentrations ranging
from 0.5 to 3.0 mg/mL.
At 3.0 mg/mL there was an increase in
Trp+ revertants (6.9 ± 1.9) in the presence
of metabolic activation compared to the
control (2.4 ± 1.3).
It was concluded that o-toluidine
was weakly positive.
Morita et al. (1989)
Genotoxicity
BALB/c-3T3 cell transformation assay.
50% cytotoxicity occurred with a
concentration of 4.33 mM. Statistical
sensitivities for the three trials were stated
to be 87/110, 106/110, and 59/110.
o-Toluidine was considered
active in the BALB/c-3T3 cell
transformation assay.
Matthews et al.
(1993)
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Table 3. Other Studies
Tests
Materials and Methods
Results
Conclusions
References
Genotoxicity
Tested in vivo mutagenicity in the bone
marrow micronucleus test with 1 to 4 ip
injections up to 1000 mg/kg (LD50 = 800
mg/kg) in male B6C3Fi mice, results were
measure 24 h after last dose.
There was a slight increase in
micronucleated polychromatic
erythrocytes in the bone marrow of dying
animals, which was not considered
biologically significant.
o-Toluidine was not considered
to be clastogenic.
Morita et al. (1997)
Genotoxicity
Tested for genotoxicity using the 24-h Syrian
hamster embryo (SHE) transformation assay at
a pH of 6.7 with concentrations up to
1200 iig/mL (higher concentrations were not
soluble).
There was a significant increase in
transformations with concentrations
ranging from 750 to 1200 ng/mL, but
there was only 38% cytotoxicity at the
highest dose tested.
o-Toluidine was positive in the
SHE transformation assay.
Kerckaert et al.
(1998)
Genotoxicity
Tested for deletion (DEL) recombination in
yeast, with concentrations ranging from 3.0 to
6.0 mg/mL.
An increase in revertants (more than
3-fold) was observed, with concentrations
that caused decreased survival (5.0 and
6.0 mg/mL, survival 3.90 and 0.49%
compared to 93% in the control). Survival
was increased and revertants decreased
when the antioxidant A-acetyl cysteine
was added to the culture.
o-Toluidine caused deletion
mutations in yeast.
Brennan and
Schiestl (1999)
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DERIVATION OF PROVISIONAL VALUES
Tables 5 and 6 below present a summary of noncancer and cancer reference values,
respectively. IRIS data are indicated in the table if available.
DERIVATION OF ORAL REFERENCE DOSE
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)
There are two subchronic-duration studies available (see Table 4). Ekman and
Strombeck (1947) did not provide sufficient details. There is no information provided on the
doses used, and there does not appear to be a control group. While the NTP (1996) study was a
peer-reviewed GLP study, only one dose was tested. The NTP (1996) study was conducted to
compare the effects of o-toluidine HC1 to o-nitrotoluene, to demonstrate the progression of toxic
effects over time (13 weeks on the diet compared to 26 weeks on the diet), and, also, to illustrate
the reversibility of effects (13 weeks on the diet followed by 13 weeks on control diet); therefore,
doses were selected to ensure a response.
A potential POD, blood methemoglobin, is suggested by a short-term study (Haskell
Laboratory, 1994) that observed a dose-dependent increase in methemoglobin levels. However,
other studies with toluidine isomers suggest that the methemoglobin response likely decreases
with longer durations of exposure, while other organ effects likely related to blood effects such
as methemoglobinemia become apparent (Malik-Brys and Senczuk, 1995). For example, some
of the splenic and hepatic effects noted in the NTP (1996a) 13-week study are consistent with
persistent chronic blood effects. The NTP (1996b) study demonstrates that the splenic effects
progress with time on the diet. Therefore, the methemoglobin levels observed in the 14-day
study (Haskell Laboratory, 1994) are supportive of some of the effects seen in the NTP (1996)
study but are not useful for deriving the subchronic p-RfD.
Table 4 below describes studies that are relevant for deriving the oral provisional RfDs.
The NTP (1996a) 13-week study provides the best data to derive a subchronic p-RfD. The NTP
(1996b) study, after 26 weeks, reported similar effects to those seen at 13 weeks in the NTP
(1996a) study at a slightly lower dose level, and supports the selection of a POD from the NTP
(1996a) 13-week study. Although both the NTP (1996a) and NTP (1996b) studies could serve as
the principal study for derivation of a subchronic p-RfD, the 13-week study (NTP, 1996a) is
selected because the exposure duration is considered to be subchronic for rodent studies.
Because there is not enough dose-response information available, the data are not amenable to
BMD modeling for derivation of the subchronic p-RfD. Further, the NTP (1996a) study
provides only a LOAEL as the POD, thus requiring four full UFs (UFA, UFH, UFD, UFL)
resulting in a composite UF (UFC) of 10,000. Consequently, the high uncertainty combined with
a very limited database precludes derivation of a subchronic p-RfD. However, the NTP (1996a)
study does provide sufficient information to derive a screening subchronic p-RfD in Appendix A.
A chronic p-RfD is not derived because no acceptable chronic-duration toxicity studies are
available and extrapolation from the subchronic study, NTP (1996a) would entail applying a full
suite of 10-fold UFs (UFA, UFD, UFH, UFL, UFS) resulting in a composite UFC of 100,000.
