vvEPA
EPA/635/R-16/161FC
www.epa.gov/iris
Toxicological Review of Trimethylbenzenes:
Executive Summary
[CASRNs 25551-13-7, 95-63-6, 526-73-8, and 108-67-8]
September 2016
Integrated Risk Information System
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC

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EXECUTIVE SUMMARY
Occurrence and Health Effects
Trimethylbenzenes (TMBs) are a commercially available mixture of three
individual isomers: 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB. TMB isomers are
produced during petroleum refining and production of aromatic hydrocarbons with
nine carbons (i.e., C9 aromatic fraction). As the vast majority of the C9 fraction is used
as a component of gasoline, vehicle emissions are expected to be the major
anthropogenic source of TMBs. TMBs are volatile hydrocarbons, and humans are
thus exposed to these isomers primarily through breathing air containing TMB
vapors, although ingestion through food or drinking water is also possible.
Effects on the nervous, respiratory, and hematological (i.e., blood) systems
have been reported in occupationally- and residentially-exposed humans, but these
effects were observed following exposure to complex mixtures containing TMB
isomers, thus making it difficult to determine the contribution of each TMB isomer to
the observed health effects. Health effects that are roughly analogous to those seen
in humans have been observed in animals exposed to the individual isomers. Effects
on the nervous system, including cognitive effects and decreased pain sensitivity, are
the most widely observed effects in animals. Effects on other systems, including the
respiratory and hematological systems, have also been observed in animals. Both
1,2,4-TMB and 1,3,5-TMB have been observed to elicit effects on pregnant animals
and developing fetuses, but at exposure levels greater than those that cause effects
on the nervous system. There is inadequate information to evaluate the
carcinogenicity of TMBs.
Effects Other Than Cancer Following Inhalation Exposure
The relationship between exposure to 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB and health
effects has been evaluated in studies of (1) exposed human adults, (2) animals exposed via
inhalation for acute, short-term, and subchronic durations, and (3) animals exposed gestationally
via inhalation.
Human studies included occupational exposure to various solvent mixtures containing
TMBs. Health effects noted in these studies were eye irritation, neurological effects (hand tremble,
abnormal fatigue, lack of coordination), and hematological effects. Residential exposure to
mixtures containing 1,2,4-TMB were observed to be associated with asthma. However, as these
studies involved exposures to mixtures containing multiple TMB isomers and other volatile organic
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compounds (VOCs), it is difficult to ascertain the specific contribution of each TMB isomer to the
specific health effects reported. Studies involving controlled exposures of healthy adult volunteers
to individual isomers also exist, although these studies generally reported little or no sensory
irritation or effects on the respiratory system. One controlled human exposure study reported
some deficits in attention following exposure to white spirit, a complex mixture containing
1,2,4-TMB.
Animal inhalation studies included acute and short-term studies of TMBs that reported
respiratory irritation (decreased respiration rates) and neurological effects (decreased pain
sensitivity, altered cognitive function, and decreased anxiety and/or increased motor function) that
are consistent with effects seen in human studies. Four subchronic inhalation studies for 1,2,3-TMB
and 1,2,4-TMB observed exposure-response effects in multiple systems, including the nervous,
hematological, and respiratory systems. In these studies, disturbances in central nervous system
(CNS) function, including decreased pain sensitivity and decreased neuromuscular function and
coordination, appear to be the most sensitive endpoints following exposure to 1,2,3-TMB or
1,2,4-TMB. No subchronic studies were found that investigated exposure to 1,3,5-TMB. One
developmental toxicity study observed maternal and fetal toxicity (i.e., decreased maternal weight
gain and fetal weight) following exposure to either 1,2,4-TMB or 1,3,5-TMB; other indices of fetal
toxicity (i.e., fetal death and malformations) were not affected by exposure.
Inhalation Reference Concentration (RfC) for TMBs for Effects Other Than Cancer
The RfC for TMBs was derived using benchmark dose (BMD) modeling coupled with
physiologically-based pharmacokinetic (PBPK) modeling or default dosimetric methods. BMD
modeling was conducted using external exposure concentrations as the dose inputs and either a
benchmark response (BMR) level of 5% change (fetal weight) or 1 standard deviation (SD) of the
control mean (all other endpoints). Once a lower confidence limit on the benchmark dose (BMDL)
(or a no-observed-adverse-effect level [NOAEL] or lowest-observed-adverse-effect level [LOAEL] in
cases where no models fit the data) was identified as the point of departure (POD), a human
equivalent concentration (HEC) was calculated for each endpoint using either a PBPK model
(1,2,4-TMB) or default dosimetric adjustments (1,2,3-TMB and 1,3,5-TMB).
