2 EPA/635/R-11/012C
3 www.epa.gov/iris
4
5
6
7
8
9
10
11
12 Toxicological review of 1,2,4- and 1,3,5-Trimethylbenzene
13
14 (CAS No. 95-63-6 and 108-67-8)
15
16 In Support of Summary Information on the
17 Integrated Risk Information System (IRIS)
18
19
20 JANUARY 2012
21
22
23
24 NOTICE
25
26 This document is an Interagency Science Consultation draft. This information is distributed
27 solely for the purpose of pre-dissemination peer review under applicable information quality
28 guidelines. It has not been formally disseminated by EPA. It does not represent and should not
29 be construed to represent any Agency determination or policy. It is being circulated for review
30 of its technical accuracy and science policy implications.
31
32
33
34
35
36 National Center for Environmental Assessment
37 Office of Research and Development
38 U.S. Environmental Protection Agency
39 Washington, DC
40
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2 DISCLAIMER
o
3
4
5 This document is a preliminary draft for review purposes only. This information is
6 distributed solely for the purpose of pre-dissemination peer review under applicable
7 information quality guidelines. It has not been formally disseminated by EPA. It does not
8 represent and should not be construed to represent any Agency determination or policy.
9 Mention of trade names or commercial products does not constitute endorsement of
10 recommendation for use.
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2
3 CONTENTS
4
5 CONTENTS 4
6 LIST OF TABLES AND FIGURES 5
7 ABBREVIATIONS AND ACRONYMS 6
8 PREAMBLE 8
9 AUTHORS | CONTRIBUTORS | REVIEWERS 18
10 PREFACE 20
11 EXECUTIVE SUMMARY 24
12 LITERATURE SEARCH STRATEGY AND STUDY EVALUATION 31
13 1.1. Synthesis of Major Toxicological Effects 34
14 1.2. Selection of Candidate Principal Studies and Critical Effects for Derivation of Reference
15 Values 61
16 1.3. Carcinogenicity Analysis 68
17 2. DOSE-RESPONSE ANALYSIS 69
18 2.1. Inhalation Reference Concentration for Effects other than Cancer 69
19 2.2. Oral Reference Dose for Effects other than Cancer 86
20 2.3. Cancer Assessment 89
21 3. REFERENCES 91
22 APPENDICES 97
23 Appendix A: Toxicological Information in Support of Hazard Identification and Dose-Response
24 Analysis for 1,2,4- and 1,3,5-Trimethylbenzene 97
25 Appendix B: Benchmark Dose Modeling Results for the Derivation of Reference Values for
26 1,2,4- and 1,3,5-Trimethylbenzene 97
27 Appendix C: Summary of External Peer Review and Public Comments and Disposition 97
28
29
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1
2 LIST OF TABLES AND FIGURES
3 Table I. Other Agency and International Assessments 22
4 Table II. Reference concentration (RfC) for 1,2,4-TMB 25
5 Table III. Reference dose (RfD) for 1,2,4-TMB 28
6 Table IV: Details of the search strategy employed for 1,2,4-TMB and 1,3,5-TMB 31
7 Table 1-1: Summary of observed in vivo neurotoxicity in subchronic and short-term studies
8 of male Wistar rats following inhalation exposure to 1,2,4-TMB or 1,3,5-TMB 40
9 Tables 1-2: Summary of observed in vivo neurotoxicity for 1,2,4-TMB and 1,3,5-TMB — oral
10 exposures 43
11 Tables 1-3: Summary of observed in vivo respiratory toxicity for 1,2,4-TMB and 1,3,5-TMB —
12 inhalation exposures 50
13 Table 1-4: Summary of observed developmental toxicity for 1,2,4-TMB and 1,3,5-TMB —
14 inhalation exposures 54
15 Tables 1-5: Summary of observed in vivo hematological toxicity and clinical chemistry
16 effects for 1,2,4-TMB and 1,3,5-TMB — inhalation exposures 56
17 Table 1-6: Non-cancer endpoints resulting from subchronic inhalation exposure to
18 1,2,4-TMB considered for the derivation of the RfC 62
19 Figure 1-1. Exposure response array for inhalation exposure to 1,2,4-TMB 64
20 Table 1-7: Non-cancer endpoints resulting from gestational inhalation exposure (GD 6-20) to
21 1,3,5-TMB considered for the derivation of the RfC 66
22 Figure 1-2. Exposure response array for inhalation exposure to 1,3,5-TMB 67
23 Table 2-1: Summary of dose-response analysis and point of departure estimation for
24 endpoints resulting from subchronic inhalation exposure to 1,2,4-TMB 70
25 Table 2-2: Candidate PODADj values, human equivalent concentrations (HECs), and applied
26 uncertainty factors used in the derivation of RfCs for 1,2,4-TMB 71
27 Figure 2-1: Array of candidate PODHEc values with applied UFs and candidate RfCs for CNS,
28 hematological, and pulmonary effects resulting from inhalation exposure to 1,2,4-TMB 75
29 Table 2-4: Duration adjusted point of departure (PODADj) estimates from short-term and
30 gestational inhalation exposures to 1,3,5-TMB 78
31 Table 2-5: Candidate PODADj values, human equivalent concentrations (HECs), and applied
32 uncertainty factors used in the derivation of RfCs for 1,3,5-TMB 80
33
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ABBREVIATIONS AND ACRONYMS
1,2,4-TMB 1,2,4-trimethylbenzene n
1,3,5-TMB 1,3,5-trimethylbenzene NCEA
AAQC Ambient air quality criterion
ACGIH American Conference of NIOSH
Governmental Industrial Hygienists
ADME Absorption, distribution, metabolism NLM
and excretion NOAEL
AEGL Acute exposure guideline limit OMOE
AIC Akaike Information Criterion OSHA
BAL bronchoalveolar lavage
BMD benchmark dose p
BMDL lower confidence limit on the p-RfC
benchmark dose PBPK
BMDS benchmark dose software
BMR benchmark response PEL
BW body weight POD
C Celsius PODADj
CAS Chemical Abstracts Service POI
CASRN Chemical Abstracts Service Registry ppm
Number RBC
CI confidence interval RDso
CNS central nervous system REL
CYP450 cytochrome P450 RfC
DAF dosimetric adjustment factor RfD
DMBA dimethylbenzoic acid RGDR
DMHA dimethylhippuric acid ROS
DNA deoxyribonucleic acid SCE
ECso half maximal effective concentration SD
EEC Electroencephalogram SOA
EPA U.S. Environmental Protection t
Agency TLV
g gram TMB
GD gestational day TSCA
Hb/g-A animal blood:gas partition coefficient TWA
Hb/g-H human blood:gas partition coefficient UF
HEC human equivalent concentration UFA
HEK Human epidermal keratinocytes UFn
HERO Health Effects Research Online UFS
HEV human epithelial keratinocytes
HSDB Hazardous Substance Database UFt
IL-8 interleukin-8 UFD
i.p. intraperitoneal
IRIS Integrated Risk Information System jig
JP-8 jet propulsion fuel 8 jil
k kilogram (imol
Km Michaelis-Menten constant UV
L liter V
LDH lactate dehydrogenase VOC
LOAEL lowest-observed-adverse-effect level W
m3 meter cubed WBC
mg milligram WS
nanogram
National Center for Environmental
Assessment
National Institute for Occupational
Safety and Health
National Library of Medicine
No-observed-adverse-effect level
Ontario Ministry of the Environment
Occupational Safety and Health
Administration
probability value
Provisional RfC
physiologically based
pharmacokinetic (model)
permissible exposure limit
point of departure
duration adjusted POD
Point of impingement
parts per million
red blood cell
50% respiratory rate decrease
Recommended exposure limit
reference concentration
reference dose
regional gas dose ratio
reactive oxygen species
sister chromatid exchange
standard deviation
Secondary organic aerosol
time
threshold limit value
trimethylbenzene
Toxic Substances Control Act
time-weighted average
uncertainty factor
interspecies uncertainty factor
intraspecies uncertainty factor
subchronic-to-chronic uncertainty
factor
LOAEL-to-NOAEL uncertainty factor
database deficiency uncertainty
factor
microgram
microliter
micromol
ultraviolet
volt
volatile organic compound
watt
white blood cell
white spirit
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chi-squared
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PREAMBLE
1. Scope of the IRIS Program
Soon after EPA was established in 1970, it was
at the forefront of developing risk assessment as a
science and applying it in decisions to protect
human health and the environment. The Clean Air
Act, for example, mandates that EPA provide "an
ample margin of safety to protect public health";
the Safe Drinking Water Act, that "no adverse
effects on the health of persons may reasonably be
anticipated to occur, allowing an adequate margin
of safety." Accordingly, EPA relies on health
assessments to identify adverse effects and
exposure levels below which these effects are not
anticipated to occur.
IRIS assessments critically review the publicly
available studies to identify adverse health effects
of chemicals and to characterize exposure-
response relationships. Exceptions are chemicals
currently used exclusively as pesticides, ionizing
and non-ionizing radiation, and criteria air
pollutants listed under section 108 of the Clean
Air Act (carbon monoxide, lead, nitrogen oxides,
ozone, particulate matter, and sulfur oxides; EPA
evaluates these in Integrated Science
Assessments). An assessment may cover a single
chemical, a group of structurally or lexicologically
related chemicals, or a complex mixture.
Once a year, the IRIS Program asks EPA
programs and regions, other federal agencies,
state governments, and the general public to
nominate chemicals and mixtures for future
assessment or reassessment. These agents may be
found in air, water, soil, or sediment. Selection is
based on program and regional office priorities
and on availability of adequate information to
evaluate the potential for adverse effects. IRIS can
assess other agents as an urgent public health
need arises. IRIS also reassesses agents as
significant new data are published.
2. Process for developing and peer-
reviewing IRIS assessments
The process for developing IRIS assessments
(revised in May 2009) involves systematic review
of the pertinent studies, opportunities for public
input, and multiple levels of scientific review. EPA
revises draft assessments after each review, and
external drafts and comments become part of the
public record (EPA 2009).
Step 1. Development of a draft Toxicological
Review (usually about 11-1/2 months
duration). The draft assessment considers all
pertinent publicly available studies and
applies consistent criteria to evaluate the
studies, identify health effects, weigh the
evidence of causation for each effect, identify
mechanistic events and pathways, and derive
toxicity values.
Step 2. Internal review by scientists in EPA
programs and regions (2 months). The draft
assessment is revised to address comments
from within EPA.
Step 3. Interagency science consultation with
other federal agencies and White House
offices (1-1/2 months). The draft assessment
is revised to address the interagency
comments. The science consultation draft,
interagency comments, and EPA's response to
major comments become part of the public
record.
Step 4. External peer review, after public
review and comment (3-1/2 months or
more, depending on the review process). EPA
releases the draft assessment for public
review and comment, followed by external
peer review. The peer review meeting is open
to the public and includes time for oral public
comments. The peer reviewers also receive
the written public comments. The peer
reviewers assess whether the evidence has
been assembled and evaluated according to
guidelines and whether the conclusions are
justified by the evidence. The peer review
draft, peer review report, and written public
comments become part of the public record.
Step 5. Revision of draft Toxicological Review
and development of draft IRIS summary
(2 months). The draft assessment is revised to
reflect the peer review comments, public
comments, and newly available studies. The
disposition of peer review comments and
public comments becomes part of the public
record.
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Step 6. Final EPA review and interagency
science discussion with other federal
agencies and White House offices [1-1/2
months). The draft assessment and summary
are revised to address EPA and interagency
comments. The science discussion draft,
written interagency comments, and EPA's
response to major comments become part of
the public record.
Step 7. Completion and posting (1 month). The
Toxicological Review and IRIS summary are
posted on the IRIS website (http://
www.epa.gov/iris/).
The remainder of this Preamble addresses
step 1, the development of a draft Toxicological
Review. IRIS assessments follow standard
practices of evidence evaluation and peer review,
many of which are discussed in EPA guidelines
(EPA 1986a, 1986b, 1991, 1996, 1998, 2000a,
2005a, 2005b) and other descriptions of "best
practices" (EPA 1994, 2000b, 2002, 2006, 2011).
Transparent application of scientific judgment is
of paramount importance. To provide a
harmonized approach across IRIS assessments,
this Preamble summarizes concepts from these
guidelines and emphasizes principles of general
applicability.
3. Identifying and selecting pertinent
studies
3.1 Identifying studies
Before beginning an assessment, EPA
conducts a comprehensive search of the primary
scientific literature. The literature search follows
standard practices and includes the PubMed and
ToxNet databases of the National Library of
Medicine and other databases listed in EPA's
HERO system (Health and Environmental
Research Online, http://hero.epa.gov/). Each
assessment specifies the search strategies,
keywords, and cut-off dates of its literature
searches. EPA posts the results of the literature
search on the IRIS website and requests
information from the public on additional studies
and ongoing research.
Each assessment also considers studies
received through the IRIS Submission Desk and
studies (typically unpublished) submitted to EPA
under the Toxic Substances Control Act. If a study
that may be critical to the conclusions of the
assessment has not been peer-reviewed, EPA will
have it peer-reviewed.
EPA also examines the toxicokinetics of the
agent to identify other chemicals (for example,
major metabolites of the agent) to include in the
assessment if adequate information is available, in
order to more fully explain the toxicity of the
agent and to suggest dose metrics for subsequent
modeling.
In assessments of chemical mixtures, mixture
studies are preferred for their ability to reflect
interactions among components (EPA 1986a,
2000a). The literature search seeks, in decreasing
order of preference:
Studies of the mixture being assessed.
Studies of a sufficiently similar mixture. In
evaluating similarity, the assessment
considers the alteration of mixtures in the
environment through partitioning and
transformation.
Studies of individual chemical components of
the mixture, if there are not adequate studies
of sufficiently similar mixtures.
3.2 Selecting pertinent epidemiologic studies
Study design is the key consideration for
selecting pertinent epidemiologic studies from the
results of the literature search.
Cohort studies and case-control studies
provide the strongest epidemiologic evidence,
as they collect information about individual
exposures and disease.
Cross-sectional studies provide useful
evidence if they relate exposures and disease
at the individual level and it is clear that
exposure preceded the onset of disease.
Ecologic studies (geographic correlation
studies) relate exposures and disease by
geographic area. They can provide strong
evidence if there are large exposure contrasts
between geographic areas, relatively little
exposure variation within study areas, and
population migration is limited.
Case reports of high or accidental exposure
lack definition of the population at risk and
the expected number of cases. They can
provide information about a rare disease or
about the relevance of analogous results in
animals.
The assessment briefly reviews ecologic
studies and case reports but includes details only
if they suggest effects not identified by other
epidemiologic studies.
3.3 Selecting pertinent experimental studies
Exposure route is a key design consideration
for selecting pertinent experimental studies from
the results of the literature search.
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Studies of oral, inhalation, or dermal exposure
involve passage through an absorption
barrier and are considered most pertinent to
human environmental exposure.
Injection or implantation studies are often
considered less pertinent but may provide
valuable toxicokinetic or mechanistic
information. They also may be useful for
identifying effects in animals if deposition or
absorption is problematic (for example, for
particles and fibers).
Exposure duration is also a key design
consideration for selecting pertinent experimental
studies.
Studies of effects from chronic exposure are
most pertinent to lifetime human exposure.
Studies of effects from subchronic exposure
are pertinent but less preferred than studies
of chronic exposure.
Short-term and acute studies are less
pertinent but are useful for obtaining
toxicokinetic or mechanistic information. The
assessment reviews short-term and acute
studies if they suggest distribution or effects
at a site not identified by longer-term studies.
For developmental toxicity and reproductive
toxicity, irreversible effects may result from a
brief exposure during a critical period of
development. Accordingly, specialized study
designs are used for these effects (EPA 1991,
1996,1998).
4. Evaluating the quality of individual
studies
4.1 Evaluating the quality of epidemiologic
studies
The assessment evaluates design and
methodologic aspects that can increase or
decrease the weight given to each epidemiologic
study in the overall evaluation (EPA 1991, 1994,
1996,1998, 2005a):
Documentation of study design, methods,
population characteristics, and results.
Definition and selection of the study and
comparison populations.
Ascertainment of exposure and the potential
for misclassification.
Ascertainment of disease or effect and the
potential for misclassification.
Duration of exposure and follow-up and
adequacy for assessing the occurrence of
effects, including latent effects.
Characterization of exposure during critical
periods for the development of effects.
Sample size and statistical power to detect
anticipated effects.
Participation rates and the resulting potential
for selection bias.
Potential confounding and other sources of
bias are identified and addressed in the study
design or in the analysis of results. The basis
for consideration of confounding is a
reasonable expectation that the confounder is
prevalent in the population and is related to
both exposure and outcome.
For developmental toxicity, reproductive
toxicity, neurotoxicity, and cancer there is further
guidance on the nuances of evaluating
epidemiologic studies of these effects (EPA 1991,
1996,1998, 2005a).
4.2 Evaluating the quality of experimental
studies
The assessment evaluates design and
methodologic aspects that can increase or
decrease the weight given to each experimental
study in the overall evaluation (EPA 1991, 1994,
1996,1998, 2005a):
Documentation of study design, animals or
study population, methods, basic data, and
results.
Relevance of the animal model or study
population and the experimental methods.
Characterization of the nature and extent of
impurities and contaminants of the
administered chemical or mixture.
Characterization of dose and dosing regimen
(including age at exposure) and their
adequacy to elicit adverse effects, including
latent effects.
Sample sizes and statistical power to detect
dose-related differences or trends.
Ascertainment of survival, vital signs, disease
or effects, and cause of death.
Control of other variables that could influence
the occurrence of effects.
