United States
Environmental Protection
1=1 m m Agency
EPA/690/R-07/037F
Final
6-11-2007
Provisional Peer Reviewed Toxicity Values for
1,2,4-Trimethylbenzene
(CASRN 95-63-6)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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Acronyms and Abbreviations
bw	body weight
cc	cubic centimeters
CD	Caesarean Delivered
CERCLA	Comprehensive Environmental Response, Compensation and
Liability Act of 1980
CNS	central nervous system
cu.m	cubic meter
DWEL	Drinking Water Equivalent Level
FEL	frank-effect level
FIFRA	Federal Insecticide, Fungicide, and Rodenticide Act
g	grams
GI	gastrointestinal
HEC	human equivalent concentration
Hgb	hemoglobin
i.m.	intramuscular
i.p.	intraperitoneal
i.v.	intravenous
IRIS	Integrated Risk Information System
IUR	inhalation unit risk
kg	kilogram
L	liter
LEL	lowest-effect level
LOAEL	lowest-observed-adverse-effect level
LOAEL(ADJ)	LOAEL adjusted to continuous exposure duration
LOAEL(HEC)	LOAEL adjusted for dosimetric differences across species to a human
m	meter
MCL	maximum contaminant level
MCLG	maximum contaminant level goal
MF	modifying factor
mg	milligram
mg/kg	milligrams per kilogram
mg/L	milligrams per liter
MRL	minimal risk level
MTD	maximum tolerated dose
MTL	median threshold limit
NAAQS	National Ambient Air Quality Standards
NOAEL	no-ob served-adverse-effect level
NOAEL(ADJ)	NOAEL adjusted to continuous exposure duration
NOAEL(HEC)	NOAEL adjusted for dosimetric differences across species to a human
NOEL	no-ob served-effect level
OSF	oral slope factor
p-IUR	provisional inhalation unit risk
p-OSF	provisional oral slope factor
p-RfC	provisional inhalation reference concentration
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p-RfD
provisional oral reference dose
PBPK
physiologically based pharmacokinetic
ppb
parts per billion
ppm
parts per million
PPRTV
Provisional Peer Reviewed Toxicity Value
RBC
red blood cell(s)
RCRA
Resource Conservation and Recovery Act
RDDR
Regional deposited dose ratio (for the indicated lung region)
REL
relative exposure level
RfC
inhalation reference concentration
RfD
oral reference dose
RGDR
Regional gas dose ratio (for the indicated lung region)
s.c.
subcutaneous
SCE
sister chromatid exchange
SDWA
Safe Drinking Water Act
sq.cm.
square centimeters
TSCA
Toxic Substances Control Act
UF
uncertainty factor
l^g
microgram
[j,mol
micromoles
voc
volatile organic compound
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PROVISIONAL PEER REVIEWED TOXICITY VALUES FOR
1,2,4-TRIMETHYLBENZENE (CASRN 95-63-6)
Background
On December 5, 2003, the U.S. Environmental Protection Agency's (EPA's) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1.	EPA's Integrated Risk Information System (IRIS).
2.	Provisional Peer-Reviewed Toxicity Values (PPRTV) used in EPA's Superfund
Program.
3.	Other (peer-reviewed) toxicity values, including:
~	Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR),
~	California Environmental Protection Agency (CalEPA) values, and
~	EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in EPA's Integrated Risk Information System (IRIS). PPRTVs are
developed according to a Standard Operating Procedure (SOP) and are derived after a review of
the relevant scientific literature using the same methods, sources of data, and Agency guidance
for value derivation generally used by the EPA IRIS Program. All provisional toxicity values
receive internal review by two EPA scientists and external peer review by three independently
selected scientific experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the
multi-program consensus review provided for IRIS values. This is because IRIS values are
generally intended to be used in all EPA programs, while PPRTVs are developed specifically for
the Superfund Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a five-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV manuscripts conclude
that a PPRTV cannot be derived based on inadequate data.
Disclaimers
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and RCRA program offices are advised to carefully review the information provided
in this document to ensure that the PPRTVs used are appropriate for the types of exposures and
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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.
It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
users are strongly encouraged to read the entire PPRTV manuscript and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center for OSRTI. Other EPA programs or external parties who may
choose of their own initiative to use these PPRTVs are advised that Superfund resources will not
generally be used to respond to challenges of PPRTVs used in a context outside of the Superfund
Program.
Questions Regarding PPRTVs
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) may be directed
to the EPA Office of Research and Development's National Center for Environmental
Assessment, Superfund Health Risk Technical Support Center (513-569-7300), or OSRTI.
INTRODUCTION
Neither a reference dose (RfD) nor a reference concentration (RfC) are available for
1,2,4-trimethylbenzene in the Integrated Risk Information System (IRIS) database (U.S. EPA,
2007) or the Health Effects Assessment Summary Tables (HEAST) (U.S. EPA, 1997). There is
no Agency for Toxic Substances and Disease Registry (ATSDR) Toxicological Profile on 1,2,4-
trimethylbenzene, other trimethylbenzene isomers, or mixtures of trimethylbenzene isomers
(ATSDR, 2006). The Chemical Assessments and Related Activities (CARA) list (U.S. EPA,
1991, 1994a) and the HEAST (U.S. EPA, 1997) list a Health and Environmental Assessment
(HEA) for trimethylbenzenes (U.S. EPA, 1987a); however, the available toxicity data were
considered inadequate for quantitative risk assessment (U.S. EPA, 1997). The CARA (U.S.
EPA, 1991, 1994a) lists a Drinking Water Health Advisory for 1,2,4-trimethylbenzene (U.S.
EPA, 1987b). Because available human and animal toxicity data were considered inadequate for
longer-term and lifetime quantitative risk assessment, the U.S. EPA (1987b) derived an RfD of
0.64 mg/kg-day for 1,2,4-trimethylbenzene based on assumptions that the Threshold Limit Value
(TLV) of 25 ppm (125 mg/cu.m) for mixed trimethylbenzenes recommended by the American
Conference of Governmental Industrial Hygienists (ACGIH, 2001, 2005) represents aNOAEL
for 1,2,4-trimethylbenzene and that exposure results in 50% absorption. The National Institute
of Occupational Safety and Health (NIOSH) adopted a recommended exposure limit (REL) time-
weighted average (TWA) of 25 ppm (123 mg/m3) for 1,2,4-trimethylbenzene (NIOSH, 2006).
The Occupational Safety and Health Administration (OSHA) has not adopted a permissible
exposure limit (PEL) for 1,2,4-trimethylbenzene (OSHA, 2006). Health assessments for 1,2,4-
trimethylbenzene are not available from other major sources, including CalEPA (2006), the
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National Toxicology Program (NTP, 2006), the World Health Organization (WHO, 2006), and
the International Agency for Research on Cancer (IARC, 2006).
A Group D (not classifiable as to human carcinogenicity) cancer classification is included
in the Drinking Water Standards and Health Advisories list (U.S. EPA, 2004). A cancer
assessment for 1,2,4-trimethylbenzene is not available on IRIS (U.S. EPA, 2007) or the HEAST
(U.S. EPA, 1997). A cancer assessment for 1,2,4-trimethylbenzene is not available from
CalEPA (2006), the National Toxicology Program (NTP, 2006), the World Health Organization
(WHO, 2006), or the International Agency for Research on Cancer (IARC, 2006). Occupational
exposure limits for 1,2,4-trimethylbenzene listed by NIOSH (2006) include no cancer notation.
