United States
Environmental Protection
1=1 m m Agency
EPA/690/R-09/065F
Final
6-16-2009
Provisional Peer-Reviewed Toxicity Values for
1,2,4-Trichlorobenzene
(CASRN 120-82-1)
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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COMMONLY USED ABBREVIATIONS
BMD
Benchmark Dose
IRIS
Integrated Risk Information System
IUR
inhalation unit risk
LOAEL
lowest-observed-adverse-effect level
LOAELadj
LOAEL adjusted to continuous exposure duration
LOAELhec
LOAEL adjusted for dosimetric differences across species to a human
NOAEL
no-ob served-adverse-effect level
NOAELadj
NOAEL adjusted to continuous exposure duration
NOAELhec
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
p-OSF
provisional oral slope factor
p-RfC
provisional inhalation reference concentration
p-RfD
provisional oral reference dose
RfC
inhalation reference concentration
RfD
oral reference dose
UF
uncertainty factor
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PROVISIONAL PEER REVIEWED TOXICITY VALUES FOR
1,2,4-TRICHLOROBENZENE (CASRN 120-82-1)
Background
On December 5, 2003, the U.S. Environmental Protection Agency's (U.S. EPA) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1.	U. S. EPA's Integrated Risk Information System (IRIS).
2.	Provisional Peer-Reviewed Toxicity Values (PPRTVs) used in U.S. EPA's Superfund
Program.
3.	Other (peer-reviewed) toxicity values, including:
~	Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR),
~	California Environmental Protection Agency (CalEPA) values, and
~	EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in U.S. EPA's IRIS. PPRTVs are developed according to a
Standard Operating Procedure (SOP) and are derived after a review of the relevant scientific
literature using the same methods, sources of data, and Agency guidance for value derivation
generally used by the U.S. EPA IRIS Program. All provisional toxicity values receive internal
review by two U.S. EPA scientists and external peer review by three independently selected
scientific experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the
multiprogram consensus review provided for IRIS values. This is because IRIS values are
generally intended to be used in all U.S. EPA programs, while PPRTVs are developed
specifically for the Superfund Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a 5-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV documents conclude that
a PPRTV cannot be derived based on inadequate data.
Disclaimers
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and Resource Conservation and Recovery Act (RCRA) program offices are advised to
carefully review the information provided in this document to ensure that the PPRTVs used are
appropriate for the types of exposures and circumstances at the Superfund site or RCRA facility
in question. PPRTVs are periodically updated; therefore, users should ensure that the values
contained in the PPRTV are current at the time of use.
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It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
users are strongly encouraged to read the entire PPRTV document and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the U.S. EPA
Office of Research and Development's National Center for Environmental Assessment,
Superfund Health Risk Technical Support Center for OSRTI. Other U.S. EPA programs or
external parties who may choose of their own initiative to use these PPRTVs are advised that
Superfund resources will not generally be used to respond to challenges of PPRTVs used in a
context outside of the Superfund Program.
Questions Regarding PPRTVs
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) may be directed
to the U.S. EPA Office of Research and Development's National Center for Environmental
Assessment, Superfund Health Risk Technical Support Center (513-569-7300), or OSRTI.
INTRODUCTION
Trichlorobenzene, specifically 1,2,4-trichlorobenzene CASRN 120-82-1, is a
high-production-volume (HPV) chemical listed in U.S. EPA's toxic release inventory (TRI)
database. Existing toxicity reference values include a chronic RfD for 1,2,4-trichlorobenzene
(verified in November 1996) which is available in IRIS (U.S. EPA, 2008). The RfD of
0.01 mg/kg-day is based on increased adrenal weights and vacuolization of the zona fasciculata
in the adrenal cortex in a multigenerational rat reproductive toxicity study (Robinson et al.,
1981). Rats were exposed from birth through three generations; a NOAEL of 14.8 mg/kg-day
was identified (Robinson et al., 1981). Uncertainty factors (UF), of 10 each, for interspecies
extrapolation, protection of sensitive humans, and lack of chronic studies were applied to the
NOAEL to derive the RfD. The two source documents were (1) the U.S. EPA (1985, 1988)
Health Assessment Document (HAD) for Chlorinated Benzenes and (2) the Drinking Water
Criteria Document (DWCD) for Trichlorobenzenes. The Drinking Water Standards and Health
Advisories list (U.S. EPA, 2006) includes the same chronic RfD of 0.01 mg/kg-day as reported
on IRIS. The Health Effects Assessment Summary Tables (HEAST; U.S. EPA, 1997) reports a
subchronic RfD of 0.01 mg/kg-day for 1,2,4-trichlorobenzene, adopting the chronic RfD from
IRIS as the subchronic RfD.
IRIS (U.S. EPA, 2008) does not report an RfC for 1,2,4-trichlorobenzene. The HEAST
(U.S. EPA, 1997) lists chronic and subchronic RfCs of 0.2 and 2.0 mg/m3 (respectively). The
RfCs were based on 6- and 26-week rat, rabbit, dog and monkey inhalation studies by
Kociba et al. (1981) and Coate et al. (1977). A nominal NOAEL of 104 ppm (772 mg/m3) and
total uncertainty factors of 100 and 1000 were used to derive the subchronic and chronic RfCs
(respectively). No U.S. EPA source document for this assessment was listed.
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Relevant documents in the Chemical Assessments and Related Activities (CARA) list
(U.S. EPA, 1991a, 1994a) include a Health Effects Assessment (HEA) for
1,2,4-Trichlorobenzene (U.S. EPA, 1987) in addition to the previously mentioned HAD for
Chlorinated Benzenes (U.S. EPA, 1985) and DWCD for Trichlorobenzenes (U.S. EPA, 1988).
The HEA includes chronic and subchronic "inhalation RfDs" of 0.18 and 1.75 mg/day for
1,2,4-trichlorobenzene based on a NOAEL of 3 ppm (22 mg/m3) in a rat study by
"3
Watanabe et al. (1978); a LOAEL of 10 ppm (74 mg/m ) was identified for increased urinary
porphyrin excretion in this study. However, this derivation is not consistent with current
U.S. EPA (1994b) methodology for RfC derivation.
The American Conference of Governmental Industrial Hygienists (ACGIH, 2007) has
adopted a Short-Term Exposure Limit (STEL) ceiling of 5 ppm (-40 mg/m ) for
1,2,4-trichlorobenzene. This value is intended to minimize the potential for ocular and upper
respiratory tract irritation in exposed workers (ACGIH, 2007). The National Institute for
Occupational Safety and Health (NIOSH, 2008) also recommends a ceiling limit of 5 ppm
"3
(-40 mg/m ) for 1,2,4-trichlorobenzene based on the same effects. The Occupational Safety and
Health Administration (OSHA, 2008) has no permissible exposure limit (PEL) for
1,2,4-trichlorobenzene.
In the IRIS, 1,2,4-trichlorobenzene is assigned to Weight-of-Evidence (WOE) Group D
(not classifiable as to human carcinogenicity), last revised in 1991, based on the U.S. EPA
(1986) Guidelines for Carcinogen Risk Assessment. The same classification is shown in the
Drinking Water Standards and Health Advisories list (U.S. EPA, 2006). A cancer assessment for
1,2,4-trichlorobenzene is not included in the HEAST (U.S. EPA, 1997). The Health Assessment
Document for Chlorinated Benzenes (U.S. EPA, 1985) and Health Effects Assessment for
1,2,4-Trichlorobenzene (U.S. EPA, 1987) also assigned the chemical to Group D.
The National Toxicology Program (NTP, 2008) has not assessed the toxicity or
carcinogenicity of 1,2,4-trichlorobenzene and this compound is not included in the 11th Report
on Carcinogens (NTP, 2005). 1,2,4-Trichlorobenzene has not been the subject of a monograph
by the International Agency for Research on Cancer (IARC, 2008) or a toxicological profile by
the Agency for Toxic Substances Disease Registry (ATSDR, 2008). An Environmental Health
Criteria document for chlorinated benzenes (WHO, 1991), a Priority Substances List Assessment
Report on trichlorobenzenes (Health Canada, 1993), and a toxicity review on halogenated
benzenes (Leber and Bus, 2001) were consulted for relevant information. To identify
toxicological information pertinent to the derivation of provisional toxicity values for
1,2,4-trichlorobenzene, literature searches were conducted in December 2007 using the
following databases: MEDLINE, TOXLINE, BIOSIS (August 2000-December 2007),
TSCATS1/2, CCRIS, DART/ETIC, GENETOX, HSDB, RTECS, and Current Contents (first
half of 2008). Except where noted, the literature searches were not limited by date.
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REVIEW OF PERTINENT DATA
Human Studies
No data were located in the review documents (WHO, 1991; Health Canada, 1993;
Leber and Bus, 2001) or the literature search (see above) regarding the effects of
1,2,4-trichlorobenzene in humans exposed by any route.
Animal Studies
Oral Exposure
Subchronic Studies—The Chemical Manufacturers Association (CMA) (1989a)
conducted a subchronic dietary study of 1,2,4-trichlorobenzene (99% pure) in F344 rats. Groups
of 10 rats/sex/dose were given diets containing nominal concentrations of 0, 200, 600, or,
1800 ppm for 94 days. Due to the volatility of the test compound, the investigators mixed diets
containing 110% of the target concentrations; diets were stored frozen and changed twice each
week. Laboratory analyses of the diet showed that the mean concentrations were ±4% of the
target concentrations. Based on estimates of weekly compound intake provided by the authors,
average intakes over the study duration were 0, 14.6, 45.6, and, 133.7 mg/kg-day in males and 0,
17.0, 52.5, and 150.6 mg/kg-day in females. Daily observations for clinical signs were made,
and both body weight and food consumption were recorded weekly. All rats were subjected to
ophthalmoscopic examination prior to termination. Blood was collected prior to sacrifice for
analysis of hematology (Hgb, Hct), erythrocyte count and morphology, platelet count and total
and differential leukocyte counts) and serum chemistry (alanine aminotransferase [ALT],
aspartate aminotransferase [AST], blood urea nitrogen [BUN], glucose, total protein, albumin,
creatinine, total bilirubin, electrolytes, calcium and phosphorus). All rats received complete
necropsies and the adrenals, brain, heart, kidneys, liver, testes with epididymides (males) and
ovaries (females) were weighed. Complete histopathology examinations (30 tissues) were
performed on control and high-dose rats of both sexes, and the kidney and liver were examined
microscopically in all dose groups.
There were no deaths during the study (CMA, 1989a). Treated rats exhibited a higher
incidence of chromodacryorrhea (red tears) and excessive lacrimation than controls, but these
effects did not exhibit a clear dose-response relationship. Body weights were not different from
controls in the treated animals, although food consumption was increased in mid- and high-dose
males. Ophthalmoscopy was not affected by treatment. Table 1 shows the hematology and
clinical chemistry findings. A borderline anemia was evident in high-dose males, with a trend
toward this effect in high-dose females as well. Mean hemoglobin and Hct values were
significantly reduced in both sexes at the high dose, and mean erythrocyte count was also
reduced in high-dose males. Platelet and total leukocyte counts were increased in high-dose
males. Clinical chemistry parameters affected by treatment included increases in BUN, total
protein, calcium and albumin in high-dose males; decreased AST levels in mid- and high-dose
males; and increased BUN in high-dose females. The authors reported that individual BUN
values were within the normal range of variability for this species.
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Table 1. Significant Changes in Rats Treated with 1,2,4-Trichlorobenzene
in the Diet for 13 Weeksa
Males

Control
200 ppm
(14.6 mg/kg-d)
600 ppm
(45.6 mg/kg-d)
1800 ppm
(133.7 mg/kg-d)
Hematology
Erythrocyte count (l()'/|iL)
8.55 ± 0.16b
8.56 ±0.17
8.51 ±0.21
8.12 ± 0.22d
Hematocrit (%)
50 ± 1
50 ± 1
49 ± 1
47 ± ld
Hemoglobin (g/dL)
16.9 ±0.2
16.9 ±0.2
16.7 ±0.4
15.7 ± 0.4d
Platelets (10>L)
6.72 ±0.32
6.71 ±0.27
6.96 ±0.45
7.80 ± 0.27d
Total leukocyte count (10 7|iL)
7.5 ± 1.0
7.9 ±1.1
8.3 ±0.9
8.9 ± 0.9°
Clinical Chemistry
AST (IU/L)
84 ± 19
77 ±7
66 ± 9°
61 ±5d
BUN (mg/dL)
17.3 ± 1.5
16.2 ± 1.7
16.3 ± 1.0
19.3 ± 1.5°
Protein (g/dL)
6.3 ±0.2
6.3 ±0.2
6.5 ±0.1
7.3 ±0.2d
Albumin (g/dL)
4.0 ±0.1
4.0 ±0.1
4.0 ±0.1
4.4 ± 0.1d
Calcium (mg/dL)
10.3 ±0.2
10.3 ±0.3
10.4 ±0.2
11.1 ± 0.3d
Organ Weights
Adrenal weight (g)
0.0425 ± 0.0063
0.0805 ±0.1124
0.0406 ± 0.0070
0.0509 ± 0.0060°
Adrenal/body weight (x 10,000)
1.57 ±0.19
3.03 ±4.41
1.53 ±0.25
1.84 ± 0.17°
Kidney weight (g)
1.932 ±0.163
2.030 ±0.181
2.113 ±0.185
2.455 ±0.199d
Kidney/body weight (x 1000)
7.13 ±0.36
7.45 ±0.31
7.92 ± 0.39d
8.85 ± 0.28d
Liver weight (g)
7.116 ±0.596
7.936 ±0.615c
8.615 ±0.678d
11.876 ± 1.063d
Liver/body weight (/ 100)
2.63 ±0.13
2.91 ± 0.10d
3.23 ±0.14d
4.28 ± 0.17d
Testes weight (g)
2.884 ± 0.072
2.989 ±0.241
2.877 ±0.129
3.059 ± 0.073d
Histopathology
Centrilobular hepatocyte
hypertrophy
0/10e
0/10
5/10f
10/108
Renal dilated tubules
0/10
0/10
0/10
10/108
Renal granular casts
0/10
0/10
0/10
10/108
Renal hyaline droplets
0/10
0/10
10/108
10/108
Interstitial nephritis
0/10
1/10
3/10
9/10s
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Table 1. Significant Changes in Rats Treated with 1,2,4-Trichlorobenzene
in the Diet for 13 Weeksa
Females