The assessment uses statistical tests to
evaluate whether the observations may be due to
chance. The standard for determining statistical
significance of a response is a trend test or
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comparison of outcomes in the exposed groups
against those of concurrent controls. In some
situations, examination of historical control data
from the same laboratory within a few years of the
study may improve the analysis. For an
uncommon effect that is not statistically
significant compared with concurrent controls,
historical controls may show that the effect is
unlikely to be due to chance. For a response that
appears significant against a concurrent control
response that is unusual, historical controls may
offer a different interpretation (EPA 2005a).
For developmental toxicity, reproductive
toxicity, neurotoxicity, and cancer there is further
guidance on the nuances of evaluating
experimental studies of these effects (EPA 1991,
1996, 1998, 2005a). In multi-generation studies,
agents that produce developmental effects at
doses that are not toxic to the maternal animal are
of special concern. Effects that occur at doses
associated with mild maternal toxicity are not
assumed to result only from maternal toxicity.
Moreover, maternal effects may be reversible,
while effects on the offspring may be permanent
(EPA 1991,1998).
4.3 Reporting study results
The assessment uses evidence tables to report
details of the design and key results of pertinent
studies. There may be separate tables for each site
of toxicity or type of study.
If a large number of studies observe the same
effect, the assessment considers the study
characteristics in this section to identify the
strongest studies or types of study. The tables
report details from these studies, and the
assessment explains the reasons for not reporting
details of other studies or groups of studies that
do not add new information. Supplemental
material provides references to all studies
considered, including those not summarized in the
tables.
The assessment discusses strengths and
limitations that affect the interpretation of each
study. If the interpretation of a study in the
assessment differs from that of the study authors,
the assessment discusses the basis for the
difference.
As a check on the selection and evaluation of
pertinent studies, EPA asks peer reviewers to
identify studies that were not adequately
considered.
5. Weighing the overall evidence of
each effect
5.1 Weighing epidemiologic evidence
For each effect, the assessment evaluates the
evidence from the epidemiologic studies as a
whole to determine the extent to which any
observed associations may be causal. Positive,
negative, and null results are given weight
according to study quality. This evaluation
considers aspects of an association that suggest
causality, discussed by Hill (1965) and elaborated
by Rothman and Greenland (1998) (EPA 1994,
2002, 2005a;DHHS 2004).
Strength of association: The finding of a large
relative risk with narrow confidence intervals
strongly suggests that an association is not
due to chance, bias, or other factors. Modest
relative risks, however, may reflect a small
range of exposures, an agent of low potency,
an increase in a disease that is common,
exposure misclassification, or other sources of
bias.
Consistency of association: An inference of
causality is strengthened if elevated risks are
observed in independent studies of different
populations and exposure scenarios.
Reproducibility of findings constitutes one of
the strongest arguments for causality.
Discordant results sometimes reflect
differences in exposure or in confounding
factors.
Specificity of association: As originally intended,
this refers to one cause associated with one
disease. Current understanding that many
agents cause multiple diseases and many
diseases have multiple causes make this a less
informative aspect of causality, unless the
effect is rare or unlikely to have multiple
causes.
Temporal relationship: A causal interpretation
requires that exposure precede development
of the disease.
Biologic gradient (exposure-response
relationship): Exposure-response
relationships strongly suggest causality. A
monotonic increase is not the only pattern
consistent with causality. The presence of an
exposure-response gradient also weighs
against bias and confounding as the source of
an association.
Biologic plausibility: An inference of causality is
strengthened by data demonstrating plausible
biologic mechanisms, if available.
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Coherence: An inference of causality is
strengthened by supportive results from
animal experiments, toxicokinetic studies, and
short-term tests. Coherence may also be
found in other lines of evidence, such as
changing disease patterns in the population.
"Natural experiments": A change in exposure
that brings about a change in disease
frequency provides strong evidence of
causality.
Analogy: Information on structural analogues or
on chemicals that induce similar mechanistic
events can provide insight into causality.
These considerations are consistent with
contemporary guidelines that evaluate the quality
and weight of evidence. Confidence is increased if
the magnitude of effect is large, if there is evidence
of an exposure-response relationship, or if an
association was observed and the plausible biases
would tend to decrease the magnitude of the
reported effect. Confidence is decreased for study
limitations, inconsistency of results, indirectness
of evidence, imprecision, or reporting bias (Guyatt
eta!2008a,b).
To make clear how much the epidemiologic
evidence contributes to the overall weight of the
evidence, the assessment may choose a descriptor
such as sufficient evidence, suggestive evidence,
inadequate evidence, or evidence suggestive of no
causal relationship to characterize the
epidemiologic evidence of each effect (DHHS
2004).
5.2 Weighing experimental evidence
For each effect, the assessment evaluates the
evidence from the animal experiments as a whole
to determine the extent to which they indicate a
potential for effects in humans. Consistent results
across various species and strains increase
confidence that similar results would occur in
humans. Although causality is not at issue in
controlled experiments, several concepts
discussed by Hill (1965) affect the weight of
experimental results: consistency of response,
dose-response relationships, strength of response,
biologic plausibility, and coherence (EPA 1994,
2002,2005a).
In weighing evidence from multiple
experiments, EPA (2005a) distinguishes
Conflicting evidence (that is, mixed positive and
negative results in the same sex and strain
using a similar study protocol) from
Differing results (that is, positive results and
negative results are in different sexes or
strains or use different study protocols).
Negative or null results do not invalidate positive
results in a different experimental system. EPA
regards all as valid observations and looks to
mechanistic information, if available, to reconcile
differing results.
It is well established that there are critical
periods for some developmental and reproductive
effects. Accordingly, the assessment determines
whether critical periods have been adequately
investigated (EPA 1991, 1996, 1998, 2005a,
2005b). Similarly, the assessment determines
whether the database is adequate to evaluate
other critical sites and effects.
5.3 Characterizing modes of action
For each effect, the assessment discusses the
available information on its modes of action and
associated key events (key events being empirically
observable, necessary precursor steps or biologic
markers of such steps; mode of action being a
series of key events involving interaction with
cells, operational and anatomic changes, and
resulting in disease). Pertinent information may
also come from studies of metabolites or of
compounds that are structurally similar or that act
through similar mechanisms. The assessment
addresses several questions about each
hypothesized mode of action (EPA 2005a).
(a) Is the hypothesized mode of action
sufficiently supported in test animals?
Strong support for a key event being
necessary to a mode of action can come from
experimental challenge to the hypothesized
mode of action, where suppressing a key
event suppresses the disease. Support for a
mode of action is meaningfully strengthened
by consistent results in different experimental
models, but not by replicate experiments in
the same model. The assessment may
consider various aspects of causality in
addressing this question.
(b) Is the hypothesized mode of action
relevant to humans? The assessment
reviews the key events to identify critical
similarities and differences between the test
animals and humans. Site concordance is not
assumed between animals and humans,
though it may hold for certain modes of
action. Information suggesting quantitative
differences is considered in dose-response
analyses but is not used to determine
relevance. Similarly, anticipated levels of
human exposure are not used to determine
relevance.
(c) Which populations or life-stages can be
particularly susceptible to the
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hypothesized mode of action? The
assessment reviews the key events to identify
populations and life-stages that might be
susceptible to their occurrence. Quantitative
differences may result in separate toxicity
values for susceptible populations or life-
stages.
The assessment discusses the likelihood that
an agent operates through multiple modes of
action. An uneven level of support for different
modes of action can reflect disproportionate
resources spent investigating them (EPA 2005a).
It should be noted that in clinical reviews, the
quality of evidence may be reduced if evidence is
limited to studies funded by one interested sector
(Guyattetal2008b).
Studies of genetic toxicity are often available,
and the assessment evaluates the evidence of a
mutagenic mode of action.
Demonstration of gene mutations,
chromosome aberrations, or aneuploidy in
humans or experimental mammals (in vivo)
provides the strongest evidence.
This is followed by positive results in lower
organisms or in cultured cells (in vitro) or for
other genetic events.
Negative results carry less weight, partly
because they cannot exclude the possibility of
effects in other tissues (IARC 2006).
For germ-cell mutagenicity, EPA has defined
categories of evidence, ranging from positive
results of human germ-cell mutagenicity to
negative results for all effects of concern (EPA
1986b).
5.4 Characterizing the overall weight of the
evidence
After weighing the epidemiologic and
experimental studies pertinent to each effect, the
assessment may select a standard descriptor to
characterize the overall weight of the evidence.
For example, the following standard descriptors
combine epidemiologic, experimental, and
mechanistic evidence of carcinogenicity (EPA
2005a).
Carcinogenic to humans: There is convincing
epidemiologic evidence of a causal association
(that is, there is reasonable confidence that
the association cannot be fully explained by
chance, bias, or confounding); or there is
strong human evidence of cancer or its
precursors, extensive animal evidence,
identification of key precursor events in
animals, and strong evidence that they are
anticipated to occur in humans.
Likely to be carcinogenic to humans: The
evidence demonstrates a potential hazard to
humans but does not meet the criteria for
carcinogenic. There may be a plausible
association in humans, multiple positive
results in animals, or a combination of human,
animal, or other experimental data.
Suggestive evidence of carcinogenic potential:
The data raise concern for effects in humans
but are not sufficient for a stronger
conclusion. This descriptor covers a range of
evidence, from a positive result in the only
available study to a single positive result in an
extensive database that includes negative
results in other species.
Inadequate information to assess carcinogenic
potential: No other descriptors apply.
Conflicting evidence can be classified as
inadequate information if all positive results
are opposed by negative studies of equal
quality in the same sex and strain. Differing
results, however, can be classified as
suggestive evidence or as likely to be
carcinogenic.
Not likely to be carcinogenic to humans: There
are robust data for concluding that there is no
basis for concern. There may be no effects in
both sexes of at least two appropriate animal
species; positive animal results and strong,
consistent evidence that each mode of action
in animals does not operate in humans; or
convincing evidence that effects are not likely
by a particular exposure route or below a
defined dose.
6. Selecting studies for derivation of
toxicity values
For each effect associated with an agent, the
assessment derives toxicity values if there are
suitable epidemiologic or experimental data. The
derivation of toxicity values may be linked to the
weight-of-evidence descriptor. For example, EPA
typically derives toxicity values for agents
classified as carcinogenic to humans or likely to be
carcinogenic, but not for agents with inadequate
information or that are not likely to be carcinogenic
(EPA2005a).
Dose-response analysis requires quantitative
measures of dose and response. Then, other
factors being equal (EPA 1994, 2005a):
Epidemiologic studies are preferred over
animal studies, if quantitative measures of
exposure are available and effects can be
attributed to the agent.
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Among experimental animal models, those
that respond most like humans are preferred,
if the comparability of response can be
determined.
Studies by a route of human environmental
exposure are preferred, although a validated
toxicokinetic model can be used to
extrapolate across exposure routes.
Studies of longer exposure duration and
follow-up are preferred, to minimize
uncertainty about whether effects are
representative of lifetime exposure.
Studies with multiple exposure levels are
preferred for their ability to provide
information about the shape of the exposure-
response curve.
Studies that show an exposure-response
gradient are preferred, as long as lack of a
monotonic relationship at higher exposure
levels can be satisfactorily explained by
factors such as competing toxicity, saturation
of absorption or metabolism, misclassification
bias, or selection bias.
Among studies that show an exposure-
response gradient, those with adequate
power to detect effects at lower exposure
levels are preferred, to minimize the extent of
extrapolation to levels found in the
environment.
If a large number of studies are suitable for
dose-response analysis, the assessment considers
the study characteristics in this section to focus on
the most informative data. The assessment
explains the reasons for not analyzing other
groups of studies. As a check on the selection of
studies for dose-response analysis, EPA asks peer
reviewers to identify studies that were not
adequately considered.
7. Deriving toxicity values
7.1 General framework for dose-response
analysis
EPA uses a two-step approach that
distinguishes analysis of the observed dose-
response data from inferences about lower doses
(EPA2005a).
Within the observed range, the preferred
approach is to use modeling to incorporate a wide
range of data into the analysis. The modeling
yields a point of departure (an exposure level near
the lower end of the observed range, without
significant extrapolation to lower doses) (sections
7.2-7.3).
Extrapolation to lower doses considers what
is known about the modes of action for each effect
(sections 7.4-7.5). An alternative to low-dose
extrapolation is derivation of reference values,
which are calculated by adjusting the point of
departure by factors that account for several
sources of uncertainty and variability (section
7.6).
Increasingly, EPA is making use of multiple
data sets or combining multiple responses in
deriving toxicity values. EPA also considers
multiple dose-response approaches when they
can be supported by robust data.
7.2 Modeling dose
The preferred approach for analysis of dose is
toxicokinetic modeling because of its ability to
incorporate a wide range of data. The preferred
dose metric would refer to the active agent at the
site of its biologic effect or to a close, reliable
surrogate measure. The active agent may be the
administered chemical or a metabolite. Confidence
in the use of a toxicokinetic model depends on the
robustness of its validation process and on the
results of sensitivity analyses (EPA 1994, 2005a,
2006).
Because toxicokinetic modeling can require
many parameters and more data than are typically
available, EPA has developed standard approaches
that can be applied to typical data sets. These
standard approaches also facilitate comparison
across exposure patterns and species.
Intermittent study exposures are
standardized to a daily average over the
duration of exposure. For chronic effects,
daily exposures are averaged over the
lifespan. Exposures during a critical period,
however, are not averaged over a longer
duration (EPA 1991,1996,1998, 2005a).
Doses are standardized to equivalent human
terms to facilitate comparison of results from
different species.
Oral doses are scaled allometrically using
mg/kg3/4-d as the equivalent dose metric
across species. As allometric scaling is
typically based on adult body weight, it is
not used for early-life exposure or for
developmental effects (EPA 2005a, 2011).
Inhalation exposures are scaled using
dosimetry models that apply species-
specific physiologic and anatomic factors
and consider whether the effect occurs at
the site of first contact or after systemic
circulation (EPA 1994).
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It can be informative to convert doses across
exposure routes. If this is done, the assessment
describes the underlying data, algorithms, and
assumptions (EPA2005a).
7.3 Modeling response in the range of
observation
Toxicodynamic ("biologically based")
modeling can incorporate data on biologic
processes leading to a disease. Such models
require sufficient data to ascertain a mode of
action and to quantitatively support model
parameters associated with its key events.
Because different models may provide equivalent
fits to the observed data but diverge substantially
at lower doses, critical biologic parameters should
be measured from laboratory studies, not by
model fitting. Confidence in the use of a
toxicodynamic model depends on the robustness
of its validation process and on the results of
sensitivity analyses. Peer review of the scientific
basis and performance of a model is essential
(EPA2005a).
Because toxicodynamic modeling can require
many parameters and more knowledge and data
than are typically available, EPA has developed a
standard set of empirical ("curve-fitting") models
(http://www.epa.gov/ncea/bmds/) that can be
applied to typical data sets, including those that
are nonlinear. EPA has also developed guidance
on modeling dose-response data, assessing model
fit, selecting suitable models, and reporting
modeling results (EPA 2000b). Additional
judgment or alternative analyses are used when
the procedure fails to yield reliable results, for
example, if the fit is poor, modeling may be
restricted to the lower doses, especially if there is
competing toxicity at higher doses (EPA 2005a).
Modeling is used to derive a point of
departure (EPA 2000b, 2005a). (See section 7.6
for alternatives if a point of departure cannot be
derived by modeling.)
For dichotomous responses, the point of
departure is the 95% lower bound on the
dose associated with a small increase of a
biologically significant effect.
If linear extrapolation to lower doses will
be used, a standard value near the low
end of the observable range is used (10%
response for animal data, 1% for
epidemiologic data, depending on the
observed response rates).
If nonlinear extrapolation will be used,
both statistical and biologic factors are
considered (10% response for minimally
adverse effects, 5% or lower for more
severe effects or for developmental
toxicity data on individual offspring).
For continuous responses, the point of
departure is ideally a level where the effect is
considered minimally adverse. In the absence
of such definition, both statistical and biologic
factors are considered in selecting a response
level.
7.4 Extrapolating to lower doses
The purpose of extrapolating to lower doses is
to estimate responses at exposures below the
observed data. Low-dose extrapolation is typically
used for known and likely carcinogens. Low-dose
extrapolation considers what is known about
modes of action (EPA 2005a).
(1) If a biologically based model has been
developed and validated for the agent,
extrapolation may use the fitted model
beyond the observed range if significant
model uncertainty can be ruled out with
reasonable confidence. Below the range
where confidence bounds on the predictions
are reasonably precise, extrapolation may
continue using a linear model.
(2) Linear extrapolation is used if the dose-
response curve is expected to have a linear
component below the point of departure. This
includes:
Agents or their metabolites that are DNA-
reactive and have direct mutagenic
activity.
Agents or their metabolites for which
human exposures or body burdens are
near doses associated with key events
leading to an effect.
Linear extrapolation is also used if the
evidence is insufficient to establish a mode of
action.
The result of linear extrapolation is described
by an oral slope factor or an inhalation unit
risk, which is the slope of the dose-response
curve at lower doses.
(3) Nonlinear extrapolation is used if there are
sufficient data to ascertain the mode of action
and to conclude that it is not linear at lower
doses, and the agent does not demonstrate
mutagenic or other activity consistent with
linearity at lower doses. If nonlinear
extrapolation is appropriate but no model is
developed, a default is to calculate reference
values.
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If linear extrapolation is used, the assessment
develops a candidate slope factor or unit risk for
each suitable data set. These results are arrayed,
using common dose metrics, to show the
distribution of relative potency across various
effects and experimental systems. The assessment
then derives an overall slope factor and an overall
unit risk for the agent, considering the various
dose-response analyses, the study preferences
discussed in section 6, and the possibility of
basing a more robust result on multiple data sets.