Additionally, the low survival seen in the chronic-duration NCI (1979a) rat study (approximately
27% at the low dose of 231 mg/kg-day in males and 55% at 264 mg/kg-day in females) suggests
that the subchronic-duration LOAEL of 301 mg/kg-day from the principal study (NTP, 1996a)
should not be extrapolated to a chronic-duration LOAEL.
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Table 4. Summary of Relevant Oral Systemic Toxicity Studies for o-Toluidine
References
Species,
# /Sex (M/F)
Exposure
(ppm unless otherwise noted)
Frequency/
Duration
NOAELadj"
(mg/kg-d)
LOAELadj"
(mg/kg-d)
Critical End point
Ekman and
Strombeck (1947)
Rat, 10 (sex not
specified)
Not specified; authors added
o-toluidine (7.5 to 12 mg/d) in
rice flour diet
At least 91 d
ND
ND
Three animals exhibited metaplasia and
early epithelial proliferation in bladder
mucous membrane
NTP (1996a)
Rat, 20 M
0,5000
13 wk in diet
None0
301d
Decreased body weight; increased
relative weight of the liver, kidney,
spleen, and testes; and increased
incidence of lesions in the liver,
kidneys, bladder, and spleen
NTP (1996b)
Rat, 20 M
0, 5000
26 wk in diet
None
285d
Decreased body weight; increased
relative weight of the liver, kidney,
spleen, and testes; and increased
incidence of lesions in the liver,
kidneys, bladder, and spleen
NCI (1979a)
Rat, 50M/50F
0, 3000, 6000
101-104 wk in
diet
None
None. 23 ld is
anFEL
Decreased survival
"NOAEL/jy = NOAEL (ppm or mg/kg food) x food consumption (kg/day) ^ body weight (kg).
bLOAELADJ = LOAEL (ppm or mg/kg food) x food consumption (kg/day) ^ body weight (kg).
°No NOAEL was identified. The NOAEL is considered equal to a LOAEL 10 for screening purposes.
dThese values were provided in the study report.
ND = Not determined.
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Table 5. Summary of Noncancer Reference Values for o-Toluidine
Toxicity Type (Units)
Species/Sex
Critical Effect
p-Reference
Value3
POD Method
POD
UFC
Principal Study
Screening subchronic p-RfD
F344 rat/M
Increased spleen weight and
incidence of spleen lesions
2 x 1(T2
LOAEL
301
10,000
NTP (1996a)
Chronic p-RfD
(mg/kg-day)
None
Subchronic p-RfC (mg/m3)
None
Chronic p-RfC (mg/m3)
None
"The screening subchronic p-RfD adjusts for the fact that o-toluidine HC1 was administered instead of o-toluidine using the following calculation: p-RfD o-toluidine =
p-RfD o-toluidine HC1 x (molecular weight of o-toluidine ^ molecular weight of o-toluidine HC1) or 3 x 10 2 mg/kg-day x (107.16 143.62) = 2 x 10 2 mg/kg-day.
Table 6. Summary of Cancer Reference Values for o-Toluidine
Toxicity Type
Species/Sex
Tumor Type
Cancer Value"
Principal Study
p-OSF
F344 rat/M
Subcutaneous fibroma and fibrosarcoma
1.6 x 10 2 (mg/kg-day) 1
Weisburger et al. (1978a)
p-IUR
None
aThe p-OSF adjusts for the fact that o-toluidine HC1 was administered instead of o-toluidine using the following calculation: p-OSF o-toluidine = p-OSF o-toluidine HC1
x (molecular weight of o-toluidine ^ molecular weight of o-toluidine HC1) or 2.2 x 10 2 (mg/kg-day) 1 x (107.16 143.62) = 1.6 x 10 2 (mg/kg-day)
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Derivation of Chronic Provisional RfD (Chronic p-RfD)
No chronic p-RfD values can be derived. The Weisburger et al. (1978a,b) study provides
only cancer incidence and does not provide information on any possible noncancer effects or on
mortality. The only chronic-duration study available that provides any nonneoplastic results
(i.e., NCI, 1979a) also had decreased survival (i.e., 27% survival in males and 55% in females at
3000 ppm) in rats relative to controls (i.e., 90% and 80% survival in control males and females,
respectively) at the lowest dose tested (see Table B.3). The decreased survival in the NCI
(1979a) study occurred at doses (231 mg/kg-day in males and 264 mg/kg-day in females) that
were equivalent to the dose that caused reduced survival in the NTP (1996) subchronic-duration
rat studies (301 mg/kg-day), which precludes using the subchronic-duration study to extrapolate
to a chronic p-RfD. In addition, using a subchronic-duration LOAEL to extrapolate to a
chronic-duration NOAEL would entail applying a full suite of 10-fold UFs (UFA, UFD, UFh,
UFl, UFs) resulting in a UFC of 100,000. Consequently, derivation of a chronic p-RfD is
precluded.
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
No subchronic or chronic p-RfC values can be derived. There are no animal inhalation
studies, and the epidemiology studies in humans do not provide any concentrations for
o-toluidine, nor can the studies make any definitive relationship between o-toluidine exposure
and any toxic effect.