Literature searches were performed to identify relevant information for 1,2,4-
trimethylbenzene for the years 1986-1998 in the databases HSDB, RTECS, TSCATS,
MEDLINE, and TOXLINE. Update literature searches were conducted in TOXLINE,
MEDLINE (plus PubMed cancer subset), and DART/ETICBACK for the time period including
January, 1998 to December, 2005. Update search of the TOXCENTER database was performed
for the time period of August, 2000 to December, 2005. Databases searched without date
limitations in December, 2005, included TSCATS, RTECS, GENETOX, HSDB and CCRIS.
Search of Current Contents encompassed July to December, 2005.
REVIEW OF PERTINENT DATA
Human Studies
Oral Exposure
No data were located regarding the oral toxicity or carcinogenicity of 1,2,4-
trimethylbenzene in humans.
Inhalation Exposure
Data regarding the inhalation toxicity of 1,2,4-trimethylbenzene in humans come from an
occupational exposure study in which workers were exposed to a mixture of trimethylbenzene
isomers. Battig et al. (1958) examined 27 workers exposed to Fleet-X DV 99 solvent in the
painting shop of a Swiss transportation plant. The solvent was analyzed spectrographically and
was found to consist primarily of aromatic hydrocarbons (97.5%) and paraffinic and naphthenic
hydrocarbons (2.5%). The aromatic hydrocarbon portion was composed of 1,2,4-
trimethylbenzene (>50%>), 1,3,5-trimethylbenzene (>30%), and possibly included 1,2,3-
trimethylbenzene, l-methyl-2-ethyl benzene, l-methyl-3-ethyl benzene, and l-methyl-4-ethyl
benzene. Based on analysis of air samples collected from the plant, the concentration of the
solvent was roughly estimated at 10-60 ppm (49-295 mg/m3). The control group consisted of 10
unskilled workers employed in a different section of the plant. Although the authors stated that
the Fleet-X DV 99 solvent was used for "a period of some ten years," the average exposure
duration of the workers was not reported. The workers reported CNS symptoms (vertigo,
headaches and drowsiness) more often than the control group (70% versus 30% in the controls).
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Chronic asthma-like bronchitis (30% of workers versus 10% of controls), anemia [defined as <
4.5 million erythrocytes/mm3 and usually combined with normal hemoglobin] (52% versus 20%)
and alterations in blood clotting (30% versus 10%) were also observed in the exposed workers.
The incidence of CNS symptoms was statistically significantly higher in the exposed workers
than in the control group (Fisher's exact test conducted for this assessment; p<0.05). For the
other effects, the incidences did not significantly differ between the groups. A higher incidence
of vitamin C deficiency was observed in the control group, suggesting that the two groups may
not have been matched for socioeconomic status. If the assumption is made that the solvent
exclusively contained trimethylbenzene isomers, then this study identifies a LOAEL in the range
of 10-60 ppm (49-295 mg/m3) for signs of neurotoxicity.
Animal Studies
Oral Exposure
The database of repeated oral exposure studies in animals for 1,2,4-trimethylbenzene is
limited to a 4-week study (Borriston Laboratories, 1984) and a chronic exposure carcinogenicity
study (Maltoni et al., 1997). No oral developmental or reproductive toxicity studies were located
for 1,2,4-trimethylbenzene.
The primary focus of the Borriston Laboratories (1984) study was the ability of 1,2,4-
trimethylbenzene to induce nephrotoxicity. In this study, groups of 10 male Fischer-344 rats
were administered doses of 0.5 or 2.0 g/kg neat 1,2,4-trimethylbenzene by gavage 5 days/week
for 4 weeks; the duration-adjusted doses were 357 and 1429 mg/kg-day, respectively. A group
of rats serving as controls were gavaged with saline. Gross necropsy was conducted in all rats,
but only the kidneys underwent histopathologic examination. Mortality rates during treatment in
the control, low-, and high-dose groups were 0/10, 1/10, and 10/10, respectively. Deaths in the
high-dose group occurred as early as the third day of treatment. Final body weight and absolute
kidney weight of low-dose rats were not significantly different than controls. Gross necropsy
findings in low-dose animals included speckled cortical surfaces in the kidneys and white
gelatinous material inside the urinary bladders. High-dose rats exhibited mottled and red
thymus, spotty kidney and liver surfaces, enlarged adrenals, gas filled and yellow intestines and
lung congestion. The presence or absence of hydrocarbon nephropathy was determined by
examining the incidence of hyaline droplet changes, regenerative epithelium and tubular dilation
with granular material. Treatment with 1,2,4-trimethylbenzene did not significantly increase the
incidence or severity of nephropathy relative to controls; however, according to the authors, it is
possible that high-dose rats died before nephropathy could develop. A NOAEL or LOAEL
could not be determined due to the limited scope of the study, although the high dose of 1429
mg/kg-day was clearly a frank effects level (FEL) for mortality.
Maltoni et al. (1997) investigated the carcinogenicity of 1,2,4-trimethylbenzene (99%
pure) in a long-term oral exposure experiment. Male and female Sprague-Dawley rats
(50/sex/group) received doses of either 0 or 800 mg/kg (4 days/week) of 1,2,4-trimethylbenzene
by gavage in 1 ml olive oil for 104 weeks. Food and water consumption and body weights were
recorded throughout the experiment. Upon death or terminal sacrifice at 123 weeks, the animals
were subjected to systemic necropsy. Histopathology was performed on brain, pituitary gland,
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Zymbal glands, salivary glands, Harderian glands, head, tongue, thymus, mediastinal lymph
nodes, lung, heart, diaphragm, liver, spleen, pancreas, kidneys, adrenal glands, esophagus,
stomach, intestine (four levels), bladder, prostrate, uterus, vagina, gonads, interscapular fat pad,
subcutaneous and mesenteric lymph nodes, sternum, femur, spinal cord and any other organs and
tissues with pathological lesions. No statistical analysis of the data was presented.
"Slight" reduction in the survival of the female Sprague-Dawley rats and an
"intermediate" reduction in the survival of male rats were reported (Maltoni et al., 1997).
However, quantitative survival data were not presented in the report and no statistical analysis of
the decreases in survival were presented. Although the study report indicated that food and
water consumption and body weight data were recorded, these data were not included in the
report. There was no significant increase in the incidence of animals bearing either malignant or
benign + malignant tumors (Table 1). Fisher's exact tests conducted for EPA indicated that the
differences in total tumors between the exposed and treated animals were not statistically
significant (p<0.05). Neuroesthesioepitheliomas were observed in the nasal cavity of 3/100
exposed animals (M + F). This tumor was not seen in concurrent controls, and a Fisher's exact
test of the data showed that the increase in incidence of neuroesthesioepitheliomas was not
statistically significant (p<0.05). The authors, however, indicated that these tumors are quite rare
in the colony of Sprague-Dawley rats used for these experiments and suggested that this finding
presents some evidence for carcinogenicity of 1,2,4-trimethylbenzene.