To each HEC, a composite uncertainty factor (UF) was applied to account for uncertainties
in the TMB database:
•	3 to account for uncertainty in extrapolating from laboratory animals to humans
(interspecies variability),
•	10 to account for variation in susceptibility among members of the human
population (interindividual variability),
•	3 to account for subchronic-to-chronic extrapolation due to the use of a subchronic
study, and
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• 3 to account for deficiencies in the database (no TMB-specific developmental
neurotoxicity studies were available).
Full details of the selection and application of the UFs are available in Section 2.1.3. Dividing the
candidate HECs by this composite UF of 300 yielded the organ/system-specific RfCs presented in
Table ES-1.
Table ES-1. Organ/system-specific chronic RfCs for individual TMB isomers
Effect
Isomer
Basis
RfC
(mg/m3)
Composite
UF
Study Exposure
Duration
Confidence
Neurological
Korsak and Rvdzvriski
(1996)
1,2,4-TMB
Decreased pain
sensitivity
6 x 10"2
300
Subchronic
Low to medium
1,2,3-TMB
5 x 10"2
300
Subchronic
Low to medium
Hematological
Korsak et al. (2000a),
Korsak et al. (2000b)
1,2,4-TMB
Decreased
clotting time
8 x 10"2
300
Subchronic
Low to medium
1,2,3-TMB
Decreased
segmented
neutrophils
6 x 10"2
300
Subchronic
Low to medium
Respiratory
Korsak et al. (2000a),
Korsak et al. (2000b)
1,2,4-TMB
Inflammatory
lung lesions
2 x 10"1
300
Subchronic
Low to medium
1,2,3-TMB
2 x 10"1
300
Subchronic
Low to medium
Developmental3
Saillenfait et al.
(2005)
1,2,4-TMB
Decreased fetal
weight
4
100
Gestational
Low to medium
1,3,5-TMB
4
100
Gestational
Low to medium
Maternal3
Saillenfait et al.
(2005)
1,2,4-TMB
Decreased
maternal weight
3
300
Subchronic
Low to medium
1,3,5-TMB
4 x 10"1
300
Subchronic
Low to medium
Chronic Overall RfC
(Neurological)
All TMB
isomers
Decreased pain
sensitivity
6 x 10"2
300
Subchronic
Low to medium
a Intended to be used for gestational exposures
Neurotoxicity is the most consistently observed endpoint in the toxicological database for
TMBs, and decreased pain sensitivity was observed in multiple studies following exposures to
1,2,3- or 1,2,4-TMB for short-term or subchronic durations. Given the consistency of this effect and
the determination that decreased pain sensitivity is an appropriate adverse effect with which to
derive reference values (see Section 2.1.5), in accordance with the EPA's Guidelines for Neurotoxicity
Risk Assessment fU.S. EPA. 19981. decreased pain sensitivity was selected as the critical effect and
Korsak and Rydzvriski f 19961 was selected as the principal study for derivation of the RfC for TMBs.
No subchronic study was available that investigated neurotoxicity endpoints following exposure to
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1,3,5-TMB, resulting in the lack of an isomer-specific neurotoxicity RfC for this isomer. However, as
discussed in Section 1.2.7, the available toxicological database for all three isomers, across all
exposure durations, indicates there are important similarities in the isomers' neurotoxicity that are
supportive of an RfC for 1,3,5-TMB that is not substantially different than the RfC derived for other
TMB isomers. Also supporting this conclusion is the observation thatTMB isomers display
important similarities with regard to chemical properties and toxicokinetics, including similarities
in blood:air partition coefficients, respiratory uptake, and absorption into the bloodstream (see
Section 1.2.7 and Appendices C.l and C.2). These similarities support the conclusion that an RfC for
1,3,5-TMB would be similar to those calculated for 1,2,3- or 1,2,4-TMB. The RfC for 1,2,4-TMB was
selected over the RfC for 1,2,3-TMB as the RfC for the entire TMB database due to increased
confidence in that it was calculated via the application of a validated PBPK model, whereas the
1.2.3-TMB	value was estimated using default dosimetric methods. Therefore, the chronic RfC for
all TMBs was set at 6 x 10"2 mg/m3 based on neurological effects following exposure to
1.2.4-TMB.	However, this overall RfC for TMBs can be used for any TMB isomer alone, or in
situations when individuals are exposed to a mixture of TMB isomers. The individual organ- or
system-specific RfCs may be useful for subsequent cumulative risk assessments that consider the
combined effect of multiple agents acting at a common site.