CANCER WEIGHT-OF-EVIDENCE (WOE) DESCRIPTOR
Table 7 identifies the cancer weight-of-evidence (WOE) descriptor for o-toluidine.
Table 7. Cancer WOE Descriptor for o-Toluidine
Possible WOE
Descriptor
Designation
Route of Entry
(Oral, Inhalation,
or Both)
Comments
"Carcinogenic to
Humans "
N/A
N/A
Occupational studies, although consistently and
positively associated with bladder cancer, cannot
establish any definitive link with exposures to
o-toluidine and are confounded with exposure to other
known or suspected carcinogens.
"Likely to Be
Carcinogenic to
Humans"
Selected
Oral
Under the Guidelines for Carcinogen Risk Assessment
(U.S. EPA, 2005), o-toluidine is "Likely to be
Carcinogenic to Humans" based on evidence of
carcinogenicity in rats and mice in the NCI (1979a,b)
and Weisburger et al. (1978a,b) oral bioassays.
"Suggestive Evidence of
Carcinogenic Potential"
N/A
N/A

"Inadequate
Information to Assess
Carcinogenic Potential"
N/A
N/A

"Not Likely to Be
Carcinogenic to
Humans "
N/A
N/A
No strong evidence of noncarcinogenicity in humans or
animals is available.
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MODE-OF-ACTION DISCUSSION
The Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005) define mode of action
(MO A) as the following: ".. .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.. .There are many examples of possible modes of carcinogenic action, such as
mutagenicity, mitogenesis, inhibition of programmed cell death, cytotoxicity with reparative cell
proliferation, and immune suppression" (p. 1-10).
Summary of Mutagenicity and Genetic Toxicology Studies
Data on the mutagenicity of o-toluidine are equivocal. o-Toluidine is not mutagenic in
standard Ames assays but has been positive in modified assays including the use of different
types of metabolic activation systems at high concentrations. It has also been generally negative
in yeast studies, but it has been found to cause deletion mutations in yeast (Brennan and Schiestl,
1999) and was weakly positive for Trp+ revertants in modified yeast (Saccaromyces cerevisiae
C658-K42) (Morita et al., 1989). There have also been mixed results with cytogenic assays.
Danford (1991) concludes that it is a clastogen but only with prolonged exposure. Morita et al.
(1997) summarize results from six independent micronuclei assays in male B6C3Fi mice
receiving one to four ip injections at doses exceeding the LD50 (800 mg/kg). There was a
small—but significant—increase in micronucleated polychromatic erythrocytes in the bone
marrow only in dying animals. The study author did not consider this to be biologically
significant, and the tests were considered negative. Chromosomal aberrations have been found
in CHO cells in vitro (Gulati et al., 1985) but not after ip injection to B6C3Fi mice in vivo
(Salamone et al., 1981). o-Toluidine has been found to cause SCE in both CHO cells in vitro
(Gulati et al., 1985) and in B6C3Fi mice in vivo (McFee et al., 1989) and to cause
transformations in BALB/c-3T3 and SHE transformation assays (Matthews et al., 1993;
Kerckaert et al., 1998). Taken together, there are insufficient data to determine the MOA of
o-toluidine.
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
There are insufficient data to determine the carcinogenic mode of action. Therefore, a
linear approach is applied.
Derivation of Provisional Oral Slope Factor (p-OSF)
The study by Weisburger et al. (1978a) is selected as the principal study. The cancer end
point is the incidence of subcutaneous fibromas and fibrosarcomas in male rats. This study is
generally well conducted, and the data from this study are able to support a quantitative cancer
dose-response assessment. There is no GLP statement, but the study otherwise meets the
standards of study design, performance, and presentation of information to examine
carcinogenicity. Details are provided in the "Review of Potentially Relevant Data" section.
Among the available, acceptable studies, Weisburger et al. (1978a) represents the highest OSF
from relevant studies in the database. A supporting study (i.e., NCI, 1979) is a peer-reviewed
technical report from the National Cancer Institute (NCI).
There were two acceptable carcinogenicity studies, each performed in rats and mice,
showing significant increases in a number of different tumor end points. All relevant tumor end
points in the Weisburger et al. (1978a,b) and NCI (1979a,b) studies were modeled using BMDS
software version 2.1.2 (see Table C.l; U.S. EPA, 2010c). Individual tumor types were modeled
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and all tumor types that could be combined were also modeled. All the remaining tumor types
that were not significantly different from the control animal frequencies were not considered
biologically relevant. Weisburger et al. (1978a) was selected as the principal study because the
increase in subcutaneous fibromas and fibrosarcomas in male rats provided the highest OSF with
adequate model fit. An increase in subcutaneous fibromas and/or fibrosarcomas was also found
in the NCI study in rats (1979a) and mice (1979b) and in the Hecht et al. (1982) study in male
rats. Although the Hecht (1982) study also found an increase in subcutaneous fibromas, with
results at a lower dose (i.e., 44.2 compared to 72.1 mg/kg-day in the Weisburger et al. [1978a]
study), the Hecht study only used one dose, and no dose-response can be established. However,
the tumor findings of these three chronic-duration studies lend internal consistency to the entire
o-toluidine database. Because the study administered o-toluidine HC1, the p-OSF is converted to
reflect the molecular weight difference between o-toluidine and the salt form. In addition,
because the study lowered the dose after 3 months, additional time-weighted adjustments were
made for the two different concentrations.