7.5 Considering susceptible populations and
life-stages
The assessment analyzes the available
information on populations and life-stages that
may be particularly susceptible to each effect. A
tiered approach is used (EPA 2005a).
(1) If an epidemiologic or experimental study
reports quantitative results for a susceptible
population or life-stage, these data are
analyzed to derive separate toxicity values for
susceptible individuals.
(2) If data on risk-related parameters allow
comparison of the general population and
susceptible individuals, these data are used to
adjust the general-population toxicity values
for application to susceptible individuals.
(3) In the absence of chemical-specific data,
application of age-dependent adjustment
factors is recommended for early-life
exposure to suspected carcinogens. There is
evidence of early-life susceptibility to various
carcinogenic agents, but most epidemiologic
studies and cancer bioassays do not include
early-life exposure. To address the potential
for early-life susceptibility, EPA recommends:
10-fold adjustment for exposures before
age 2 years.
3-fold adjustment for exposures between
ages 2 and 16 years.
These adjustments are generally applied only
for a mutagenic mode of action, though early-
life susceptibility has been observed for
several carcinogens that are not mutagenic
(EPA2005b).
7.6 Reference values and uncertainty factors
An oral reference dose or an inhalation
reference concentration is an estimate of an
exposure (including in susceptible subgroups)
that is likely to be without an appreciable risk of
adverse health effects over a lifetime (EPA 2002).
Reference values are typically calculated for
effects other than cancer and for suspected
carcinogens if a well characterized mode of action
indicates that a threshold can be based on
prevention of an early key event. Reference values
provide no information about risks at exposures
above the reference value.
The assessment characterizes effects that
form the basis for reference values as adverse,
considered to be adverse, or a precursor to an
adverse effect. For developmental, reproductive,
and neurotoxicity there is guidance on adverse
effects and their biologic markers (EPA 1991,
1996,1998).
To account for uncertainty and variability in
the derivation of a lifetime human exposure where
effects are not anticipated to occur, reference
values are calculated by adjusting the point of
departure by a series of uncertainty factors. If a
point of departure cannot be derived by modeling,
a no-observed-adverse-effect level or a lowest-
observed-adverse-effect level is substituted. The
assessment discusses scientific considerations
involving several areas of variability or
uncertainty.
Human variation. A factor of 10 is applied to
account for variation in susceptibility across
the human population and the possibility that
the available data may not be representative
of individuals who are most susceptible to the
effect. This factor is reduced only if the point
of departure is derived specifically for
susceptible individuals (not for a general
population that includes both susceptible and
non-susceptible individuals) (EPA 1991,1994,
1996,1998,2002).
Animal-to-human extrapolation. A factor of 10
is applied if animal results are used to make
inferences about humans. This factor is often
regarded as comprising toxicokinetics and
toxicodynamics in equal parts. Accordingly, if
the point of departure is based on
toxicokinetic modeling, dosimetry modeling,
or allometric scaling across species, a factor of
101/2 (rounded to 3) is applied to account for
the remaining uncertainty involving
toxicodynamic differences. An animal-to-
human factor is not applied if a biologically
based model adjusts fully for toxicokinetic
and toxicodynamic differences and residual
uncertainty across species (EPA 1991, 1994,
1996,1998,2002).
Adverse-effect level to no-observed-adverse-
effect level. If a point of departure is based on
a lowest-observed-adverse-effect level, the
assessment must infer a dose where such
effects are not expected. This can be a matter
of great uncertainty, especially if there is no
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evidence available at lower doses. A factor of
10 is applied to account for the uncertainty in
making this inference. A factor other than 10
may be used, depending on the magnitude
and nature of the response and the shape of
the dose-response curve (EPA 1991, 1994,
1996,1998,2002).
Subchronic-to-chronic exposure. If a point of
departure is based on subchronic studies, the
assessment considers whether lifetime
exposure would have effects at lower levels. A
factor of 10 is applied to account for the
uncertainty in using subchronic studies to
make inferences about lifetime exposure. This
factor may also be applied for developmental
or reproductive effects if exposure covered
less than the full critical period. A factor other
than 10 may be used, depending on the
duration of the studies and the nature of the
response (EPA 1994,1998, 2002).
Incomplete database. If an incomplete database
raises concern that further studies might
identify a more sensitive effect, organ system,
or life-stage, the assessment may apply a
database uncertainty factor (EPA 1991, 1994,
1996, 1998, 2002). EPA typically follows the
suggestion that a factor of 10 be applied if
both a prenatal toxicity study and a two-
generation reproduction study are missing,
and a factor of 1Q1/2 if either is missing (EPA
2002).
In this way, the assessment derives candidate
reference values for each suitable data set and
effect that is plausibly associated with the agent.
These results are arrayed, using common dose
metrics, to show where effects occur across a
range of exposures (EPA 1994). The assessment
then selects an overall reference dose and an
overall reference concentration for the agent to
represent lifetime human exposure levels where
effects are not anticipated to occur.
The assessment may also report reference
values for each effect. This would facilitate
subsequent cumulative risk assessments, where it
may be important to consider the combined effect
of chemicals acting at a common site or operating
through common mechanisms (EPA 2002).
7.7 Confidence and uncertainty in the
reference values
The assessment selects a standard descriptor
to characterize the level of confidence in each
reference value, based on the likelihood that the
value would change with further testing.
Confidence in reference values is based on quality
of the studies used and completeness of the
database, with more weight given to the latter.
The level of confidence is increased for reference
values based on human data supported by animal
data (EPA 1994).
High confidence: The reference value is not likely
to change with further testing, except for
mechanistic studies that might affect the
interpretation of prior test results.
Medium confidence: This is a matter of
judgment, between high and low confidence.
Low confidence: The reference value is especially
vulnerable to change with further testing.
These criteria are consistent with
contemporary guidelines that evaluate the quality
of evidence. These also focus on whether further
research would be likely to change confidence in
the estimate of effect (Guyatt et al 2008a).
All assessments discuss the significant
uncertainties encountered in the analysis. EPA
provides guidance on characterization of
uncertainty (EPA 2005a). For example, the
discussion distinguishes model uncertainty (lack
of knowledge about the most appropriate
experimental or analytic model), parameter
uncertainty (lack of knowledge about the
parameters of a model), and human variation
(interpersonal differences in biologic
susceptibility or in exposures that modify the
effects of the agent).
For other general information about this
assessment or other questions relating to IRIS, the
reader is referred to EPA's IRIS Hotline at (202)
566-1676 (phone), (202) 566-1749 (fax), or
hotline.iris@epa.gov (email address).
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AUTHORS | CONTRIBUTORS | REVIEWERS
Authors
J. Allen Davis, M.S.P.H. (Chemical Manager)
Eva McLanahan, Ph.D. (LCDR, USPHS)
Paul Schlosser, Ph.D.
John Cowden, Ph.D.
Gary Foureman, Ph.D. (Currently ICF Int.)
Andrew Kraft, Ph.D
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
Research Triangle Park, NC
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
Washington, DC
Ray Antonelli, B.S.
Oak Ridge Institute for Scientific Education
Research Triangle Park, NC
Contributors
Reeder Sams, Ph.D.
John Stanek, Ph.D.
Rob Dewoskin, Ph.D.
George Woodall, Ph.D.
Geniece Lehmann, Ph.D.
Connie Meacham, M.S.
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
Research Triangle Park, NC
Technical Support
Ellen Lorang, M.S.
Deborah Wales
Gerald Gurevich
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
Research Triangle Park, NC
Contractor Support
Battelle Memorial Institute, Pacific Northwest Division, Richmond, WA
Karla D. Thrall, Ph.D.
Battelle Memorial Institute, Columbus, OH
Jessica D. Sanford, Ph.D.
Maureen A. Wooton
Robert A. Lordo, Ph.D.
Anthony Fristachi
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Toxicology Excellence for Risk Assessment (TERA)
Under Battelle Memorial Institute Contract EP-C-09-006
Lisa M. Sweeney, Ph.D., DABT
Melissa J. Kohrman-Vincent, B.A.
Executive Direction
Reeder Sams Ph D U'S' Environmental Protection Agency
,,.,,' _, _. Office of Research and Development
John Vandenberg, Ph.D. I^^FC- ,. i A
' , ... , , .° National Center for Environmental Assessment
Debra Walsh, M.S.
Research Triangle Park, NC
Vincent Cogliano, Ph.D. U.S. Environmental Protection Agency
Samantha Jones, Ph.D. Office of Research and Development
Lynn Flowers, Ph.D. National Center for Environmental Assessment
Jamie Strong Ph.D. Washington, DC
Reviewers
This document has been provided for review to EPA scientists.
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PREFACE
Background of Current Toxicological Review
There is currently no entry on the IRIS Database for either 1,2,4-TMB or 1,3,5-TMB.
The current assessment has undergone draft development in which an RfC and RfD were
derived for both 1,2,4-TMB and 1,3,5-TMB. No cancer values are derived for either isomer.
Chemical and Physical Information
The commercially available substance known as trimethylbenzene (TMB}, CAS No.
25551-13-7, is a mixture of three isomers in various proportions, namely CAS No. 526-73-8
(1,2,3-trimethylbenzene or hemimellitene], CAS No. 108-67-8 (1,3,5-trimethylbenzene or
mesitylene}, and CAS No. 95-63-6 (1,2,4-trimethylbenzene or pseudocumene}. The focus of
this EPA review is two of these isomers: 1,2,4-trimethylbenzene (1,2,4-TMB} and 1,3,5-
trimethylbenzene (1,3,5-TMB}.
The TMBs are aromatic hydrocarbons with three methyl substituents attached to a
benzene ring and the chemical formula C9H12. The chemical and physical properties of the
TMB isomers are similar to one another. TMB is a colorless, flammable liquid with a strong
aromatic odor; an odor threshold of 0.4 parts per million (ppm} of air has been reported
(U.S. EPA. 1994}. It is insoluble in water but miscible with organic solvents such as ethyl
alcohol, benzene, and ethyl ether [OSHA. 1996}.
Vehicle emissions are a major anthropogenic source of 1,2,4-TMB and 1,3,5-TMB, due to
the widespread use of the C9 fraction as a gasoline additive (U.S. EPA. 1994}. Other uses of
1,2,4-TMB and 1,3,5-TMB include solvents in research and industry, uses as a dyestuff
intermediate, paint thinner, and as a UV oxidation stabilizer for plastics (HSDB. 2011a. b}.
Production and use of 1,2,4-TMB and 1,3,5-TMB may result in their release to the
environment through various waste streams. If released to the atmosphere, 1,2,4-TMB and
1,3,5-TMB will exist solely in the vapor phase in the ambient atmosphere, based on
measured vapor pressures of 2.10 and 2.48 mm Hg at 25QC, respectively (HSDB. 2011a. b}.
Both isomers are expected to have limited mobility through soil based on their Log KOC -
values, but are expected to volatilize from both moist and dry soil surfaces and surface
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waters based on their respective Henry's Law constants and vapor pressures (see Table A.l-
1}. Degradation of both isomers in the atmosphere occurs by reaction with hydroxyl
radicals, the half-life of which is 11-12 hours (HSDB. 2011a. bj. Non-volatilized 1,2,4-TMB
and 1,3,5-TMB may be subject to biodegraelation under aerobic conditions (HSDB. 2011a. bj.
The estimated bio-concentration factors (439 and 234} and high volatility of 1,2,4-TMB and
1,3,5-TMB suggest that bioaccumulation of these chemicals will not be significant (U.S. EPA.
19871
Additional information on the chemical identities and physicochemical properties of
1,2,4-TMB and 1,3,5-TMB are listed in Table A. 1-1.
Table 1. Physical properties and chemical identity of 1,2,4-TMB and 1,3,5-TMB
CAS Registry Number
Synonym(s)
Molecular formula
Molecular weight
Chemical structure
Melting point, °C
Boiling point, °C @ 760 mm Hg
Vapor pressure, mm Hg @ 25°C
Density, g/mL at 20 °C relative to
the density of H2O at 4 °C
Flashpoint, °C
Water solubility, mg/L at 25 °C
Other solubilities
Henry's Law Constant, atm-
m3/mole
Log KOW
Log KOC
Bioconcentration Factor
95-63-6
1,2,4-
Trimethylbenzene,
pseudocumene,
asymmetrical
trimethylbenzene
108-67-8
1,3,5-Trimethyl benzene,
mesitylene, symmetrical
trimethylbenzene
C9H12
120.19
CH3
^^./CH3
k^^
CH3
-43.8
168.9
2.10
0.8758
44
57
Miscible with ethanol,
benzene, ethyl ether,
acetone, carbon
tetrachloride, petroleum
ether
6.16 x 10"3
3.78
2.73
439
CH3
H3C CH3
-44.8
164.7
2.48
0.8637
50
48.2
Miscible with alcohol,
ether, benzene, acetone,
and oxygenated and
aromatic solvents
8.77 x 10"3
3.42
2.70-3.13
234
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CAS Registry Number
Synonym(s)
Conversion factors
95-63-6
1,2,4-
Trimethylbenzene,
pseudocumene,
asymmetrical
trimethylbenzene
108-67-8
1,3,5-Trimethyl benzene,
mesitylene, symmetrical
trimethylbenzene
1 ppm = 4.92 mg/m3
1 mg/m3 = 0.2 ppm
Source: [HSDB. 2011a. b; U.S. EPA. 1987]
Programmatic Interest
1,2,4-trimethylbenzene (1,2,4-TMB) and 1,3,5-trimethybenzene (1,3,5-TMB) are
industrial solvents found at Superfund sites. This IRIS assessment is being developed due
to the potential for human environmental exposure to these compounds. The related
isomer, 1,2,3-trimethylbenzene, is not included in this Toxicological Review.
Other Agency and International Assessments
Table 2. Other Agency and International Assessments
Agency
National Institue of
Occupational Safety and
Health (NIOSH. 1992)
American Conference of
Governmental Industrial
Hygienists (ACGIH. 2002)
National Advisory Committee
for Acute Exposure Guideline
Levels for Hazardous
Substances (U.S. EPA, 2007)
Ontario Ministry of the
Environment (MOE. 2006)
Inhalation value
Recommended Exposure Limit (REL) for TMBs- 25 ppm (123 mg/m3)
time weighted average for up to a 10 hour work day and a 40 hour
work week, based on the risk of skin irritation, central nervous system
depression, and respiratory failure (reference not provided)
Threshold Limit Value (TLV) for VOC mixture containing 1,2,4-TMB and
1,3,5-TMB- 25 ppm (123 mg/m3) time weighted average for a normal
8-hour work day and a 40-hour work week, based on the risk of
irritation and central nervous system effects (Battig et al., 1956)
For TMBs: AEGL-1 (nondisabling) - 180 ppm (890 mg/m3) to 45 ppm
(220 mg/m3) (10 minutes to 8 hours, respectively) (Korsak and
Rydzynski, 1996)
AEGL-2 (disabling) - 460 ppm (2300 mg/m3) to 150 ppm (740 mg/m3)
(10 minutes to 8 hours, respectively) (Gage, 1970)
For TMBs: 24 hour Ambient Air Quality Criterion (AAQC) - 0.3 mg/m3
based on CNS effects; half-hour Point of Impingement (POI) - 0.9
mg/m3 based on CNS effects (Wiaderna et al., 2002; Gralewicz and
Wiaderna, 2001; Gralewicz et al., 1997a; Korsak and Rydzynski, 1996)
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EXECUTIVE SUMMARY
Effects other than cancer observed following inhalation exposure to
1,2,4-TMB
The relationship between exposure to 1,2,4-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 and
controlled human exposures to 1,2,4-TMB or solvent mixtures containing 1,2,4-TMB.
Health effects noted in these studies were limited to irritative (eye irritation] and
neurological effects (hand tremble, abnormal fatigue, lack of coordination] (Lammers et al..
2007: Chenetal.. 1999: Norseth et al.. 1991: Battigetal.. 19561 No human studies were
found for 1,3,5-TMB.
Animal inhalation studies included acute and short-term studies for both isomers that
reported respiratory irritative (decreased respiration rates] and neurological effects
(decreased pain sensitivity and decreased neuromuscular function and coordination] that
supported effects seen in human studies (Wiaderna et al.. 2002: Gralewicz and Wiaderna.
2001: Gralewicz etal.. 1997a: Gralewicz etal.. 1997b: Korsaketal.. 1995]. Three
subchronic inhalation studies were found for 1,2,4-TMB that observed exposure-response
effects in multiple organ systems, including the nervous, hematological, and pulmonary
systems fKorsaketal.. 2000: Korsaketal.. 1997: Korsakand Rydzynski. 19961 In these
studies, disturbances in CNS function, including decreased pain sensitivity and decreased
neuromuscular function and coordination appear to be the most sensitive endpoints
following exposure to 1,2,4-TMB. No subchronic studies were found that investigated
exposure to 1,3,5-TMB. One developmental study that was found (Saillenfait et al.. 2005]
that observed similar levels of maternal and fetal toxicity (i.e., decreased maternal weight
gain and fetal weight] following exposure to either isomer; other indices of fetal toxicity
(i.e., fetal death and malformations] were not affected by exposure.