Control
200 ppm
(17.0 mg/kg-d)
600 ppm
(52.5 mg/kg-d)
1800 ppm
(150.6 mg/kg-d)
Hematology
(%)Hematocrit
50 ± 1
50 ± 1
50 ± 1
48 ± 1°
Erythrocyte count(106/uL)
8.09 ±0.23
7.98 ±0.15
8.00 ± 0.22
7.89 ±0.11
Hemoglobin (g/dL)
17 ±0.4
16.8 ±0.4
16.8 ±0.3
16.4 ± 0.1°
Clinical Chemistry
BUN (mg/dL)
16.2 ± 1.3
17.2 ± 1.8
18.2 ± 1.8
19.4 ± 2.1d
Organ Weights
Kidney/body weight (xlOOO)
7.76 ±0.31
7.83 ±0.45
8.34 ± 0.39°
8.34 ± 0.59°
Liver weight (g)
4.139 ±0.298
4.278 ±0.101
4.863 ± 0.408d
5.381 ±0.294d
Liver/body weight (x 100)
2.63 ±0.15
2.71 ±0.10
3.14 ± 0.37d
3.54 ± 0.20d
Histopathology
Centrilobular hepatocyte
hypertrophy
0/10
0/10
0/10
10/108
Renal tubular mineral deposition
9/10
10/10
10/10
10/10
aCMA, 1989a
bMean ± standard deviation
"Significantly different from control atp< 0.05
d/?<0.01
"Number affected/number examined
(p < 0.05 by Fischer's exact test conducted for this review
sp < 0.0001 by Fischer's exact test conducted for this review
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Significant changes in organ weights were observed in the adrenal glands, liver, kidneys,
and testes of male rats and in the kidneys and liver of female rats (CMA, 1989a). Table 1 details
the organ-weight changes in all treatment groups. As the table indicates, absolute and relative
adrenal weights were increased over control values (20% and 17%, respectively) in the high-dose
males but not in females. Absolute and relative liver weights were significantly increased in
males at all doses (with >60% increases at the high dose) and in females exposed to 600 or
1800 ppm (-30-35%) increase at the high dose). Absolute kidney weight was increased in
high-dose males but not at any dose in females; relative kidney weight was increased in both
sexes at doses >600 ppm. Testes weights were modestly increased in high-dose males. The
histopathology findings were consistent with the liver and kidney weight changes (see Table 1).
Centrilobular hepatocyte hypertrophy was observed in all rats of both sexes at the high dose and
in some mid-dose males, and may underlie the changes in observed organ weights. The authors
characterized this finding as "minimal" in females and mid-dose males and "mild" in high-dose
males, but severity scores are not reported. Kidney findings were largely restricted to males,
although there was a suggestion of increased severity (but not increased incidence) of kidney
tubular mineral deposition in high-dose females. All high-dose males exhibited dilated renal
tubules and granular casts. Hyaline droplets were observed in all mid- and high-dose males.
Finally, a dose-related increase in the incidence of interstitial nephritis was reported in male rats.
The constellation of kidney changes occurring only in male rats (especially hyaline
droplets, dilated tubules, granular casts) is suggestive of male rat-specific alpha 2[j,-globulin
nephropathy, an endpoint that is not considered relevant to human health (U.S. EPA, 1991b).
The absence of kidney effects in mice treated subchronically (CMA, 1989b) or chronically (with
similar dosing ranges) (CMA, 1994b) and the finding of related kidney histopathology in male
rats treated via the diet for 104 weeks (renal pelvis mineralization, transitional cell hyperplasia,
increased severity of chronic progressive nephropathy; CMA, 1994a) or via inhalation for
26 weeks ("hyaline degeneration"; Coate et al., 1977) provide support for a potential relationship
with alpha 2[j,-globulin nephropathy. In female rats in this study (CMA, 1989b), kidney weights
were increased and there was a suggestion of increased severity of renal tubular mineralization.
In female rats exposed chronically via the diet, a statistically significant increase in the incidence
of renal pelvis mineralization was observed (CMA, 1994a). The biological significance of these
slight changes is uncertain. In the absence of data demonstrating the accumulation of
alpha 2[j,-globulin, it is not possible to conclusively demonstrate this mechanism. However, the
closely related compound, 1,4-dichlorobenzene, is considered a model inducer of
alpha 2[j,-globulin nephropathy (U.S. EPA, 1991b). As available data provide suggestive support
for a finding that the male rat kidney effects may not be relevant to humans, these effects are not
considered as the basis for the LOAEL.
The authors identified the low dose (200 ppm) as a NOAEL for female rats and reported
that a NOAEL for male rats was not identified (CMA, 1989a). The effect occurring at the low
dose was an 11% increase in liver weights only in male rats. In the absence of accompanying
histopathology, this response may be considered an adaptive physiologic change. However, at
the mid-dose (600 ppm) the incidence of hepatocellular hypertrophy was increased in males and
liver weights were increased significantly (-20%) in both sexes. U.S. EPA (2002) considers
these two endpoints to be adverse when there is a known mode of action (MOA) for toxicity. In
the case of 1,2,4-trichlorobenzene, there is evidence to suggest that liver enlargement is related
to induction of liver enzymes that may result in secondary toxicity. Specifically,
1,2,4-trichlorobenzene is a potent inducer of liver enzymes, including cytochrome P-450,
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cytochrome c reductase, benzpyrene hydroxylase, azoreductase, aniline hydroxylase,
aminopyrine demethylase, heme oxygenase, and others (Carlson and Tardiff, 1976;
Black et al., 1988; Cote et al., 1988; Ariyoshi et al., 1981). Induction of liver enzymes may be
associated with secondary toxic effects (U.S. EPA, 2002). Several studies have shown toxic
effects of 1,2,4-trichlorobenzene that may be attributable to enzyme induction: increased
excretion of porphyrins (Carlson, 1977; Kociba et al., 1981; Watanabe et al., 1977, 1978),
hematologic effects (CMA, 1989a,b; Black et al., 1988), reduced circulating levels of thyroid
hormones (den Besten et al., 1991a) and thyroid histopathology (Cote et al., 1988;
Black et al., 1988). Porphyrinuria may be the result of the induction of liver enzymes and/or
disruption of liver function and heme metabolism and, as such, may be related to observations of
anemia. Alterations in hepatic thyroxine metabolism may contribute to the reduction in
circulating thyroid hormone levels and induction of thyroid histopathology. For the purpose of
this review, based on the suggestive evidence that induction of liver enzymes by
1,2,4-trichlorobenzene may be associated with secondary toxicity, the mid dose
(45.6 mg/kg-day) is considered a LOAEL based on hepatocellular hypertrophy and increased
liver weight in male rats; the low dose (14.6 mg/kg-day) is considered a NOAEL.
CMA (1989b) conducted a parallel subchronic study in B6C3Fl/CrlBR hybrid mice.
Groups of 10 mice/sex/dose were given diets containing target concentrations of 0, 200, 3500, or
7000 ppm 1,2,4-trichlorobenzene (99.48% pure) for 13 weeks. Due to the volatility of the test
compound, the investigators mixed diets containing 110% of the target concentrations; diets
were stored frozen and changed twice each week. Tests for stability of the diet material showed
a predictable rate of loss over time; using this loss constant, the authors estimated that a 10%
increase in nominal concentration would result in average concentrations near that of the target
levels. Analysis of the administered diets was not performed. Based on estimates of weekly
compound intake provided by the authors, average intakes over the study duration were 0, 67,
851, and 1222 mg/kg-day in males and 0, 87, 1184 and 1346 mg/kg-day in females.
Observations for clinical signs were made daily; body weight and food consumption were
measured weekly. Ophthalmoscopic examinations were administered before the study and prior
to termination. Blood was collected prior to sacrifice for analysis of hematology (Hgb, Hct,
erythrocyte count, platelet count, total and differential leukocyte counts, mean cell volume
[MCV], mean cell hemoglobin [MCH], and mean cell hemoglobin concentration [MCHC]) and
serum chemistry (sorbitol dehydrogenase [SDH], ALT, BUN, glucose, total protein, albumin,
globulin, gamma glutamyl transferase [GGT], electrolytes, calcium and phosphorus). Mice were
given complete necropsies, and the adrenals, brain, kidneys, liver, spleen and testes with
epididymides were weighed. Complete histopathology examinations (38 tissues) were
performed on control and high-dose mice, and the lung, kidney, and liver (as well as any gross
lesions) were examined microscopically in all dose groups.
Treatment did not affect survival, incidence of clinical signs or ophthalmoscopic findings
(CMA, 1989b). Food consumption was consistently lower than control values in mid- and
high-dose mice of both sexes throughout most of the study. The study reported that average
cumulative body weight gain was significantly lower than controls throughout the study in males
of all doses. However, examination of individual body weight data revealed an apparent error in
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the initial body weight recorded for one male control mouse1; this error resulted in a falsely
inflated estimate of cumulative body weight gain for male control mice and distorted the body
weight gain comparisons with treated male mice (leading to inflated differences from control).
Average cumulative body weight gain was significantly lower than controls in high-dose females
(female body weight gain was not subject to this error) and, notwithstanding the error in baseline
male control weights, may have also been reduced in high-dose males. Terminal body weight
values (which were unaffected by the error) were significantly (p < 0.05) lower than controls in
mid- and high-dose males (14% lower for both doses) and in high-dose females (8% lower).
Weekly body weight measures were significantly (p < 0.05) lower than controls in low dose
males during Weeks 11 and 12 (7% lower each time) but not at termination. Hematology
findings were unremarkable except in high-dose females, which exhibited reduced Hct, Hgb,
MCV, and MCH (see Table 2); these changes are similar to those observed in the subchronic rat
study (CMA, 1989a). Serum chemistry changes, shown in Table 2, include marked increases in
AST and SDH in mice of both sexes exposed to >3500 ppm, with more pronounced effects
evident in males than in females.
Statistically significant organ-weight changes were observed in the liver, spleen, kidney,
and brain of both sexes and in the adrenals and testes of males (CMA, 1989b). Absolute and
relative liver weights were markedly increased (57-128% above control values), despite body
weight reductions, in the mid- and high-dose mice of both sexes (see Table 2). Liver was also
the site of the only significant histopathology findings, consisting of cytomegaly/karyomegaly
multinucleation, atrophy, degeneration, and microcytosis, as well as coagulative necrosis.
Table 2 shows the incidence of these lesions. As shown in Table 2, the absolute and
relative spleen weights were reduced in high-dose females, which may be correlated with the
hematology changes in this group. Absolute—but not relative—spleen weights were also
decreased in high-dose males. Weight changes in the kidney, adrenal, brain, and testes were
small increases in relative weight and/or decreases in absolute weight consistent with, and
probably secondary to, decreased body weight in the mid- and high-dose groups. The kidney,
adrenal, brain, and testes weight changes were not associated with toxicological or
histopathological correlates.
The authors identified the low dose as a NOAEL for females and reported that a NOAEL
was not identified for males given the reductions in cumulative body weight gain observed at all
doses. However, as noted above, the cumulative body weight gain comparisons were distorted
by the inclusion of an erroneously low initial body weight in one control mouse. Weekly
measures of body weight in males exposed at the low dose were reduced slightly (7%) during
Weeks 11 and 12 but not at termination; no other statistically significant changes were observed
at this dose. In contrast, both food consumption and body weights were reduced at the mid dose
in males, and clear evidence of liver toxicity (including 3-fold increases in ALT and/or SDH,
histopathology and liver weight increases) was observed in both males and females at the mid
dose. Thus, for the purpose of this review, the mid dose serves as a LOAEL (851 and
1154 mg/kg-day in males and females, respectively), and the low dose serves as a NOAEL (67
and 87 mg/kg-day in males and females).
1 The initial (Week 0) body weight measurement for male control mouse #31937 was reported as 12.9 g, while other
male controls weighed from 19.6-22.7 g. The Week 1 body weight for mouse #31937 was reported as 21.5 g,
resulting in a 1-week body weight gain of 8.6 g. Such a weight gain is highly implausible, as is the chance that a
mouse with such an unusually low body weight would be included in the study.
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Table 2. Selected Changes in Mice Treated with 1,2,4-Trichlorobenzene
in the Diet for 13 Weeksa
Males

Control
200 ppm
(67 mg/kg-d)
3500 ppm
(851 mg/kg-d)
7000 ppm
(1222 mg/kg-d)
Clinical Chemistry
ALT (IU/L)
44 ± 8.8b
56 ± 13.5
150 ± 71.4°
146 ± 47d
SDH (IU/L)
33.3 ±4.09
33.1 ±5.10
130.0 ±72.38c
152.2 ±37.75°
Protein (g/dL)
5.9 ±0.25
6.1 ±0.29
6.7 ± 0.34°
7.4 ±0.15°
Albumin (g/dL)
3.5 ±0.09
3.7 ±0.31
3.8 ±0.29
4.5 ±0.32°
Terminal Body Weight (g)
25.0 ± 1.9
23.3 ± 1.8
21.6 ± 1.4°
21.5 ± 1.4°
Organ Weights
Liver weight (g)
1.249 ±0.068
1.209 ±0.069
1.957 ±0.232c
2.338 ±0.261°
Liver/body weight (x 100)
5.017 ±0.368
5.201 ±0.232
9.034 ± 0.678°
10.860 ±0.638°
Histopathology of Liver
Coagulative necrosis
0/10d
1/10
0/10
2/10
Cytomegaly/karyomegaly with
multinucleation, atrophy,
degeneration, microcytosis
0/10e
0/10
10/10e
10/10°
Females

Control
200 ppm
(87 mg/kg-d)
3500 ppm
(1184 mg/kg-d)
7000 ppm
(1346 mg/kg-d)
Hematology
Hematocrit (%)
54.1 ± 1.39
55.5 ±3.58
53.8 ± 1.24
49.1 ±2.33°
Hemoglobin (g/dL)
15.71 ±0.41
15.8 ±0.97
15.7 ±0.67
14.5 ± 0.26°
MCV (FL)
51 ±0.5
51 ±0.9
50 ± 1.3
48 ± 1.5°
MCH (pg)
14.7 ±0.46
14.4 ±0.19
14.7 ±0.22
14.0 ±0.22°
Clinical Chemistry
ALT (IU/L)
51 ± 10.3
51 ±29.2
73 ± 18.3
199 ± 124.4°
SDH (IU/L)
26.9 ±3.43
29.9 ± 10.54
74.8 ± 23.62°
86.5 ± 26.26°
Protein (g/dL)
6.1 ±0.15
5.9 ±0.13
6.3 ±0.42
6.7 ±0.17°
Terminal Body Weight (g)
21.2 ±0.7
21.6 ± 1.9
20.2 ± 1.2
19.6 ±0.6°
Organ Weights
Spleen weight (g)
0.0772 ± 0.0098
0.0785 ±0.0111
0.0700 ±0.0106
0.0626 ± 0.0075°
Spleen/body weight (x 1000)
0.3629 ± 0.0424
0.3618 ±0.0229
0.3454 ±0.0398
0.3189 ±0.0300°
Liver weight (g)
1.088 ±0.066
1.178 ±0.139
1.734 ±0.116°
2.292 ±0.184°
Liver/body weight (x 100)
5.119 ±0.223
5.443 ±0.317
8.597 ±0.499°
11.714 ± 1.057°
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Table 2. Selected Changes in Mice Treated with 1,2,4-Trichlorobenzene

in the Diet for 13 Weeksa


Females


200 ppm
3500 ppm
7000 ppm

Control
(87 mg/kg-d)
(1184 mg/kg-d)
(1346 mg/kg-d)
Histopathology of Liver
Coagulative necrosis
0/9
0/10
0/10
2/9
Cytomegaly/karyomegaly with
0/9
0/10
10/10e
9/9e
multinucleation, atrophy,