The following dosimetric adjustments were made for dietary treatment in adjusting doses
for oral cancer analysis for the first 3 months (90 days) of treatment:
(DOSEaDJ, HED)\¥eisburger et al., 1978a = (concentration)
Weisburger et al., 1978a x food
consumption per day x (1 -h body weight) x (days
dosed ^ total days) x body-weight adjustment
Body-weight adjustment	= (BWa^BWh)1'4
BWa	= 0.267 kg (average body weight for male rats in
subchronic-duration study) (U.S. EPA, 1994b)
BWh	= 70 kg (human reference body weight) (U.S. EPA,
1997)
Body-weight adjustment	= (0.267 ^ 70)1/4 = 0.2485
(DOSEadj, HED)weisburger et ai., 1978a = 8000 mg/kg x (0.023 kg/day) x (1 -H 0.267 kg) X
(90 days ^ 90 days) x 0.2485
= 689.08 mg/kg-day x 1 x 0.2485
(DOSEadj, HED)weisburger et ai., 1978a = 171.26 mg/kg-day
The following dosimetric adjustments were made for dietary treatment in adjusting doses
for oral cancer analysis for the last 15 months (455 days) of treatment:
(DOSEadj, HED)weisburgeretai., 1978a = (concentration)
Weisburger et al., 1978a
x food
consumption per day x (1 -h body weight) x (days
dosed ^ total days) x body-weight adjustment
Body-weight adjustment	= (BWa^BWh)1'4
BWa	= 0.523 kg (average body weight for male rats in
chronic-duration study) (U.S. EPA, 1994b)
BWh	= 70 kg (human reference body) (U.S. EPA, 1997)
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Body-weight adjustment	= (0.523 /70)1/4 = 0.294
(DOSEadj, m:i))we,sburgcretaL 1978a = 4000 mg/kg x (0.036 kg/day) X (1 H- 0.523 kg) x
(455 days ^ 455 days) x 0.294
= 275.328 mg/kg-day x 1 x 0.294
= 275.328 mg/kg-day x 0.294
(DOSEadj, HED)weisburger et al., 1978a = 80.95 mg/kg-day
The time-weighted average HED including the 6 months on the control diet =
[(DOSEadj, hed3 months x 90 days) + (DOSEadj, hed15 months x 455 days)] ^ study duration =
[(171.26 x 90) + (80.95 x 455)] - (90 + 455 + 180) = 72.1 mg/kg-day; similar calculations for
the high dose (16,000 ppm reduced to 8000 ppm) derived a value of 144.1 mg/kg-day.
Table 8 presents BMD input data for incidence of subcutaneous fibromas and
fibrosarcomas in male F344 rats exposed to o-toluidine in the diet for 18 months.
Table 8. BMD Input for Incidence of Subcutaneous Fibromas and Fibrosarcomas in
Male F344 Rats Exposed to o-Toluidine via the Diet3
(Dose)n
(ppm)
(DOSEadj,hed),,
(mg/kg-d)
Number of Subjects
Responseb
0
0
16
0(0)
8000 reduced to 4000
72.1
23
18(78)c
16,000 reduced to 8000
144.1
24
21(87.5)c
aWeisburger et al. (1978a).
bNumber of rats with tumors, () = percentage of rats with tumors.
Statistically significant in pairwise test versus control.
Table 9 shows the modeling results. Adequate model fit is obtained for the subcutaneous
fibroma and fibrosarcoma incidence data using the BMDS version 2.1.2 (U.S. EPA, 2010c)
multistage cancer model. The BMD modeling results with 10% extra risk for subcutaneous
fibroma and fibrosarcoma yield a BMDio of 6.08 mg/kg-day and a BMDLio of 4.50 mg/kg-day
(see Table 13).
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Table 9. Model Predictions for Subcutaneous Fibroma and Fibrosarcomas3
Model
Goodness of Fit
/>-Valucb
AIC for Fitted
Model
BMDl0
(mg/kg-day)
BMDLl0
(mg/kg-day)
Conclusions
Multistage cancer
0.57
45.25
6.08
4.50
Lowest AIC
Lowest BMDL
aWeisburger et al. (1987).
Values <0.10 fail to meet conventional goodness-of-fit criteria.
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMD10 = BMD at a response rate of
10% incidence, extra risk; BMDL = lower confidence limit (95%) on the benchmark dose.
The curve and BMD output for the selected model, only, are provided in the BMD
supplement to the document (Appendix C).
The p-OSF0_toiuidine hci = 0.1^- BMDLio
= 0.1 ^ 4.50 mg/kg-day
= 2.2 x 10~2 (mg/kg-day)"1
Conversion of the p-OSF based on o-toluidine HCI salt (molecular weight of 143.62) to
o-toluidine (molecular weight of 107.16) is as follows:
p~OSFo-toiuidine =	p-OSF0.toiuidineHci x (MW of o-toluidine ^ MW of o-toluidine HCI)
=	2.2 x 10~2 (mg/kg-day)-1* (107.16 - 143.62)
=	2.2 x 10~2 (mg/kg-day)_1x 0.746
=	1.6 x 10~2 (mg/kg-day)-1
Derivation of Provisional Inhalation Unit Risk (p-IUR)
No human or animal studies examining the carcinogenicity of o-toluidine following
inhalation exposure have been located. Therefore, derivation of an IUR is precluded.