Inhalation Reference Concentration (RfC) for 1,2,4-TMB
The chronic RfC of 2 x 10-2 mg/m3 for 1,2,4-TMB was calculated from a BMDL of 84.0
mg/m3 (resulting in an PODADJ of 0.085 mg/L blood] for decreased pain sensitivity in
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male rats exposed to 1,2,4-TMB via inhalation for 90 days (6 hours/day, 5 days/week]
[Korsakand Rydzynski. 1996). A PBPK model was then used to estimate a human
equivalent concentration of 15.6 mg/m3, which was used as the POD to derive the RfC. A
total UF of 1000 was used: 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], 10 to account for
subchronic-to-chronic extrapolation due the lack of a suitable chronic study, and 3 to
account for deficiencies in the database (no chronic study, no two-generation
reproductive/developmental toxicity study].
Table II. Reference concentration (RfC) for 1,2,4-TMB
Critical Effect
Decreased pain sensitivity
90 day rat study
Korsak and Rydzynski (1996)
Point of Departure (mg/m3)
PODHEc = 15.6
A PBPK model was used to
calculate an internal blood dose
from the rat inhalation study and
then a human equivalent
concentration was calculated. This
HEC served as the POD.
UF
1,000
Chronic RfC (mg/m3)
2 x 10"2
Confidence Levels for the Derivation of the RfC 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. 1994]. Confidence in the study from which the critical
effect was identified, Korsak and Rydzynski [1996] is medium. The study is a well-
conducted peer-reviewed study that utilized three dose groups plus untreated controls, an
appropriate number of animals per dose group, and performed statistical analyses. The
critical effect on which the RfC is based is well-supported as the weight of evidence for
1,2,4-TMB-induced neurotoxicity is coherent across multiple animals species (i.e., human,
mouse, and rat] and consistent across multiple exposure durations (i.e., acute, short-term,
and sub-chronic] (Gralewicz and Wiaderna. 2001: Chen etal.. 1999: Wiaderna etal.. 1998:
Gralewicz etal.. 1997a: Gralewicz etal.. 1997b: Korsak and Rydzynski. 1996: Norseth et al..
1991]. Confidence in the database for 1,2,4-TMB is low to medium as the database includes
acute, short-term, subchronic, and developmental toxicity studies in rats and mice. The
database lacks a chronic and multigenerational reproductive study, and the studies
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supporting the critical effect predominately come from the same research institute. Overall
confidence in the RfC for 1,2,4-TMB is low to medium.
Inhalation Reference Concentration (RfC) for 1,3,5-TMB
No chronic or subchronic studies exist that would support the derivation of an RfC for
1,3,5-TMB, however two short-term neurotoxicity studies and one developmental toxicity
study were identified as potential studies from which to identify a critical effect for RfC
derivation. Ultimately, the two short-term neurotoxicity studies were inappropriate for the
derivation of an RfC due to the magnitude of uncertainty associated with those data sets: in
order to use the endpoints from these studies, a total uncertainty factor of 10,000 would be
necessary: 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], 10 to account for extrapolation from a
LOAEL to a NOAEL, 10 to account for subchronic-to-chronic extrapolation due the lack of a
suitable chronic study, and 3 to account for deficiencies in the database (no chronic study,
no two-generation reproductive/developmental toxicity study]. Using the Saillenfait et al.
[2005] study and the most sensitive endpoint in that study, decreased maternal weight
gain, would result in an RfC 15-fold greater than that derived for 1,2,4-TMB (3 x 10-1 vs. 2
x 10-2 mg/m3]. This was deemed to not be scientifically justified as the toxicological
database for 1,2,4-TMB and 1,3,5-TMB, demonstrates that the two isomers are similar to
one another regarding respiratory, neurological, and developmental toxicity in acute and
developmental studies (Saillenfait et al.. 2005: Korsak and Rydzynski. 1996: Korsak et al..
1995], although 1,3,5-TMB was observed to induce neurotoxicity at a slightly greater
magnitude and earlier onset of effect compared to 1,2,4-TMB at the same exposure
concentration (Wiaderna etal.. 2002: Gralewicz and Wiaderna. 2001]. Additionally,
available toxicokinetic data regarding blood:air partition coefficients, respiratory uptake,
and absorption into the bloodstream in humans and rats do not suggest any appreciable
differences can be expected between the two isomers (Meulenberg and Vijverberg. 2000:
larnberg et al.. 1996: Dahletal.. 1988]. Therefore, given the apparent similarities between
the two isomers (see Section 2.1.6], it was determined that the scientific database
supported adopting the RfC for 1,2,4-TMB for the RfC for 1,3,5-TMB. Thus, the chronic
RfC of 2 x 10-2 mg/m3 derived for 1,2,4-TMB was adopted as the RfC for 1,3,5-TMB
based on the determination of sufficient similarity regarding chemical properties,
kinetics, and toxicity between the two isomers.
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Confidence Levels for the Derivation of the RfCfor 1,3,5-TMB
As noted previously, 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 EPA (1994}.
Section 4.3.9.2. The chronic RfC of 2 x 10-2 mg/m3 derived for 1,2,4-TMB was adopted as
the RfC for 1,3,5-TMB based on the conclusion that the two isomers were sufficiently
similar regarding chemical properties, kinetics, and toxicity. Thus, confidence in the study
from which the critical effect was identified, Korsakand Rydzynski (1996) is medium (see
above}. Confidence in the database is low to medium as the database includes acute, short-
term, and developmental toxicity studies in rats and mice. The database lacks a chronic,
subchronic, and multigenerational reproductive study. Additionally, the studies supporting
the critical effect predominately come from the same research institute Overall confidence
in the RfC for 1,3,5-TMB is low due to uncertainties surrounding the adoption of the RfC
derived for 1,2,4-TMB as the RfC for 1,3,5-TMB.
Effects other than cancer observed following oral exposure
No chronic, subchronic, or short-term studies were identified that examined the
noncancer effects of oral exposure to 1,2,4-TMB or 1,3,5-TMB. A series of oral or i.p.
injection studies were identified that investigated the acute neurotoxic effects of 1,2,4-TMB
and 1,3,5-TMB exposure fTomas et al.. 1999a: Tomasetal.. 1999b: Tomasetal.. 1999cj. In
these studies exposed rats demonstrated changes in electrocortical arousal, altered EEC
activity in the cortical and hippocampal regions of the brain, and increase locomotor
activity (possible due to difficulty maintain balance due to motor ataxia}. As these effects
were only observed in studies investigating acute exposures, they were not deemed
sufficient for derivation of human health values.
Oral Reference Dose (RfD) for 1,2,4-TMB
A PBPK model (Hissink et al.. 2007}. modified by EPA to include an oral compartment
was available for estimating the oral dose that would yield a blood concentration equal to
the blood concentration at the POD used in the derivation of the RfC for 1,2,4-TMB. Under
the assumption of constant oral ingestion and 100% absorption of 1,2,4-TMB via constant
infusion rate into the liver, a PODHED of 6.2 mg/kg-day was derived. Hepatic first pass
metabolism was also evaluated in humans using the modified PBPK model; at low daily
doses, inhalation doses were estimated to result in steady state venous blood
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concentrations 4-fold higher than blood concentrations resulting from equivalent oral
doses, due to hepatic first pass metabolism.
The same total UF of 1,000 as was used for the RfC derivation was applied: 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], 10 to account for subchronic-to-chronic
extrapolation due the lack of a suitable chronic study, and 3 to account for deficiencies in
the database (no multigeneration reproductive/developmental toxicity study].
Table 3. Reference dose (RfD) for 1,2,4-TMB
Critical Effect
Decreased pain sensitivity
90 day rat study
Korsak and Rydzyriski (1996)
Point of Departure (mg/kg-
day)
Route to route
extrapolation using Korsak
and Rydzyriski (1996)
subchronic inhalation study
in Wistar rats
UF
1000
Chronic RfD (mg/kg-
day)
6 x 10"3
Confidence Levels for the Derivation of the RfD for 1,2,4-TMB
A PBPK model was utilized to perform a route-to-route extrapolation to determine a
POD for the derivation of the RfD from the Korsak and Rydzynski [1996] inhalation study
and corresponding critical effect. The confidence in the study from which the critical effect
was identified, Korsak and Rydzynski [1996] is medium (see above]. Confidence in the
database for 1,2,4-TMB is low to medium as the database includes acute, short-term,
subchronic, and developmental toxicity studies in rats and mice. The database lacks a
multigenerational reproductive study, and the studies supporting the critical effect
predominately come from the same research institute. Overall confidence in the RfD for
1,2,4-TMB is low due to uncertainties surrounding the application of the available PBPK
model for the purposes of a route-to-route extrapolation.
Oral Reference Dose (RfD) for 1,3,5-TMB
The oral database for 1,3,5-TMB includes no chronic, subchronic, or short-term oral
exposure studies. However, as determined for the RfC derivation for 1,3,5-TMB, the
toxicokinetic and toxicological similarities between 1,3,5-TMB and 1,2,4-TMB demonstrate
sufficient similarity between the two isomers to support adopting the RfD for 1,2,4-TMB for
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the RfD for 1,3,5-TMB. In addition to the previously discussed similarities in toxicokinetics,
the qualitative metabolic profiles for the two isomers are similar to such a degree that first-
pass metabolism through the liver is not expected to differ greatly between 1,2,4-TMB and
1,3,5-TMB. Therefore, the chronic RfD of 6 x 10-3 mg/kg-day derived for 1,2,4-TMB
was adopted as the RfD for 1,3,5-TMB based on the determination of sufficient
similarity regarding toxicokinetics and toxicity between the two isomers.
Confidence Levels for the Derivation of the RfD for 1,3,5-TMB
As noted previously, a confidence level of high, medium, or low is assigned to the study
used to derive the RfD, the overall database, and the RfD itself, as described in EPA (1994}.
Section 4.3.9.2. The chronic RfD of 6 x 10-3 mg/kg-day derived for 1,2,4-TMB was adopted
as the RfD for 1,3,5-TMB based on the conclusion that the two isomers were sufficiently
similar regarding chemical properties, kinetics, and toxicity. Thus, confidence in the study
from which the critical effect was identified, Korsakand Rydzynski (1996} is medium (see
above}. Confidence in the database is low to medium as the database includes acute, short-
term, and developmental toxicity studies in rats and mice. The database lacks a
multigenerational reproductive study, and the studies supporting the critical effect
predominately come from the same research institute. Overall confidence in the RfD for
1,3,5-TMB is low due to uncertainties surrounding the adoption of the RfD derived for
1,2,4-TMB as the RfD for 1,3,5-TMB.
Evidence for human carcinogenicity
No chronic inhalation studies that investigated cancer outcomes were identified in the
literature for 1,2,4-TMB or 1,3,5-TMB. One oral cancer study was found in which rats were
exposed via oral gavage to one experimental dose of 800 mg/kg-day (Maltoni etal.. 1997}.
This study observed marginal increases in total malignant tumors and head tumors (e.g.,
neuroesthesioepithelioma} and provided no statistical analyses of results. A number of
methodological issues limit the utility of this study (only one dose group, no discussion of
histopathological analyses}. When Fisher's exact test was performed by EPA on the
incidences calculated from the reported percentages of animals bearing tumors in the
control and 800 mg/kg dose groups no statistically significant associations were observed.
Therefore, in accordance with the Guidelines for Carcinogen Risk Assessment (U.S. EPA.
2005aj. the database for 1,2,4-TMB was deemed to provide inadequate information to
assess carcinogenic potential, and thus, no cancer values for 1,2,4-TMB are derived in this
document. No studies of carcinogenicity were available for 1,3,5-TMB and thus the
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database was deemed inadequate to assess carcinogenic potential; no cancer values were
derived for 1,3,5-TMB.
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LITERATURE SEARCH STRATEGY AND STUDY
EVALUATION
Literature Search Strategy and Study Selection
The literature search strategy employed for 1,2,4-TMB and 1,3,5-TMB was conducted
with the keywords listed in Table VI. Primary, peer-reviewed literature identified through
December 2011 was included where that literature was determined to be relevant to the
assessment. Potentially relevant publications on 1,2,4-TMB and 1,3,5-TMB were identified
through a literature search conducted with the EBSCO Discovery Service feature of Health
and Environmental Research On-Line (HERO], a meta-search engine with access to
numerous databases including the Science Citation Index (SCI], Toxicology Literature
Online (TOXLINE), The National Library of Medicine (NLM, PubMed/Medline], and Web of
Science (WOS}. Other peer-reviewed information, including health assessments developed
by other organizations, review articles, literature necessary for the interpretation of TMB-
induced health effects, and independent analyses of the health effects data was retrieved
and included in the assessment where appropriate. A data call-in was announced by EPA in
April, 2008 and any pertinent scientific information submitted by the public to the IRIS
Submission Desk was also considered in the development of this document.
Table IV: Details of the search strategy employed for 1,2,4-TMB and 1,3,5-TMB
Databases
Keywords
Limits
EBSCO DISCOVERY
SERVICE:
HERO
SCI
NLM
TOXLINE
WOS
1,2,4-trimethylbenzene OR pseudocumene OR 95-63-6
1,3,5-trimethylbenzene OR mesitylene OR 108-67-8
Additional search terms: neurotoxicity, genotoxicity,
developmental toxicity inflammation, irritation, toxicokinetics,
pbpk, mode of action, white spirit, C9, C9 fraction, JP-8
Also, specific searches were performed on specific
metabolites: 2,4-dimethylbenzoic acid OR 611-01-8; 2,4-
dimethylhippuric acid OR 41859-41-0; 2,5-dimethylbenzoic acid
OR 610-72-0; 2,5-dimethylhippuric acid OR 41859-40-9; 3,4-
dimethylbenzoic acid OR 619-04-5; 3,4-dimethylhippuric acid OR
23082-12-4; 2,4,5-trimethylphenol OR 496-78-6; 2,3,5-
Search
constraints:
none
Last search:
December
2011
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trimethylphenol OR 697-82-5; 2,3,6-trimethylphenol OR 2416-
94-6; 2,4,6-trimethylphenol OR 527-60-6; 3,5-dimethylbenzoic
acid OR 499-06-9; 3,5-dimethylhippuric acid OR 23082-14-6
The first step in the review of the available literature is the identification of studies
pertinent to the development of the document. These references include, but are not
limited to, studies related to: toxicokinetics, toxicity, carcinogenicity, mode of action, and
physiologically-based pharmacokinetic modeling. The pertinent studies are then identified
for inclusion in the hazard identification based on a preliminary review of the overall study
design, with particular attention to the exposure route and duration. The available
epidemiological and toxicological studies are further evaluated and identified for
consideration for quantitative analysis based on a more specific evaluation of the study
design, methods, and data quality.
Approximately 2500 references were obtained from the literature searches for
1,2,4-TMB and 1,3,5-TMB including references retrieved from specific literature searches
necessary for the interpretation of TMB-induced health effects (e.g., literature on specific
modes of action, PBPK analysis}. The comprehensive, unedited list of studies captured in
the literature search can be found on the HERO website. From this full list of references,
there are 583 references that were considered for inclusion in the Toxicological Review.
From this list of "considered" references, 112 full text publications were identified as
providing relevant information for use in the development of this document.
The references that are cited in the document, as well as those that were considered but
not included in the Toxicological Review of 1,2,4-TMB and 1,3,5-TMB, can be found within
the HERO website (http(s]:hero.epa.gov/tmb}. This site contains HERO links to lists of
references, including bibliographic information and abstracts, which were considered for
inclusion in the Toxicological Review of 1,2,4-TMB and 1,3,5-TMB. This document is not
intended to be a comprehensive treatise on the chemical or toxicological nature of 1,2,4-
TMB and 1,3,5-TMB.
Study Evaluation for Hazard Identification
In general, the quality and relevance of health effects studies were evaluated as outlined
in the Preamble to this assessment. In addition, A Review of the Reference Dose and
Reference Concentration Processes [U.S. EPA. 2002) and Methods for Derivation of
Inhalation Reference Concentrations and Application of Inhalation Dosimetry [U.S. EPA.
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1994) were consulted for guidance in evaluating scientific quality of the available studies.
All studies that were considered to be of acceptable quality, whether yielding positive,
negative, or null results, were considered in assessing the totality of the evidence for health
effects in humans. The hazard identification analyses for each health endpoint in Section 1
discuses the breadth of the available literature and the extent to which the studies
informed the conclusions concerning hazard. The available studies examining health
effects of 1,2,4-TMB and 1,3,5-TMB exposure in humans (three occupational exposure
studies, one cross-section residential study, and two controlled experiments of acute
exposures] are discussed and evaluated in the hazard identification sections of the
assessment (Section 1], with specific limitations of individual studies and of the collection
of studies noted. The evaluation of the effects seen in the experimental animal studies
focuses on the available acute, short-term, subchronic, and developmental toxicity studies,
as no chronic inhalation exposure studies were found and the only identified chronic oral
exposure study did not include data on effects other than cancer.
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1. HAZARD IDENTIFICATION
1.1. Synthesis of Major Toxicological Effects
1.1.1. Neurotoxic Effects
There is evidence in humans and animals that inhalation exposure to TMBs induces
neurotoxic effects. Occupational exposure studies in humans provide evidence of
neurotoxicity following inhalation exposure to complex volatile organic compound (VOC)
mixtures containing 1,2,4-TMB and 1,3,5-TMB. Prevalence rates of neuropsychological
symptoms increased with exposure duration in dockyard painters [Chen etal.. 1999):
similarly, a significant positive association between 1,2,4-TMB exposure and exposure
symptoms (e.g., abnormal fatigue] was reported in asphalt workers (Norseth etal.. 1991}.