degeneration, microcytosis




aCMA, 1989b
bMean ± standard deviation
Significantly different from control atp< 0.05
dNumber affected/number examined
ep < 0.0001 by Fischer's exact test conducted for this review
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Cote et al. (1988) administered 1,2,4-trichlorobenzene (>99% pure, in corn oil) in the diet
to groups of Sprague-Dawley rats (10/sex/dose) for 13 weeks. Dietary concentrations of 0, 1, 10,
100, or 1000 ppm were used. Diets were freshly made and stored in airtight containers to
prevent evaporative loss of the test material. Based on measured body weights and food
consumption, the authors estimated doses of 0, 0.07, 0.78, 7.8, or, 82 mg/kg-day in males and 0,
0.11, 1.4, 15, or 101 mg/kg-day in females. Clinical observations were performed daily, body
weights were recorded weekly, and food consumption was measured monthly. Urine was
collected monthly for analysis of pH, protein, and nitrite. At sacrifice after 13 weeks of
exposure, all animals were subjected to complete necropsy and blood was collected for
hematology (Hgb, Hct, erythrocyte count, total and differential leukocyte count, platelet count,
prothrombin time, MCH, and MCHC) and clinical chemistry (electrolytes, inorganic phosphate,
total bilirubin, ALP, AST, total protein, calcium, cholesterol, glucose, uric acid and LDH)
evaluations. Weights of the brain, heart, liver, kidney, and spleen were recorded and
comprehensive histopathology evaluations were conducted. Hepatic mixed function oxidase
(aniline hydroxylase [AH] and aminopyrine demethylase [APDM]) activities were measured and
bone marrow cytology evaluated.
One high-dose female rat died during the fourth week on study; the authors did not
identify a cause of death but did not consider it to be related to exposure to
1,2,4-trichlorobenzene (Cote et al., 1988). The authors reported that treatment did not otherwise
affect survival, incidence of clinical signs, body weight gain, urinalysis parameters, serum
chemistry, or hematology in either sex (only body weight data shown in the original report). At
the highest dose, activities of AH and ADPM were increased in males and ADPM was increased
in females. In males exposed to 1000 ppm, relative liver weight and absolute and relative kidney
weights were significantly (p < 0.05) increased over control values (20%, 31%, and 36%,
respectively). Organ weights of female rats were not affected by treatment. Gross necropsy
revealed nephrosis in one male rat of the highest-dose group. Histopathology findings were
reported generally for the three trichlorobenzene isomers tested, without incidence data or, in
most cases, specific reference to the dose or isomer involved. Liver changes included a
mild-to-moderate increase in cytoplasmic volume and anisokaryosis of hepatocytes in "most"
treated groups. Rats exposed to 1000 ppm 1,2,4-trichlorobenzene were reported to have marked
liver changes characterized by aggregated basophilia and fatty infiltration leading to widespread
midzonal vacuolation. All treated groups were reported to exhibit thyroid changes (reduced
follicular size, increased epithelial height, reduced colloid density) with increasing severity at the
highest doses. For both liver and thyroid changes, the authors considered the effects to be
biologically significant only at the highest dose. Based on the authors' conclusions regarding
liver and thyroid histopathology, the highest dose (82 and 101 mg/kg-day in males and females,
respectively) is considered a LOAEL and the NOAEL is 7.8 or 15 mg/kg-day (males or females,
respectively).
Carlson and Tardiff (1976) evaluated selected parameters in adult male albino rats given
1,2,4-trichlorobenzene (reagent grade, purity not reported) orally (presumably via gavage), in
corn oil at doses of 0, 10, 20, or 40 mg/kg-day every day for 90 days, with or without a 30-day
recovery period, in groups of 6 rats. Body weight gain was measured in all groups. Liver
enzyme levels were assessed and blood was collected for hemoglobin and hct determinations.
Upon sacrifice, livers were weighed and examined microscopically. Body weights were not
affected by treatment. Liver weights were significantly (p < 0.05) increased at the high
dose—both in the group sacrificed immediately after dosing (10% higher than control) and in the
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recovery group (14% higher). Several liver enzyme levels (including cytochrome c reductase,
cytochrome P-450, benzpyrene hydroxylase, and azoreductase) were increased by treatment. No
effects were observed on hematology parameters or liver histopathology. Effect levels were not
determined from this study due to the limited parameters evaluated.
Carlson (1977) evaluated the ability of chlorinated benzenes to induce hepatic porphyria
in female rats (strain not reported). Groups of five rats were given gavage doses of 0, 50, 100, or
200 mg/kg 1,2,4-trichlorobenzene (purity not specified) in corn oil daily for 30, 60, 90, or
120 days prior to sacrifice. Liver porphyrins (total); urinary excretion of porphyrins, delta
aminolevulinic acid (ALA) and porphobilinogen; and liver weights were the only endpoints
measured. Absolute liver weights were significantly (p < 0.05) increased at the high dose after
30 days (20% over controls) and at all doses after 60 and 90 days of exposure (17 to 42%);
however, liver weights were not different from control at any dose after 120 days of exposure.
Liver porphyrins were slightly, but statistically significantly, increased at 100 and 200 mg/kg
after 30, 90, and 120 days of exposure, but not after 60 days. The maximum increase in liver
porphyrins, occurring after 90 days of exposure to 200 mg/kg, was 86%. Urinary porphyrin
excretion was increased at the highest dose after 30 and 90 days (59% and 2-fold higher,
respectively) but not after 60 or 120 days; urinary porphyrins were increased at the mid dose
after 120 days. The increases in urinary porphyrin levels were small relative to the 10- to
100-fold increases seen with hexachlorobenzene; the authors characterized the effects of
1,2,4-trichlorobenzene on this parameter as "minimal." Other parameters were not affected by
treatment. Based on increases in both liver and urinary porphyrins, as well as increased liver
weights, the mid-dose (100 mg/kg-day) is considered a LOAEL for the purpose of this review.
At the low dose (50 mg/kg-day), increases in absolute liver weight (up to 32%) were reported
without effects on porphyrins; this is considered a NOAEL.
The U.S. EPA conducted a 1-month study in rats (Cicmanec, 1991). The study was
designed to confirm the effects of 1,2,4-trichlorobenzene on the adrenal glands seen in a
multigeneration reproductive toxicity study (Robinson et al., 1981). Groups of five rats (sex,
strain, and age not specified) were given doses of 0 or 53 mg/kg-day in corn oil by daily gavage
for 30 days. Purity of the compound was not reported. Body weights were recorded at study
initiation and termination. Urine samples were collected for porphyrin analysis and blood was
analyzed for serum corticosterone levels as a measure of adrenal cortical function. At sacrifice,
adrenal gland weights and histopathology were evaluated. The results of porphyrin analysis
were not reported. When compared with controls, treated rats had decreased serum
corticosterone levels (32% lower) and increased absolute (14.8% higher) and relative (13.8%)
adrenal gland weights (see Table 3). No statistical analyses were performed by the authors;
however, statistical analysis (t-test) performed for this review indicated that the decrease in
corticosterone levels was significant, while the adrenal weight differences were not.
Microscopic examination of the adrenals showed moderate vacuolization of the zona fasciculata
in all treated animals with only slight vacuolization in controls. Increased adrenal weight of
greater than 10%, with accompanying histopathology, does constitute an adverse effect. No
other study details are available. This study identifies a LOAEL of 53 mg/kg-day for effects on
the adrenal glands; no NOAEL can be identified.
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Table 3. Adrenal Effects in Rats Exposed to 1,2,4-Trichlorobenzene via

Gavage for 30 Daysa


Absolute Adrenal Weight
Adrenal Weight Relative to
Serum Corticosterone Level
Dose (mg/kg-d)
(mg)
Body Weight (mg/100 g)
(ng/mL)
0
5.4 ± 0.60b
18.8 ± 1.7
483.9 ±62.0
53
6.2 ±0.58
21.4 ±3.1
330.2 ±42.9C
aCicmanec, 1991
bMean ± standard deviation
Significantly different from control (p = 0.002) by t-test conducted for this review
In a study reported only in abstract form, Smith et al. (1978) administered oral daily
doses (presumably via gavage) ranging from 1 to 173.6 mg/kg 1,2,4-trichlorobenzene (purity not
given) to rhesus monkeys (number and sex not reported) for unspecified periods of time (at least
20-30 days). While endpoints were not enumerated in the abstract, it appears that mortality,
clinical signs, body weight, and serum chemistry (including BUN, electrolytes, calcium,
phosphate, creatinine phosphokinase, AST, ALT, ALP, and LDH) were monitored. The authors
reported that doses of 25 mg/kg-day were nontoxic, while doses of 90 mg/kg-day were toxic and
doses of 173.6 mg/kg-day were lethal within 20-30 days. At the highest dose, monkeys
exhibited fine tremors and severe weight loss, as well as serum chemistry changes, prior to death.
This study does not provide enough information to identify effect levels other than a Frank
Effect Level (FEL) of 173.6 mg/kg-day associated with mortality and emaciation.
In another study published only in abstract form, Cragg et al. (1978) reported a
subchronic study in rhesus monkeys. Groups of four rhesus monkeys (sex not specified) were
given oral (presumably gavage) doses of 1,2,4-trichlorobenzene (purity not given) from
1-25 mg/kg-day for 120 days. The authors reported that body weight, clinical observations,
hematology, and clinical chemistry (parameters not specified) were not affected at doses up to
25 mg/kg-day, and that cytochrome P-450 and P-448 were not induced at these doses; no data
were presented to support these findings. A dose of 125 mg/kg was reported to be lethal for
1/4 monkeys and to cause temporary weight loss and cytochrome P-450 induction in survivors.
The information presented in the abstract is insufficient to identify effect levels other than a FEL
of 125 mg/kg-day associated with mortality.
Chronic Studies—In a carcinogenicity bioassay in rats, groups of F-344 rats (50 per sex
per group) were fed basal diets containing target concentrations of 0, 100, 350, or 1200 ppm of
1,2,4-trichlorobenzene (98.9% pure) for 104 weeks (CMA, 1994a). Due to the volatility of the
test compound, the investigators mixed diets containing 110% of the target concentrations. Diets
were prepared weekly and frozen until use; in addition, the diet was analyzed for actual
concentrations weekly for the first month and then monthly thereafter. Measured concentrations
averaged over the 7 days between diet changes were within 4% of the target concentrations.
Based on food intake, body weight, and measured compound concentrations in the diet, the mean
daily consumed doses were reported by the authors to be 0, 5.6, 19.4, and 66.5 mg/kg-day for
males and 0, 6.9, 23.5, and 81.4 mg/kg-day for females. Rats were examined for mortality and
moribundity twice daily, for clinical signs daily, and they were given a thorough physical
examination weekly. Body weights and food consumption were measured weekly during
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Weeks 1-16 and monthly thereafter. Blood samples for hematology were collected during
Weeks 52 and 78 of treatment and at termination. Blood smears were used to determine cellular
morphology and leukocyte differentials for the controls and high-dose animals. All animals were
given a complete necropsy upon death or sacrifice. Organ weights were recorded for the brain,
kidneys, liver, and testes of 10 rats/sex/group and 36 tissues were collected for histopathology.
Microscopic examination was carried out on all tissues in control and high-dose rats surviving to
104 weeks and in rats dying prematurely. Gross lesions were examined in the low- and mid-dose
groups and the liver and kidney in the mid-dose group.
Survival was significantly (p-value not reported) reduced in high-dose males after
83 weeks of exposure, but was unaffected in other treatment groups; survival at termination was
84, 80, 84, and 60% in males and 76, 78, 72, and 72% in females in the control, low-, mid- and
high-dose groups, respectively. The authors did not indicate the cause of the reduced survival,
but they reported that there was no evidence that treatment-related histopathology was the cause.
During the first 24 weeks, body weight and body weight gain were significantly lower in
high-dose rats compared to controls, but the differences were small and overtaken by a rebound
effect during the last half of the study. Overall (Weeks 1-104) mean total body weight gain was
not affected by treatment. Food consumption was consistently significantly (p < 0.05) lower in
treated rats than in controls; mean total food consumption was 4—7% lower than controls across
all treatment groups. There are no biologically significant hematology findings. The only
compound-related organ weight changes were significant (p < 0.05) increases in absolute and
relative liver weights in the high-dose rats of both sexes (19—21% higher than controls for both
parameters in both sexes). There was histopathologic evidence for compound-associated toxicity
in the liver (slight-to-moderate centrilobular hepatocellular hypertrophy and diffuse fatty change
in both sexes and focal cystic degeneration in males) at the high dose (see Table 4). Similarly,
renal toxicity (mineralization of the renal pelvis in both sexes, transitional cell hyperplasia of the
pelvic urothelium in males, and increased severity of chronic rat nephropathy in males) was
observed at the high dose (see Table 4). The incidence of renal pelvis mineralization was also
significantly increased in mid-dose males. The adrenal glands were not weighed in this study,
but there were no treatment-related histopathologic changes in these organs. The incidences of
adrenal cortical vacuolization were 11/50 and 13/50 in control and high-dose males
(respectively) and 15/50 and 20/50 in control and high-dose females. The authors identified the
mid dose (350 ppm) as a NOAEL for systemic toxicity. Although the male rats exhibited an
increased incidence of renal pelvis mineralization (without an increase in severity) at the mid
dose, available evidence suggests this effect may be related to male rat-specific hyaline droplet
nephropathy (see additional discussion of this in the discussion of CMA 1989a above). For the
purpose of this review, a LOAEL of 1200 ppm (66.7 mg/kg-day in males and 81.4 mg/kg-day in
females) is identified from this study based on reduced survival (males only), reduced body
weight (first 24 weeks of the study), and liver histopathology. The NOAEL is 350 ppm
(19.4 mg/kg-day in males and 23.5 mg/kg-day in females).
There was no statistically significant increase in the incidence of neoplasia in any
tissue—including liver and kidney (CMA, 1994a). The incidence of hematopoietic neoplasia
was slightly elevated in males (15/50 and 22/50 in control and high-dose males; 10/50 and 10/50
in control and high-dose females), but the difference is not statistically significant. This study is
adequate to assess the carcinogenicity of 1,2,4-trichlorobenzene in rats. Although survival is
reduced at the high dose, adequate numbers of animals survived to termination. In addition, the
high dose appears to have been a Maximum Tolerated Dose (MTD).
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Table 4. Incidence of Significant Liver and Kidney Effects in Rats Exposed to
1,2,4-Trichlorobenzene in the Diet for 104 Weeksa
Male

Control
350 ppm
(19.4 mg/kg-d)
1200 ppm
(66.5 mg/kg-d)
Liver
Grossly enlarged liver
1/5 0b
5/50
7/50
Hepatocellular hypertrophy
2/50
5/50
30/49°
Diffuse fatty change
5/50
5/50
14/49°
Focal cystic degeneration
9/50
4/50
19/49°
Kidney
Granular/ pitted appearance
11/50
11/50
34/50°
Renal pelvis mineralization
34/50 (0.7)d
44/50° (1.0)
49/50° (3.0)
Transitional cell hyperplasia
2/50 (0.1)
2/50 (0.0)
34/50° (1.0)
Moderate chronic progressive nephropathy
8/50 (2.7)
4/50 (2.6)
29/50° (3.6)
Female

Control
350 ppm
(23.5 mg/kg-d)
1200 ppm
(81.4 mg/kg-d)
Liver
Grossly enlarged liver
0/50
0/50
0/50
Hepatocellular hypertrophy
6/50
5/50
37/50°
Diffuse fatty change
15/50
21/50
30/50°
Focal cystic degeneration
0/50
0/50
0/50
Kidney
Renal pelvis mineralization
39/48 (0.8)
47/50 (1.0)
48/50° (1.3)
aCMA, 1994a
bNumber affected/total number examined
Significantly different from control incidence (p < 0.05) by Fisher's exact test conducted for this review
dMean severity score is given in parentheses
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CMA (1994b) also evaluated potential carcinogenicity in mice. Groups of
50 mice/sex/dose were given 1,2,4-trichlorobenzene (98.9% pure) in the diet at target
concentrations of 0, 150, 700, or 3200 ppm for 104 weeks. Due to the volatility of the test
compound, the investigators mixed diets containing 110% of the target concentrations and
analyzed the diet for actual concentrations. Diets were prepared weekly and frozen until use; in
addition, the diet was analyzed for actual concentrations weekly for the first month and then
monthly thereafter. Measured concentrations averaged over the 7 days between diet changes
were within 2% of the target concentrations. Based on measured food consumption and body
weight, as well as analysis of the actual diet concentrations, the authors estimated mean doses of
0, 21.0, 100.6, or 519.9 mg/kg-day in males and 0, 26.3, 127.0, or 572.6 mg/kg-day in females.
Animals were observed twice daily for mortality and moribundity and daily for clinical signs of
toxicity. Food consumption and body weights were measured weekly through Week 16 and
monthly thereafter. Blood was collected for limited hematology analysis (cellular morphology
and differential leukocyte counts) at Weeks 52 and 78 and at termination. Clinical chemistry
was not evaluated in this study. Upon sacrifice, all mice were necropsied and the brain, liver
with gall bladder, kidneys, and testes with epididymides were weighed. Comprehensive
histopathology examinations were performed on all control and high-dose mice, while the liver,
adrenals, and testes with seminal vesicles from all dose groups were examined microscopically.
Reduced survival was observed in high-dose animals beginning after Week 48. Survival
to termination was significantly (p < 0.05) reduced in high-dose males and females; survival to
the beginning of Week 105 was 90, 88, 82, and 10% (control through high dose) in males and 78,
76, 84, and 0% in females. A high incidence of hepatocellular carcinoma in high dose animals of
both sexes explains the mortality at this dose. High-dose animals also exhibited reduced body
weight, reduced total body weight gain, and reduced food consumption. In low and mid-dose
mice, body weight and body weight gain were either increased or unchanged from control
values. Similarly, there were no consistent dose-related effects on food consumption at doses
other than the high dose. Clinical signs were observed only in moribund animals prior to death.
There were no significant hematology findings in the limited analyses performed; sporadic,
statistically significant changes in differential leukocyte counts did not exhibit a
treatment-related pattern. Animal observations revealed dose-related increases in the incidence
of distended abdomen in all treatment groups; the authors attributed this observation to liver
enlargement and liver masses, with associated accumulation of ascitic fluids. Organ weights
were not evaluated in high-dose females due to premature death. Absolute and relative liver
weights were increased in a dose-related fashion in both males and females of all remaining
groups (except relative liver weight in low-dose males), with marked changes (2-fold or higher)
at the mid- and high doses (see Table 5). Other statistically significant organ weight changes
were unrelated to treatment or attributable to body weight differences. Nonneoplastic
histopathology findings were restricted to hepatocyte hypertrophy, the incidence of which was
increased in mid- and high-dose males. The incidence of hepatocellular carcinoma was
significantly increased in males and females of the mid- and high-dose groups, while the
incidence of hepatocellular adenoma was increased only at the mid-dose in both sexes (CMA,
1994b). The low incidence of adenoma at the high dose is attributable to the high incidence of
carcinomas at this dose; nearly 100% of high dose animals of both sexes exhibited carcinomas.
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Table 5. Liver Changes in Mice Treated with 1,2,4-Trichlorobenzene in the Diet for
104 Weeksa
Males