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APPENDIX A. PROVISIONAL SCREENING VALUES
For reasons noted in the main PPRTV document, it is not possible to derive a provisional
subchronic p-RfD for o-toluidine. 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.
DERIVATION OF SCREENING SUBCHRONIC ORAL REFERENCE DOSE
The 13-week component of the NTP (1996a) report is selected as the principal study for
the derivation of the screening subchronic p-RfD. The critical end point is spleen toxicity as
measured by increased relative spleen weight accompanied by increased incidence of lesions in
the spleen in male F344 rats. The effects progressed with longer duration and were not entirely
reversible after a 13-week recovery period. This study is peer reviewed and performed
according to GLP principles. Details are provided in the "Review of Potentially Relevant Data"
section. BMD analysis is not possible with these data because there was only one dose level
presented in addition to the control. Among the available, acceptable studies, NTP (1996a)
represents the lowest POD for developing a subchronic p-RfD. The POD in this study is a
LOAEL of 301 mg/kg-day.
Dosimetric adjustments for daily exposure
No dosimetric adjustments were made for the dose in the principal study for dietary
treatment, as the study authors report the average daily dose of 301 mg/kg-day.
The screening subchronic p-RfD for o-toluidine, based on the LOAEL of 301 mg/kg-day
in male rats exposed for 13 weeks, is derived as follows:
Screening Subchronic p-RfD0_t0iuidineHci = LOAELadj ^ UF
= 301 mg/kg-day ^ 10,000
= 3 x 10~2 mg/kg-day
This value is normalized to account for the actual amount of o-toluidine from the
compound used, o-toluidine HC1:
Screening Subchronic p-RfD
o-toluidine	P~RfDo-toluidineHCl ^ (MW of O-toluidine •
MW of o-toluidine HC1)
= 3 x 10~2 mg/kg-day x (107.16 - 143.62)
= 3 x 10~2 mg/kg-day x 0.746
= 2 x 10~2 mg/kg-day
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Table A.l summarizes the uncertainty factors for the screening subchronic p-RfD for
o-toluidine.
DERIVATION OF SCREENING CHRONIC ORAL REFERENCE DOSE
A screening chronic p-RfD is not derived because the only available chronic-duration
study that reported noncancer effects (i.e., NCI, 1979) also reported low survival at both tested
doses. Further, deriving a screening chronic p-RfD based on the same subchronic study (NTP,
1996) that was used to derive the screening subchronic p-RfD would result in a UFC of 100,000
based on 10-fold values for each of the following UFs: UFA, UFD, UFH, UFL, UFS.
Consequently, the derivation of a screening chronic p-RfD is precluded. See the section titled
"Derivation of Chronic Provisional RfD (Chronic p-RfD)" for further discussion.
Table A.l. Uncertainty Factors for Screening Subchronic p-RfD for o-Toluidine
UF
Value
Justification
Notes
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 the
liver, spleen, kidney, and bladder toxicity of o-toluidine.

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 to humans.

ufl
10
A UFl of 10 is applied for using a POD based on a LOAEL.
Only one dose
was tested.
UFS
1
A UFS of 1 is applied because a subchronic-duration study was utilized.

UFC
<3000
10,000


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APPENDIX B. DATA TABLES
Table B.l. Results in Male F344 Rats Exposed to Oral (in Feed) o-Toluidine for 13 Weeks3
Parameter
Adjusted Daily Dose (mg/kg-d)b
Control
301
Relative Body-weight (g)
331
293 (11%)C
Food consumption (g/d)
14.9
13.5 (91%)
Necropsy body weight (g)
345 ± 5d
298 ± 4 (86%)**
Relative right kidney weight (mg/g)
3.27 ±0.03
3.50 ±0.05 (107%)**
Relative liver weight (mg/g)
34.91 ±0.31
42.57 ± 0.36 (122%)**
Relative spleen weight (mg/g)
2.12 ±0.03
7.43 ±0.16 (350%)**
Relative right testis weight (mg/g)
4.61 ±0.03
5.07 ±0.07 (110%)**
Hemosiderin pigmentation in the liver
0/10e
20/20**
Kidney pigmentation
0/10
20/20**
Transitional epithelium hyperplasia in
the urinary bladder
0/10
10/20**
Congestion in the spleen
0/10
20/20**
Hematopoietic cell proliferation in the
spleen
2/10
20/20**
Hemosiderin pigmentation in the
spleen
0/10
20/20**
Thrombosis in the spleen
0/10
3/20
Fibrosis capsule in the spleen
0/10
20/20**
aNTP (1996a).
bAnimals were administered diets with 5000 ppm; adjusted daily doses were provided in the study report.
°Number in parentheses is the percent of control.
dMean ± standard error.
eNumber of animals with lesions/number of animals.