Nervousness, tension, headaches, vertigo, and anxiety were reported in paint shop workers
exposed to 49-295 mg/m3 of a solvent mixture containing 50% 1,2,4-TMB and 30%
1,3,5-TMB (Battig et al. (1956). as reviewed by [MOE. 2006)). Increased fatigue, decreased
vigor, and increased reaction time were noted in controlled, acute volunteer studies in
which humans were exposed to mixtures containing 1,2,4-TMB (Lammers etal.. 2007).
although it is unclear whether 1,2,4-TMB or other constituents within the mixtures were
responsible for the observed effects. Uptake of 1,2,4-TMB and 1,3,5-TMB was reported in
volunteers exposed for 2 hours to 11 or 123 mg/m31,2,4-TMB, or 123 mg/ m31,3,5-TMB;
however, effects on the CNS, based on measures of overt CNS depression (heart rate during
exposure and pulmonary ventilation) and a subjective rating of CNS symptoms (data not
reported), were not observed (Jarnbergetal.. 1996). The Jarnberg et al. (1997a: 1996]
studies are limited for evaluating neurotoxicity to TMBs due to a lack of methods to
adequately assess CNS function and lack of no-exposure controls, short exposure duration,
and exposure of individual subjects to multiple, different concentrations of TMB isomers
and/or mixtures containing TMBs.
In animals, there is consistent evidence of neurotoxicity following inhalation exposure
to either 1,2,4-TMB or 1,3,5-TMB. Decreased pain sensitivity has been observed following
inhalation exposure to 1,2,4-TMB and 1,3,5-TMB in multiple studies conducted in male
Wistar rats. To test pain responses following TMB exposure, animal studies have employed
the hot plate test. In this test, a thermal stimulus is applied to determine pain sensitivity, as
indicated by the animals' latency to paw-lick following introduction of the stimulus.
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Decreases in pain sensitivity have been observed at concentrations > 492 mg/m3 following
subchronic and short-term exposure to 1,2,4-TMB (Wiaderna etal.. 2002: Gralewicz and
Wiaderna. 2001: Korsak and Rydzynski. 1996} and short-term exposure to 1,3,5-TMB
(Wiaderna et al.. 2002: Gralewicz and Wiaderna. 2001}. In the subchronic study (Korsak
and Rydzynski. 1996}. 1,2,4-TMB inhalation resulted in reduced pain sensitivity which
occurred in a concentration-dependent manner. In short-term studies that examined a
range of concentrations [Wiaderna etal.. 2002: Gralewicz etal.. 1997aj these decreases in
pain sensitivity following exposure to 1,2,4-TMB or 1,3,5-TMB were non-monotonic.
Differences in experimental design may account for the lack of monotonicity in these short-
term studies, in contrast to the observations in Korsak and Rydzynsky, (1996}. Similar to
the subchronic study, acute exposures to 1,2,4-TMB or 1,3,5-TMB induced concentration-
dependent decreases in pain sensitivity, with ECso values of 5,682 and 5,963 mg/m3,
respectively (Korsak and Rydzynski. 1996: Korsak etal.. 1995}.
A second, somewhat different measure of pain sensitivity was reported in studies
evaluating performance in the hot plate test (before and after footshock} several weeks
following short-term, inhalation exposure to 1,2,4-TMB or 1,3,5-TMB (Wiaderna et al..
2002: Gralewicz and Wiaderna. 2001: Gralewicz etal.. 1997aj. In these studies, treatment-
related, statistically significant changes in pain sensitivity at > 492 mg/m31,2,4-TMB or
1,3,5-TMB were observed 24 hours after rats were given a footshock; no statistically
significant effects at any exposure concentration were observed prior to or immediately
following footshock. These findings indicate that inhalation exposure to TMBs may prolong
footshock-induced reductions in pain sensitivity. It is also plausible that an amplification of
responses associated with classically conditioned analgesia (i.e., decreased pain sensitivity}
occurs following 1,2,4-TMB and 1,3,5-TMB exposure. Specifically, footshock can cause
contextual cues (e.g., hot plate test} to become associated with the noxious stimulus
(footshock}, inducing stress or fear-related responses in the shocked animal such that,
subsequently, both footshock itself as well as the contextual cues associated with
footshock, can reduce sensitivity to pain (possibly via the release of endogenous opiods}.
Thus, exposure to the hot plate apparatus immediately following footshock may associate
this test environment with the footshock, such that subsequent re-exposure to the hot plate
apparatus can, itself, produce analgesia. From the data available, the relative contribution
of these behaviors to the observed effects cannot be easily distinguished.
The decreases in pain sensitivity measured in the subchronic and acute studies were
observed immediately after exposure, with no significant effects persisting two weeks after
exposures were terminated (Korsak and Rydzynski. 1996: Korsak et al.. 1995}. In contrast,
performance in the hot plate test was significantly impaired following short-term exposure
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to either 1,2,4-TMB or 1,3,5-TMB when tested 50-51 days after exposure (Wiaderna et al..
2002: Gralewicz and Wiaderna. 2001: Gralewicz etal.. 1997a). indicating a persistence of
these effects. The ability of male Wistar rats to respond to a thermal stimulus in the hot
plate test was consistently impaired following inhalation exposure to TMBs. In these
studies, 1,2,4-TMB and 1,3,5-TMB similar in their capacity to decrease pain sensitivity
(Table 1-1}. Pain sensitivity was not examined following oral exposure.
Human exposures to solvent mixtures containing 1,2,4-TMB (Lammers etal.. 2007) or
1,2,4-TMB and 1,3,5-TMB (Battig etal.. 1956). as reviewed by MOE (2006) suggest possible
effects on the neuromuscular system, as effects reported included increased reaction time
and vertigo, respectively. Animal studies using rotarod performance, which tests motor
coordination, balance, and overall neuromuscular function, indicate that inhalation of 1,2,4-
TMB or 1,3,5-TMB can affect neuromuscular system function. Significant decreases in
rotarod performance were observed at 1230 mg/m31,2,4-TMB when tested immediately
after exposure for either 8 or 13 weeks (Korsakand Rydzynski. 1996). This impaired
function was still evident at 2 weeks post-exposure and, while not statistically significant,
may indicate long-lasting neuromuscular effects of subchronic exposures to 1,2,4-TMB.
Acute inhalation exposure studies support this observation. Effects such as loss of reflexes
and righting responses have been observed following acute inhalation exposure to 1250-
45,000 mg/m3 of 1,2,4-TMB (MOE. 2006: Henderson. 2001). Similarly, acute exposure to
1,2,4-TMB or 1,3,5-TMB resulted in decreased performance in rotarod tests immediately
following exposure, with ECso values of 4693 mg/m3 and 4738 mg/m3, respectively
(Korsak and Rydzynski. 1996: Korsaketal.. 1995): these results indicate the 1,2,4-TMB and
1,3,5-TMB may be similar in their ability to impair neuromuscular function, balance, and
coordination following acute inhalation exposure. No studies evaluating oral exposure to
TMB isomers address this endpoint.
The neurobehavioral tests administered (i.e., hot plate and rotarod) in the subchronic
and acute studies by Korsak and Rydzynsky, (1996) and Korsak et al. (1995) appear to
have been administered on the same days; however, it is unclear whether the tests were
performed sequentially in the same cohorts of animals. Performing the hot plate test
immediately following the rotarod test could introduce a potential confounder, as shock
alone (such as that used as negative reinforcement following rotarod failure) can cause
reductions in pain sensitivity. Thus, if the tests were performed sequentially in the same
animals, TMB-exposed animals failing more often in the rotarod test may exhibit increases
in paw-lick latency unrelated to treatment, as compared to controls receiving less shock
reinforcement. However, the observations by Korsak and Rydzynsky, (1996) and Korsak et
al. (1995) are supported by 2.8 and 2.9-fold increases in latency to paw-lick that, although
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not statistically significant, were observed weeks subsequent to short-term exposures to
492 mg/m31,2,4-TMB or 1,3,5-TMB fGralewicz and Wiaderna. 20011
Effects in open field testing have been consistently reported in oral and inhalation
studies of exposure to 1,2,4-TMB and 1,3,5-TMB in male rats. Altered behaviors in open
field tests can involve contributions not only from elevated anxiety due to open spaces and
bright light but also from changes in motor function. Decreased anxiety and/or increased
motor function at > 492 mg/m31,2,4-TMB or 1,3,5-TMB has been reported in short-term
studies, as evidenced by increases in horizontal locomotion or grooming activities
(Gralewicz and Wiaderna. 2001: Gralewicz etal.. 1997a}. Statistically significant increases
in horizontal locomotion were observed in short-term studies assessing open field
behavior following inhalation exposure to either isomer [Gralewicz and Wiaderna. 2001).
Non-monotonic increases in grooming were reported following short-term exposure to
1,2,4-TMB (with a statistically significant increase only in the mid-exposure group};
changes in horizontal locomotion were not statistically significant (increases of 3-35%
were non-monotonic) (Gralewicz etal.. 1997a). As open field testing was conducted 25
days after termination of exposure in these studies, the results suggest latency for the
effects on anxiety and/or motor function.
Slight increases in locomotor activity were also observed in open field tests
immediately following acute, oral exposure to 1,2,4-TMB or 1,3,5-TMB. Significant
increases were observed at 3850 mg/kg for 1,2,4-TMB and at > 1,920 mg/kg for 1,3,5-TMB,
with minimal dose-effect or time-effect relationships and negligible differences in the
magnitude of the change in activity between isomers (Tomas et al., 1999b). The study
authors attributed these changes to discomfort arising from difficulty in breathing and/or
maintaining balance due to the onset of motor ataxia; notably, by 90 minutes following
exposure, the rats were reported to be completely inactive and several rats in the high dose
group died within 24 hours (Tomas etal.. 1999b). Open field tests cannot easily distinguish
between anxiety-related responses and changes in motor activity. However, effects on
motor activity were observed following inhalation exposure to elevated concentrations of
TMBs in several acute studies, although the results are somewhat inconsistent with
observations in open field tests. Decreased motor activity was observed in male rats
immediately after exposure at 5000 mg/m31,2,4-TMB (McKee etal.. 2010). Decreased
motor activity was also reported in rats acutely exposed to a mixture containing TMB
isomers (Lammers etal.. 2007). but the use of a mixture precludes a determination of the
toxicity specifically associated with 1,2,4-TMB or 1,3,5-TMB. As biphasic changes in
activity are frequently observed following exposures to solvents, it is likely that the timing
of the evaluations conducted in the short-term versus acute studies may influence the
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consistency of these results. Overall, exposure to 1,2,4-TMB and 1,3,5-TMB affects anxiety
and/or motor function at concentrations above 492 mg/m3, although the exact, potentially
biphasic, concentration-response relationship remains unclear.
Cognitive function following exposure to 1,2,4-TMB or 1,3,5-TMB has not been
evaluated in humans or following oral exposure in animals; exposure of human volunteers
to mixtures containing TMBs did not indicate any effects on short-term learning and
memory tests (Lammers etal.. 2007). Similarly, short-term spatial memory (radial maze
performance] was unaffected by exposure to either TMB isomer in animal studies
(Wiaderna etal.. 2002: Gralewicz and Wiaderna. 2001: Gralewicz etal.. 1997a}. In contrast,
effects on cognitive function in different neurobehavioral tests, observed as altered
conditioning behaviors, were consistently observed in multiple studies in male rats weeks
following short-term inhalation exposure to 1,2,4-TMB or 1,3,5-TMB, although clear
concentration-effect relationships were not observed. Comparing the results of the
behavioral tests reveals that there are differences in these neurological effects reported for
1,2,4-TMB and 1,3,5-TMB, as well as differences in the exposure concentrations at which
the cognitive effects were observed. Decreased step-down latency in passive avoidance
tests 35-45 days after short-term inhalation exposure to > 492 mg/m31,2,4-TMB or > 123
mg/m31,3,5-TMB was observed in treated male rats (Wiaderna etal.. 2002: Gralewicz and
Wiaderna. 2001: Gralewicz etal.. 1997a}: decreases may be attributed to a reduced ability
to inhibit motor reactions (or a lowered motor threshold] in response to stress. These
responses were consistently observed and similar in magnitude across all studies at 7 days
post footshock. Statistically significant changes were not observed < 24 hours following
footshock and were not consistently observed 3 days following footshock, suggesting that
1,2,4-TMB and 1,3,5-TMB exposure may affect adaptive behaviors associated with the
persistence of stress or fear-related responses. Reduced active avoidance learning was also
observed in male rats following short-term inhalation exposure to 492 mg/m31,2,4-TMB
(Wiaderna etal.. 2002: Gralewicz and Wiaderna. 2001]: however, these changes were not
observed in the other 1,2,4-TMB short-term study (Gralewicz etal.. 1997a]. Decreased
performance in active avoidance tests was consistently observed following short-term
exposure to > 492 mg/m31.3.5-TMB(Wiaderna etal.. 2002: Gralewicz and Wiaderna.
20011 Similar to 1,2,4-TMB fWiaderna etal.. 2002: Gralewicz and Wiaderna. 20011 the
effects of 1,3,5-TMB were particular to the learning component of the test (acquisition
training], rather than the memory component (retention session 7 days later]. It is unclear
whether potential TMB-induced alterations in locomotor activity would affect performance
in these tests. Acute inhalation exposure studies provide some support for the observed
effects of TMBs on learned behaviors. Significant increases in response latency in
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psychomotor tasks, observed immediately after exposure (effects did not persist to 24
hours later], were reported in male rats following acute exposure to 5000 mg/m31,2,4-
TMB (McKee et al., (2010}} or to 4800 mg/m3 of a mixture containing TMBs (Lammers et
al.. 2007}. The effects on active and passive avoidance behaviors indicate that learning
and/or long-term memory processes are affected by exposure to 1,2,4-TMB or 1,3,5-TMB.
The data suggest that 1,3,5-TMB may be a more potent inducer of toxic effects on cognitive
function than 1,2,4-TMB, as the effects following exposure to 1,3,5-TMB occurred at lower
concentrations (123 mg/m3 vs. 492 mg/m3, were of greater intensity and occurred earlier
than those reported following exposure to 1,2,4-TMB.
Controlled human exposure studies suggest that exposures of < 123 mg/m31,2,4-TMB
or 1,3,5-TMB do not cause overt CNS depression (larnberg et al.. 1996}. although
symptoms related to this effect (e.g., lightheadedness, fatigue} have been reported in
workers occupationally exposed to mixtures containing TMBs. In animals, CNS depression
has been observed following acute inhalation exposure to 25,000-44,000 mg/m31,3,5-TMB
(ACGIH. 2002}. Neurophysiological evidence from short-term inhalation studies, as well as
supportive evidence from acute oral and injection studies, suggests that exposures to 1,2,4-
TMB or 1,3,5-TMB at lower concentrations (at least for 1,2,4-TMB} may affect parameters
associated with brain arousal. Concentration-dependent decreases (statistically significant
at 1230 mg/m3} in electrocortical arousal (i.e., spike-wave discharge activity in recordings
from cortical-hippocampal electroencephalograms, EEGs} were observed in male rats 120
days after short-term exposure to > 492 mg/m31,2,4-TMB, suggesting persistent functional
changes in the rat CNS (Gralewicz etal.. 1997b}. In recordings from rats that were awake,
but immobile (not exhibiting pronounced exploratory activity, as determined by EEC
morphology}, statistically significant decreases in spike wave discharge activity were
observed at 24 hours following short-term exposure to 492 mg/m31,2,4-TMB (Gralewicz
etal.. 1997b}. Dose-related decreases in spike wave discharges were observed at > 240
mg/kg 1,2,4-TMB or 1,3,5-TMB subsequent to acute oral exposure (Tomas etal.. 1999a}:
stronger and more persistent effects on electrocortical arousal were observed following
oral exposure to 1,3,5-TMB compared with 1,2,4-TMB (Tomas etal.. 1999a}. Similar effects
were observed followingi.p. injection of 1,2,4-TMB and 1,3,5-TMB (Tomas etal.. 1999c}.
The observed EEC abnormalities following inhalation and oral exposure to 1,2,4-TMB or
1,3,5-TMB provide supportive evidence of the CNS depressant effects of these compounds
(Gralewicz etal.. 1997b}.
A summary of the observed neurotoxicity for 1,2,4-TMB and 1,3,5-TMB is shown in
Tables 1-1 and 1-2 for inhalation and oral exposures, respectively.
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Table 1-1: Summary of observed in vivo neurotoxicity in subchronic and short-term
studies of male Wistar rats following inhalation exposure to 1,2,4-TMB or 1,3,5-TMB
Health Effect
Study Design and Reference
Results
Lowest Level
at which
Significant
Effects were
observed
(mg/m3)3
1,2,4-TMB
Decreased Pain
Sensitivity
0, 123, 492, 1,230 mg/m
recovery (1,230 mg/ m3 at 2
weeks post-exposure)
90 days; 10/group
Korsak & Rydzynsky (1996)
0, 492 mg/m3
4 weeks; 11/group
Gralewicz & Wiaderna (2001)
0, 123, 492, 1,230 mg/m3
4 weeks; 15/group
Gralewicz et al. (1997a)
Exposure-dependent increases in paw-lick
latency which recover by two weeks post-
exposure.
Response relative to control: 0,18, 79*, 95*%
(recovery= 12%)
Increased paw-lick latency 24 hours after
intermittent footshockb.
Response relative to control: 0,191*%
Non-monotonic increases in paw-lick latency
24 hours after intermittent footshockb.
Response relative to control: 0, 9, 61*, 46*%
492
492
492
Impaired
Neuromuscular
Function and
Coordination
0, 123, 492, 1,230 mg/m
recovery (1,230 mg/m3 at 2
weeks post-exposure)
90 days; 10/group
Korsak & Rydzynsky (1996)
Exposure-dependent increases in rotarod
failures which do not recover by two weeks
post exposure.