Control
150 ppm
(21.0 mg/kg-d)
700 ppm
(100.6 mg/kg-d)
3200 ppm
(519.9 mg/kg-d)
Terminal Body Weight (g)
32.5 ±4.1
35.3 ±4.6C
32.1 ±3.6
26.7 ± 2.0°
Organ Weights at Termination
Liver weight (g)
1.59 ± 0.60b
1.77 ± 0.43°
3.05 ± 1.72°
7.37 ± 1.84°
Liver/body weight (%)
4.962 ± 2.268
5.022 ± 1.392
9.522 ± 5.508°
27.364 ± 5.740°
Nonneoplastic Lesions
Hepatocyte hypertrophy
0/4 9 d
0/50
27/50e
20/50e
Neoplastic Lesions
Hepatocellular adenoma
4/49
7/50
16/50e
2/50
Hepatocellular carcinoma
8/49
5/50
27/50e
50/50e
Females

Control
150 ppm
(26.3 mg/kg-d)
700 ppm
(127.0 mg/kg-d)
3200 ppm
(572.6 mg/kg-d)
Terminal Body Weight (g)
28.6 ±3.4
31.6 ± 5.8°
30.7 ± 3.8°
No survivors
Organ Weights at Termination
Liver weight (g)
1.42 ±0.23
1.87 ± 0.43°
3.98 ± 2.32°
No survivors
Liver/body weight (%)
5.057 ±0.504
5.994 ±0.910c
12.734 ±6.817c
No survivors
Nonneoplastic Lesions
Hepatocyte hypertrophy
0/50
0/50
1/50
8/5 0e
Neoplastic Lesions
Hepatocellular adenoma
3/50
4/50
16/50e
8/50
Hepatocellular carcinoma
1/50
1/50
28/50e
46/50e
aCMA, 1994b
bMean ± standard deviation
Significantly different from control atp< 0.05
dNumber affected/number examined (including premature deaths)
ep < 0.01 by Fischer's exact test conducted for this review
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The authors characterized the low dose (150 ppm) as a NOAEL for systemic toxicity,
presumably due to liver weight changes and liver histopathology at the mid-dose. However, the
incidence of neoplasia in the livers of both male and female mice was markedly increased at the
mid-dose, so the interpretation of the nonneoplastic changes in this organ is complicated by the
prevalence of tumors. As a consequence, effect levels were not defined for this study.
In a review of a paper published in German, WHO (1991) reported that liver weight was
not affected and gross and histological lesions of the liver were not observed in 20 male
ICR-JCL mice given 78 mg/kg-day 1,2,4-trichlorobenzene via dietary administration for
6 months (Goto et al., 1972, as cited in WHO, 1991). No other information was available in the
secondary source.
Reproductive/Developmental Studies—Robinson et al. (1981) conducted a
multigeneration reproductive toxicity study using rats (strain not specified). Beginning at birth
of the Fo generation and continuing through postnatal day (PND) 32 of the F2 generation, rats
(17-23 litters per group for all generations) were continuously exposed to concentrations of 0,
25, 100, or 400 ppm 1,2,4-trichlorobenzene (purity not specified, solubilized in Tween 20) in the
drinking water. Both water and vehicle control groups were included. Body weights, food
consumption, and water intake were recorded regularly. Locomotor activity was assessed at
intervals up to 90 days of age. F0 and Fi rats were bred to same-group animals at 90 days of age.
Fertility, litter size, and neonatal sex, weight, and postnatal viability were monitored. Vaginal
opening was assessed in F2 female rats. Interim sacrifices were performed on each generation;
the Fo and F2 interim sacrifices occurred on days 27 and 95 of age and the Fi interim sacrifice
occurred only on day 95 of age. At each time, 10 rats/sex/group were sacrificed; blood samples
were collected for chemistry determinations (i.e., glucose, BUN, creatinine, electrolytes, uric
acid, Ca, P, cholesterol, triglyceride, bilirubin, ALP, ALT, AST, LDH, creatinine phosphokinase
[CPK], total protein, globulin, and albumin) and selected organs (liver, kidney, uterus, adrenals,
lungs, heart, and gonads) were weighed. Livers and kidneys from control and high-dose Fi rats
sacrificed at 95 days of age were examined microscopically.
The authors indicated that body weight was not changed by treatment at any dose or in
any generation; data were not shown (Robinson et al., 1981). Food intake of the Fo and
Fi generations was not adversely affected by treatment at any dose, although a transient increase
in food intake was seen in Fo high dose males at 29 days of age. Water intake was significantly
(p < 0.05) reduced at the high dose in F0 females at 35 days of age (data not reported) and at both
sexes of the Fo generation at 83 days of age (12% and 17% lower than controls in males and
females, respectively). It was not clear from the report whether the control water consumption
data reported were for water or vehicle controls. Water intake was not affected in the
Fi generation. Representative compound intake rates estimated by the authors based on the
water consumption and body weight data (Fo generation at 83 days of age) were 2.5, 8.9, and
33 mg/kg-day in males and 3.7, 14.8, and 53.6 mg/kg-day in females in the 25-, 100-, and
400-ppm groups. The authors reported that no treatment-related changes were observed in serum
chemistry analyses in any generation (data not shown). Gestation rate, maternal weight (data not
shown), litter size, postnatal viability, growth (data not shown), and locomotor activity were not
affected by treatment in any generation. Vaginal opening of the F2 females was unchanged by
exposure; this endpoint was not assessed in other generations. The only organ weight that was
significantly affected by treatment was that of the adrenal glands, which were significantly
(p < 0.05) increased at the high dose in both sexes of both Fo and Fi generations when assessed
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at 95 days of age. Adrenal weights were 9-11% higher than water controls in males and 8-22%
higher in females (see Table 6). No histopathology was observed in the liver or kidneys of high
dose Fi animals; adrenal histopathology was not assessed. U.S. EPA (2008) identified the high
dose as a LOAEL based on increased adrenal gland weights, noting that this effect had been
confirmed in an acute toxicity study using i.p. administration (also reported in Robinson et al.,
1981) as well as in an unpublished U.S. EPA study (Cicmanec, 1991, as cited in U.S. EPA,
2008). The latter study, a 1-month rat gavage study using a comparable dose (53 mg/kg-day),
also observed adrenal histopathology (increased vacuolization of the zona fasciculata), as well as
decreased serum corticosterone levels. Increased adrenal weights, were also observed in male
rats given 1,2,4-trichlorobenzene in the diet for 13 weeks at an average dose of 133.7 mg/kg-day
(CMA, 1994a). For the purpose of this review, the mid dose (8.9 or 14.8 mg/kg-day in males or
females, respectively) is, thus, identified as a NOAEL and the high dose (33 or 53.6 mg/kg-day
in males or females, respectively) is a LOAEL for adrenal effects.
Table 6. Adrenal Weight Changes (Left Adrenal Only) at 95 Days of Age in Rats Treated
with 1,2,4-Trichlorobenzene in the Drinking Water for Three Generations51

Control
(water)
Control
(Tween 20)
25 ppm
(2.5 mg/kg-d)
100 ppm
(8.9 mg/kg-d)
400 ppm
(33 mg/kg-d)
F0 Male Adrenal weight (mg)
28.7 ± 1.78b
28.6 ± 1.09
28.8 ± 1.59
28.2 ± 1.10
31.8 ± 2.43°
F, Male Adrenal weight (mg)
27.1 ± 1.71
28.0 ± 1.57
28.8 ± 1.46
26.6 ±0.82
29.6 ± 1.66°

Control
(water)
Control
(Tween 20)
25 ppm
(3.7 mg/kg-d)
100 ppm
(14.8 mg/kg-d)
400 ppm
(53.6 mg/kg-d)
F0 Female Adrenal weight (mg)
34.1 ± 1.87
36.8 ± 1.14
35.6 ± 1.70
36.6 ± 1.08
41.5 ± 1.99°
Fi Female Adrenal weight (mg)
35.8 ± 1.89
37.0 ± 1.36
34.8 ± 1.77
35.3 ± 1.07
38.5 ± 1.89°
"Robinson etal., 1981
bMean ± standard error
Significantly different from control atp< 0.05
In a study of embryotoxicity, Kitchin and Ebron (1983; also U.S. EPA, 1982)
administered gavage doses of 0, 36, 120, 360, or, 1200 mg/kg-day 1,2,4-trichlorobenzene
(>99% pure) in corn oil to timed-pregnant Sprague-Dawley rats (at least six per dose) on
gestation days (GDs) 9-13. Upon sacrifice by decapitation on GD 14, maternal livers were
weighed, sectioned, and examined microscopically; liver homogenates were analyzed for
enzyme content. Uteri of control and 360 mg/kg-day rats were examined for implantations and
resorptions; rats from lower dose groups were not examined. Live embryos were examined
grossly and the presence of a beating heart, somite number and embryo size were noted.
rd
None of the rats treated with 1200 mg/kg-day survived beyond the 3 day of treatment
and 2/9 rats given 360 mg/kg-day died (Kitchin and Ebron, 1983, U.S. EPA, 1982). Body
weight gain was markedly reduced in survivors given 360 mg/kg-day (17 g weight loss versus
37 g gain in controls). Liver weight and hepatic microsomal protein content were not affected by
treatment; however, hepatic enzyme induction was observed at 120 and 360 mg/kg-day.
Microscopic examination of the livers revealed slight hepatocellular hypertrophy in 1/9 rats
exposed to 120 mg/kg and moderate hypertrophy in 7/8 rats (presumably including one rat that
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died prematurely) exposed to 360 mg/kg-day. Treatment at 360 mg/kg-day resulted in fewer
implantations, an increase in the incidence of dead embryos (this increase was not significant on
a litter basis), reduced head and crown-rump lengths, decreased number of somites, and
decreased embryonic protein content (see Table 7) when compared with controls. The decrease
in implantations was not likely to be caused by the treatment, since implantation occurs on GD 6,
and the dosing started on GD 9. Uterine and embryonic parameters were not evaluated in
lower-dose groups, precluding the clear identification of embryotoxic effect levels from this
study.
Table 7. Significant Changes in Uterine and Embryonic Parameters in Rats Exposed to
1,2,4-Trichlorobenzene from GDs 9-13a

Uterine parameters
(12 litters examined)
Embyronic parameters
(24 treated and 26 control embryos examined)
Dose
(mg/kg-d)
Implantations
Dead
Embryos/
Total
Number of
Embryos
Litters
With Dead
Embryos/
Total
Number of
Litters
Embryonic
Head
Length
(mm)
Embryonic
Crown-Rump
Length (mm)
Somites
Protein
(Hg)
0
14.16 ±0.31b
0/161
0/12
4.72 ± 0.06
8.17 ±0.09
49.6 ±0.6
2670 ± 108
360
12.5 ± 0.79°
25/138d
3/12
4.43 ± 0.10°
7.69 ± 0.17°
47.7 ± 0.6°
2053 ± 12le
'Kitchin and Ebron. 1983
bMean ± standard error of mean
Significantly different from control atp< 0.05
dp< 0.001
ep < 0.01
Black et al. (1988; Ruddick et al., 1983) exposed timed-pregnant Sprague-Dawley rats
(about 14/group) to gavage doses of 0, 75, 150, or 300 mg/kg-day 1,2,4-trichlorobenzene
(99.5% pure, in corn oil) on GD 6-15. The dams were sacrificed on GD 22, whereupon the
uterus with ovaries was removed for examination and liver, kidney, spleen, heart, and brain were
weighed. Body weights were recorded before and after uterine removal. Maternal blood
collected at sacrifice was analyzed for hematology (Hgb, Hct, erythrocyte count, total and
differential leukocyte count, MCV, MCH, and MCHC) and serum chemistry (electrolytes,
inorganic phosphorus, total bilirubin, ALP, AST, total protein, calcium, cholesterol, glucose, uric
acid and LDH). Liver samples were homogenized for analysis of liver enzymes. Finally,
24 maternal tissues were subjected to histopathology evaluation. Uterine contents were
examined for resorptions and live or dead pups. Pups were weighed and examined grossly for
abnormalities; live fetuses were prepared for skeletal or visceral examination and histopathology.
There were no treatment-related deaths or clinical signs during the study
(Black et al., 1988; Ruddick et al., 1983). Body weights of treated dams were not statistically
distinguishable from control values, although they were lower in high-dose animals (data not
provided in publication). Absolute and relative liver weights were significantly (p < 0.05)
increased in the high-dose dams (6% increase in relative weight; absolute weight increase not
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reported). No other organ weights were affected by treatment. Maternal mixed function oxidase
activity was increased at 150 and 300 mg/kg-day. Maternal hemoglobin concentration (7% and
6% below control, at 150 and 300 mg/kg-day, respectively) and Hct (31% at both doses) were
reduced at 150 and 300 mg/kg-day, but reticulocytes were not increased. Mild histopathology
changes consisting of follicle size reduction were observed in the thyroids of dams treated at the
high dose. The authors reported mild hepatic lesions (increased periportal cytoplasmic
eosinophilia and mild anisokaryosis of hepatocellular nuclei) at the mid and high doses.
Increased splenic hematopoiesis was reported to occur in some animals, but details were not
provided. Incidences of histopathology changes were not reported. Treatment with
1,2,4-trichlorobenzene did not affect pregnancy rate, resorptions, incidence of dead fetuses, litter
size, fetal weight or the incidences of gross, skeletal, or visceral abnormalities. In the absence of
any fetotoxic or teratogenic effects, the high dose (300 mg/kg-day) is considered a freestanding
NOAEL for developmental toxicity. Based on mild anemia (reduced Hgb and Hct) and mild
hepatic lesions in dams, the mid dose (150 mg/kg-day) is considered a systemic LOAEL; the
NOAEL is 75 mg/kg-day.
In a series of papers reporting the development of a teratology-screening assay
(Chernoff and Kavlock, 1983; Gray et al., 1983; Gray and Kavlock, 1984; Gray et al., 1986),
investigators administered gavage doses of 0 or 130 mg/kg-day 1,2,4-trichlorobenzene to groups
of 25 pregnant CD-I mice on GD8-12, after which dams were allowed to deliver. Maternal
survival, body weight change and pregnancy rate were recorded, as well as the postnatal viability
and body weight of offspring on PND 1 and 3. Dams that had not delivered 3 days after
expected parturition date were sacrificed and uteri were examined for implantation sites. Dead
pups were necropsied and evaluated for gross abnormalities. Groups of three male and three
female pups were randomly assigned to same-treated dams for fostering on PND 6. At 30 days
of age, offspring number and weights were recorded, as was the number of females with patent
vaginas. Locomotor activity in the pups was assessed at 21, 58, and 210 days of age. Female
offspring that became pregnant were removed for delivery and their litter size, weight, and sex
ratio were evaluated. At about 250 days of age, the male mice were sacrificed and necropsied;
body, liver, kidney, testes, and seminal vesicle weights were recorded. Tabular data presented in
the studies showed that treatment did not result in differences from control for any parameter
assessed. This series of studies identified a NOAEL of 130 mg/kg-day for postnatal effects of
gestational exposure to 1,2,4-trichlorobenzene in mice.
Inhalation Exposure
Subchronic Studies—Kociba et al. (1981) exposed groups of 20 male Sprague-Dawley
rats, four male New Zealand rabbits and two male beagle dogs by inhalation to 0, 30, or,
100 ppm (0, 223, or, 742 mg/m3) of 1,2,4-trichlorobenzene (99.41% pure) vapor for 7 hours/day,
5 days/week, for a total of 30 exposures in 44 days. The following endpoints were monitored:
body weight, clinical signs, hematology (total erythrocytes, differential leukocytes, packed cell
volume [PCV] and Hgb), clinical chemistry parameters (BUN, ALT, and ALP), organ weights
(liver, kidneys, spleen, adrenals [in dogs and rabbits], heart, brain, thymus [rats] and testes), and
gross and microscopic examination of most tissues and organs in all dogs and rabbits as well as
five control and high-dose rats. Urine samples, taken from control and high-concentration rats
only, were analyzed for coproporphyrin and uroporphyrin as indicators of hepatotoxicity. At the
end of the experiment, these levels were observed to be elevated, so a second, confirmatory
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experiment was undertaken with 10 male rats per exposure at 0, 30, and 100 ppm; urine samples
were collected after 15 and 30 days of exposure and analyzed for coproporphyrin and
uroporphyrin.
Analysis of chamber concentrations indicated values close to the nominal concentrations
(30 ± 4 ppm and 104 ± 14 ppm) (233 ±31 mg/m3 and 771 ± 104 mg/m3) (Kociba et al., 1981).
Exposure to 1,2,4-trichlorobenzene did not result in clinical signs or statistically significant
effects on body weight gain in any of the species tested (statistical analysis not provided).
Results from hematologic examinations and clinical chemistry tests revealed no
compound-related effects in any of the three species. In rats, changes in organ weights included
statistically significant (p < 0.05) increases in absolute and relative liver weight (each
11% higher than control) and increases in relative kidney weight (9%) at the 100-ppm level.
Urinary uroporphyrin and coproporphyrin levels were significantly (p < 0.05) elevated in rats in
the original experiment. Furthermore, these levels were significantly elevated at both time points
and at both exposure levels in the confirmatory experiment (see Table 8). In rabbits, relative
liver weight was significantly decreased at the 30- and 100-ppm levels (each 83% of control) and
absolute and relative testes weights were increased (30% and 43% higher than control,
respectively) at the 100-ppm level. While there was a statistically significant decrease in BUN in
rabbits, the authors considered the measure within the range of normal variability. In dogs,
absolute and relative liver weights were nominally increased over control values at the 100-ppm
level, but the small number of animals precluded statistical comparison. Exposure to
1,2,4-trichlorobenzene did not result in gross or microscopic alterations in any tissues of any
species tested.
Table 8. Significant Changes at Urinary Porphyrins in Rats Inhaling