In addition to providing an RfC for chronic exposures in multiple systems, this document
also provides an RfC for subchronic-duration exposures. In the case of TMBs, all of the studies used
to calculate the chronic RfCs were subchronic or gestational in duration. Therefore, the methods to
calculate subchronic RfCs are identical to those used for calculation of chronic RfCs, minus the
application of a subchronic-to-chronic UF (UFs) (see Table ES-1). It should be noted that the
subchronic RfC values for the developing fetus are identical to the chronic RfC values for the
developing fetus as gestation represents a critical window of susceptibility and no UFs was applied
to account for less-than-chronic exposure in either case. The subchronic inhalation RfC is intended
for use with exposures for more than 30 days, up to approximately 10% of the lifespan in humans.
The subchronic RfC for TMBs was set to 2 x 10"1 mg/m3 based on neurological effects
following exposure to 1,2,4-TMB.
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Table ES-2. Organ/system-specific subchronic RfCs for individual TMB
isomers
Effect
Isomer
Basis
RfC
(mg/m3)
Composite
UF
Study Exposure
Duration
Confidence
Neurological
Korsak and
Rvdzvriski (1996)
1,2,4-TMB
Decreased pain
sensitivity
2 x 10"1
100
Subchronic
Low to medium
1,2,3-TMB
2 x 10"1
100
Subchronic
Low to medium
Hematological
Korsak et al.
(2000a), Korsak et
al. (2000b)
1,2,4-TMB
Decreased
clotting time
2 x 10"1
100
Subchronic
Low to medium
1,2,3-TMB
Decreased
segmented
neutrophils
2 x 10"1
100
Subchronic
Low to medium
Respiratory
Korsak et al.
(2000a), Korsak et
al. (2000b)
1,2,4-TMB
Inflammatory
lung lesions
6 x 10"1
100
Subchronic
Low to medium
1,2,3-TMB
6 x 10"1
100
Subchronic
Low to medium
Developmental3
Saillenfait et al.
(2005)
1,2,4-TMB
Fetal weight
4
100
Gestational
Low to medium
1,3,5-TMB
4
100
Gestational
Low to medium
Maternal3
Saillenfait et al.
(2005)
1,2,4-TMB
Decreased
maternal weight
8
100
Subchronic
Low to medium
1,3,5-TMB
1
100
Subchronic
Low to medium
Subchronic
Overall RfC
(Neurological)
All TMB
isomers
Decreased pain
sensitivity
2 x 101
100
Subchronic
Low to medium
a Intended to be used for gestational exposures
Confidence in the Chronic Inhalation RfC for 1,2,4-TMB
A confidence level of high, medium, or low is assigned to the study used to derive the RfC,
the overall database, and the RfC itself, as described in Section 4.3.9.2 of EPA's Methods for
Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA.
19941.
Confidence in the study from which the critical effect was identified is low to medium. The
study is a peer-reviewed study that utilized three dose groups plus untreated controls, employed an
appropriate number of animals per dose group, and performed appropriate statistical analyses.
However, sources of uncertainty exist that reduce confidence in this study.
One area of uncertainty regarding this study is the lack of reported actual concentrations.
However, as the methods by which the test atmosphere was generated and analyzed were reported
in sufficient detail, and given the fact that this laboratory has used this methodology in subsequent
studies and achieved appropriate actual concentrations (i.e., within 10% of target concentrations),
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the concern regarding the lack of reported actual concentrations is reduced. Another source of
uncertainty is the fact that the principal study does not explicitly state that the reported measures
of variance in Table 1 of that reference are SDs. However, careful analysis of the reported levels of
variance and magnitude of statistical significance indicate that the measures of variance are SDs.