Table 1. Incidences of Benign and Malignant Tumors in Male and Female Sprague-
Dawley Rats after a Long-term (104 week) Oral Exposure to 1,2,4-Trimethylbenzene.a
Dose

Animals
Percent of animals with tumors
(mg/kg bw)b
Sex
Number
Benign +
Malignant
Malignant

M
50
62
26
800
F
50
66
24

M + F
100
64
25

M
50
54
24
0
F
50
70
22

M + F
100
62
23
aSource: Maltoni etal., 1997
bGavage dose administered 4 days/week for 104 weeks and animals were terminated after 123 weeks.
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Inhalation Exposure
Korsak and Rydzynski (1996) examined the neurotoxic effects of acute exposure of male
Wistar rats (10/group) to 1,2,4-trimethylbenzene (>97% pure) and other trimethylbenzene
isomers, and also examined the neurotoxic effects of subchronic exposure to 1,2,4-
trimethylbenzene and 1,2,3-trimethylbenzene. In the acute experiment, rats were exposed to
concentrations of 250-2000 ppm (1227-9816 mg/m3) for 4 hours. Acute exposure to 1,2,4-
trimethylbenzene caused concentration-related impairment in a rotarod performance test (EC50 =
4693 mg/m3) and concentration-related decreased pain sensitivity (as measured by increased
paw-lick response latency; EC50 = 5682 mg/m3).
In the subchronic experiment, rats were exposed to 1,2,4-trimethylbenzene at
concentrations of 0, 25, 100 or 250 ppm (0, 123, 491 or 1227 mg/m3), 6 hours/day, 5 days/week
for 3 months and observed for exposure-related clinical signs and body weight effects (Korsak
and Rydzynski, 1996). Rotarod performance and hot-plate behavior were measured as indices of
the neurotoxicity of trimethylbenzene isomers. Rotarod performance was tested prior to start of
the study, weekly during exposure, and 2 weeks after the termination of the exposure. Hot-plate
behavior was tested immediately after termination of the exposure. Fisher's exact test was used
for analysis of rotarod performance and the Kruskall-Wallis test used for changes in pain
sensitivity (hot plate behavior). Exposures to 1,2,4-trimethylbenzene did not result in any
apparent body weight effects or clinical signs of toxicity. However, exposure-related indicators
of neurotoxicity were noted. Rotarod performance failure increased in a concentration-related
manner in the groups exposed to 1,2,4-trimethylbenzene, but reached the level of statistical
significance (40% failure; p<0.05) only in the highest (1227 mg/m3) exposure group following 8
or 13 weeks of exposure. The incidence of rotarod performance failure in control rats was 0%
throughout the study period. Although the mean rotarod performance failure rate in the highest
exposure group remained at 30% after a 2-week recovery period, the rate was not significantly
different from controls. Pain-sensitivity was also decreased in a concentration dependent manner
(evidenced by increased latency of the paw-lick response). As shown in Table 2, the increased
latency reached the level of statistical significance in the 491- and 1227-mg/m3 groups. After a
2-week recovery period, the highest (1227 mg/m3) exposure group no longer exhibited a
significant difference in pain sensitivity, relative to controls. This study identified a NOAEL of
123 mg/m3 and a LOAEL of 491 mg/m3 (6 hours/day, 5 days/week) for significantly decreased
pain sensitivity.
Table 2. Exposure-Related Effect on Latency of the Paw-Lick Response in Rats Exposed to
1,2,4-Trimethylbenzene Vapors 6 Hours/Day, 5 Days/Week for 3 Months.3
Number of rats
Exposure level (mg/m3)
Mean latency of paw-lick response (seconds)
9
0
15.4 ± 5.8b
10
123
18.2 ± 5.7
9
491
27.6 ± 3.2C
10
1227
30.1 ±7.9C
a Source: Korsak and Rydzynski, 1996
b The authors did not specify whether standard deviation or standard error of the mean is presented
c Statistically significantly different from controls (p<0.01)
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Gralewicz et al. (1997a) investigated 1,2,4-trimethylbenzene-induced behavioral effects
on groups of male Wistar rats (15/group) exposed to vapor concentrations of 0, 50, 100 or 250
ppm (0, 123, 491 or 1227 mg/m3) for 6 hours/day, 5 days/week for 4 weeks. To assess the effect
of exposure on short-term working memory, choice accuracy in a radial arm maze was tested.
Effects on spontaneous activity were evaluated with an open field test. Effects on long-term
memory and learning ability were assessed on the basis of conditioned passive and active
avoidance tests. The hot-plate test was performed to compare the groups with respect to the
decrease in responsiveness to a thermal stimulus following a brief intermittent foot shock.
Animals were subjected to the following sequence of behavioral testing:
1.	radial maze: 2 weeks before exposure and on days 14-18 after exposure,
2.	open field activity: day 25 after exposure,
3.	passive avoidance: days 35-45 after exposure,
4.	hot-plate test: days 50 and 51 after exposure,
5.	active avoidance: day 54 after exposure.
The data were analyzed by ANOVA and comparisons among treatments were made using
Sheffe's test, or Tukey's test for 2-way ANOVA.
There was no significant effect of 1,2,4-trimethylbenzene exposure on body weight gain
during the 4-week exposure. Passive-avoidance learning was significantly (p<0.001) retarded in
groups exposed to 491 or 1227 mg/m3 of 1,2,4-trimethylbenzene and tested 35-45 days after the
end of the exposure period. Retardation of passive-avoidance learning was more pronounced in
the 491 mg/m3 exposure group than in the 1227 mg/m3 group. In the hot-plate test following
foot shock, evaluation of rats 50 days following termination of exposures to 491 or 1227 mg/m3
of 1,2,4-trimethylbenzene revealed significantly (p<0.01) increased paw-lick latency times, in
comparison to unexposed controls. There was no significant change in the active avoidance test,
although there was a trend toward decreased avoidance responses with increasing 1,2,4-
trimethylbenzene exposure concentration. Short-term working memory did not appear to be
adversely affected by 1,2,4-trimethylbenzene exposure. In the open field test there was no
significant effect on spontaneous movement or on rearing behavior; however, there was a
significant (p<0.05) increase in grooming behavior of animals exposed to 1,2,4-trimethylbenzene
at 491 mg/m3. Although grooming behavior also was increased above controls in the 123 and
1227 mg/m3 groups, the difference was not statistically significant. The results of these
experiments suggest that 4-week exposures at concentrations that produced no overt clinical
signs of toxicity can produce long-term effects on the functional state of the rat central nervous
system. Based on findings of significantly retarded passive avoidance learning and increased
paw-lick latency in rats of the 491 and 1227 mg/m3 exposure groups, the 123 mg/m3 group
represented a NOAEL and the 491 mg/m3 group represented a LOAEL (6 hours/day, 5
days/week) for persistent neurotoxic effects.