1,2,4-Trichlorobenzene for 15 or 30 Daysa


15 Exposure Days
30 Exposure Days

Coporphyrin
Uroporphyrin
Coporphyrin
Uroporphyrin
Exposure (ppm)
Gig/24 h)
Oig/24 h)
Oig/24 h)
Oig/24 h)
First Experiment
0
Not measured
Not measured
4.2 ± 1.4
0.4 ±0.2
100
Not measured
Not measured
12.4 ± 2.8a
11.5 ± 0.4b
Second Experiment
0
3.6 ±0.9
4.6 ± 1.0
6.2 ±2.1
6.3 ±3.9
30
16.2 ± 2.7b
11.6 ± 1.3b
15.8 ± 3.0b
12.8 ± 1.3b
100
33.8 ± 2.9b
10.8 ± 4.6b
15.2 ± 2.7b
13.5 ± 2.3b
aKociba et al., 1981
bSignificantly different from control, p < 0.05
The authors considered the testes weight increases in rabbits to be unrelated to treatment
(Kociba et al., 1981), but they provided no rationale for this conclusion. While the investigators
considered the increased urinary excretion of porphyrins to be a physiological effect of
1,2,4-trichlorobenzene inducing hepatic microsomal enzymes (cytochrome P-450), rather than a
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toxic effect on destruction of heme-containing cytochromes or inhibition of heme synthesis, no
data were provided to support this hypothesis. For the purpose of this review, the exposure level
"3
of 30 ppm of 1,2,4-trichlorobenzene (223 mg/m ) is defined as a LOAEL for rats in this study
based on increased excretion of porphyrins; no NOAEL can be defined for rats from these data.
Due to the small numbers of rabbits and dogs used in this study, effect levels for these species
cannot be reliably defined.
In a subsequent (albeit published earlier) study by the same group of investigators
(Watanabe et al., 1977, 1978), the effects of inhaled 1,2,4-trichlorobenzene (99.6% pure) on both
liver and urinary excretion of porphyrins were evaluated in groups of 10 male and 26 female
Sprague-Dawley rats. The rats were exposed to 0, 2.8, or 10.2 ppm (0, 21, or 76 mg/m3)
6 hours/day, 5 days/week, for 3 months. Animals were observed for clinical signs daily and
body weights were recorded weekly. Between four and five females/group were sacrificed after
2 weeks, 1 month, or 2 months of exposure and 2 or 4 months post-exposure for assessment of
total liver porphyrins. Urine was collected at these same intervals from the rats maintained for
the entire experiment; these samples were analyzed for porphyrin, coporphyrin, and creatinine.
All animals received gross necropsy and weights of brain, kidney, liver, and lung were recorded.
Histopathological examinations were not performed because the Kociba et al. study (1981),
conducted earlier, publication delayed, had reported no histopathological findings at higher
exposure levels.
Measured chamber concentrations of 1,2,4-trichlorobenzene averaged 2.8 and 10.2 ppm
over the duration of the experiment (Watanabe et al., 1977, 1978). Only one female control and
one female of the 3-ppm group died; no treatment-related effect on mortality was observed.
Likewise, treatment did not affect clinical signs, body weight, organ weights, gross necropsy
findings, or total liver porphyrins. Statistically significant increases (over control levels) in
urinary porphyrin excretion were observed sporadically over the 3-month period in rats exposed
to 10 ppm (see Table 9 for results at 3 months of exposure); levels were not different from
controls 2 or 4 months after exposure ceased. While the authors maintained that the increased
urinary porphyrin excretion was of minor toxicological significance, they suggested that this
effect might directly precede toxic manifestations of exposure to 1,2,4-trichlorobenzene noted by
other investigators (Rimington and Ziegler, 1963). As noted previously with the oral dosing
studies, the scenario of hepatic enzyme induction, tissue damage, possibly mediated by quinine
metabolites, hepatic hypertrophy, and increased liver weight, appears to be the progression
(Carlson and Tardiff, 1976; Carlson, 1977; Watanabe et al., 1977, 1978; Ariyoshi et al., 1981;
Kociba et al., 1981; Black et al., 1988; Cote et al., 1988; CMA, 1989a,b). Consequently, the
"3
authors considered the exposure concentration of 2.8 ppm (21 mg/m ) to be the NOAEL for rats,
as this level did not increase urinary porphyrin excretion. For the purpose of this review, a
minimal LOAEL of 10.2 ppm (76 mg/m ) is identified based on increased porphyrin excretion;
the NOAEL is 2.8 ppm (21 mg/m3).
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Table 9. Significant Changes at Urinary Porphyrins in Rats Inhaling
1,2,4-Trichlorobenzene (Results after 3 Months of Exposure)51