Response relative to control: 0,10, 20, 40*%
(recovery= 30%)
1,230
Decreased Anxiety
and/or Increased
Motor Function
0, 492 mg/m
4 weeks; 11/group
Gralewicz & Wiaderna (2001)
Increased horizontal locomotion in open field
activity tests.
Response relative to control: 0, 62*%
0, 123, 492, 1,230 mg/m
4 weeks; 15/group
Gralewicz et al. (1997a)
Non-monotonic increases in grooming in open
field activity tests at middle concentration; no
change in horizontal locomotion.
Response relative to control: 0, 82,147*, 76%
492
492
Altered Cognitive
Function
0, 492 mg/m
4 weeks; 11/group
Gralewicz & Wiaderna (2001)
Decreased step down latency in passive
avoidance tests and decreased performance in
active avoidance tests.
Response relative to control: 0, 43*%c; 0,
60*%d
0,123, 492, or 1,230 mg/m
4 weeks; 15/group
Gralewicz et al. (1997a)
Non-monotonic decreases in step down latency
in passive avoidance tests.
Response relative to control: 0, 21, 81*, 49*%c;
492
492
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Decreased Cortico-
Hippocampal
Activity
0, 123, 492, 1,230 mg/m3
4 weeks; 9/group
Gralewicz et al. (1997b)
0,30, 27, 34% e
Non-monotonic decreases in spike-wave
discharges at 24 hours post-exposure (EEG) at
middle concentration.
Response relative to control: 0, 31, 64*, 19%
492
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Decreased Pain
Sensitivity
0, 492 mg/m
4 weeks; 11/group
Gralewicz & Wiaderna (2001)
1,3,5-TMB
Increased paw-lick latency 24 hours after
intermittent footshockb.
Response relative to control: 0, 250*%
0, 123, 492, 1,230 mg/m3
4 weeks; 12/group
Wiaderna et al. (2002)
Non-monotonic increases in paw-lick latency
24 hours after intermittent footshock at middle
concentration11.
Response relative to control: 0, 4, 70*, 17%
492
492
Decreased Anxiety
and/or Increased
Motor Function
0, 492 mg/m
4 weeks; 11/group
Gralewicz & Wiaderna (2001)
Increased horizontal locomotion in open field
activity tests.
Response relative to control: 0, 70*%
492
0, 123, 492, 1,230 mg/m
4 weeks; 12/group
Wiaderna et al. (2002)
Altered Cognitive
Function
Non-monotonic decreases in step down latency
in passive avoidance tests and in performance
in active avoidance tests.
Response relative to control: 0, 48*, 55*, 46*%c
; 0, 40*, 35*, 50*%d
0, 492 mg/m3
4 weeks; 11/group
Gralewicz & Wiaderna (2001)
Decreased step down latency in passive
avoidance tests and decreased performance in
active avoidance tests.
Response relative to control: 0, 57*%c;
0, 70*%d
123
492
* Significantly different from controls
For studies other than Korsak and Rydzynsky, 1996, % change from control calculated from digitized data using Grablt
software
a Lowest effect level at which statistically significant changes were observed
b This effect was only observed 24 hours following intermittent foot shock; no significant effects at any
exposure were observed prior to or immediately following foot shock
c Decreased step down latency in passive avoidance tests at 7 days post footshock
Increased number of trials to reach avoidance criteria
e Decreased avoidance response % in trials 25-30 of training
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Tables 1-2: Summary of observed in vivo neurotoxicity for 1,2,4-TMB and 1,3,5-TMB
— oral exposures
Health Effect
Study Design and Reference
Results
Lowest Level
at which
Significant
Effects were
observed
(mg/kg)a
1,2,4-TMB
Decreased Cortico-
Hippocampal
Activity
Decreased Anxiety
and/or Increased
Motor Function
Decreased Cortico-
Hippocampal
Activity
240, 960, 3,850 mg/kg, single
oral gavage
Rat, Wag/Rij, male, 18/group
Tomas et al. (1999a)
960, 1920, 3850 mg/kg single
oral gavage
Rat, Wag/Rij, male, 10/group
Tomas et al. (1999b)
790 mg/kg, single i.p injection
Rat, Wistar, Male, 4
Tomas etal. (1999c)
Inhibition of the number and duration of high
voltage spindle activity in the hippocampus and
cortex.
Response relative to control: All doses
produced differences from control during at
least one of the measured time points
Slight increase in spontaneous locomotor
activity in open field test.
Response relative to control: Significant
difference from control reported at 3,850
mg/kg when data were considered by time
points (i.e., dose x time interaction)
Significant differences in hippocampal and
cortical brain wave amplitude following
injection.
Response relative to control: cortical wave
amplitude decreased up to 6.5%; hippocampal
wave amplitude decreased up to 59.6%
240
3,850
790
1,3,5-TMB
Decreased Cortico-
Hippocampal
Activity
Decreased Anxiety
and/or Increased
Motor Function
Decreased Cortico-
Hippocampal
Activity
240, 960, 3,850 mg/kg, single
oral gavage
Rat, Wag/Rij, male, 18/group
Tomas et al. (1999a)
960, 1920, 3850 mg/kg single
oral gavage
Rat, Wag/Rij, male, 10/group
Tomas et al. (1999b)
790 mg/kg, single i.p injection
Rat, Wistar, Male, 4
Tomas et al. (1999c)
Inhibition of high voltage spindle activity in the
hippocampus and cortex.
Response relative to control: All doses
produced differences from control during at
least one of the measured time points
Slight increase in spontaneous locomotor
activity in open field test.
Response relative to control: Significant
difference from control reported at 1,920 and
3,850 mg/kg
Significant differences in hippocampal and
cortical brain wave amplitude following
injection.
Response relative to control: cortical wave
amplitude decreased up to 13.9%;
hippocampal wave amplitude decreased up to
38%
240
1920
790
* Significantly different from controls
a Lowest effect level at which statistically significant changes were observed
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> Mode of Action Analysis for neurotoxicity
The observation of neurotoxicity following acute-, short-term-, and subchronic-
duration exposure to TMB (Lammers etal.. 2007: Wiaderna etal.. 2002: Gralewicz and
Wiaderna. 2001: Wiaderna et al.. 1998: Gralewicz et al.. 1997a: Gralewicz et al.. 1997b:
Korsak and Rydzynski. 1996: Korsaketal.. 1995} may indicate that TMB perturbs normal
neurotransmission in exposed animals (see Table 1-3], although the specific key events
necessary for TMB-induced neurotoxicity are not established. Although limited
mechanistic data for TMBs exists, structurally similar compounds like toluene and xylene
have been more thoroughly characterized and it is hypothesized that TMBs would operate
through a similar mechanism in producing the resultant neurotoxicological effect.
Aromatic hydrocarbons are known to interact with catecholaminergic systems [Kyrklund.
1992). Inhalation exposures to toluene and xylene have been shown to significantly change
concentration and turnover rate of both dopamine and norepinephrine in various regions
of the rat brain (Rea etal.. 1984: Andersson etal.. 1983: Andersson etal.. 1981: Andersson
etal.. 1980). These changes have been hypothesized to be due to potential metabolites
with affinity to catecholamine receptors that would, in turn, influence the uptake and
release of neurotransmitters [Andersson etal.. 1983: Andersson etal.. 1981: Andersson et
al.. 1980).
Catecholaminergic changes with toluene have been reported and are similar to that
observed with TMBs which would therefore increase the plausibility that the mechanisms
of neurotoxicity are similar between the two compounds. For example, subchronic
inhalation exposures of rats to low concentrations of toluene (as low as 80 ppm [300
mg/m3]) have been shown to decrease spatial learning and memory, increase dopamine-
mediated locomotor activity, increase in the number of dopamine D2 receptors, and
increase dopamine D2 agonist receptor binding (Hillefors-Berglund et al.. 1995: von Euler
etal.. 1994: von Euler et al.. 1993). These effects were observed to persist up to four weeks
after the termination of the toluene exposure. Activation of the dopaminergic system may
also result in an inability to inhibit locomotor responses normally suppressed by
punishment (Jackson and Westlind-Danielsson. 1994). Direct application of dopamine to
the nucleus accumbens of rats has been observed to result in retardation of the acquisition
of passive avoidance learning at concentrations that also stimulated locomotor activity
(Braes etal.. 1984). Increases in catecholaminergic neurotransmission (through exposure
to norepinephrine or dopamine agonists) result in dose-dependent reductions in the
duration of spike wave discharges in rats (Snead. 1995: Warter etal.. 1988). These
observations and findings are in concordance with those resulting from exposure to
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1,2,4-TMB and 1,3,5-TMB fWiaderna etal.. 2002: Gralewicz and Wiaderna. 2001:
Gralewiczetal.. 1997a: Gralewicz etal.. 1997b1 fTomas etal.. 1999a: Tomas etal.. 1999d.
Additionally, with regards to toluene and related aromatic hydrocarbons, it is known that
there is direct interaction with these compounds on various ion channels (ligand and
voltage gated] that are present in the central nervous system (Bowen etal.. 2006: Balster.
1998). There is not enough information to ascertain the specific molecular sites and how
the changes correlate to the observed neurotoxicological effects. However, it is widely
believed that the interactions with the neuronal receptors in the brain (e.g. ion channels,
catecholaminergic systems] may influence these changes.
Aromatic hydrocarbons may also affect the phospholipids in the nerve cell membrane
[Andersson etal.. 1981]. Pertubation of the phospholipids on the cell membrane could
indirectly affect the binding of neurotransmitters to the catecholamine receptors and
potentially lead to alterations in receptor activity or uptake-release mechanisms. Uneven
distribution of metabolites within differing regions of the brain, or spatial variations in
phospholipid composition of nerve cell membranes may explain the differential effects
seen in regard to catecholamine levels and turnover. Based on effect levels with other
related solvents (e.g., toluene - see Balster (1998]]. it is overall hypothesized that with
TMBs there may be an initial interaction with the neuronal receptors (e.g.,
catecholaminergic systems, ion channels] followed by, at much higher exposures,
interaction with the lipid membrane when the available sites on the neuronal receptors are
completely occupied.
Additional mechanisms that may play a role in TMB neurotoxicity include production of
reactive oxygen species (ROS]. Myhre et al. (2000] found increased respiratory burst in
neutrophils after 1,2,4-TMB exposure demonstrated by fluorescence spectroscopy,
hydroxylation of 4-hydroxybenzoic acid, and electron paramagnetic resonance
spectroscopy. The authors suggest the observation of solvent-induced ROS production may
relevant to brain injury as microglia cells have a respiratory burst similar to neutrophils.
Stronger evidence of potential ROS-related mechanisms of neurotoxicity was observed in a
related study by Myhre and Fonnum (2001] in which rat neural synaptosomes exposed to
1,2,4-TMB produced a dose-dependent increase in reactive oxygen and nitrogen species
demonstrated by the formation of the fluorescence of 2'7'-dichlorofluorescein (DCF]. This
observation of ROS production in rat synaptosomes may explain the observed TMB-
induced neurotoxicity in acute, short-term, and subchronic inhalation studies.
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7s the hypothesized mode of action sufficiently supported in test animals?
The hypothesis that TMB exposure results in abnormal neurotransmission in animals is
supported by the available literature, including the observation that related methylated
aromatic compounds (i.e., toluene and xylene} perturb the catecholaminergic system and
elicit similar neurological effects as TMB isomers.
7s the hypothesized mode of action relevant to humans?
The observed neurotoxic effects in animals are relevant to humans, especially given the
observation of similar neuropsychological effects in humans exposed to complex solvent
mixtures containing TMB isomers.
In summary, neurotoxicity is associated with exposure to 1,2,4-TMB and 1,3,5-TMB
based on evidence in humans and animals. The information regarding neurotoxicity in
humans is limited and most of the available studies evaluating these effects involve
exposure to mixtures containing TMB isomers and not specific exposure to the individual
isomers. Additionally, none of the available studies have addressed the potential for latent
neurological effects or effects in sensitive populations. However, the available information
shows uptake of 1,2,4-TMB and 1,3,5-TMB in humans and suggests an association between
TMB exposure in humans and neurotoxic effects. The observation of neurotoxicity in
1,2,4-TMB-exposed male Wistar rats was consistent across multiple exposure
concentrations, including subchronic, short-term, and acute exposures. Similar indices of
neurotoxicity were observed in male rats exposed to 1,3,5-TMB for short-term and acute
durations. Although the oral database is limited, similar effects were observed (e.g., EEC;
open field] across inhalation and oral study paradigms.
All of the available animal studies were conducted in male rats (Wistar or Wag/Rij} and
by the same research group (The Nofer Institute of Occupational Medicine, Lodz Poland}.
No chronic studies are available, although there is consistent evidence of neurotoxicity
following inhalation and oral exposure to 1,2,4-TMB and 1,3,5-TMB. Most of the
neurotoxicity tests incorporated the application of footshock which could involve multiple
neurological functions. The spectrum of effects suggests that TMBs affect multiple, possibly
overlapping, CNS systems rather than a single brain region or neuronal nuclei (suggested
by the solvent activity of the compounds}. Almost all tests (other than pain} involve a
contributing component of motor system function. Some endpoints exhibited clear
exposure-response relationships (e.g., pain sensitivity and rotarod, although the former
was not consistent across studies with different experimental design (i.e., varying exposure
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durations and timing of endpoint analyses}. Other endpoints did not show a clear
concentration-effect relationship, suggesting either that exposures below a threshold value
were not tested or do not exist, or that the presence of TMBs alone was sufficient to elicit a
response in these tests, possibly via irritation or stress-related phenomena. However,
irritation is highly unlikely given the latency between the exposures and the effects.
Multiple neurotoxic effects were observed weeks to months after cessation of
inhalation exposure despite rapid clearance of these chemicals from blood and CNS tissues
(see Appendix A], indicating that these effects are persistent. Although the reported human
symptoms do not directly parallel the animal data, some similar effects were observed in
both humans and rats. The majority of the neurotoxicity evidence available for TMBs was
observed in laboratory animals as neurobehavioral effects including decreased pain
sensitivity, impaired neuromuscular function and coordination, altered cognitive function,
and decreased anxiety and/or increased motor function; and neurophysiological effects
including decreased cortico-hippocampal activity. These effects are recognized in the U.S.
EPA's Guidelines for Neurotoxicity Risk Assessment (U.S. EPA, 1998} as possible indicators of
neurotoxicity. EPA considered the neurotoxic effects to be biologically plausible and
analogous to effects that could occur in humans; and concluded that the available evidence
for 1,2,4-TMB and 1,3,5-TMB identified neurotoxicity as a toxicity hazard.
1.1.2. Respiratory Effects
There is evidence in humans and animals that inhalation exposure to TMBs induces
respiratory toxicity. Occupational and residential exposure studies in humans provide
evidence of respiratory toxicity following inhalation exposure to complex VOC mixtures
containing 1,2,4-TMB and 1,3,5-TMB. While controlled human exposures (lones et al..
2006: Jarnbergetal.. 1997a: Jarnberg et al.. 1996} have failed to observe substantial
irritative symptoms following acute (less than 4 hours} inhalation exposures of up to 25
ppm (123 mg/m3} 1,2,4-TMB or 1,3,5-TMB, occupational exposures have been shown to be
associated with increased measures of sensory irritation, such as laryngeal and/or
pharyngeal irritation (Norseth etal.. 1991} and asthmatic bronchitis (Battig et al.. 1956}.
as reviewed in (MOE. 2006}. Residential exposures have demonstrated significant
associations between 1,2,4-TMB and asthma (Billionnet et al.. 2011}. However, these
studies evaluated TMB exposures occurring as exposures to complex solvent or VOC
mixtures, thereby precluding a determination of the ultimate etiological agent.
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In animals, there is consistent evidence of respiratory toxicity following inhalation
exposure of rodents to 1,2,4-TMB (Table 1-3}. Markers of pulmonary inflammation and
irritation in the lungs of rats have been observed following subchronic inhalation
exposures of Wistar rats to 1,2,4-TMB. Increases in populations of immune and
inflammatory cells in bronchoalveolar lavage (BAL) fluid have been observed at
concentrations > 123 mg/m3 following subchronic exposures of male Wistar rats to 1,2,4-
TMB [Korsak et al.. 1997). Specifically, the amount of cells in the BAL fluid of exposed rats
was increased for total cells (> 123 mg/m3, increased 2.3-3.0 fold] and macrophages (> 492
mg/m3, increased 2.1-2.7 fold}. However, some attenuation of these effects was observed
at high concentrations (i.e., 1230 mg/m3} compared to lower doses. For example, the
number of macrophages was increased 2.7-fold relative to control at 492 mg/m3, but only
2.2-fold at 1,230 mg/m3. This may indicate either adaptation to the respiratory irritation
effects of 1,2,4-TMB during exposure, saturation of metabolic pathways, or immune
suppression at higher doses. Subchronic exposure of male Wistar rats also significantly
increased the BAL numbers of polymorphonuclear leukocytes and lymphocytes; however
the specific exposure concentrations eliciting these significant increases were not reported
by study authors. A small, but not significant, decrease in cell viability was observed at >
123 mg/m3 following subchronic exposure to 1,2,4-TMB (Korsak et al.. 1997}.
In addition to increased populations of immune and inflammatory cells,
histopathological alterations described as peribronchial, lung parenchymal, and
perivascular lymphocytic infiltration in the lower respiratory tract have also been observed
following subchronic exposures of 1,2,4-TMB to male and female Wistar rats (Korsak et al..