Gralewicz and Wiaderna (2001) employed the same general protocol used by Gralewicz
et al. (1997a) in a comparative study of the behavioral effects of repeated inhalation exposure to
individual trimethylbenzene isomers or m-xylene. The study included a group of 11 adult male
Wistar rats exposed to 100 ppm (491 mg/m3) of 1,2,4-trimethylbenzene (purity not stated) and a
control (air only) group of 10 male rats. Exposures were for 6 hours/day, 5 days/week for 4
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weeks. The sequence of behavioral testing varied slightly from that employed by Gralewicz et
al. (1997a) and included:
1.	radial maze: 1 week before exposure and on days 14-18 after exposure,
2.	open field activity: day 8 before exposure and day 25 after exposure,
3.	passive avoidance: days 39-48 after exposure,
4.	hot-plate test: days 50 and 51 after exposure,
5.	active avoidance: days 54 and 60 after exposure.
No significant exposure-related effects were seen regarding body weights or short-term
working memory (as determined in the radial arm maze test) for any of the trimethylbenzene
isomers or m-xylene. Acquisition, but not retention, of the two-way active avoidance response
was significantly impaired in all solvent-exposed groups. Results of other behavioral tests
demonstrated exposure-related effects for each of the solvents. In the case of 1,2,4-
trimethylbenzene, significantly increased spontaneous locomotor activity in the open field,
impaired passive avoidance learning and significantly longer paw-lick latencies in the hot-plate
test 24 hours after foot shock were observed. These results support the findings of the earlier
study (Gralewicz et al., 1997a) in which the 491 mg/m3 (6 hours/day, 5 days/week) exposure
level represented a LOAEL for neurotoxic effects in 1,2,4-trimethylbenzene-exposed rats.
Gralewicz et al. (1997b) investigated the effect of a 4-week (6 hours/day, 5 days /week)
inhalation exposure to 1,2,4-trimethylbenzene (purity not stated) at concentrations of 0, 25, 100
or 250 ppm (0, 123, 491 or 1227 mg/m3) on the occurrence of spike-wave discharges (SWD) in
the neurocortex of male Wistar rats (9-10/group). Bursts of SWD increase in number and/or
duration with advancing age and it was hypothesized that exposure to neurotoxic solvents may
accelerate the aging process in the brain. Electrodes were implanted into the fronto-parietal
cortex and into the dorsal hippocampus. One-hour EEG recordings were performed immediately
before initiation of exposure, at the end of the exposure period, 1 month later and 3 months later.
The occurrence of SWD bursts is limited to the state of awake immobility. The number and total
duration of SWD bursts were determined from each EEG. The data were analyzed by ANOVA
and multiple comparisons among treatments was performed with Tukey's test. The study results
included information regarding mean body weights, but the study report did not provide details
of body weight data collection.
The study authors (Gralewicz et al., 1997b) indicated that 1,2,4-trimethylbenzene
exposure resulted in no statistically significant body weight effects. In the control and lowest
(123 mg/m3) exposure groups, the total duration of SWD showed an increasing trend with time,
in comparison to pre-exposure SWD and reached statistical significance (p<0.05) at 3 months
after exposure. In contrast, the total duration of SWD tended to decline with time in the mid-
and high-exposure groups after exposure. The decrease in SWD occurrence, however, was
statistically significant only for the measurements performed 1 month after the end of exposure
in the mid-exposure (491 mg/m3) group. A similar trend was seen when the number of SWD
bursts per hour was determined. The frequency of SWD bursts increased with age in the control
and lowest exposure groups and tended to decline with time in the mid- and high-exposure
groups. The data, however, were highly variable and were statistically significantly different
from pre-exposure SWD only for the highest exposure level at 3 months after exposure. Thus,
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there were no clear 1,2,4-trimethylbenzene induced concentration-related effects on SWD,
although the results are suggestive that long-term effects on brain activity may have occurred.
Korsak et al. (2000) exposed groups of male and female rats (10/sex/group; 20/sex/group
at the highest exposure concentration) of outbred Imp:WIST to 1,2,4-trimethylbenzene (97%
pure) vapors at target concentrations of 0, 123, 492 or 1230 mg/m3 for 6 hours/day, 5 days/week
for 3 months. Animals were observed twice daily for clinical signs of toxicity. Body weights
were recorded prior to initiation of exposures and weekly thereafter and food consumption was
measured weekly. Blood was drawn for hematological examination prior to initiation of
exposures and 1 week prior to exposure termination. Clinical chemistry testing was performed at
the end of the 3-month exposure period. Organ weights were determined for lungs, liver, spleen,
kidneys, adrenals, heart and gonads. Histopathological examinations were performed on tissues
from brain, nose, larynx, trachea, thymus, lungs, heart, liver, spleen, kidney, adrenals, thyroid,
pancreas, gonads, urinary bladder, stomach, duodenum, small and large intestines and salivary
glands.
Clinical findings were unremarkable (Korsak et al., 2000). No significant exposure-
related effects were seen regarding food consumption or body weights. The few differences in
some relative or absolute organ weights did not appear to be of toxicological relevance.
Significant concentration-related trends (p<0.01) for decreased numbers of red blood cells and
increased numbers of white blood cells were noted for male (but not female) rats. In the male
rats of the highest 1,2,4-trimethylbenzene exposure group (1230 mg/m3), both red and white
blood cell counts were significantly different (p<0.01) from those of control males. A significant
trend (p<0.01) for concentration-related decreased reticulocyte counts was observed in female
rats and the difference was significant (p<0.05) in the 1230 mg/m3 group. Hematological testing
also revealed a significant trend (p<0.01) for decreased clotting time in female (but not male)
rats; the decrease reached the level of statistical significance (p<0.05) in the 492 and 1230 mg/m3
groups. Clinical chemistry results were unremarkable, with the exception of significantly
increased serum sorbitol dehydrogenase in all 1,2,4-trimethylbenzene-exposed groups of male
rats (not concentration related). Histopathological examinations revealed exposure-related
significantly increased severity of pulmonary lesions, which included increased proliferation of
peribronchial lymphatic tissue in males of the mid- (but not highest) exposure level, increased
alveolar macrophages in males of the highest exposure level and increases in interstitial
lymphocytic infiltrations in males of the mid- (but not highest) exposure level and females of the
highest exposure level. No significant exposure-related changes were seen in the other examined
organs and tissues. The mid exposure level of 492 mg/m3 can be considered a LOAEL for
hematological and respiratory effects in this study. The low exposure level of 123 mg/m3 is a
NOAEL.
Korsak et al. (1997) exposed male Wistar rats of IMP:DAK outbreed stock (10/group) to
1,2,4-trimethylbenzene (>97% pure) at concentrations of 0, 25, 100 or 250 ppm (0, 123, 491 or
1227 mg/m3) for 90 days (6 hours/day, 5 days/week). Lung lavage fluid was collected 24 hours
after termination of the subchronic exposure and centrifuged at 400 g for 10 minutes.
Differential counts of bronchoalveolar lavage (BAL) cell smears were determined by light
microscopy after staining and the trypan blue test was used to determine cell viability. Total
9

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protein concentration, mucoprotein concentration, lactate dehydrogenase and acid phosphatase
activity were determined in the BAL supernatant.