Male
Female
24-h Excretion
Exposure (ppm)
Coproporphyrin
Oig/24 h)
Uroporphyrin
Gig/24 h)
Coproporphyrin
Gig/24 h)
Uroporphyrin
Gig/24 h)
0
6.1 ±3.4
1.3 ± 1.0
5.3 ±9.4
0.6 ±0.3
3
8.5 ±5.4
2.2 ± 1.7
2.4 ±0.5
0.7 ±0.1
10
11.4 ± 5.9b
4.1 ±2.4b
3.1 ± 1.0
1.0 ± 0.2b
Excretion Adjusted for Creatinine
Exposure (ppm)
Coproporphyrin
(jig/mg creatinine)
Uroporphyrin
(jig/mg creatinine)
Coproporphyrin
(jig/mg creatinine)
Uroporphyrin
(jig/mg creatinine)
0
0.58 ±0.33
0.11 ±0.05
0.67 ± 1.23
0.07 ± 0.04
3
0.78 ±0.42
0.17 ±0.09
0.35 ±0.14
0.10 ±0.04
10
1.0 ±0.5
0.31 ±0.12b
0.32 ± 0.11
0.10 ±0.02
"Watanabc et al., 1977
bSignificantly different from control, p < 0.05
Chronic Studies—Coate et al. (1977) exposed groups of 30 male Sprague-Dawley rats,
16 male New Zealand rabbits and 9 male cynomolgus monkeys (Macaca fascicularis) to
nominal concentrations of 0, 25, 50, or 100 ppm (0, 186, 371, or 742 mg/m3) of
1,2,4-trichlorobenzene vapor (99.07% pure) 7 hours/day, 5 days/week, for 26 weeks. Average
measured concentrations of 1,2,4-trichlorobenzene were 0, 25.3, 49.2, and 92.8 ppm (0, 188,
365, and 689 mg/m3) for rats and rabbits and 0, 24.8, 49.2, and 94.5 ppm (0, 184, 365, and
701 mg/m3) for monkeys. Daily observations were performed and body weights were measured
weekly for the first 4 weeks, biweekly for the next 8 weeks, and monthly for the remainder of the
study. Hematology (complete blood count) and clinical chemistry parameters (BUN, total
bilirubin, ALT, AST, ALP, and LDH) were monitored in rabbits and monkeys throughout the
study and in rats at sacrifice. The following tests were conducted before and during the exposure
period: ophthalmoscopy, pulmonary function, and operant behavior (in monkeys) and
ophthalmoscopy (in rabbits). Selected rats (five/group) were sacrificed after 1, 3, and 6 months
of exposure. All animals that died during the study and/or were sacrificed were subjected to a
complete necropsy with gross pathological examination followed by histopathological
examination of selected organs (brain, lungs, heart, liver, kidneys, spleen, eyes, spinal cord, bone
marrow, and abdominal skin).
Coate et al. (1977) reported that no compound-related effects were observed on body
weights, survival, hematology, or clinical chemistry tests, ophthalmic tests in rabbits and
monkeys, or histologic examinations in rabbits and monkeys (data not shown). Tabular data on
pulmonary function and operant behavior tests in monkeys also indicated no effect of exposure.
Several rabbits (16/64 across all groups including control) and monkeys (4/36, also distributed
across all groups) died during the study from pneumonia, but the lack of a
concentration-response relationship suggests that this is not a treatment-related effect.
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Histopathologic findings were restricted to the liver and kidney of rats; the incidences of these
effects are not reported. Exposed rats are reported to have enlarged hepatocytes, a finding that
was more apparent at 4 weeks than at 13 weeks and greater at 50 and 100 ppm than at 25 ppm
(no additional details were provided). The authors reported a slight increase in the degree of
vacuolization of hepatocytes at 4 and 13 weeks, a slight increase in the incidence and degree of
granuloma of the liver at 4 weeks, and an increase in the degree of biliary hyperplasia at 4 and
13 weeks. The authors did not indicate the levels at which the latter findings were observed;
however, according to the report, these effects do not exhibit a concentration-response
relationship. All groups of rats (presumably including controls) exhibited "hyaline
degeneration" in kidney sections. The authors indicated that the severity of this lesion was
"slightly increased"—although not in a concentration-related manner—in all exposed rats at
4 weeks of exposure but only in the 100-ppm group at 13 weeks. Neither liver nor kidney
changes were present in rats sacrificed after 26 weeks of exposure.
Details that would enable a determination of whether the renal lesion constituted hyaline
droplet (alpha 2[j.-globulin) nephropathy were not provided (Coate et al., 1977), but its
description as hyaline degeneration, transient nature and occurrence in male rats are consistent
with the description of this lesion (U.S. EPA, 1991b); further, evidence for hyaline droplet
nephropathy has also been observed in other rat studies (CMA, 1989a, 1994a). The absence of
information on the incidence and/or severity of the hepatic lesions observed in the rats, coupled
with the reported lack of concentration-response relationship and the absence of effects in the
rats treated longest, preclude the identification of a reliable LOAEL value from the rat data. For
rabbits and monkeys, the highest exposure levels of 92.8 ppm (689 mg/m3) and 94.5 ppm
"3
(701 mg/m ), respectively, represent NOAELs.
Summary of Oral and Inhalation Studies
Table 10 provides a summary of the cited studies in the Derivations Section.
Other Studies
Other Routes
den Besten et al. (1991a) compared the liver, kidney, and thyroid toxicity of several
chlorinated benzenes after a single intraperitoneal exposure of male Wistar rats to 1,2, or
4 mmol/kg of each compound (181, 362, or 543 mg/kg of trichlorobenzene). Of the compounds
tested, 1,2,4-trichlorobenzene induced the greatest hepatotoxicity as measured by increase in
serum ALT and evidence of liver histopathology 72 hours after dosing. In addition, only
1,2,4-trichlorobenzene exposure resulted in severe degenerative damage to the kidney. Serum
thyroxine (T4) levels plunged rapidly after exposure to 2 or 4 mmol/kg 1,2,4-trichlorobenzene;
levels were significantly below controls after only 5 hours and reached a nadir 24 hours after
dosing. Serum triiodothyronine levels (T3) were not affected by treatment. Among the
compounds tested, the decline in T4 levels was correlated with the relative binding affinity of the
phenolic metabolites to the plasma transport protein for thyroxine (transthyretin). The authors
suggested that alterations in hepatic thyroxine metabolism may also have contributed to the
reduction in T4 levels.
In a study published in Japanese with an English abstract and tables,
Yamamoto et al. (1982) applied 1,2,4-trichlorobenzene in acetone to the dorsal skin of Slc:ddy
mice twice weekly for 2 years. The solution of 1,2,4-trichlorobenzene was 60% for the high
dose and 30% for the low dose; the volume applied was 0.03 mL/application. Each treated group
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contained 75 animals of each sex and there were 50 vehicle control animals of each sex.
Mortality, growth rate, urinalysis, hematology, clinical chemistry, organ weights, and
histopathology were evaluated. Growth rates in treated and control mice were comparable
through 83 weeks. Mean survival days were significantly (p < 0.05) reduced in the
60% 1,2,4-trichlorobenzene groups of males and females and, also, in the 30% treatment group
of females. Spleen weights were increased in males of both treatment groups and adrenal
weights were increased in high-dose females. Dermal effects (e.g., keratinization, edema, cell
infiltration, fibrosis, etc.) were apparent in all treated groups with evidence of dose-related
changes. Males treated at the highest concentration exhibited increases in AST, ALT, and BUN;
other serum chemistry and hematology findings were unremarkable. The incidences of several
nonneoplastic changes were reportedly increased in treated rats, including lung inflammation;
liver degeneration, inflammation and amyloidoisis; kidney inflammation and amyloidosis;
adrenal amyloidosis and spleen amyloidosis. Further information on the nature of these lesions
is not available. Treatment did not increase the incidence of any single tumor type.
Toxicokinetics
WHO (1991) summarized the toxicokinetics of chlorobenzenes, reporting that these
compounds are readily absorbed across both the respiratory and gastrointestinal tracts.
WHO (1991) noted that data on hexachlorobenzene suggest that oral absorption of
chlorobenzenes is likely affected by exogenous factors including the presence of bile and/or
lipids in the gastrointestinal tract. Koss and Koransky (1975) reported greatly enhanced
absorption of orally administered hexachlorobenzene when administered to rats in olive oil when
compared with administration in an aqueous solution (80% absorption from olive oil vs. 6% in
aqueous solution). Based on this information, it is possible that, in toxicity studies of
1,2,4-trichlorobenzene administered in corn oil, absorption was enhanced by the vehicle.
Carlson and Tardiff (1976) have shown that 1,2,4-trichlorobenzene is a potent inducer of
liver enzymes including P-450; it has also been demonstrated to induce delta-aminolevulinic acid
synthetase, the rate-limiting enzyme in heme synthesis and heme oxygenase, which is the
rate-limiting enzyme in heme degradation (Ariyoshi et al., 1981).
Only two studies have examined the importance of quinone metabolites in the
hepatotoxicity of 1,2,4-trichlorobenzene. den Besten et al. (1991b) demonstrated that secondary
metabolism to hydroquinones (after initial epoxide formation) was strongly correlated with
covalent binding to protein in rat liver microsomes in vitro. The authors observed complete
inhibition of protein binding with the addition of the reducing agent ascorbic acid, providing
support for quinones as the sole reactive species formed. Mizutani and Miyamoto (1999)
examined the role of quinone metabolites in vivo using male ddY mice exposed to a single i.p.
dose of 1,2,4-trichlorobenzene. The mice were pretreated with either butylated hydroxyanisole
(BHA, an inducer of DT-diaphorase, the enzyme that detoxifies quinone intermediates) or
dicoumarol (an inhibitor of DT-diaphorase). Pretreatment with BHA markedly suppressed
hepatotoxicity (as measured by serum ALT levels), while dicoumarol enhanced hepatotoxicity in
mice treated with 1,2,4-trichlorobenzene. This study provided evidence for the importance of
quinone metabolites in the liver toxicity of 1,2,4-trichlorobenzene.
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Mutagenicity
Mutagenicity and in vitro clastogenicity studies of 1,2,4-trichlorobenzene have yielded
uniformly negative results, while studies of in vivo clastogenicity have been positive. With or
without metabolic activation, the chemical did not induce reverse mutation in Salmonella
typhimurium strains TA98, TA100, TA1535, TA1537, or TA1538 or mitotic recombination in
Saccharomyces cerevisiae strain D3 (Ethyl Corp., 1975; Schoeny et al., 1979;
Lawlor et al., 1979). In a hepatocyte primary culture DNA repair assay, 1,2,4-trichlorobenzene
gave negative results (CMA, 1984). With or without metabolic activation,
1,2,4-trichlorobenzene did not increase the frequency of chromosomal aberrations in cultured
Chinese hamster ovary cells (Bioassay Systems Corp., 1982). Cell transformation was induced
by 1,2,4-trichlorobenzene treatment of adult rat liver epithelial cells (CMA, 1984). In a study
published in Italian (Parrini et al., 1990; English abstract reviewed), micronuclei were reportedly
induced by intraperitoneal administration of 1,2,4-trichlorobenzene in male Swiss CD-I mice; no
other details are available in the English abstract. In another test of clastogenicity, exposure to
doses of 210 to 840 mg/kg 1,2,4-trichlorobenzene via intraperitoneal injection also increased the
frequency of micronucleated polychromatic erythrocytes in the bone marrow of male NMRI
mice (Mohtashamipur et al., 1987).
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
ORAL RfD VALUES FOR 1,2,4-TRICHLOROBENZENE
Subchronic p-RfD
The toxicological database for 1,2,4-trichlorobenzene includes several oral studies that
are potentially relevant to the derivation of a subchronic p-RfD for this chemical. These include
subchronic (CMA, 1989a; Cote et al., 1988; Carlson, 1977) studies and a 30-day (Cicmanec,
1991) study in rats; a multigeneration reproductive toxicity study in rats (Robinson et al., 1981);
and one adequate developmental toxicity study in rats (Black et al., 1988). In addition, there is a
subchronic study in mice (CMA, 1989b). Other developmental toxicity studies were performed;
however, effect levels could not be identified for one (Kitchin and Ebron, 1983), and the
remaining studies were teratology screening assays that failed to identify a LOAEL for
1,2,4-trichlorobenzene (Chernoff and Kavlock, 1983; Gray et al., 1983; Gray and Kavlock, 1984;
Gray et al., 1986). Carlson and Tardiff (1976) conducted a subchronic study in rats, but few
endpoints are examined and the effect levels are not defined. In addition, Smith et al. (1978) and
Cragg et al. (1978) studied the effects of subchronic exposure in monkeys; however, these
studies are reported only in abstract form without enough information to define effect levels.
Table 10 provides a summary of the effect levels and critical effects in the studies that were
considered for derivation of a subchronic p-RfD. A review of these studies suggests that
1,2,4-trichlorobenzene affects the liver in mice, and results in liver, thyroid, adrenal and
hematologic effects in rats. Among the available studies, LOAELs for liver and adrenal effects
in rats were of the same order of magnitude, including a LOAEL for liver and thyroid
histopathology in rats (82-101 mg/kg-day from Cote et al., 1988); a LOAEL for liver effects in
male rats (45.6 mg/kg-day from CMA, 1989a); a freestanding LOAEL for adrenal effects in rats
(53 mg/kg-day from Cicmanec, 1991); and a LOAEL for adrenal effects in rats(33 or
54 mg/kg-day for males and females, respectively, from Robinson et al., 1981). LOAELs for the
other studies (CMA, 1989b and Black et al., 1988) were higher (150-1184 mg/kg-day); thus,
these studies were not considered for use in deriving the subchronic p-RfD.
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Table 10. Summary of Oral Noncancer Dose-Response Information Suitable for Subchronic p-RfD Derivation
Species, Sex,
Number
Dose (mg/kg-d)
Exposure
Regimen
NOAEL
(mg/kg-d)
LOAEL
(mg/kg-d)
Adjusted3
LOAEL
(mg/kg-d)
Responses at the LOAEL
Reference
Subchronic Studies
Rat
M/F
10/sex/dose
0, 14.6. 45.6, or 133.7 (M)
0, 17.0, 52.5, or 150.6 (F)
Diet for
13 wk
14.6 (M)
45.6 (M)
45.6 (M)
Increased liver weight and
increased incidence of hepatocyte
hypertrophy in males only
CMA, 1989a
Rat
M/F
10/sex/dose
0, 0.07, 0.78, 7.8, or 82 (M)
0,0.11, 1.4, 15, or 101 (F)
Diet for
13 wk
7.8 (M)
15(F)
82 (M)
101 (F)
82 (M)
101 (F)
Liver and thyroid histopathology
Cote et al., 1988
Rat
F
5/dose
0, 50, 100, or 200
Daily
gavage for
30, 60, 90,
or 120 d
50
100
100
Increased liver weight with
transiently increased liver and
urinary porphyrins
Carlson, 1977
Rat
Sex not
specified
5/dose
0 or 53
Daily
gavage for
30 d
NA
53
53
Decreased serum corticosterone
levels, vacuolization of zona
fasciculata and nonsignificant
increase in adrenal weights
Cicmanec, 1991
Mouse
M/F
10/sex/dose
0, 67, 851, or 1222 (M)
0, 87, 1184, or 1346 (F)
Diet for
13 wk
67 (M)
87(F)
851 (M)
1184(F)
851 (M)
1184(F)
Liver toxicity in both males and
females
CMA, 1989b
Reproductive/Developmental Toxicity Studies
Rat
M/F
17-23 litters/
group
0,2.5, 8.9, or 33.0 (M)
0,3.7, 14.8, or 53.6(F)
Drinking
water for 3
generations
8.9 (M)
14.8 (F)
33.0 (M)
53.6 (F)
33.0 (M)
53.6 (F)
Increased adrenal weights in both
sexes of F0 and F, generations at
95 days of age
Robinson et al.,
1981
Rat
F
14/dose
0, 75, 150, or 300
Daily
gavage on
GD 6-15
75 (maternal)
300 (fetal)
150 (maternal)
NA (fetal)
150 (maternal)
NA (fetal)
Mild anemia and mild hepatic
lesions in dams
Black et al.,
1988;
Ruddick et al.,
1983
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Table 10. Summary of Oral Noncancer Dose-Response Information Suitable for Subchronic p-RfD Derivation
Species, Sex,
Number
Dose (mg/kg-d)
Exposure
Regimen
NOAEL
(mg/kg-d)
LOAEL
(mg/kg-d)
Adjusted3
LOAEL
(mg/kg-d)
Responses at the LOAEL
Reference
Reproductive/Developmental Toxicity Studies
Mice
F
25/dose
0 or 130
Daily
gavage on
GD 8-12
130
NA
NA
None
Chernoff and
Kavlock, 1983;
Gray et al., 1983,
1986; Gray and
Kavlock, 1984
'Adjusted for continuous exposure
NA = Not applicable
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In order to select a point-of-departure (POD) for the subchronic p-RfD, each of the
selected studies (CMA, 1989a; Cote et al., 1988; Cicmanec, 1991; Robinson et al., 1981) was
evaluated to determine whether the data for the critical endpoint(s) were amenable to benchmark
dose (BMD) modeling. Cote et al. (1988) did not report incidences or severity of liver and
thyroid histologic findings, so these endpoints could not be modeled. Because Cicmanec (1991)
used only one dose, the data on adrenal weight changes in this study could not be modeled.
CMA (1989a) reported dose-related changes in liver weights and hepatocellular hypertrophy
incidences (see Table 1), so these endpoints were modeled. Finally, adrenal weight changes in
male and female Fo rats (see Table 6) reported by Robinson et al. (1981) were modeled. Adrenal
weight changes were also reported in Fi rats; however, dose estimates were provided only for
Fo rats. Appendix A provides details of the modeling efforts and selection of best fitting models.
The best fit is defined as those with the best goodness of fit and Akaike's information criterion
(AIC) scores. The benchmark response level is defined as 1 standard deviation (SD) above the
control mean. Table 11 shows the BMD and BMDL predictions from the best-fitting models for
each of these data sets.
Table 11. Comparison of BMD and BMDL Predictions for Available Data sets
Reference
Endpoint Modeled
Best-fitting Model
BMD
(mg/kg-d)
BMDL
(mg/kg-d)
CMA, 1989a
Male Rat Centrilobular Hepatocyte
Hypertophy Incidence
Gamma
33.09
14.35
CMA, 1989a
Male Rat Absolute Liver Weight
Linear
(constant variance)
21.28
17.49
CMA, 1989a
Male Rat Relative Liver Weight
Linear
(constant variance)
11.27
9.41
Robinson et al.,
1981
Male Rat Absolute Adrenal Weight
Power
(modeled variance)
31.27
16.63
Robinson et al.,
1981
Female Rat Absolute Adrenal Weight
Polynomial
(constant variance)
28.60
25.51
Among the available data sets, the data for increased relative liver weight in male rats
(CMA, 1989a) gave the lowest BMDL (9.41 mg/kg-day). While the hepatocellular hypertrophy
that probably underlies this change in liver weight might be a better endpoint, the analysis of the
BMD of this endpoint yields a slightly higher value. Being conservative, this value for increased
liver weights is selected as the POD for derivation of the subchronic p-RfD. A composite
uncertainty factor (UF) of 100 is applied to the BMDL to calculate a subchronic p-RfD of
0.09 mg/kg-day as follows:
Subchronic p-RfD = BMDL UF
= 9.41 mg/kg-day -M00
= 0.09 mg/kg-day or 9 x 10"2 mg/kg-day
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The composite UF of 100 is composed of the following:
A full UF of 10 is applied for interspecies extrapolation to account for potential
pharmacokinetic and pharmacodynamic differences between rats and humans. Data to
define these parameters for 1,2,4-Trichlorobenzene are unavailable.
A full UF of 10 for intraspecies differences is used to account for potentially susceptible
individuals in the absence of information on the variability of response in humans. Data
to define these parameters for 1,2,4-Trichlorobenzene are unavailable.
• No database UF is applied. The toxicological database for oral exposure to
1,2,4-trichlorobenzene includes high-quality chronic and subchronic bioassays in two
species and adequate developmental toxicity and multigeneration reproduction studies.
Although there are multigeneration reproductive toxicity data in only one species,
available information suggests that systemic maternal toxicity occurs at lower doses than
reproductive or developmental effects. Available studies have not identified
neurotoxicity at high doses, indicating that the lack of a neurotoxicity study is not a
significant concern.
Confidence in the principal study (CMA, 1989a) is medium. This subchronic toxicity
study used an acceptable number of animals and an appropriate range of dose levels. A variety
of endpoints were measured and the study identified both a NOAEL (one sex) and LOAEL (both
sexes). Confidence in the database, which includes high-quality chronic and subchronic
bioassays in two species, developmental toxicity studies, and a multigeneration reproduction
study, is medium. Confidence in the subchronic p-RfD is, therefore, medium.
Chronic p-RfD
Because a chronic RfD of 0.01 mg/kg-day (1E-2 mg/kg-day) exists on IRIS (U.S. EPA,
2008), a chronic p-RfD is not derived in this assessment.
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION RfC VALUES FOR 1,2,4-TRICHLOROBENZENE
The animal studies provide sufficient information for derivation of p-RfCs for
1,2,4-trichlorobenzene. Table 12 summarizes the noncancer dose-response data from available
"3
inhalation studies. In the study by Kociba et al. (1981), the low concentration of 223 mg/m
serves as a LOAEL for the increased urinary excretion of porphyrins in rats, while the study by
"3
Watanabe et al. (1978) defines a LOAEL of 74 mg/m for increased urinary excretion of
porphyrins in rats; a NOAEL of 21 mg/m3 is established, as well. In the Coate et al. (1977)
"3
study, the highest exposure concentration (689-701 mg/m ) is a freestanding NOAEL for rabbits
and monkeys exposed 7 hours/day, 5 days/week, for 4 or 13 weeks. Due to the lack of
information on the incidence and/or severity of hepatic lesions reported in rats, the reported lack
of concentration-response relationship, and the absence of these effects in rats treated for
26 weeks in the same study, effect levels for this species could not be identified.
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Table 12. Summary of Inhalation Noncancer Dose-Response Information
Species,
Sex,
Number
Exposure
Concentration
(ppm)
Exposure
NOAEL
LOAEL
Responses
Reference
Subchronic Studies
Rat
M
20/conc.
0, 30 or 100
7 h/d,
5 d/wk for
44 d
NA
30 ppm
(223 mg/m3)
Increased urinary excretion of porphyrins.
Kocibaetal., 1981
Rat
M/F
10 M and
26 F/conc.
0,2.8 or 10.2
6 h/d,
5 d/wk for
3 mo
2.8 ppm
(21 mg/m3)
10.2 ppm
(76 mg/m3)
Increased urinary excretion of porphyrins; minimal LOAEL.
Watanabe et al., 1977,
1978
Chronic Studies
Rat
M
30/conc.
0, 25.3, 49.2, or
92.8
7 h/d,
5 d/wk for 4,
13 or 26 wk
Cannot be
determined
Cannot be
determined
Hepatic lesions were reported to occur in treated rats at
13 wk; however, there was not a clear
concentration-response, neither incidence nor severity was
reported, and no effects were observed at 26 wk.
Coate et al., 1977
Rabbit
M
16/conc.
0, 25.3, 49.2, or
92.8
7 h/d,
5 d/wk for
26 wk
92.8 ppm
(689 mg/m3)
NA
None
Coate et al., 1977
Monkey
M
9/conc.
0, 24.8, 49.2, or
94.5
7 h/d,
5 d/wk for
26 wk
94.5 ppm
(701 mg/m3)
NA
None
Coate et al., 1977
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The NOAELs and LOAELs from Watanabe et al. (1977, 1978) were first adjusted to an
equivalent continuous exposure concentration, then converted to human equivalent
concentrations (NOAEL[hec] and LOAEL[hec]) based on the guidance provided in U.S. EPA
(1994b). The human equivalent concentration was then calculated using the appropriate
dosimetric adjustment (U.S. EPA, 1994b). As increased urinary excretion of porphyrins is an
extrarespiratory effect, 1,2,4-trichlorobenzene was treated as a Category 3 gas and the blood:gas
partition coefficients were used to make the dosimetric adjustment. In the absence of blood:gas
partition coefficients for 1,2,4-trichlorobenzene, the default ratio of 1.0 was used U.S. EPA
(1994b). Table 13 shows a summary of the NOAEL and LOAEL values and the corresponding
HEC values.
Table 13. Calculation of Human Equivalent Concentrations
Study
Species
Effect
Effect Level
(mg/m3)
Adjusted Effect
Level3 (mg/m3)
Dosimetric
Adjustment13
Human Equivalent
Concentration
(mg/m3)
Watanabe
et al., 1977,
1978
Rat
Increased
urinary
excretion of
porphyrins
NOAEL = 21
LOAEL = 76
BMCL = 26
NOAELradji = 3.8
LOAEL[adj] = 14
BMCL[adj] = 4.6
1.0
NOAELpjEcj = 3.8
LOAEL[hec] = 14
BMCLjhec] = 4.6
aExposure concentration adjusted to equivalent continuous concentration based on exposure regimen (number of
hours/day and days/week; see Table 12)
bRatio of blood:gas partition coefficients
In order to identify a POD for both the subchronic and chronic p-RfCs, BMD modeling
was performed on the significant changes in urinary coproporphyrin and uroporphyrin excretion
in male and female rats exposed for 3 months (see Table 9) using the nominal exposure
concentrations. Appendix B provides details of the modeling and results. The recommended
Benchmark Response (BMR) of 1 SD from the control mean (U.S. EPA, 2000) was used in the
absence of a biologically-based benchmark response level. No model fit was achieved with
female uroporphyrin data, the male coproporphyrin data, or the male uroporphyrin results when
reported in |ig/mg creatinine. However, adequate fit was achieved with the male uroporphyrin
data when reported in jj.g/24 hours. For this data set, the test for homogenous variance indicated
adequate fit to the variance data and the linear model provided adequate fit to the means. The
benchmark concentration (BMCisd) and the 95% lower confidence limit (BMCLisd) resulting
"3
from this model were 5.76 and 3.47 ppm (43 and 26 mg/m ), respectively. The BMCLisd[HEC]
calculated as shown in Table 13 was 4.6 mg/m3. This value was used as the POD for both the
subchronic and chronic p-RfCs for 1,2,4-trichlorobenzene.
Subchronic p-RfC
For the subchronic p-RfC derivation, the BMCLisd [hec] was divided by a composite UF
of 300, as shown below:
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Subchronic p-RfC = BMCLisdrHEq UF
= 4.6 mg/m 300
= 0.02 mg/m3 or 2 x 10"2 mg/m3
The composite UF of 300 is composed of the following:
A UF of 3 (10°5) is used to account for the extrapolation from rats to humans using
dosimetric adjustments. The interspecies UF includes a factor of 1 for species differences
in pharmacokinetic considerations (as a dosimetric adjustment was used) and 3 for
pharmacodynamic considerations in accordance with U.S. EPA (1994b).
A full UF of 10 is used for the protection of sensitive individuals in the absence of
information to determine potentially susceptible populations.
A UF of 10 is used to account for database deficiencies; the toxicological database for
inhaled 1,2,4-trichorobenzene contains a limited chronic study in three species and two
subchronic studies, but it does not contain any reproductive or developmental toxicity
studies by the inhalation route of exposure. Available oral studies do not indicate that the
fetus is especially sensitive to the effects of 1,2,4-trichlorobenzene; in the one adequate
developmental toxicity study, maternal toxicity was observed at a dose below that
affecting the fetus (Black et al., 1988). Likewise, a multigeneration oral reproductive
toxicity study did not indicate effects on reproductive endpoints.
Confidence in the key study is medium. The study was well designed, thoroughly
documented, and carefully conducted. It identifies both a NOAEL and LOAEL. However, only
two exposure concentrations are included and only a limited number of toxicological endpoints
are evaluated. Confidence in the database is medium. Though it includes two subchronic
studies, a limited chronic study in three species, and oral data on both reproductive and
developmental toxicity, the database lacks inhalation data on reproductive and developmental
toxicity, as well as a well documented chronic inhalation toxicity study in rats, a sensitive
species. Available studies have not identified neurotoxicity at high concentrations, indicating
that the lack of a neurotoxicity study is not a significant concern. Medium confidence in the
subchronic p-RfC follows.
Chronic p-RfC
For the chronic p-RfC derivation, the BMCL[Hec] was divided by a UF of 3000, including
3 for extrapolation from rats-to-humans using dosimetric adjustments, 10 for protection of
sensitive individuals and 10 for database deficiencies (each discussed above under Subchronic
p-RfC Derivation), as well as an additional UF of 10 for use of a subchronic study. The absence
of a well documented chronic study in a sensitive species (such as rat) is accounted for by the use
of a full 10-fold UF for extrapolation from sub chronic-to-chronic effects. The chronic p-RfC is
calculated below:
Chronic p-RfC = BMCL[hec] ^ UF
= 4.6 mg/m3 3000
= 0.002 mg/m3 or 2 x 10 3 mg/m3
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Confidence in the key study is medium. The study was well designed, thoroughly
documented, and carefully conducted. It identifies both a NOAEL and LOAEL. However, only
two exposure concentrations are included and only a limited number of toxicological endpoints
are evaluated. Confidence in the database is medium. Though it includes two subchronic
studies, a limited chronic study in three species, and oral data on both reproductive and
developmental toxicity, the database lacks inhalation data on reproductive and developmental
toxicity as well as a well documented chronic toxicity study in rats. Medium confidence in the
chronic p-RfC follows.
PROVISIONAL CARCINOGENICITY ASSESSMENT FOR
1,2,4-TRICHLOROBENZENE
Weight-of-Evidence Classification
Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005),
1,2,4-trichlorobenzene is considered "Likely to Be Carcinogenic to Humans" by the oral route of
exposure based on a finding of increased tumor incidence in more than one sex of mouse.
Chronic (2-year) dietary administration of 1,2,4-trichlorobenzene at a concentration of >700 ppm
produced statistically significant increased incidences of hepatocellular carcinomas and
adenomas in male and female B6C3F1 mice (CMA, 1994b). The incidence of carcinoma
approached 100% in both sexes treated at 3200 ppm (see Table 5) and led to marked reductions
in survival at this dose. Holsapple et al. (2006) reviewed the relevance of mouse liver tumors.
They conclude that rodent liver tumors induced by porphyrogenic compounds may be relevant as
a predictor of human toxicity, particularly if the metabolism of the compound is similar in
rodents and humans. However, they also conclude that the MOA for the induction of tumors by
such agents is probably cytotoxicity, not mutagenicity. Short-term tests in bacteria and yeast
have given negative evidence for mutagenicity both with and without metabolic activation. A
test for DNA repair in mammalian cells was negative, while in vivo tests for clastogenicity
(micronucleus formation) were positive.
Mode of Action Discussion
The U.S. EPA (2005) Guidelines for Carcinogen Risk Assessment defines MOA as "A
sequence of key events and processes, starting with the interaction of an agent with a cell,
proceeding through operational and anatomical changes and resulting in cancer formation.
Toxicokinetic processes leading to the formation or distribution of the active agent (i.e., parent
material or metabolite) to the target tissue are not part of the mode of action." Examples of
possible carcinogenic MO As include mutagenic, mitogenic, anti-apoptotic (inhibition of
programmed cell death), cytotoxic (with reparative cell proliferation), and immunologic
suppression.
Very little information is available on the potential mode by which
1,2,4-trichlorobenzene increases the incidence of liver tumors in mice. Only two studies have
been conducted (both in mice): a subchronic toxicity study (CMA, 1989b) and the chronic
bioassay (CMA, 1994b) that reported an increased incidence of hepatocellular adenomas and
carcinomas in male and female B6C3F1 mice exposed to 1,2,4-trichlorobenzene for 2 years. The
subchronic study reports increased absolute and relative liver weights, increased serum ALT,
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and/or SDH and liver histopathology (including cytomegaly, karyomegaly, multinucleation,
atrophy, degeneration, microcystis) at dietary concentrations of 3500 ppm and higher (CMA,
1989b). The chronic study (CMA, 1994b) reports increased absolute and relative liver weight at
>150 ppm and hepatocellular hypertrophy, adenomas and carcinomas at >700 ppm; liver
enzymes were not analyzed. No mechanistic studies examining cancer endpoints were located.
Quinone metabolites have been implicated in the hepatotoxicity of 1,2,4-trichlorobenzene in
mice (den Besten et al., 1991b; Mizutani and Miyamoto, 1999) and may play a role in liver
carcinogenesis as well. Quinones alkylate cellular nucleophiles (e.g., glutathione or tissue
macromolecules) and can cause oxidative stress via redox cycling between the semiquinone
anion radical and the corresponding hydroquinone or benzoquinone (den Besten et al., 1994).
These limited data are inadequate for outlining potential key events in the MOA for
1,2,4-trichlorobenzene-induced hepatocellular tumors.
Quantitative Estimates of Carcinogenic Risk
Oral Exposure
Oral data are sufficient to derive a quantitative estimate of cancer risk from
1,2,4-trichlorobenzene; this derivation is shown below. Male and female B6C3F1 mice both
exhibited increased incidences of hepatocellular carcinomas and adenomas in a chronic bioassay
(CMA, 1994b). The incidence of combined adenomas and carcinomas was not reported, nor
could the incidences be combined. Although the incidence of adenomas was increased at the
mid dose in both sexes, there was no increase in adenoma incidence at the high dose due to the
high incidence of carcinomas at this dose; thus, only the carcinoma incidence was used in
quantitative estimates of cancer risk. The MOA for liver tumors produced by
1,2,4-trichlorobenzene has not been fully elucidated. Existing data do not confirm a mutagenic
MOA for 1,2,4-trichlorobenzene, however, the contribution of a linear MOA to induction of
liver tumors cannot be ruled out based on available data; thus, a linear assessment was
conducted.
The dose-response data used in the quantitative cancer assessment are shown in Table 5
and also below in Table 14. Animal doses in the CMA (1994b) mouse study were first converted
to human equivalent doses (HEDs) by adjusting for differences in body weight between humans
and mice. In accordance with U.S. EPA (2005) guidelines for carcinogen risk assessment, a
factor of BW3 4 was used for cross-species scaling. Using this scaling factor, the straight dose
(mg) in humans is obtained by multiplying the straight animal dose (mg) by the ratio of
human:animal body weight raised to the 3/4 power. For doses expressed per unit body weight
(mg/kg or mg/kg-day), the relationship is reciprocal and the human dose (mg/kg) is obtained by
multiplying the animal dose (mg/kg) by the ratio of animal :human body weight raised to the
1/4 power. Because the test article was administered in the diet ad libitum for 2 years, no
adjustment for discontinuous exposure or less-than-lifetime administration is necessary. The
equation used to calculate the HEDs is shown below and the HEDs are presented in Table 14.
HED = Dose x (W/70 kg)1/4
where:
Dose = average daily animal dose (mg/kg-day)
W = average mouse body weight during the study (kg)
70 kg = reference human body weight (U.S. EPA, 1988)
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Table 14. Dose-Response Data for Liver Tumors in Male and Female Micea
Male
Female
Animal
Dose
(mg/kg-d)
Average
Body
Weightb
(kg)
Human
Equivalent
Dose
(mg/kg-d)
Incidence of
Hepatocellular
Carcinoma
Animal
Dose
(mg/kg-d)
Average
Body
Weightb
(kg)
Human
Equivalent
Dose
(mg/kg-d)
Incidence of
Hepatocellular
Carcinoma
0
NA
0
8/49
0
NA
0
1/50
21
0.0373
3.19
5/50
26.3
0.0327
3.87
1/50
100.6
0.0365
15.2
27/50
127.0
0.0322
18.6
28/50
519.9
0.0302
74.9
50/50
572.6
0.0286
81.4
46/50
aCMA, 1994b
bCalculated from body weight measurements made during the study
Dose-response modeling of the data in Table 14 was performed to obtain a POD for a
quantitative assessment of cancer risk. The POD is an estimated dose (expressed in
human-equivalent terms) near the lower end of the observed range that marks the starting point
for extrapolation to lower doses. Appendix C provides details of the modeling effort. Table 15
shows the BMPmpn] and BMDLi0[hed] predicted by the multistage model for the liver tumor
data in males and females.
Table 15. Summary of Human Equivalent BMDs and BMDLs Based on Hepatocellular
Carcinoma Incidence Data in Male and Female Mice