**p < 0.01
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Table B.2. Results in Male F344 Rats Exposed to Oral (in Feed) o-Toluidine for 26 Weeks or
for 13 Weeks with a 13-Week Recovery Period3
Parameter
Adjusted Daily Dose (mg/kg-d)b
Control
26 Wk
(285)
13 Wk with 13-Wk
Recovery
(304)
Body-weight (g)
382
314 (18%)°
342 (11%)
Food consumption (g/d)
14.7
13.4 (91%)
13.9 (95%)
Necropsy body weight (g)
389 ± 5d
316 ±3 (81%)**
351 ±9 (90%)**
Relative right kidney weight (mg/g)
3.55 ±0.08
3.99 ±0.09 (112%)**
3.56 ±0.05 (100%)
Relative liver weight (mg/g)
37.09 ±0.69
45.71 ±0.88 (123%)**
39.58 ± 1.13 (107%)
Relative spleen weight (mg/g)
2.08 ±0.05
9.01 ±0.26 (433%)**
3.23 ±0.16 (155%)**
Relative right testis weight (mg/g)
4.16 ±0.06
4.84 ±0.05 (116%)**
4.28 ±0.12 (103%)
Hemosiderin pigmentation in the liver
0/10e
20/20 **
11/20**
Placental glutathione .S'-transfcrasc-positivc foci in
the liver
17 ±2
145 ±61**
Not conducted
Kidney pigmentation
0/10
20/20**
20/20**
Transitional epithelium hyperplasia in the urinary
bladder
0/10
17/20**
0/20
Congestion in the spleen
0/10
20/20**
20/20**
Hematopoietic cell proliferation in the spleen
3/10
20/20**
1/20
Hemosiderin pigmentation in the spleen
3/10
20/20**
18/20**
Thrombosis in the spleen
0/10
2/20
0/20
Fibrosis capsule in the spleen
0/10
20/20**
20/20**
Capsule, lymphatic, angiectasis in the spleen
0/10
6/20
15/20**
aNTP (1996b,c).
bAnimals were administered diets with 5000 ppm; adjusted daily doses were provided in the study report.
°Number in parentheses is the percent of control.
dMean ± standard error.
"Number of animals with lesions/number of animals.
**p < 0.01.
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Table B.3. Incidence of Selected Noncancer Parameters in the F344 Rat Exposed to Oral
(in Feed) o-Toluidine for 101 to 104 Weeks3
Parameter
Exposure Group (ppm)
(Average Daily Dose, mg/kg-d)
Males
0
3000 (231)
6000 (525)
Probability of survival13
90%
27%
0%
Liver Necrosis
2/20 (10%)c
12/50 (24%)
17/49 (35%)*
Bladder epithelial hyperplasia
0/20 (0%)
9/50 (18%)*
7/44 (16%)
Females
0
3000 (264)
6000 (600)
Probability of survival13
80%
55%
21%
Liver Necrosis
0/20 (0%)
1/50 (2%)
15/49 (31%)*
Bladder epithelial hyperplasia
0/20 (0%)
21/45 (47%)*
13/47 (28%)*
aNCI (1979a).
bActual mortality was not provided in the study report; results were estimated for the study termination from
Kaplan and Meier curves provided in the study report. There were significant positive dose-response trends in
mortality.
°Number of animals with lesions/total number of animals (%).
*p < 0.05 Fischer's Exact Test performed for this PPRTV document.
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Table B.4. Incidence of Tumors in the F344 Rat Exposed to Oral (in Feed)
o-Toluidine for 101 to 104 Weeks3
Parameter
Exposure Dose (ppm)
(Human Equivalent Dose, mg/kg-d)
Males
0
3000 (63.0)
6000 (138.8)
Subcutaneous fibroma
0/20 (0%)b
28/50 (56%)**
27/49 (55%)**
Spleen sarcoma, NOS°
0/20
1/49 (2%)
3/42 (7%)
Mesotheliomas of the tunica vaginalis or multiple
organs
0/20
17/50 (34%)**
9/49 (18%)*
Multiple organ osteosarcoma
0/20 (0%)
3/50 (6%)
5/49 (10%)
Multiple organ fibrosarcoma
0/20 (0%)
8/50 (16%)
20/49 (41%)**
Multiple organ sarcoma, NOS
0/20 (0%)
3/50 (6%)
11/49 (22%)*
Multiple organ sarcoma, NOS, fibrosarcoma,
angiosarcoma, or osteosarcoma
0/20
15/50 (30%)**
37/49 (76%)**
Females
0
3000 (63.7)
6000 (140.4)
Mammary fibroadenoma
6/20 (30%)
20/50 (40%)
35/49 (71%)*
Bladder transitional-cell carcinoma
0/20 (0%)
9/45 (20%)*
22/47 (47%)**
Multiple organ osteosarcoma
0/20 (0%)
0/50 (0%)
18/49 (37%)**
Multiple organ sarcoma, NOS
0/20 (0%)
1/50 (2%)
2/49 (4%)
Multiple organ sarcoma, NOS, fibrosarcoma,
angiosarcoma, or osteosarcoma
0/20
3/50 (6%)
21/49 (43%)**
Spleen sarcoma, NOS, angiosarcoma, or osteosarcoma
0/20
9/49 (18%)*
12/49 (24%)*
Spleen fibroma
0/20 (0%)
4/49 (8%)
6/49 (12%)
Spleen angiosarcoma
0/20 (0%)
7/49 (14%)
9/49 (18%)*
Spleen sarcoma, NOS
0/20 (0%)
1/49 (2%)
3/49 (6%)
aNCI (1979a).
bNumber of animals with tumor/number of animals (%).