All rats exposed to 1,2,4-trimethylbenzene for 90 days survived the experiment and there
were no significant differences in final body weight. Statistically significant increases were
observed in total cell and macrophage numbers in BAL of all treated groups after 90 days in
comparison to controls. Significant increases were also observed in total protein, lactate
dehydrogenase (LDH) and acid phosphatase (AP) in BAL fluid of all treated groups. However,
the observed increases in these parameters were either at or near their highest observed response
at the lowest exposure concentration, and there was no indication of further concentration-related
increases. For the observed effects, the lowest exposure level used (123 mg/m3) would be a
LOAEL; however, the toxicological significance of these effects is not clear.
In a study by IBT (1981), groups of 5 male and 5 female COBS rats were exposed to 49
or 480 mg/m3 MCS-1809 6 hours/day, 5 days/week for 4 weeks (IBT, 1981). MCS-1809 was
identified as a compound containing 75% 1,2,4-trimethylbenzene and 25% C9 aromatics
(Monsanto, 1992). The test atmosphere was generated by passing the MCS-1809 through a
nebulizer; no information on the particle size distribution was reported. Based on the vapor
pressure of 1,2,4-trimethylbenzene, it is likely that the animals were predominantly exposed to
1,2,4-trimethylbenzene vapors rather than a mist. The following parameters were used to assess
toxicity: daily observations, weekly body-weight measurements, organ weights (adrenal glands,
brain, gonads, heart, kidneys, liver, lungs, spleen and thyroid gland), gross necropsy and
histopathological examination of adrenal glands, brain, bronchi, gonads, heart, kidneys, liver,
lungs, pancreas, pituitary glands, lymph nodes, spleen, trachea and thyroid gland of the control
and 480 mg/m3 groups (tissues from the 49 mg/m3 group were examined if significant findings
were found in the 480 mg/m3 group).
Exposure to MCS-1809 did not result in deaths. Clinical signs of toxicity in the 480
mg/m3 group included ataxia and hypoactivity that persisted between exposures, ptosis, red
ocular discharge, and ruffed fur. Less pronounced hypoactivity and ruffed fur were observed in
the 49 mg/m3 group. In the 480 mg/m3 group, significant decreases in body weight gain (35%
lower in the males; no significant alteration in females), increases in absolute (females only) and
relative liver weights and decreases in absolute and relative spleen weights (females only) were
observed. A significant increase in absolute liver weight was also observed in the 49 mg/m3
female rats. Histological alterations were limited to focal or diffuse testicular atrophy in 3/5
male rats exposed to 480 mg/m3 in the absence of statistically significant changes in testis
weight; no testicular effects were observed in the 49 mg/m3 (testes examined in four rats from
this group) or control groups. This study identified a NOAEL of 49 mg/m3 and LOAEL of 480
mg/m3 (6 hours/day, 5 days/week) for clinical signs of toxicity (persistent ataxia and
hypoactivity, ptosis, ocular discharge), decreased body weight gain, and histopathological
evidence of testicular atrophy. The increased absolute liver weight observed in the 49 mg/m3
female rats was not considered adverse because no histological alterations were observed at the
49 or 480 mg/m3 concentrations.
Battig et al. (1958) exposed 8 male rats (strain not reported) to air concentrations of 1700
ppm of Fleet-X DV 99 solvent for 4 months (8 hours/day, 5 days/week). Other rats (sex, strain,
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and number not specified) were exposed to 500 ppm for 70 days (8 hours/day, 5 days/week). As
described earlier, Fleet-X DV 99 is a solvent containing 97.5% aromatic hydrocarbons (>50%
1,2,4-trimethylbenzene and >30% 1,3,5-trimethylbenzene) and 2.5% of paraffinic and
naphthenic hydrocarbons. The 500- and 1700-ppm concentrations would be approximately 2523
and 8155 mg/m3, respectively, if the aromatic hydrocarbon fraction of the vapors were
comprised exclusively of trimethylbenzenes. Within 2 weeks of exposure, 4 of the 8 rats
exposed to 1700 ppm died and were replaced, while none of the animals in the 500-ppm group
died. Histopathologic examinations, performed only on 1700-ppm animals that died, revealed
cloudy swelling and fatty infiltration in the kidneys, peripheral fatty infiltration in the liver, an
increase in secondary nodules in the spleen and marked congestion of the pulmonary capillaries
with alveolar wall thickening. Alterations in differential white blood cell counts (increase in the
percentage of segmented neutrophilic granulocytes and a decrease in the percentage of
lymphocytes) were reported at 500 ppm. Increases in drinking water consumption (43-45%
higher than in the control group) were observed in the 1700-ppm group. The authors reported
that during the exposure period, the 1700-ppm animals were initially "highly excited and
aggressive" followed by a period of narcosis and ataxia. Because histopathology was only
performed on the animals that died, no histopathology data are available on the 500-ppm rats.
Due to the limited scope of the study, a NOAEL or LOAEL cannot be identified. The high
concentration of 1700 ppm (8155 mg/m3) is a FEL for mortality.
Bernshtein (1972) exposed rats (number, sex and strain not specified) to 1000 mg/m3
(200 ppm) of a mixture of trimethylbenzenes for 6 months (4 hours/day, 6 days/week). An
inhibition of phagocytic activity of the leukocytes was reported. This study was summarized by
Sandmeyer (1981) and further experimental details were not provided.
Korsak et al. (1997) examined the effect of acute exposures (6 min) to the
trimethylbenzene isomers, 1,2,3-trimethylbenzene (90-95% pure), 1,3,5-trimethylbenzene (99%
pure) and 1,2,4-trimethylbenzene (97% pure) on the respiratory rate of Balb/C male mice (8-
10/group) at concentrations ranging from 253 to 1591 ppm (1926-9453 mg/m3). The
concentration depressing mouse respiratory rate by 50% (RD50) was calculated by least squares
regression and the Kruskall-Wallis test was applied for evaluation of protein and enzyme levels
in the BAL fluid. All three trimethylbenzene isomers showed irritating effects on the respiratory
tract and caused concentration-dependent decreases in respiratory rate. The concentration
depressing the respiratory rate in mice to 50% was 519 ppm (2547 mg/m3) for 1,2,4-
trimethy lb enzene.
The developmental toxicity of 1,2,4-trimethylbenzene was assessed by Saillenfait et al.
(2005). Groups of mated (sperm-positive) Sprague-Dawley rats (24/group) were exposed (whole
body) to 1,2,4-trimethylbenzene (97% pure) at vapor concentrations of 0, 100, 300, 600 or 900
ppm (0, 491, 1475, 2950 or 4425 mg/m3) for 6 hours/day on gestation days 6 through 20.
Maternal food consumption was recorded for the intervals of gestation days 6-13 and 13-21.
Maternal body weights were recorded weekly during gestation. At necropsy on gestation day 21,
the uterus was weighed and numbers of corpora lutea, implantation sites, resorptions and dead
and live fetuses were recorded. Live fetuses were weighed, sexed and examined for external
anomalies. Half of the live fetuses from each litter were prepared for visceral examination, the
others were subjected to skeletal examination.
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All dams survived to scheduled necropsy (Saillenfait et al., 2005). No clinical signs of
1,2,4-trimethylbenzene-induced toxicity were observed. Maternal food consumption was
significantly (p<0.01) depressed in the 600- and 900-ppm groups (approximately 12-14% and
15-19%, respectively, relative to controls). The 900-ppm dams exhibited significantly reduced
mean body weight gain (22-52%) lower than controls) throughout the exposure period.