BMDio[hed]
(mg/kg-d)
BMDLio[hed]
(mg/kg-d)
Male
6.23
3.50
Female
6.84
5.00
As the table shows, the BMD estimates for both sexes are very similar. The
BMDLio[hed] for liver tumors in male mice (3.50 mg/kg-day) is slightly lower than that for
females (5.00 mg/kg-day) and is selected as the POD for the provisional oral slope factor
(p-OSF). A provisional oral slope factor of 0.029 or 2.9 x 10"2 (mg/kg-day)"1 was calculated
by dividing 0.1 (10%) by the BMDLio[hed] of 3.50 mg/kg-day. The p-OSF for
1,2,4-trichlorobenzene should not be used with exposures exceeding the POD (BMDLi0[hed] =
3.50 mg/kg-day), because below this level the fitted dose-response model better characterizes
what is known about the carcinogenicity of 1,2,4-trichlorobenzene. Table 16 shows the doses
associated with specific levels of cancer risk based on the provisional oral slope factor estimated
herein.
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Table 16. Doses of 1,2,4-Trichlorobenzene Associated With Specific Levels of Cancer

Risk
Risk Level
Dose (mg/kg-d)
lO"4
0.003
10"5
0.0003
lO"6
0.00003
Inhalation Exposure
Inhalation studies of 1,2,4-trichlorobenzene are inadequate for the purpose of estimating
an inhalation unit risk for this compound. Only one chronic inhalation study is identified
(Coate et al., 1977) and, in the study, the neoplastic changes are not reported.
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42

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U.S. EPA. 1982. Three Manuscripts Describing the Dose-related Maternal and Embryonic
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(English translation).
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APPENDIX A. DETAILS OF BENCHMARK DOSE MODELING
FOR SUBCHRONIC ORAL p-RfD
Model-Fitting Procedure for Continuous Data:
The model fitting procedure for continuous data is as follows. The simplest model
(linear) is first applied to the data while assuming constant variance. If the data are consistent
with the assumption of constant variance (p> 0.1), then the fit of the linear model to the means is
evaluated and the polynomial, power and Hill models are fit to the data while assuming constant
variance. Among the models providing adequate fit to the means (p> 0.1), the one with the
lowest AIC for the fitted model is selected for BMD derivation. If the test for constant variance
is negative, the linear model is run again while applying the power model integrated into the
BMDS to account for nonhomogenous variance. If the nonhomogenous variance model provides
an adequate fit (p > 0.1) to the variance data, then the fit of the linear model to the means is
evaluated and the polynomial, power and Hill models are fit to the data and evaluated while the
variance model is applied. Among those providing adequate fit to the means (p > 0.1), the one
with the lowest AIC for the fitted model is selected for BMD derivation. If the test for constant
variance is negative and the nonhomogenous variance model does not provide an adequate fit to
the variance data, then the data set is considered unsuitable for modeling. If after these attempts,
no model provides an adequate fit to the data, the highest dose is dropped, if appropriate, and the
entire procedure is repeated. If no fit is obtained after dropping the highest dose, the next highest
dose is dropped, if appropriate and the procedure is repeated. Dose-dropping continues until:
(1) adequate fit is obtained; (2) there are only controls and two dose groups remaining. If no fit
is obtained following application of this procedure, then the data set is not considered to be
amenable to BMD modeling.
Model-Fitting Results for Male Rat Absolute and Relative Liver Weights (CMA, 1989a)
Data on male rat absolute and relative liver weights were modeled according to the
procedure outlined above using BMDS version 1.4.1 with default parameter restrictions. In the
absence of data regarding a biologically meaningful change in liver weight, the BMR was chosen
to be 1 SD from the control mean, as recommended by U.S. EPA (2000). The linear model with
constant variance provided adequate fit to both data sets. Table A-l shows the modeling results
for each data set. A BMDisd and BMDLisd of 21.28 and 17.49 mg/kg-day, respectively, were
predicted using the absolute liver weight data. A BMDisd and BMDLisd of 11.27 and
9.41 mg/kg-day, respectively, were predicted using the relative liver weight data. Figures A-l
and A-2 show the fit of the linear model with constant variance to these two data sets.
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Table A-l.
Model Predictions for Liver Weight Changes in Male Rats3