°NOS = Not otherwise stated.
*p < 0.05; **p < 0.01.
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Table B.5. Incidence of Tumors in Male CD Rats Exposed to Oral (in Feed)
o-Toluidine for 18 Months3
Parameter
Human Equivalent Dose (mg/kg-d)b
Concurrent
Control
Pooled Control
72.1
144.1
Subcutaneous fibroma and
fibrosarcoma
0/16 (0%)c
18/111 (16%)
18/23 (78%)d
21/24 (88%)d
Bladder tumors
0/16 (0%)
5/111 (5%)
3/23 (13%)
4/24 (17%)
Multiple tumors
3/16 (19%)
14/111 (13%)
6/23 (26%)
8/24 (33%)e
aWeisburger etal. (1978a).
bAnimals were administered diets with 8000 or 16,000 ppm for 3 months followed by 4000 or 8,000 ppm for an
additional 15 months, then placed on control diet for 6 months.
°Number of animals with tumor/number of animals (%).
Ap < 0.025 denotes significant difference from all controls.
ep < 0.025 denotes significant difference from pooled controls only
Table B.6. Incidence of Tumors in the B6C3Fi Mice Exposed to Oral (in Feed)
o-Toluidine for 101 to 104 Weeks3
Parameter
Exposure Group (ppm)
(Human Equivalent Dose, mg/kg-d)
Males
0
1000 (23.0)
3000 (73.8)
Hepatocellular adenoma
1/20 (5%)b
3/50 (6%)
3/50 (6%)
Hepatocellular carcinoma
4/19 (21%)
16/50 (32%)
11/50 (22%)
Hepatocellular adenoma and carcinoma
5/19 (26%)
19/50 (38%)
14/50 (28%)
Hemangiosarcoma (all sites)
1/20 (5%)
1/50 (2%)
10/50 (20%)°
Females
0
1000 (26.5)
3000 (91.9)
Hepatocellular adenoma
0/20 (0%)
2/49 (4%)
6/50 (12%)
Hepatocellular carcinoma
0/20 (0%)
2/49 (4%)
7/50 (14%)°
Hepatocellular adenoma and carcinoma
0/20 (0%)
4/49 (8%)
13/50 (26%)**
Hemangiosarcoma (all sites)
1/20 (5%)
1/49 (2%)
2/50 (4%)
aNCI (1979b).
bNumber of animals with tumor/number of animals (%).
Significant (p < 0.05) trend but incidence not significantly different from the control.
**p < 0.01.
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Table B.7. Incidence of Vascular Tumors in the CD-I Mice Exposed to Oral (in Feed)
o-Toluidine for 18 Months3
Parameter
Human Equivalent Dose (mg/kg-d)b
Males
Concurrent control
Pooled control
210.5
420.9
Vascular tumors
0/14 (0%)c
5/99 (5%)
5/14 (36%)**
9/11 (82%)**
Females
Concurrent control
Pooled control
211.8
423.7
Vascular tumors
0/15 (0%)
9/102 (9%)
5/18 (28%)*
9/21 (43%)**
Males and females
combined
Concurrent control
Pooled control
211.2
422.3
Vascular tumors
0/29 (0%)
14/201 (7%)
10/32 (31%)
18/32 (56%)
aWeisburger et al. (1978b).
bAnimals were administered diets with 16,000 or 32,000 ppm for 3 months followed by 8000 or 16,000 ppm for an
additional 15 months, then were on control diet for an additional 3 months.
°Number of animals with tumor/number of animals (%).
*p < 0.05; **p < 0.025.