Significantly (p<0.01) reduced body weight gain (30%> lower than controls) was observed in the
600-ppm group, but only during the first week of 1,2,4-trimethylbenzene exposure. At necropsy
on gestation day 21, mean body weight gain (corrected for gravid uterine weight) was
significantly depressed in both 600- and 900-ppm dams (approximately 50% lower than
controls). Mean fetal body weight was significantly lower in both 600- and 900-ppm exposure
groups (approximately 5 and 11% lower, respectively, than controls). There were no other
significant indications of maternal or fetal toxicity. This study identified a NOAEL of 300 ppm
(1475 mg/m3) and a LOAEL of 600 ppm (2950 mg/m3, 6 hours/day on gestation days 6 through
20) for maternal and fetal body weight effects. However, the observed fetal toxicity was likely
secondary to maternal toxicity because the decreased fetal body weight was noted only at
exposure levels resulting in significantly depressed maternal body weight gain.
Other Studies
Limited genotoxicity data suggest that 1,2,4-trimethylbenzene is not mutagenic. 1,2,4-
trimethylbenzene produced negative results in the Ames test with Salmonella typhimurium
strains TA97a, TA98, TA100 and TA102 both in the presence and absence of rat liver S9
metabolic activation (Janik-Spiechowicz et al., 1998). 1,2,4-Trimethylbenzene was not
cytogenic in the mouse micronucleus test, but elicited a positive response in sister chromatid
exchange (SCE) tests with bone marrow cells of Imp:Balb/c mice treated in vivo (Janik-
Spiechowicz et al., 1998).
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC RfDs
FOR 1,2,4-TRIMETHYLBENZENE
Limited information is available regarding the oral toxicity of 1,2,4-trimethylbenzene in
humans and animals. The nephrotoxicity study by Borriston Laboratories (1984) is too limited
in scope to be used to identify a NOAEL or LOAEL for 1,2,4-trimethylbenzene, although the
2000 mg/kg dose (1429 mg/kg-day) is clearly a FEL for increased mortality. The study of
Maltoni et al. (1997) is also unsuitable for derivation of an RfD, as only one dose level was
employed, decreased survival occurred at this dose level and reporting of the results was
inadequate. Thus, the database for 1,2,4-trimethylbenzene is inadequate to derive a provisional
RfD.
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC RfCs
FOR 1,2,4-TRIMETHYLBENZENE
Several studies have examined the inhalation toxicity of mixtures predominantly
containing one or more trimethylbenzene isomers, or pure 1,2,4-trimethylbenzene. Significant
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increases in the incidence of CNS toxicity (vertigo, dizziness, headaches) and non-significant
increases in the incidences of respiratory effects (bronchitis) and hematological effects
(hyperchromic anemia and blood clotting alterations) have been observed in workers exposed to
10-60 ppm (49-295 mg/m3) of a solvent containing >80% trimethylbenzene isomers (Battig et
al., 1958). Many of these effects reported in humans have been observed in experimental
animals repeatedly exposed to 1,2,4-trimethylbenzene or other trimethylbenzene isomers or
trimethylbenzene mixtures. For example, hematological effects have been reported in
experimental animals exposed to a trimethylbenzene mixture (Bernshtein, 1972) or 1,2,4-
trimethylbenzene (Korsak et al., 2000). Signs of adverse CNS effects have been observed in
animals exposed to mixtures containing 1,2,4-trimethylbenzene (Battig et al., 1958; IBT, 1981)
or 1,2,4-trimethylbenzene alone (Gralewicz and Wiaderna, 2001; Gralewicz et al., 1997a, 1997b;
Korsak and Rydzynski, 1996). Results of other animal studies provide evidence of 1,2,4-
trimethylbenzene-induced respiratory effects (Korsak et al., 1997, 2000). Other effects observed
in animal studies include testicular atrophy in rats exposed to 480 mg/m3 (98 ppm) of a mixture
containing 75% 1,2,4-trimethylbenzene for 4 weeks (6 hours/day, 5 days/week) (IBT, 1981) and
lung, liver, kidney and spleen effects in rats exposed to 1700 ppm (8155 mg/m3) of a solvent
containing >80%) trimethylbenzene isomers for 4 months (8 hours/day, 5 days/week) (Battig et
al., 1958).
The lowest estimated level of occupational exposure to the solvent Fleet-X DV 99 (>80%>
1,2,4- and 1,3,5-trimethylbenzene) in the study of Battig et al. (1958) was 10 ppm (49 mg/m3).
Assuming that the solvent exclusively contained trimethylbenzene isomers, the 49 mg/m3
exposure concentration can be considered to represent a LOAEL. Although the Battig et al.
(1958) report provides the lowest inhalation LOAEL (49 mg/m3) of any study, it may be an
inappropriate study for consideration as the principal study for a number of reasons.
Importantly, Battig et al. (1958) identified spectrophotographically the presence of various
aromatic hydrocarbons, to include naphthenic and paraffenic compounds, in addition to 1,2,4-
trimethylbenzene in the solvent mixture. While 1,2,4-trimethylbenzene comprised up to 50% of
the Fleet-X DV 99 mixture, it is virtually impossible to confidently attribute human toxicities
solely to 1,2,4-trimethylbenzene (i.e. the study LOAEL is for the mixture not the individual
compound). Additional concerns that warrant exclusion of the Battig et al. (1958) human study
from consideration include inappropriate selection of a human control population [e.g. nutritional
status (Vit. C deficient)], and the fact that average Fleet-X DV 99 solvent exposure duration, for
the 27 exposed workers examined, was not reported.
An advantage of some of the animal models of 1,2,4-trimethylbenzene inhalation
exposure over the Battig et al. (1958) human study is that controlled atmospheres involved the
compound of interest at relatively high purities (e.g. 97%> 1,2,4-trimetylbenzene in the Korsak et
al., 2000 study). However, available repeated exposure inhalation studies in animals are limited
to subchronic exposure duration (4 weeks to 3 months) in which the lowest identified LOAEL
for 1,2,4-trimethylbenzene was 491 mg/m3 (Gralewicz and Wiaderna, 2001; Gralewicz et al.,
1997a; Korsak and Rydzynski, 1996); furthermore, many of the effects observed in these rodent
studies are of unclear toxicological significance and/or have concentration-responses that are
difficult to interpret.
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Provisional RfCs may be derived based on adverse pulmonary or hematological effects
reported in male or female rats, respectively, exposed to 1,2,4-trimethylbenzene (97% pure) for 3
months (Korsak et al., 2000). The selection of the Korsak et al. (2000) study as the basis for
deriving RfCs is supported by previous observations in rats (Korsak et al., 1997) and humans
(Battig et al., 1958) exposed to pure 1,2,4-trimethylbenzene or a mixture of trimethylbenzenes,
respectively, for > 90 days. Indeed, pulmonary lesions and hematological abnormalities in rats
exposed to pure 1,2,4-trimethylbenzene for 3 months (Korsak et al., 2000) are consistent with
observations in humans following presumably longer duration exposure to a mixture containing
1,2,4-trimethylbenzene (Battig et al., 1958).