Variance
Means
BMDlsd
BMDLlsd
Model

/j-valuc'
/>-value'
(mg/kg-d)
(mg/kg-d)
Absolute Liver Weight
Linear (constant variance)0
0.1802
0.4346
21.28
17.49
Relative Liver Weight
Linear (constant variance)0
0.4288
0.2090
11.27
9.41
aCMA, 1989a
bValues <0.10 fail to meet conventional goodness-of-fit criteria
°Betas restricted to >0
Linear Model with 0.95 Confidence Level
13
12
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Linear Model with 0.95 Confidence Level
4.5
Linear
4
3.5
3
2.5
BMDL
BMD
0
20
40
60
80
100
120
140
Dose
12:22 04/23 2008
Figure A-2. Fit of Linear Model (Homogenous Variance) to Data on Male Rat Relative
Liver Weight (CMA, 1989a)
BMDs and BMDLs indicated are associated with a change of 1 SD from the control and are in units of mg/kg-day.
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Model-Fitting Results for Male and Female Rat Absolute Adrenal Weights
(Robinson et al., 1981)
Data on male and female rat absolute adrenal weights were modeled according to the
procedure outlined above using BMDS version 1.4.1 with default parameter restrictions. In the
absence of data regarding a biologically meaningful change in adrenal weight, the BMR was
chosen to be 1 SD deviation from the control mean, as recommended by U.S. EPA (2000).
Table A-2 shows the modeling results for both data sets. Using the male adrenal weight data, the
test for constant variance was negative, indicating that the variance should be modeled; the
variance model in the software provided adequate fit. The linear model did not provide adequate
fit to the means, so the polynomial and power models were fit to the data (there were not enough
data points to fit the Hill model). Both of these models provided adequate fit to the means in the
male adrenal weight data, but the power model provided better fit based on lower AIC. The
BMD isd and BlVEDLisd predicted by the power model were 20.57 and 16.84 mg/kg-day,
respectively. Figure A-3 shows the fit of the power model (modeled variance) to the male
adrenal weight data. Using the female adrenal weight data, the test for constant variance was
positive, but the linear model did not provide adequate fit to the means. The polynomial and
power models were applied, but only the polynomial model provided adequate fit to the means.
This model predicted a BMDisd and BMDLisd of 28.60 and 25.51 mg/kg-day, respectively, for
the female adrenal weight data. Figure A-4 shows the fit of the polynomial model with constant
variance to the female rat adrenal weight data.
Table A-2. Model Predictions for Adrenal Weight Changes in Male and Female Ratsa
Model
Variance
/j-valuc'
Means
/j-valuc'
AIC
BMDlsd
(mg/kg-d)
BMDLiSd
(mg/kg-d)
Male adrenal weight
Linear (constant variance)0
0.02696
0.1062
86.12
16.25
11.83
Linear (modeled variance)0
0.593
0.0272
82.70
14.12
9.26
Polynomial (modeled variance)°'d
0.593
0.1753
78.98
20.57
16.84
Power (modeled variance) °'f
0.593
0.3385
77.66
31.27
16.63
Hill (modeled variance)6
NA°
NA
NA
NA
NA
Female adrenal weight
Linear (constant variance)0
0.1522
0.01715
83.73
15.64
12.34
Polynomial (constant variance)0,6^
0.1522
0.1800
79.03
28.60
25.51
Power (constant variance)0
0.1522
0.0652
81.00
30.31
17.62
Hill (constant variance)0
NA
NA
NA
NA
NA
aRobinsonetal., 1981
bValues <0.10 fail to meet conventional goodness-of-fit criteria
°Betas restricted to >0
dPolydegree = 2 (lowest degree with adequate fit)
ePower restricted to >1
fBest fitting model
gNA = not applicable (insufficient degrees of freedom available to fit this model)
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Power Model with 0.95 Confidence Level
34
Power
33
32
31
30
29
28
27
BMDL
BMD
0
5
10
15
20
25
30
Dose
12:56 04/23 2008
Figure A-3. Fit of Power Model (Modeled Variance) to Data on Male Rat Absolute
Adrenal Weight (Robinson et al., 1981)
BMDs and BMDLs indicated are associated with a change of 1 SD from the control and are in units of mg/kg-day.
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Polynomial Model with 0.95 Confidence Level
Polynomial
42
40
38
36
34
BMDL
BMD
0
10
20
30
40
50
Dose
13:00 04/23 2008
Figure A-4. Fit of Polynomial Model (Homogenous Variance) to Data on Female Rat
Absolute Adrenal Weight (Robinson et al., 1981)
BMDs and BMDLs indicated are associated with a change of 1 SD from the control and are in units of mg/kg-day.
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Model-Fitting Procedure for Quantal Data:
The model fitting procedure for dichotomous data is as follows. All available
dichotomous models in the U.S. EPA BMDS are fit to the incidence data using the extra risk
option. The multistage model is run for all polynomial degrees up to n - 1 (where n is the
number of dose groups including control); the lowest degree polynomial providing adequate fit is
used for comparison with the other models, per U.S. EPA (2000) guidance. Goodness of fit is
2	2
assessed by the % test. When several models provide adequate fit to the data (x p > 0.1), models
are compared using the AIC. The model with the lowest AIC is considered to provide the best fit
to the data. When several models have the same AIC, the model resulting in the lowest BMDL
is selected. In accordance with U.S. EPA (2000) guidance, BMDs and lower bounds on the
BMD (BMDLs) associated with an extra risk of 10% are calculated for all models.
Model-Fitting Results for Male Rat Centrilobular Hepatocyte Hypertrophy (CMA, 1989a)
Data on the incidence of centrilobular hepatocyte hypertrophy in male rats (see Table 1)
were modeled according to the procedure outlined above using BMDS version 1.4.1 with default
parameter restrictions. Table A-3 shows the modeling results. With the exception of the quantal
linear model, which the software indicated was an invalid model choice, all of the models in the
software provided adequate fit to the data (p > 0.1). The gamma model provided the best fit as
assessed by AIC. The BMDio and BMDLio predicted by this model for the data on incidence of
centrilobular hepatocyte hypertrophy in male rats are 33.09 and 14.35 mg/kg-day, respectively.
Table A-3. Model Predictions for Incidence of Male Rat Centrilobular
Hepatocyte Hypertrophy51
Model
Degrees of
Freedom
x2
x2
Goodness-
of-Fit
p-\alueb
AIC
BMD10
(mg/kg-d)
BMDL10
(mg/kg-d)
Gamma (power >1)°
3
0.00
1.0000
15.8635
33.09
14.35
Log-logistic (slope >1)
2
0.00
1.0000
17.8629
40.29
16.74
Logistic
2
0.00
1.0000
17.8629
41.94
18.95
Multistage (degree = l)d
3
3.99
0.2626
23.0471
6.43
4.04
Log-probit (slope >1)
2
0.00
1.0000
17.8629
35.74
16.04
Probit
2
0.00
1.0000
17.8629
38.66
17.28
Quantal Linear
Invalid model choice per BMDS software
Weibull (power >1)
2
0.00
0.9999
17.8631
37.60
13.40
aCMA, 1989a
bValues <0.10 fail to meet conventional goodness-of-fit criteria
°Best-fitting model
dDegree of polynomial initially set to (n -1) where n = number of dose groups including control; model selected is
lowest-degree model providing adequate fit. Betas restricted to >0.
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Gamma Multi-Hit Model with 0.95 Confidence Level
Gamma Multi-Hit
0.8
| 06
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APPENDIX B. DETAILS OF BENCHMARK DOSE MODELING
FOR SUBCHRONIC AND CHRONIC INHALATION p-RfCs
Model-Fitting Procedure for Continuous Data:
The model fitting procedure for continuous data is as follows. The simplest model
(linear) is first applied to the data while assuming constant variance. If the data are consistent
with the assumption of constant variance (p> 0.1), then the fit of the linear model to the means is
evaluated and the polynomial, power and Hill models are fit to the data while assuming constant
variance. Among the models providing adequate fit to the means (p> 0.1), the one with the
lowest AIC for the fitted model is selected for BMD derivation. If the test for constant variance
is negative, the linear model is run again while applying the power model integrated into the
BMDS to account for nonhomogenous variance. If the nonhomogenous variance model provides
an adequate fit (p > 0.1) to the variance data, then the fit of the linear model to the means is
evaluated and the polynomial, power and Hill models are fit to the data and evaluated while the
variance model is applied. Among those providing adequate fit to the means (p > 0.1), the one
with the lowest AIC for the fitted model is selected for BMD derivation. If the test for constant
variance is negative and the nonhomogenous variance model does not provide an adequate fit to
the variance data, then the data set is considered unsuitable for modeling. If after these attempts,
no model provides an adequate fit to the data, the highest dose is dropped, if appropriate, and the
entire procedure is repeated. If no fit is obtained after dropping the highest dose, the next highest
dose is dropped, if appropriate and the procedure is repeated. Dose-dropping continues until:
(1) adequate fit is obtained; (2) there are only controls and two dose groups remaining. If no fit
is obtained following application of this procedure, then the data set is not considered to be
amenable to BMD modeling.
Model-Fitting Results for Urinary Porphyrin Levels in Rats (Watanabe et al., 1977,1978):
Data on male coproporphyrin levels (reported in |ig/24 hours) and uroporphyrin levels
(reported in |ig/24 hours as well as in |ig/mg creatinine), as well as data on female uroporphyrin
levels (reported in |ig/24 hours), were modeled according to the procedure outlined above using
BMDS version 1.4.1 with default parameter restrictions. In the absence of data regarding a
biologically meaningful change in urinary porphyrin levels, the BMR was chosen to be 1 SD
from the control mean, as recommended by U.S. EPA (2000). Although modeling of the male
coproporphyrin levels indicated adequate fit to both the variance and means using the linear
model with constant variance, the initial test for a difference among the responses was not
significant (p > 0.05), indicating that modeling of the data set would not be appropriate. The
linear model with constant variance provided adequate fit to the male data on uroporphyrin levels
when reported in |ig/24 hours. Using the data on male uroporphyrin levels reported in |ig/mg
creatinine, adequate fit of the variance data was achieved with the homogenous variance model,
but the linear model did not provide adequate fit to the means data. Because only three dose
groups were available for modeling, other model forms could not be used for this data set. Using
the data on female uroporphyrin levels reported in |ig/24 hours, the test for homogenous variance
was negative and the nonhomogenous variance model in the BMDS did not provide adequate fit
to the variance data either, indicating that this data set is not suitable for BMD modeling.
Table B-l shows the modeling results for each data set.
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Table B-l. Model Predictions for Urinary Porphyrins


in Male and Female Ratsa


Variance
Means
BMClsd
BMCLlsd
Model
/>-value'
/j-valuc'
(ppm)
(ppm)
Male coproporphyrin in jig/24 hc
Linear (constant variance/
0.4636
0.7486
8.84
4.54
Male uroporphyrin in jig/24 h
Linear (constant variance/
0.1769
0.947
5.76
3.47
Male uroporphyrin in jig/mg creatinine
Linear (constant variance/
0.1802
<0.0001
4.08
2.69
Polynomial (constant variance/
NAe
NA
NA
NA
Power (constant variance)6
NA
NA
NA
NA
Hill (constant variance)6
NA
NA
NA
NA
Female uroporphyrin in jig/24 h
Linear (constant variance/
0.07468
0.8540
4.78
3.03
Linear (modeled variance/
0.0347
0.8213
5.33
3.20
"Watanabe et al., 1977, 1978
bValues <0.10 fail to meet conventional goodness-of-fit criteria
Test for a difference among the responses was not significant (p = 0.32)
dBetas restricted to >0
"Power restricted to >1
fNA = not applicable (insufficient degrees of freedom available to fit this model)
In summary, BMD modeling was successful only for the male uroporphyrin data reported
in |ig/24 hours. A BMCisd and BMCLisd of 5.76 and 3.47 ppm, respectively, were predicted
using these data. Figure B-l shows the fit of the linear model with constant variance to these
data.
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Linear Model with 0.95 Confidence Level
Linear
7
6
5
4
3
2
1
0
BMDL
BMD
0
2
4
6
8
10
Dose
13:35 02/08 2008
Figure B-l. Fit of Linear Model (Homogenous Variance) to Data on Male Uroporphyrin
Levels (measured in jig/24 hours)
BMCs and BMCLs indicated are associated with a change of 1 SD from the control and are in units of ppm.
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APPENDIX C. DETAILS OF BENCHMARK DOSE MODELING
FOR ORAL SLOPE FACTOR
Model-Fitting Procedure for Cancer Incidence Data:
The model-fitting procedure for dichotomous cancer incidence data is as follows. The
multistage-cancer model in the U.S. EPA BMDS is fit to the incidence data using the extra risk
option. The multistage model is run for all polynomial degrees up to n - 1 (where n is the
number of dose groups including control); the lowest degree polynomial providing adequate fit is
selected, per U.S. EPA (2000) guidance. Goodness of fit is assessed by the % test; adequate fit is
indicated by ap-value greater than 0.1. In accordance with U.S. EPA (2000) guidance,
benchmark doses (BMDs) and lower bounds on the BMD (BMDLs) associated with an extra risk
of 10% are calculated.
Model-Fitting Results for Hepatocellular Carcinomas in Mice (CMA, 1994b):
Table 14 shows the dose-response data on liver carcinoma incidence in male and female
mice (CMA, 1994b). The incidence and human equivalent dose data were modeled according
the procedure outlined above using BMDS version 1.4.1 with default parameter restrictions. As
assessed by the % goodness-of-fit test, the multistage model with 2-degree polynomial was the
lowest degree polynomial providing adequate fit to the male tumor data (x p> 0.1) (see
Table C-l). The multistage model did not provide adequate fit to the female hepatocellular
carcinoma data using the full data set. After exclusion of the high-dose group, adequate fit was
achieved with the 2-degree multistage model. The liver tumor data in males generated the lower
of the two BMDLsio[HF.r>]. Figure C-l shows the fit of the multistage model (2-degree) to the
data set for males, while Figure C-2 shows the fit of the multistage model to the reduced data set
for females.
Table C-l. Model Predictions for Hepatocellular Carcinomas in Male and Female Micea
Model
Degrees
of
Freedom
2
X
X2 Goodness-
of-Fit
7?-Valueb
AIC
BMD10[hed]
(mg/kg-d)
BMDL10[hed]
(mg/kg-day)
Multistage Cancer
Slope Factor
(mg/kg-d)1
Male Mice: All Doses
Multistage
(degree = 2)°
2
1.62
0.4456
150.778
6.2274
3.5040
0.029
Female Mice: All Doses
Multistage
(degree = 3)°
2
7.62
0.0221
130.175
3.2527
2.6036
0.038
Female Mice: High Dose Excluded
Multistage
(degree = 2)°
1
0.98
0.3213
93.3518
6.8444
5.0006
0.020
aCMA, 1994b
bValues <0.10 fail to meet conventional goodness-of-fit criteria
Degree of polynomial initially set to (n -1) where n = number of dose groups including control; model selected is
lowest degree model providing adequate fit. Betas restricted to >0.
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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
EfMDL
BMD
0
10
20
30
40
50
60
70
Dose
15:44 02/12 2008
Figure C-l. Fit of Multistage (2-degree) Model to Data on Hepatocellular Carcinomas in
Male Mice (CMA, 1994b)
BMDs and BMDLs indicated are human equivalent doses associated with an extra risk of 10% and are in units of
mg/kg-day.
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Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0.7
0.6
0.5
o
>2
c 0.4
o
o
CO
0.3
LL
0.2
BMDL
BMD
0
5
10
15
Dose
15:49 02/12 2008
Figure C-2. Fit of Multistage (2-degree) Model to Data (excluding high dose) on
Hepatocellular Carcinomas in Female Mice (CMA, 1994b)
BMDs and BMDLs indicated are human equivalent doses associated with an extra risk of 10% and are in units of
mg/kg-day.
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