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APPENDIX C. BMD OUTPUTS
Table C.l. Multistage Cancer Model Predictions for Tumor Data for o-Toluidine
Tumor
Type/Site
Gender/Species
Goodness-of-Fit
p-Valuea
AICb
for
Fitted
Model
BMDL10C
(mg/kg-d)
Slope Factor
Reference
Subcutaneous
fibroma and
fibrosarcoma
Male rat
0.57
45.25
4.50
0.0222316
Weisburger et
al. (1978a)
Vascular tumor
data
Female mouse
0.96
52.02
50.00
0.00199986
Weisburger et
al. (1978b)
Vascular tumor
data
Male mouse
0.54
31.99
23.00
0.00434693
Weisburger et
al. (1978a)
Bladder tumors
Male rat
0.85
41.74
40.3
0.00248149
Weisburger et
al. (1978a)
Multiple tumors
Male rat
0.64
71.32
34.25
0.00292001
Weisburger et
al. (1978a)
Subcutaneous
fibroma
Male rat
0.01
146.48
10.41
0.00960838
NCI (1979a)
Multiple organ
sarcomas,
NOSd,
fibrosarcoma,
angiosarcoma,
or osteosarcoma
Male rat
1.00
119.64
13.77
0.00726365
NCI (1979a)
Multiple organ
sarcoma, NOS,
fibrosarcoma,
angiosarcoma,
or osteosarcoma
Female rat
0.59
92.80
47.03
0.00212622
NCI (1979a)
Spleen sarcoma,
NOS,
angiosarcoma,
or osteosarcoma
Female rat
0.55
100.40
32.71
0.00305746
NCI (1979a)
Multiple organ
osteosarcoma
Male rat
0.95
57.09
73.66
0.00135766
NCI (1979a)
Multiple organ
osteosarcoma
Female rat
0.09
74.80
61.92
0.00161487
NCI (1979a)
Multiple organ
sarcoma, NOS
Male rat
0.62
77.90
45.27
0.00220876
NCI (1979a)
Multiple organ
sarcoma, NOS
Female rat
1.00
28.52
137.23
0.000728707
NCI (1979a)
Multiple organ
fibrosarcoma
Male rat
0.76
112.81
22.89
0.00436794
NCI (1979a)
Spleen fibromas
Male rat
0.01
73.52
Failed
failed
NCI (1979a)
Spleen fibromas
Female rat
0.85
66.45
61.62
0.00162297
NCI (1979a)
Spleen
angiosarcomas
Female rat
0.56
86.85
42.73
0.00234007
NCI (1979a)
Spleen
sarcomas
Female rat
0.76
25.71
132.25
0.000756154
NCI (1979a)
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Table C.l. Multistage Cancer Model Predictions for Tumor Data for o-Toluidine
Tumor
Type/Site
Gender/Species
Goodness-of-Fit
p-Valuea
AICb
for
Fitted
Model
BMDLioC
(mg/kg-d)
Slope Factor
Reference
Spleen sarcoma,
NOS
Male rat
0.81
24.93
122.54
0.000816071
NCI (1979a)
Mammary
fibroadenoma
Female rat
0.86
154.40
14.99
0.00667223
NCI (1979a)
Rat bladder
transitional-cell
carcinoma
Female rat
0.82
112.41
19.10
0.0052354
NCI (1979a)
Hepatocellular
carcinoma
Female mouse
1.00
59.21
39.22
0.00254972
NCI (1979b)
Hemangio-
sarcomas, all
sites
Male mouse
0.26
72.92
38.35
0.00260727
NCI (1979b)
Hemangio-
sarcomas, all
sites
Female mouse
0.21
31.41
100.95
0.000990631
NCI (1979b)
Hepatocellular
adenoma and
carcinoma
Female mouse
1.00
87.02
22.20
0.00450356
NCI (1979b)
Hepatocellular
adenoma
Female mouse
0.99
55.43
43.29
0.00231009
NCI (1979b)
"Values <0.10 fail to meet conventional goodness-of-fit criteria.
bAIC = Akaike's Information Criteria.
cBMDLio = lower confidence limit (95%) on the benchmark dose at 10% incidence, extra risk.
dNOS = not otherwise stated.
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Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
1
0.8
0.6
0.4
0.2
0
BMDL
BMD
0	20	40	60	80	100 120 140
dose
14:12 01/13 2011
Figure C.l.° Multistage Cancer BMD Model for Subcutaneous Fibroma and Fibrosarcoma
in Male F344 Rats Data (Weisburger et al., 1978)
Text Output for 1° Multistage cancer BMD Model for Subcutaneous Fibroma and
Fibrosarcoma in Male F344 Rats Data (Weisburger et al., 1978)
Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)
Input Data File:
C:/18/Jan2011/Weisburger_1978_fibroma_fibrosarcoma_rat_m_MultiCancl_l.(d)
Gnuplot Plotting File:
C:/18/Jan2011/Weisburger_1978_fibroma_fibrosarcoma_rat_m_MultiCancl_l.pit
Thu Jan 13 14:12:20 2011
Male rat subcutaneous fibroma and fibrosarcoma
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl) ]
The parameter betas are restricted to be positive
40
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Dependent variable = DichPerc
Independent variable = Dose
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
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 =	0.14965 4
Beta(1) =	0.014428
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Beta(1)
Beta (1)	1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable	Estimate	Std. Err.	Lower Conf. Limit Upper Conf.
Limit
Background	0	*	*	*
Beta(1)	0.0173338	*	*	*
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
#
Log(likelihood)
-21.085
-21.6251
-41.8653
Param's
3
1
1
Deviance Test d.f.
1.08023
41.5606
P-value
0.5827
<.0001
AIC:
45 .2502
Goodness of Fit
Scaled
Dose	Est._Prob. Expected Observed	Size	Residual
0.0000	0.0000	0.000	0.000	16	0.000
72.0626	0.7132	16.405 18.000	23	0.736
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144.1251	0.9178	22.026 21.000	24	-0.763
Chi^2 = 1.12	d.f. = 2	P-value = 0.5704
Benchmark Dose Computation
Specified effect =	0.1
Risk Type =	Extra risk
Confidence level =	0.95
BMD =	6.07834
BMDL =	4.4 9811
BMDU =	8 .32855
Taken together, (4.49811, 8.32855) is a 90	% two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor =	0.0222316
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