Subchronic p-RfC
The subchronic p-RfC for 1,2,4-trimethylbenzene is derived from the NOAEL of 25 ppm
(123 mg/m3) identified in the Korsak et al. (2000) rat subchronic inhalation study. Two different
toxic effects (pulmonary or hematological) were identified in male or female rats, respectively,
in this study at the same LOAEL/NOAEL. As such, two separate subchronic p-RfC derivations
are presented below to identify the most sensitive endpoint. Under an assumption of category 3
for decreased clotting time in female Imp:WIST rats, an adjusted experimental NOAEL can be
derived using the NOAEL of 123 mg/m3 and the exposure duration data from Korsak et al.
(2000) as follows:
NOAEL[adj] (mg/m3) = rat NOAEL (mg/m3) x 6hr/24hr x 5 days/7 days
= 123 mg/m3 x 0.25 x 0.71
= 21.8 mg/m3
According to equation (4-48) for extrarespiratory effects [Methods for Derivation of Inhalation
Reference Concentrations and Application of Inhalation Dosimetry (EPA/600/8-90/066F
October 1994)], a human equivalent concentration (NOAEL[Hec]) can be calculated as follows:
NOAEL[hec] (mg/m3) = NOAEL[adj] (mg/m3) x (Hb/g)A/(Hb/g)H
*blood:gas (b/g) partition coefficients for 1,2,4-trimethylbenzene could not be located, therefore
a default value of 1 is used for the term (Hb/g)A/(Hb/g)H • The human NOAEL[Hec] is equivalent to
the duration adjusted rat NOAEL of 21.8 mg/m3. A subchronic p-RfC of 7E-2 mg/m3 based on
a hematological effect is derived by dividing the NOAEL[Hec] of 21.8 mg/m3 by a composite UF
of 300, as follows:
UF (animal to human) = 3
UF (interindividual variability) =10
UF (database deficiencies) =10
Subchronic p-RfC = NOAEL[Hec] / UF
= 21.8 mg/m3/ 300
= 0.07 mg/m3 or 7E-2 mg/m3
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Under an assumption of category 1 for pulmonary toxicity in male rats, the same duration
adjusted ratNOAEL of 21.8 mg/m3 is obtained as shown above. Histopathological observations
in lung tissue of male Imp:WIST rats exposed to 1,2,4-trimethylbenzene for 3 months indicated
inflammatory lesions primarily in the bronchiolar region. Therefore, according to equation 4-22
for Category 1 tracheobronchial effects [Methods for Derivation of Inhalation Reference
Concentrations and Application of Inhalation Dosimetry (EPA/600/8-90/066F October 1994)], a
NOAEL[hec] can be calculated as follows:
NOAEL[Hec] (mg/m3) = NOAEL[ADJ] (mg/m3) x RGDRn»:
= 21.8 mg/m3 x 1.6
= 34.9 mg/m3
^ Derivation of the RGDRTB can be found in Appendix 1
A subchronic p-RfC of 1E-1 mg/m3 based on pulmonary effects is derived by dividing the
NOAEL[hec] of 34.9 mg/m3 by the same composite UF of 300 identified above:
Subchronic p-RfC = NOAEL[Hec] / UF
= 34.9 mg/m3 / 300
= 0.1 mg/m3 or 1E-1 mg/m3
The composite UF includes a factor of 3 for extrapolation from animal to human, 10 for
interindividual variability, and 10 for database deficiencies. The reduced uncertainty of 3 for
animal to human extrapolation is due in part to both the conversion of the rat NOAEL to a
human equivalent concentration as well as the consistency of hematological and pulmonary
toxicity between rats and humans exposed to 1,2,4-trimethylbenzene. The database deficiencies
include lack of developmental toxicity studies in a second species, multigeneration reproductive
toxicity studies, and a lack of confidence in the large majority of available animal studies
reporting effects of undetermined toxicological significance with concentration-responses that
are difficult to interpret. The derivations shown above clearly indicate that decreased clotting
time in female rats due to subchronic inhalation exposure to 1,2,4-trimethlybenzene is the more
sensitive or health protective endpoint under consideration.
Chronic p-RfC
The chronic p-RfC of 7E-3 mg/m3 based on decreased clotting time in female rats
(Korsak et al., 2000) is derived by dividing the NOAEL[Hec] of 21.8 mg/m3 by a composite UF
of 3000, as follows:
Chronic p-RfC = NOAEL[Hec] / UF
= 21.8 mg/m3 /3000
= 0.007 or 7E-3 mg/m3
As for the chronic RfC, the composite UF includes a factor of 10 for extrapolation from
subchronic to chronic exposure, 3 for extrapolation from animal to human, 10 for interindividual
variability, and 10 for database deficiencies.
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Confidence in the principal study (Korsak et al., 2000) is low. While it is remarkable that
hematological and pulmonary effects are apparently conserved from rats (Korsak et al., 2000) to
humans (Battig et al., 1958), the concentration-response for either compartment in rats
(particularly male rats) is difficult to interpret. Specifically, the low inhalation concentration
(123 mg/m3) in female rats from the Korsak et al. (2000) study was clearly a NOAEL for
decreased clotting time (hematological compartment); this NOAEL was also identified for
pulmonary effects (e.g. proliferation of peribronchial lymphatic tissue, interstitial lymphocytic
infiltration of parenchyma, bronchitis and bronchopneumonia) in male rats. Interestingly, female
rats seemed slightly more resistant to these pulmonary effects. However, the overall
commutative score, following statistical trend analysis, of all pulmonary lesions suggested that
the lungs of male and female Imp:WIST rats are significantly affected by inhalation exposure to
1,2,4-trimethylbenzene at the mid dose of 492 mg/m3 (Korsak et al., 2000). However,
paradoxically, in male rats of the high dose group the pulmonary effects decreased compared to
animals in the mid dose group. This counter-intuitive concentration-response relationship might
suggest a concentration-dependent transition in mode of action for pulmonary toxicity (note the
increase in absolute lung weight of male Imp:WIST rats at the mid concentration of 492 mg/m3,
which is the concentration at which inflammatory foci were identified in lung tissue, and yet in
the high concentration group lung weight decreased back to control levels (Korsak et al., 2000);
more work would be required to verify.).
According to the derivations provided above it appears that the hematological endpoint
(i.e. decreased clotting time in female rats) may be a more appropriate endpoint to consider for
inhalation exposure to 1,2,4-trimethylbenzene. Further work in this area is certainly warranted.
The human occupational report from Battig et al. (1958) identified a lower inhalation effect level
(e.g. LOAEL = 49 mg/m3) compared to any of the available animal data. However, the utility of
this study in derivation of RfCs is limited by poor reporting of results, undetermined exposure
levels, the lack of statistical analysis of results, the lack of information on the exposed and
control groups (e.g., age, education level, length of employment), small group sizes and possibly
a poorly matched control group (as evidenced by increased incidence of vitamin C deficiency in
controls). Also, the controls worked in adjacent rooms and the possibility that they also may
have been exposed to trimethylbenzene cannot be excluded. Confidence in the database is low
because the database is lacking developmental toxicity data in a second species and reproductive
toxicity studies. Reflecting low confidence in the principal study and database, confidence in the
provisional subchronic and chronic RfC values is low.