Supporting this conclusion is the observation that all other papers from this laboratory report
variance as SDs. The critical effect on which the RfC is based is well-supported as the weight of
evidence for TMB-induced neurotoxicity is coherent across species (i.e., human, mouse, and rat),
coherent across isomers, and consistent across multiple exposure durations (i.e., acute, short-term,
and subchronic).
The database for TMBs includes acute, short-term, subchronic, and developmental toxicity
studies in rats and mice. However, confidence in the overall database is low to medium because it
lacks chronic and developmental neurotoxicity studies, and the studies supporting the critical effect
predominantly come from the same research institute. The overall confidence in the RfC for TMBs
is low to medium.
Effects Other Than Cancer Observed Following Oral Exposure
Only one subchronic study was identified that examined the effects of oral exposure to
1,3,5-TMB. Effects in the hematological system, including changes in clinical chemistry parameters
and differential white blood cell (WBC) numbers, were observed following exposure to 1,3,5-TMB
via gavage in rats. Altered organ weights were also observed in multiple systems (kidneys, liver).
The alterations to clinical chemistry parameters and organ weights were observed in the absence of
histopathological changes in relevant systems, and were thus considered to be compensatory in
nature. Discounting effects that could be non-adverse or compensatory in nature left an observed
increase in monocytes in male rats as the only statistically significant effect on which to base the
reference dose (RfD) derivation. While a slight increase in monocytes may be of questionable
adversity if taken with no context of the larger TMB database, a number of endpoints involving the
alteration of WBC counts have been observed in the inhalation toxicity database. It was therefore
deemed that the observed increase in monocytes following oral exposures was possibly indicative
of an underlying toxicity to the hematological system also evident following inhalation exposure.
Oral Reference Dose (RfD) for TMBs for Effects Other Than Cancer
The RfD for TMBs was derived using BMD modeling coupled with default dosimetric
methods. BMD modeling was conducted using external exposure concentrations as the dose inputs
and a BMR level of 1 SD of the control mean. Once a BMDL was identified as the POD, a human
equivalent dose (HED) of 3.0 mg/kg-day was calculated for increased monocytes using default
dosimetric adjustments (i.e., body weight to the % power).
To the estimated HED, a composite UF was applied to account for uncertainties in the TMB
database: 3 to account for uncertainty in extrapolating from laboratory animals to humans
(interspecies variability), 10 to account for variation in susceptibility among members of the
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human population (interindividual variability), 3 to account for subchronic-to-chronic
extrapolation due to the use of a subchronic study, and 3 to account for deficiencies in the database
(no TMB-specific developmental neurotoxicity studies were available). Full details of the selection
and application of the UFs are available in Section 2.2.3. Dividing the HED by this composite UF
of 300 yielded an RfD of 1 x 10-2 mg/kg-day that can be applied to any TMB isomer
individually or to mixtures of TMB isomers.
In addition to the RfD calculated for TMBs from oral data, an RfD was calculated from
inhalation data using a route-to-route extrapolation to address the lack of suitable neurotoxicity
data in the oral TMB database. It is clear from the inhalation database for TMB that neurotoxicity is
an important endpoint for derivation of reference values, especially given the consistency with
which neurotoxicity is observed in the TMB database, across all isomers following acute oral and
acute, short-term, and subchronic inhalation exposures. Ultimately, the fact that oral and inhalation
neurotoxic endpoints are comparable, and that neurotoxic endpoints resulted in the most strongly
supported RfCs in the inhalation database, it is reasonable to expect that neurotoxicity-based PODs
would be critical for deriving RfDs. The available database for 1,2,4-TMB supports the use of route-
to-route extrapolation; sufficient evidence exists that demonstrates similar qualitative profiles of
metabolism (i.e., observation of dimethylbenzoic and hippuric acid metabolites) and patterns of
parent compound distribution across exposure routes (Section C.2, Appendix C).