2000). Significant proliferation of peribronchial lymphatic tissue and interstitial
lymphocytic infiltrations were observed in male rats exposed to 492 mg/m3, although
trend-analysis demonstrated these increases were not concentration-dependent. The
bronchial epithelium lost its cuboidal shape in some rats with peribronchial lymphatic
proliferation, and was observed to form lymphoepithelium. Interstitial lymphocytic
infiltrations were also observed in female rats exposed to 1230 mg/m3. Although unlike
male rats, this increase in females was concentration-dependent as determined by a trend
analysis. Alveolar macrophages were increased in both sexes at 1,230 mg/m3 with trend
analysis demonstrating concentration-dependence across the entire concentration range.
However, when the incidences of all pulmonary lesions were analyzed in aggregate, trend-
analysis demonstrated significant increases in pulmonary lesions in both sexes across the
entire concentration range. Pulmonary lesions were significantly increased in males at >
492 mg/m3, but not at any exposure concentration in females. Studies on the respiratory
effects of subchronic exposures to 1,3,5-TMB were not available.
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Additional effects on clinical chemistry including increased total protein (37% increase
at both 123 and 492 mg/m3), decreased mucoprotein (13% decrease, 123 mg/m3),
increased lactate dehydrogenase (170% and 79% increase at 123 and 492 mg/m3,
respectively] and increased acid phosphatase activity (47-75% increase at > 123 mg/m3}
were observed in 1,2,4-TMB-exposure animals; suggesting pulmonary irritation or
inflammation. All of these effects also exhibited either some attenuation of effect at high
concentrations compared to lower concentrations, or no increase in effect as exposure
concentration increased. Therefore, some adaptation to the respiratory irritation effects of
1,2,4-TMB may be occurring.
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Tables 1-3: Summary of observed in vivo respiratory toxicity for 1,2,4-TMB and
1,3,5-TMB — inhalation exposures
Health Effect
Study Design and Reference
Results
Lowest Level
at which
Significant
Effects were
observed
(mg/m3)a
1,2,4-TMB
Pulmonary
inflammation/
irritation
Clinical Chemistry
Effect
Sensory Irritation
(Decreased
respiration)
123-1,230 mg/m3, 90 days
(6h/day, 5 days/week)
Rat, Wistar, male, 6-7
Korsaketal. (1997)
123-1,230 mg/m3, 90 days
(6h/day, 5 days/week)
Rat, Wistar, male, 6-7
Korsaketal. [1997]
123-1,230 mg/m3, 90 days
(6h/day, 5 days/week)
Rat, Wistar, male and female,
6-7
Korsaketal. [2000]
123-1,230 mg/m3, 90 days
(6h/day, 5 days/week)
Rat, Wistar, male, 6-7
Korsaketal. [1997]
1,245-9,486 mg/m3, single
exposure, 6 minutes
Mouse, BALB/C, male, 8-10
Korsaketal. [1997]: Korsak
etal. [1995]
Increased total bronchoalveolar cell count
with evidence of attenuation at high
exposure
Response relative to control: 0, 202*, 208*,
231*%
Increased macrophage count with evidence
of attenuation at high exposure
Response relative to control:0, 107, 170*,
116*%
Increase in number of pulmonary lesions
Response relative to control: Incidences not
reported, calculation of response relative to
control not possible; authors report
statistically significant increases at 492 and
1,230 mg/m3
Increased acid phosphatase activity with
evidence of attenuation at high exposure
Response relative to control:0, 47*, 74*, 45*%
Decreased respiratory rate as measured
during first minute of exposure
Response relative to contro/;RDso = 2,844
123
492
492
123
2,844
1,3,5-TMB
Sensory Irritation
(Decreased
respiration)
1,245-9,486 mg/m3, single
exposure, 6 minutes
Mouse, BALB/C, male, 8-10
Korsaketal. (1997)
Decreased respiratory rate as measured
during first minute of exposure
Response relative to control: RDso = 2,553
2,553
* Significantly different from controls
a Lowest effect level at which statistically significant changes were observed
Decreased respiration, a symptom of sensory irritation, has been observed in male
BALB/C mice following acute inhalation exposures either 1,2,4-TMB or 1,3,5-TMB for six
minutes. These acute exposures were observed to result in dose-dependent depression of
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respiratory rates, with the maximum decrease in respiration occurring in the first one or
two minutes of exposure [Korsak et al.. 1997: Korsaketal.. 1995). The concentration of
1,2,4-TMB and 1,3,5-TMB that was observed to result in a 50% depression in the
respiratory rate (RDso) was similar between the two isomers: 578 and 519 ppm (2,844 and
2,553 mg/m3}, respectively.
> Mode of Action Analysis for respiratory toxicity
Data regarding the potential mode of action for the respiratory effects resulting from
TMB exposure are limited and the key events for TMB-induced respiratory toxicity are not
established. However, the available toxicological data suggest that TMB isomers act as
potent acute respiratory irritants and induce inflammatory responses following longer
exposures (i.e., subchronic) in animals. The study authors (Korsaketal.. 1997: Korsak et
al.. 1995) suggested that the decreased respiratory rate is indicative of irritation, and
proposed that respiratory irritants such as TMB may activate a "sensory irritant receptor"
on the transgeminal nerve ending in the nasal mucosa leading to an inflammatory
response. Korsak et al. (1997:1995) further suggest that activation of this irritant receptor
follows either adsorption of the agonist, or adsorption and chemical reaction with the
receptor. The authors reference a proposed model for the receptor protein that includes
two main binding sites for benzene moieties and a thiol group. Further, the study authors
suggest that in the case of organic solvents (i.e., toluene, xylene, TMB) a correlation
between the potency of the irritating effect and the number of methyl groups is likely given
the observation that RDso values for depressed respiratory rates following exposure to
TMB isomers is approximately 8-fold lower than toluene and 4-fold lower than xylene.
Following subchronic exposure of rats to 1,2,4-TMB, inflammatory cell (macrophages,
polymorphonuclear leukocytes, and lymphocytes) numbers were increased along with
markers of their activation (total lactate dehydrogenase and acid phosphatase activity in
BAL) (Korsaketal.. 1997). further indicating the inflammatory nature of responses in the
respiratory tract of TMB-exposed animals. Inflammatory pulmonary lesions were also
observed following subchronic exposures in rats. However, many of these effects were not
observed to be concentration-dependent in repeated exposure studies (i.e., no progression
of effect over an order of magnitude of doses), suggesting that there may be adaptation to
respiratory irritation that occurs following extended exposure to TMB. The processes
responsible for the respiratory inflammatory responses in sub chronically exposed animal
are unknown. However, a major inflammatory mediator, interleukin 8 (IL-8), was
increases following exposure of porcine and human macrophages to secondary organic
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aerosol (SOA} particles derived from 1,3,5-TMB [Gaschen etal.. 2010). The observation
that IL-8 levels increase following exposure to 1,3,5-TMB-derived SOA is noteworthy as a
major function of IL-8 is to recruit immune cells to sites of inflammation. Therefore, the
observation of inflammatory lesions involving immune cells (i.e., macrophages, leukocytes]
may be partially explained by increases in inflammatory cytokines following TMB
exposures. Additionally, ROS-generation has been observed in cultured neutrophil
granulocytes and rat neural synaptosomes exposed to TMB [Myhre and Fonnum. 2001:
Myhre etal.. 2000}. and the related compounds benzene and toluene have been shown to
induce oxidative stress in cultured lung cells (Mogel etal.. 2011}. Although pulmonary
ROS-generation has not been observed following in vivo or in vitro TMB exposures, there is
suggestive evidence that it could play a role in the irritative and inflammatory responses
seen in exposed animals.
In a study investigating jet fuel-induced cytotoxicity in human epidermal keratinocytes
(HEK}, aromatic hydrocarbons were more potent inducers of cell death than aliphatic
constituents, even though the aromatic compounds only accounted for less than one-fourth
of aliphatic constituents (Chouetal.. 2003}. Of the single aromatic ring hydrocarbons,
1,2,4-TMB and xylene were the most lethal to HEK. Increased cytotoxicity may explain the
small, but insignificant, decrease in BAL cell viability observed in Korsak et al. (1997}.
7s the hypothesized mode of action sufficiently supported in test animals?
Data that would allow for the determination of a mode of action for respiratory effects
due to TMB exposure are limited. However, the observation of ROS generation in cultured
neutrophils and ROS generation in lung cells exposed to related aromatic compounds (i.e.,
benzene and toluene}, suggests that oxidative stress may play a role in the observed TMB-
induced respiratory effects in exposed animals.
7s the hypothesized mode of action relevant to humans?
The respiratory effects in animals are relevant to humans, especially given the
observation of irritative and inflammatory respiratory effects (e.g., asthma} in humans
exposed to complex solvent mixtures containing TMB isomers.
In summary, respiratory toxicity is associated with exposure to 1,2,4-TMB and 1,3,5-
TMB based on evidence in humans and animals. The information in humans is limited for a
number of reasons including: all studies investigating exposure to 1,2,4-TMB and 1,3,5-
TMB were conducted using a complex VOC mixture. However, the available information
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demonstrates uptake of 1,2,4-TMB and 1,3,5-TMB by humans, and suggests an association
between TMB exposure in humans and respiratory toxicity. The observation of respiratory
irritation and inflammation in Wistar rats and BALB/C mice following exposure to 1,2,4-
TMB was consistent across multiple exposure concentrations, and subchronic and acute
exposure durations. All of the available animal studies were conducted in rodents (Wistar
rats or BALB/C mice] and by the same research group (The Nofer Institute of Occupational
Medicine, Lodz Poland}. No chronic studies are available. Although some endpoints (BAL
macrophages, alkaline phosphatase] showed dose-dependence at low and mid exposure
concentrations, all effects were observed to exhibit some attenuation of effect at high
doses, potentially indicating either adaptation to the respiratory irritation effects,
saturation of metabolic and/or toxicity pathways, or immune suppression at higher doses.
Although the reported human symptoms (laryngeal and/or pharyngeal irritation,
asthmatic bronchitis, asthma] do not directly parallel the effects observed in animal
studies, the observation of irritative and/or inflammatory responses in multiple species
(including humans] demonstrates a consistency in TMB-induced respiratory toxicity. EPA
considered the observed respiratory effects in animals to be biologically plausible and
analogous to effects that could occur in humans; and concluded that the available evidence
for 1,2,4-TMB identified respiratory toxicity as a toxicity hazard.
1.1.3. Reproductive and Developmental Toxicity
There are no studies in humans that investigated the reproductive or maternal toxicity
of either 1,2,4-TMB or 1,3,5-TMB. Maternal toxicity in the form of decreased corrected
body weight (i.e., maternal body weight minus the weight of the gravid uterus] was
observed in dams following exposure during gestational exposure to 1,2,4-TMB or 1,3,5-
TMB (Saillenfait et al., 2005]. Dams exposed to 2,952 mg/m3 1,2,4-TMB gained only 50%
of the weight gained by control animals, whereas dams exposed to 2,952 mg/m3 1,3,5-TMB
gained only 25% of the weight gained by controls. Decreased maternal food consumption
(across GD 6-21] was also observed at 2,952 mg/m3 1,2,4-TMB and 1,476 mg/m3
1,3,5-TMB, although the difference compared to controls (9-13%] was modest compared to
the observed decreases in maternal weight gain. The decrease in food consumption at
1,476 mg/m3 1,3,5-TMB was determined to not be a marker of adversity given no
accompanying decrease in maternal weight gain at that concentration.
There are no studies in humans that investigated the developmental toxicity of either
1,2,4-TMB or 1,3,5-TMB. Developmental toxicity (reported as decreased fetal body weight]
has been observed in male and female rat fetuses following gestational exposure to 1,2,4-
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TMB and 1,3,5-TMB on gestational days 6 through 20 via inhalation for 6 hours a day
(Saillenfait et al., 2005} (Table 1-4}. Fetal body weights were decreased (statistically
significant} by 5-13% at concentrations of > 2,952 mg/m3 of 1,2,4-TMB and 1,3,5-TMB. No
adverse effects were noted on embryo/fetal viability and no increase in skeletal, visceral, or
external morphology (i.e., teratogenesis} was observed up to the highest concentrations for
either isomer.
Table 1-4: Summary of observed developmental toxicity for 1,2,4-TMB and
1,3,5-TMB — inhalation exposures
Health Effect
Developmental
Toxicity
Maternal Toxicity
Developmental
Toxicity
Maternal Toxicity
Study Design and Reference
0, 492, 1,476, 2,952, 4,428
mg/m3, GD 6-20 [6h/day]
Rat, Sprague-Dawley, female
&male, 24-25 dams
Saillenfait et al. [2005]
0, 492, 1,476, 2,952, 4,428
mg/m3, GD 6-20 [6h/day]
Rat, Sprague-Dawley, female
&male, 24-25 dams
Saillenfait et al. [2005]
0, 492, 1,476, 2,952, 4,428
mg/m3, GD 6-20 [6h/day]
Rat, Sprague-Dawley, female
&male, 24-25 dams
Saillenfait et al. [2005]
0, 492, 1,476, 2,952, 4,428
mg/m3, GD 6-20 [6h/day]
Rat, Sprague-Dawley, female
&male, 24-25 dams
Saillenfait et al. [2005]
Results
1,2,4-TMB
Decreased fetal body weight of male and
female fetuses
Response relative to control:
Male: 0, 1, 2, 5*, 11*%
Female: 0, 1, 3, 5*, 12*%
Decreased corrected maternal weight gain
Response relative to control: 0, +7, 7, 51*,
100*% [weight gain = 0 g]
1,3,5-TMB
Decreased fetal body weight of male and
female
Response relative to control:
Male: 0, 1, 5, 7*, 12*%
Female: 0, 1, 4, 6, 13*%
Decreased corrected maternal weight gain
Response relative to control: 0, +3, 31, 76*,
159*% [weight gain = -12 g]
Lowest Level
at which
Significant
Effects were
observed
(mg/m3)a
2,952
2,952
2,952
2,952
* Significantly different from controls
a Lowest effect level at which statistically significant changes were observed
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> Mode of Action Analysis for developmental toxicity
The mode of action for 1,2,4-TMB- and 1,3,5-TMB-induced developmental toxicity is
unknown. The database for developmental toxicity following exposure to 1,2,4-TMB is
limited to one animal study; no studies in humans are available. Although there is only one
study available, 1,2,4-TMB and 1,3,5-TMB demonstrated effects on fetal and maternal body
weights. The developmental and maternal toxicity in animals was considered by the
Agency to be biologically plausible and potentially analogous to effects that could occur in
humans. EPA concluded that the available evidence for 1,2,4-TMB and 1,3,5-TMB identified
maternal and developmental toxicity as a toxicity hazard.
1.1.4. Hematological Toxicity and Clinical Chemistry Effects
There is limited evidence in humans, and stronger evidence in animals, that exposure to
TMBs induces hematological toxicity. Alterations in blood clotting and anemia in workers
exposed to a paint solvent containing 1,2,4-TMB and 1,3,5-TMB was reported by Battig et
al. (1956). as reviewed by MOE (2006). A LOAEL of 295 mg/m3 was identified from this
study. However, as workers were exposed to a solvent mixture containing TMB isomers, it
is impossible to ascertain the ultimate contribution of either isomer to the observed health
effects.
In animals, there is evidence of hematological toxicity following subchronic exposure to
1,2,4-TMB and short-term exposure to 1,3,5-TMB (Table 1-5). Subchronic exposures to
1,2,4-TMB have been shown to result in hematological effects and changes in serum
chemistry in exposed rats (Korsak et al.. 2000). In male rats at termination of exposure,
RBC counts were decreased by 23% and WBC counts increased 180% at 1,230 mg/m3; the
observed alteration in blood cell counts were exposure concentration-dependent as
determined by trend analysis. A concentration-dependent increase in WBC count was also
observed in female rats, pair-wise comparisons of individual doses failed to reach
statistical significance at any concentration. WBC counts were observed to be slightly
decreased (18%) relative to controls two weeks after the termination of exposure, whereas
RBC counts were still decreased by 24% relative to control (although this decreased failed
to reach statistical significance). Significant decreases in reticulocytes (71% of controls)
and clotting time (37% of controls) were observed in female rats exposed to 1,230 mg/m3
and 492 mg/m3 1,2,4-TMB, respectively. Both of these effects were concentration-
dependent when analyzed over the entire range of exposure concentrations with values
60-65% greater than controls after end of exposure; animals fully recovered within two
weeks after termination of exposure. The only clinical chemistry parameter significantly
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altered was an increase in sorbitol dehydrogenase at > 123 mg/m3 in exposed male rats,
although these increases were not exposure-dependent. Sorbitol dehydrogenase activity
was also higher in exposed female rats, but the increases in activity were not significantly
higher when compared to controls.
An increase in aspartate aminotransferase, but no other substantial hematological
effects, was observed in rats 14 days following short-term exposure (6 hours/day, 6
days/week for five weeks] [Wiglusz etal.. 1975a: Wiglusz etal.. 1975b]. The adversity of
aspartate aminotransferase is unclear given the lack of a clear pattern in temporality
(effects at some days post-exposure, but not others] and the lack of accompanying liver
histopathology.
Tables 1-5: Summary of observed in vivo hematological toxicity and clinical
chemistry effects for 1,2,4-TMB and 1,3,5-TMB — inhalation exposures
Health Effect
Study Design and
Reference
Results
Lowest
Level at
which
Significant
Effects
were
observed
(mg/m3)3
1,2,4-TMB
Hematological
toxicity
123-1,230 mg/m3,90 days
(6h/day, 5days/week)
Rat, Wistar, female & male,
6-7
Korsaketal. (2000)
123-1,230 mg/m3,90 days
(6h/day, 5days/week)
Rat, Wistar, female & male,
6-7
Korsaketal. [2000]
123-1,230 mg/m3,90 days
(6h/day, 5days/week)
Rat, Wistar, female & male,
6-7
Korsaketal. f20001
123-1,230 mg/m3,90 days
(6h/day, 5days/week)
Rat, Wistar, female & male,
6-7
Korsaketal. [2000]
Decreased red blood cells in males only.