PROVISIONAL CARCINOGENICITY ASSESSMENT FOR
1,2,4-TRIMETHYLBENZENE
Weight-of-evidence Classification
No information was located regarding the carcinogenicity of 1,2,4-trimethylbenzene or
mixtures of trimethylbenzene isomers in humans. The database of information regarding the
carcinogenicity of trimethylbenzene in animals is limited to a single carcinogenicity study in
which male and female Sprague-Dawley rats (50/sex/group) were administered 1,2,4-
trimethylbenzene via oral gavage at doses of 0 or 800 mg/kg for 104 weeks (Maltoni et al.,
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1997). Although quantitative survival data were not included in the study report, the authors
noted "intermediate" and "slight" reduction in the survival of 1,2,4-trimethylbenzene treated
male and female rats, respectively. Under the conditions of the study, oral exposure to 1,2,4-
trimethylbenzene did not cause a statistically significant increase in the incidence of animals
bearing either malignant tumors or benign and malignant tumors (combined) or in the incidence
of neuroesthesioepitheliomas. The study of Maltoni et al. (1997) included a single animal
species (rat) and a single 1,2,4-trimethylbenzene dose level (800 mg/kg). Based on limitations in
study design and reporting of results and the lack of additional carcinogenicity data in animals,
the database of information for 1,2,4-trimethylbenzene is inadequate to establish the potential
carcinogenicity of 1,2,4-trimethylbenzene. Limited genotoxicity data demonstrated that 1,2,4-
trimethylbenzene was not mutagenic in several strains of Salmonella typhimurium, and did not
elicit cytogenicity in the mouse micronucleus test, but did elicit a positive response in sister
chromatid exchange (SCE) tests with bone marrow cells of Imp:Balb/c mice treated in vivo
(Janik-Spiechowicz et al., 1998). These data provide inadequate evidence for genotoxic activity.
Collectively, the available carcinogenicity and genotoxicity data do not adequately assess
the carcinogenic potential of 1,2,4-trimethylbenzene in humans or animals. Under the current
U.S. EPA (2005) cancer guidelines, the human and animal data are inadequate for a
determination of the human carcinogenic potential of 1,2,4-trimethylbenzene.
Quantitative Estimates of Carcinogenic Risk
There are no appropriate human or animal data from which to derive an oral slope factor
or inhalation unit risk for 1,2,4-trimethylbenzene.
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major gasoline aromatics on rats. Ann. New York Acad. Sci. 837:15-52.
Monsanto. 1992. Cover letter submitted to EPA's Office of Toxic Substances with the IBT
(1981) study.
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NIOSH (National Institute for Occupational Safety and Health). 2006. Online NIOSH Pocket
Guide to Chemical Hazards. Available at http://www.cdc.gov/niosh/npg
NTP (National Toxicology Program). 2006. Management Status Report. Available at
http://ntp-server.niehs.nih.gov/
OSHA (Occupational Safety and Health Administration). 2006. OSHA Standard 1910.1000
Table Z-l. Part Z, Toxic and Hazardous Substances. Available at
http://www.osha.gov/pls/oshaweb/owadisp.show docuinent'.'p table=STANDARDS&p id=9992
Saillenfait, A., F. Gallissot, J. Sabate and G. Morel. 2005. Developmental toxicity of two
trimethylbenzene isomers, mesitylene and pseudocumene, in rats following inhalation exposure.
Food Chem. Toxicol. 43:1055-1063.
Sandmeyer, E.E. 1981. Aliphatic hydrocarbons. In: Patty's Industrial Hygiene and Toxicology.
Vol.3. G.D. Clayton and R.E. Clayton, Eds. John Wiley and Sons, Inc., New York. pp. 3300-
3302.
U.S. EPA. 1987a. Health Effects Assessment for Trimethylbenzenes. Prepared by the Office of
Health and Environmental Assessment, Environmental Criteria and Assessment Office,
Cincinnati, OH for the Office of Solid Waste and Emergency Response, Washington, DC.
U.S. EPA. 1987b. Drinking Water Health Advisory for 1,2,4-Trimethylbenzene. Prepared by
the Environmental Criteria and Assessment Office, Office of Health and Environmental
Assessment, Cincinnati, OH for the Office of Drinking Water, Washington, DC.
U.S. EPA. 1991. Chemical Assessments and Related Activities (CARA). Office of Health and
Environmental Assessment, Washington, DC. April.
U.S. EPA. 1994a. Chemical Assessments and Related Activities (CARA). Office of Health and
Environmental Assessment, Washington, DC. December.
U.S. EPA. 1994b. Methods for Derivation of Inhalation Reference Concentrations and
Application of Inhalation Dosimetry. Prepared by the Office of Health and Environmental
Assessment, Research Triangle Park, NC. EPA/600/8-90/066F.
U.S. EPA. 1997. Health Effects Assessment Summary Tables. Annual Update. FY-1997.
Office of Research and Development, Office of Emergency and Remedial Response,
Washington, DC. July 1997. EPA/540/R-97/036. NTIS PB97-921199.
U.S. EPA. 2004. 2004 Edition of the Drinking Water Standards and Health Advisories. Office
of Water, Washington, DC. EPA/822/R-02/038. Available at
http://www.epa.gov/waterscience/drinking/standards/dwstandards.pdf
U.S. EPA. 2005. Guidelines for Carcinogen Risk Assessment. Risk Assessment Forum,
Washington, DC. EPA/630/P-03/00 IB. Available at www .epa.gov/cancerguidelines
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U.S. EPA. 2007. Integrated Risk Information System (IRIS). Online. Office of Research and
Development, National Center for Environmental Assessment, Washington, DC.
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WHO (World Health Organization). 2006. Online Catalogs for the Environmental Criteria
Series. Available at http://www.inchem.org/pages/ehc.html
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APPENDIX 1
According to equation 4-22 for Category 1 tracheobronchial effects [Methods for Derivation of
Inhalation Reference Concentrations and Application of Inhalation Dosimetry (EPA/600/8-
90/066F October 1994)], a NOAEL[Hec] is calculated using the duration adjusted animal NOAEL
and a dosimetric adjustment factor (DAF). In this case the DAF is the RGDR for the
tracheobronchial region of the lung (RGDRTB). The RGDRTB is calculated as follows:
RGDRtb = (RGDTb)a/(RGDTb)h = (Ve/SAtb)a (e SAet/Ve)a
(Ve/SATb)h (e SAet/Ve)h
where,
Rat
VE = 160.07 ml/min or 0.16 L/min (derived using equation 4-4, default body wt. for Wistar
rats, and the default intercept and coefficient values
provided in Table 4-6)
SAtb = 22.5 cm2 (Table 4-4)
SAet= 15.0 cm2 (Table 4-4)
Human
Ve = 13.8 L/min (default value based on human body weight of 70 kg)
SAtb = 3,200 cm2 (Table 4-4)
SAet = 200.0 cm2 (Table 4-4)
= (0.16 L/min/ 22.5 cm2)A (e -i^^/o.ieL/mm^ *
(13.8 L/min / 3,200 cm2)H (e "2000cm2/13 8L/min)H
* the exponential portion of the equation is much smaller than 1; thus this half of the equation is
negligible.
= 0.007/0.0043
RGDRtb = 16
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