Therefore, assuming that oral exposure would result in the same systemic effect as
inhalation exposure (i.e., altered CNS function, measured as decreased pain sensitivity), an oral
exposure component was added to the PBPK model by EPA (Section C.3.3.5, Appendix C), assuming
100% absorption of the ingested 1,2,4-TMB by constant infusion of the oral dose into the liver and
an idealized pattern of six ingestion events (see Section 2.2.3). Using the modified PBPK model
resulted in an HED of 3.5 mg/kg-day, which was divided by the composite UF of 300 to
estimate an RfD of 1 x 10-2 mg/kg-day. Although identical to the RfD calculated from the oral
1,3,5-TMB data for increased monocytes, this value of 1 x 10~2 mg/kg-day was ultimately selected
as the RfD for TMB isomers based on multiple lines of evidence in the oral and inhalation database,
including commonalities in the pattern of neurotoxic effects observed following oral and inhalation
exposures, similarities in blood:air and tissue:air partition coefficients and absorption into the
bloodstream between TMB isomers, and qualitative metabolic profiles that suggest that first-pass
metabolism through the liver is not expected to differ greatly between the three isomers.
In addition to providing RfDs for effects in the hematological and nervous systems, this
document also provides values for subchronic RfD values for exposures that may be of concern in a
less-than-lifetime context In the case of TMBs, the oral 1,3,5-TMB study and the inhalation
1,2,4-TMB study used for the route-to-route extrapolation for the calculation of the chronic RfDs
were both of subchronic duration. Therefore, the methods used to calculate subchronic RfDs is
identical to that used for calculation of chronic RfCs, minus the application of a UFs. This results in a
composite UF of 100 (interspecies UF [UFa] of 3, intraspecies UF [UFh] of 10, UFs of 1, and database
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UF [UFd ] of 3). Dividing the POD for hematological effects (3.01 mg/kg-day) and neurotoxicity
effects (3.5 mg/kg-day) by the composite UF of 100 results in RfDs of 3 x 10~2 and
4 x 10"2 mg/kg-day for decreased monocytes and decreased pain sensitivity, respectively. The
subchronic RfD was set to 4 x 10-2 mg/kg-day based on neurological effects following
exposure to 1,2,4-TMB. The subchronic oral RfD is intended for use with exposures for more than
30 days, up to approximately 10% of the lifespan in humans.
Confidence in the Chronic Oral RfD for 1,2,4-TMB
The confidence in the oral database for TMB is low because it only contains acute oral
studies investigating neurotoxicity endpoints for multiple isomers, and one subchronic study
investigating general toxicity endpoints for one isomer (1,3,5-TMB). This database was used to
derive an RfD, but given the concern over the lack of a suitable neurotoxicity study, the confidence
in this RfD is low. A PBPK model was utilized to perform a route-to-route extrapolation to
determine a POD for the derivation of the RfD from inhalation data. The confidence in the study
from which the critical effect was identified is low to medium (see Section 2.1.7). The inhalation
database for 1,2,4-TMB includes acute, short-term, subchronic, and developmental toxicity studies
in rats and mice. However, confidence in the overall database for TMB is low to medium because it
lacks chronic and developmental neurotoxicity studies, and the studies supporting the critical effect
predominantly come from the same research institute. Reflecting the confidence in the study and
the database and the uncertainty surrounding the application of the available PBPK model for the
purposes of a route-to-route extrapolation, the overall confidence in the RfD for TMB is low.
Evidence of Carcinogenicity
Under EPA's Guidelines for Carcinogen Risk Assessment fU.S. EPA. 20051. there is "inadequate
information to assess carcinogenic potential" of TMBs. No chronic inhalation studies that
investigated cancer outcomes were identified in the literature for 1,2,3-TMB, 1,2,4-TMB, or
1,3,5-TMB. One cancer study in which rats were exposed to 1,2,4-TMB via gavage at one
experimental dose of 800 mg/kg-day reported marginal increases in total malignant tumors and
head tumors (e.g., neuroesthesioepitheliomas), but provided no statistical analyses of the results. A
number of methodological issues limit the utility of this study (e.g., only one dose group and no
discussion of histopathological analyses). Therefore, a quantitative cancer assessment for TMBs
was not conducted.
Susceptible Populations and Lifestages
No TMB-specific data that would allow for the identification of populations or lifestages
with increased susceptibility to TMB exposure exist. However, some data gleaned from the related
compound, toluene, do provide some suggestive evidence that periods in early life represent
periods of susceptibility to solvent exposure. Therefore, it can be reasonably assumed that
exposures in early life to individual TMB isomers are of particular concern.
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