Response relative to control:0,1,15, 23*%
(recovery = 24%)
Increased white blood cells in males only.
Response relative to control:0, 2, 4, 80*%
(recovery = 18% decrease)
Decreased reticulocytes in females only.
Response relative to control:0, 51, 49,71*%
(recovery = 65% increase)
Non-monotonic decreases in clotting time in
females only.
Response relative to control:0, 23,37*,27*%
(recovery = 60% increase)
1,230
1,230
1,230
492
Clinical Chemistry
Effects
123-1,230 mg/m3,90 days
(6h/day, 5days/week)
Rat, Wistar, female & male,
6-7
Non-monotonic increases in sorbitol
dehydrogenase in males only.
Response relative to control:0, 73*, 74* 73*
123
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Korsaketal. f20001
1,3,5-TMB
Hematological
Effect
1,500-6,000 mg/m3, single
exposure, 6 hours
Samples collected 0,1, 7,14,
and 28 days post exposure
Rat, Wistar, male, 6
Wiglusz et al. [Wiglusz et al..
1975a)
Increased segmented neutrophilic
granulocytes (1-28 days post exposure).
Response relative to control: Increased across
all days of exposure.
3,000
Clinical Chemistry
Effects
3,000 mg/m3, 5 weeks (6
h/day, 6days/week)
Samples collected 1, 3, 7,14,
and 28 days during exposure
Rat, Wistar, male, 6
Wiglusz et al. [1975b]
Increased aspartate aminotransferase on day
14
Response relative to control (day 14):12*%
3,000
300-3,000 mg/m3, single
exposure, 6 hours
Samples collected 0, 2, 7,14
and 28 days post exposure
Rat, Wistar, male, 6
Wiglusz et al. fl975bj
Increased alkaline phosphatase on day 7 post-
exposure
Response relative to control (on day 7):Q, -0.1,
0.03, 84*%
3,000
* Significantly different from controls
a Lowest effect level at which statistically significant changes were observed
Acute exposures of male Wistar rats to 1,500-6,000 mg/m3 1,3,5-TMB for six hours did
not result in substantial effects on hemoglobin or RBC or WBC count [Wiglusz et al..
1975a}. However, the number of segmented neutrophilic granulocytes was increased in
1,3,5-TMB-exposed rats up to 28 days following exposure (statistics not reported}. The
greatest increase in granulocyte numbers (100%} was observed the day of exposure and
one day following in rats exposed to 6,000 mg/m3, although attenuation was seen 7-28
days following exposure, possibly indicating induction of metabolizing enzymes or
saturation of toxicity pathways. Investigation of clinical chemistry parameters in rats
acutely exposed to 300- 3,000 mg/m3 for six hours did not reveal any consistent pattern in
the levels of aspartate or alanine aminotransferases, although alkaline phosphatase was
statistically increased 84% in rats seven days following exposure to 3,000 mg/m3 (Wiglusz
etal.. 1975bj. NOAELs and LOAELs determined from these acute exposure studies are
provided in Table 1-5.
> Mode of Action Analysis for hematological toxicity and clinical chemistry
effects
The mode of action for 1,2,4-TMB-induced hematological and clinical chemistry effects
is currently unknown. Increased sorbitol dehydrogenase activity is a marker for hepatic
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injury [Ramaiah. 2007) and therefore, underlying hepatotoxicity could explain its increase
in exposed males. However, absolute and relative liver weights were not observed to
increase with exposure, and microscopic histopathological analysis of the liver did not
demonstrate any observable changes. The increases in WBC counts in exposed animals
could be secondary to the observed respiratory irritative and inflammatory effects of
1,2,4-TMB exposure (2000; 1997}.
In summary, hematological toxicity was observed with exposure to 1,2,4-TMB and
1,3,5-TMB in humans and animals. The information regarding hematological toxicity in
humans is limited to one study involving exposure to a complex VOC mixture containing
both 1,2,4-TMB and 1,3,5-TMB, but it did report hematological effects (alterations in
clotting an anemia] that are roughly analogous to those effects (decreased RBCs and
decreased clotting time] observed in rats following subchronic exposure to 1,2,4-TMB.
Although both databases are limited (there are no chronic studies in animals], there is
some consistency in effects across species (i.e., rats and humans]. EPA considered the
hematological effects to be biological plausible and analogous to effects that could occur in
humans; and concluded that the available evidence for 1,2,4-TMB identified hematological
toxicity as a toxicity hazard.
1.1.5. Other lexicological Effects
One animal study was identified that investigated the association of chronic oral
exposure (via gavage] to 1,2,4-TMB and cancer endpoints (Maltoni etal.. 1997]. Male and
female Sprague-Dawley rats were exposed to a single dose of 800 mg/kg-day of 1,2,4-TMB
in olive oil by stomach tube for four days/week starting at 7 weeks of age. Exposures were
terminated at the end of 104 weeks (i.e. at 111 weeks of age] and the animals were kept
under observation until natural death. The authors report that chronic oral exposure to
1,2,4-TMB resulted in an "intermediate" reduction of survival in male rats and a "slight"
reduction in females (no quantitative information on survival was reported]. A slight
increase in total malignant tumors in both sexes of rats was observed, with the incidence of
head cancers being specifically increased in male rats. The predominant type of head
cancer identified was neuroesthesioepithelioma, which arises from the olfactory
neuroepithelium and is normally rare in Sprague-Dawley rats. Other head cancers
observed included those in the Zymbal gland, ear duct, and nasal and oral cavities. No tests
of statistical significance were reported for these data. When Fisher's exact test was
performed (by EPA] on the incidences calculated from the reported percentages of animals
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bearing tumors in the control and exposed animals, no statistically significant associations
were observed.
Janik-Spiechowicz et al. (1998} investigated the genotoxicity of the trimethylbenzene
isomers 1,2,4-TMB, and 1,3,5-TMB by measuring three genotoxic endpoints: mutation
frequency in bacteria, micronucleus formation in mice, and sister chromatid exchanges in
mice. Neither isomer induced gene mutations in any Salmonella typhimurium strain tested
(TA102, TA100, TA98, and TA97a}. Both isomers were also negative for the formation of
micronuclei in Imp:BALB/c mice following i.p. injection. Males in the high dose groups for
1,2,4-TMB and 1,3,5-TMB exposures had a statistically significant reduction in the ratio of
PCEs to NCEs indicating bone marrow cytotoxicity. However, both isomers significantly
increased the frequency of sister chromatid exchanges (SCEs} in Imp:BALB/c mice
following i.p. injection, with 1,2,4-TMB having the more significant response. These results
appeared to have occurred at doses that did not induce significant bone marrow
cytotoxicity except at the highest dose.
In summary, although very little genotoxicity data are available on 1,2,4-TMB and
1,3,5-TMB, Janik-Spiechowicz et al. (1998) observed negative results in several key
mutagenicity assays, including the Ames mutation assay in Salmonella and in vivo assays
for micronucleus formation in mouse bone marrow cells. However, Janik-Spiechowicz et al.
(1998} did observe increased incidence of SCE in mice exposed to both TMB isomers.
Increased frequency of SCEs indicates that DNA damage has occurred as a result of
exposure to these isomers, but it does not provide a specific indication of mutagenic
potential, as there is no known mechanistic association between SCE induction and a
transmissible genotoxic effect. With only one positive SCE result, and all other data
showing negative results for gene and chromosomal mutation in vitro and in vivo, there is
not enough evidence to conclude that either isomer is directly genotoxic.
1.1.6. Similarities between 1,2,4-TMB and 1,3,5-TMB regarding observed inhalation
and oral toxicity
In the existing toxicological database for 1,2,4-TMB and 1,3,5-TMB, important
similarities have been observed in the potency and magnitude of effect resulting from
exposure to the two isomers, although some important differences also exist. In acute
studies investigating respiratory irritative effects, the RDso of the two isomers were very
similar: 2,844 mg/m3 for 1,2,4-TMB and 2,553 mg/m3 for 1,3,5-TMB fKorsak et al.. 19971
The similarity regarding toxicity was also observed in acute inhalation neurotoxicity
studies: the ECso for decreased coordination, balance, and neuromuscular function was
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4,694 mg/m3 for 1,2,4-TMB and 4,738 mg/m3 for 1,3,5-TMB. The EC5o for decreased pain
sensitivity were also similar for both isomers: 5,683 mg/m3 for 1,2,4-TMB and 5,963
mg/m3 for 1,3,5-TMB (Korsakand Rydzynski. 1996}. Neurotoxic endpoints were also
similarly affected by oral exposure to individual isomers: increased electrocortical arousal
was observed in rats exposed to 240 mg/kg 1,2,4-TMB or 1,3,5-TMB, and altered brain
EEGs were observed in rats exposed to 790 mg/kg 1,2,4-TMB or 1,3,5-TMB [Tomas et al..
1999a: Tomas etal.. 1999c}. Although these effects were seen at the same dose levels in
animals exposed to either isomer, these doses were LOAELs, and it is unclear whether this
similar potency would be observed at lower doses. Additionally, there were differences in
the magnitude of effect between the isomers, with 1,2,4-TMB inducing larger abnormalities
in brain EEGs and 1,3,5-TMB affecting electrocortical arousal to a greater degree than
1,2,4-TMB.
In short-term neurotoxicity studies, a similar pattern of effects (inability to learn
passive and/or active avoidance and decreased pain sensitivity] indicating altered
neurobehavioral function were observed for both isomers (Wiaderna etal.. 2002:
Gralewicz and Wiaderna. 2001: Gralewicz etal.. 1997a}. In these studies, 1,3,5-TMB was
shown be more toxic than 1,2,4-TMB: rats exposed to 123 mg/m3 1,3,5-TMB displayed
significantly decreased abilities to learn passive or active avoidance (Wiaderna et al..
2002}. whereas 1,2,4-TMB elicited the inability to learn passive avoidance in rats exposed
to 492 mg/m3 1,2,4-TMB and did not affect active avoidance at any exposure concentration
[Gralewicz etal.. 1997a}. Additionally, in animals exposed to either isomer at 492 mg/m3,
exposure to 1,3,5-TMB decreased the ability to learn passive avoidance to a greater degree
than 1,2,4-TMB (approximately 50% decrease vs. 40%}, and the effect of 1,3,5-TMB
manifested at earlier time points than 1,2,4-TMB (three vs. seven days} (Gralewicz and
Wiaderna. 20011
Lastly, similarities were observed in 1,2,4-TMB- and 1,3,5-TMB-induced developmental
and maternally toxic effects (Saillenfait et al.. 2005}. Male fetal weights were significantly
reduced in animals exposed gestationally to 2,952 mg/m3 1,2,4-TMB (5% decrease} or
1,3,5-TMB (7% decrease}. 1,2,4-TMB also significantly decreased female fetal weights by
approximately 5% in animals exposed to the same concentration. Although, 1,3,5-TMB
significantly reduced female fetal weights by 13% in animals exposed to 5,904 mg/m3,
female weights were decreased at 2,952 mg/m3 to a similar magnitude (6%} as animals
exposed to the same concentration of 1,2,4-TMB. Maternal toxicity, measured as decreased
corrected maternal weight gain, was significantly decreased in animals exposed to 2,952
mg/m3 1,2,4-TMB or 1,3,5-TMB. However, 1,3,5-TMB exposure resulted in a 75%
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reduction of maternal weight gain compared to controls, whereas 1,2,4-TMB exposure
reduced maternal weight gain by 50%.
1.1.7. Susceptible Populations orLifestages
Although there are no chemical-specific data that would allow for the definitive
identification of susceptible subpopulations, the reduced metabolic and elimination
capacities in children relative to adults may be a source of susceptibility [Ginsberg etal..
2004). TMB isomers are metabolized via side-chain oxidation to form alcohols and
aromatic carboxylic/mercapturic acids or by hydroxylation to form phenols, which are
then conjugated with glucuronic acid, glycine, or sulfates for urinary excretion. The
activities of multiple cytochrome P450 (GYP P450) mono-oxygenase isozymes have been
shown to be reduced in children up to 1 year of age compared to adult activities [Ginsberg
etal.. 2004). Additionally, the rate of glucuronidation and sulfation is decreased in
children. Therefore, as both GYP P450 mono-oxygenase activities and the rate of
glucuronidation and sulfation appear to be decreased in early life, newborns and young
infants may experience higher and more persistent blood concentrations of 1,2,4-TMB,
1,3,5-TMB, and/or their respective metabolites compared with adults at similar exposure
levels. Reduced renal clearance in children may be another important source of potential
susceptibility. TMB isomers and their metabolites are excreted in the urine of exposed
laboratory animals and occupationally exposed humans. Data indicating reduced renal
clearance for infants up to 2 months of age [Ginsberg et al.. 2004) may suggest a potential
to affect TMB excretion, thus possibly prolonging its toxic effects. Additionally, those with
pre-existing respiratory diseases [e.g., asthma) may be more sensitive to the respiratory
irritative and inflammatory effects of TMB isomers.
1.2. Selection of Candidate Principal Studies and Critical Effects for
Derivation of Reference Values
1.2.1. Inhalation Exposure - Effects Other Than Cancer - 1,2,4-TMB
While literature exists on the noncancer effects of 1,2,4-TMB exposure in humans,
including neurological, respiratory, and hematological toxicities, no human studies are
available that would allow for the quantification of subchronic or chronic noncancer
effects. The available human studies evaluated TMB exposures occurring as complex
solvent or VOC mixtures, and this consideration along with other uncertainties including
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high imprecision in effect measures due to low statistical power, lack of quantitative
exposure assessment, and lack of control for co-exposures, limit their utility in derivation
of quantitative human health toxicity values. However, these studies provide supportive
evidence for the neurological, respiratory, and hematological toxicity of TMB isomers in
humans and determination of coherency of effect in both humans and laboratory animals
for deriving toxicity values.
Studies investigating 1,2,4-TMB noncancer effects in experimental animal models were
identified in the literature. Acute inhalation studies observing neurotoxicity and
respiratory toxicity in exposed rodents were identified, but the high exposure
concentrations used and acute exposure duration of these studies limit their applicability
for quantitation of chronic human health effects. However, as with the human mixture
studies, these studies do provide qualitative information regarding consistency and
coherence of effect that is supportive of the development of quantitative human health risk
values.
1,2,4-TMB-induced toxicity across several organ systems was observed in three
subchronic studies by Korsak et al., [2000:1997) and Korsak and Rydzynski (1996). Data
from these studies were considered as candidate critical effects for the purpose of
determining the point of departure (POD] for derivation of the inhalation RfC for
1,2,4-TMB. These studies were determined to be adequate as evaluated using study quality
characteristics related to study populations (studies used rats as an appropriate laboratory
animal species and utilized appropriate sham-exposed controls], exposure (the purity of
1,2,4-TMB was reported as > 97% pure (impurities not reported], the studies utilized an
appropriate route [inhaled air] and duration [subchronic] of exposure, and the studies used
a reasonable range of appropriately-spaced dose levels to facilitate dose-response
analysis], and data (appropriate latency between exposure and development of
toxicological outcomes was used, the persistence of some outcomes after termination of
exposure was investigated, adequate numbers of animals per dose group were used, and
appropriate statistical tests including pair-wise and trend analyses were performed].
When considered together, these subchronic studies examined 1,2,4-TMB-induced toxicity
in multiple organ systems including CNS, hematological, and pulmonary effects (Table 1-6].
Table 1-6: Non-cancer endpoints resulting from subchronic inhalation exposure to
1,2,4-TMB considered for the derivation of the RfC
Endpoint
Species/
Sex
Exposure Concentration (mg/m3)
0
123
Neurotoxicological Endpoints
492
1,230
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Decreased pain sensitivity (measured
as latency to paw-lick in seconds)
Impaired neuromuscular function
and coordination (% failures on
rotarod)
Rat, male
15.4±5.8a
(n = 9)
0
(n = 10)
18.2 ±5.7
(n = 10)
10
(n = 10)
27.6±3.2d
(n = 9)
20
(n = 10)
30.1 ± 7.9d
(n = 10)
40e
(n = 10)
Hematological Endpoints
Decreased RBCs (106/cm3)
Increased WBCs (106/cm3)
Decreased reticulocytes (%)
Decreased clotting time (seconds)
Rat, male
Rat, female
9.98 ±1.68
(n = 10)
8.68 ±2.89
(n = 10)
3.5 ±2. 6
(n = 10)
30 ±10
(n = 10)
9.84 ±1.82
(n = 10)
8.92 ± 3.44
(n = 10)
1.7 ±2.0
(n = 10)
23 ±4
(n = 10)
8.50 ±1.11
(n = 10)
8.30 ±1.84
(n = 10)
1.8 ±0.9
(n = 10)
19±5d
(n = 10)
7.70 + 1.38d
(n = 10)
15.89 ± 5.74d
(n = 10)
1.0±0.6C
(n = 20)
22±7C
(n = 20)
Pulmonary Endpoints
Increased BAL macrophages
(106/cm3)
Increased BAL total cells (106/cm3)
Increased inflammatory lung lesions
Rat, male
1.83 ± 0.03
(n = 6)
1.93 ± 0.79
(n = 6)
b
(n = 10)
3.78 ±0.8
(n = 6)
5.82 ± 1.32f
(n = 6)
b
(n =10)
4.95 ± 0.2d
(n = 7)
5.96±2.80d
(n = 7)
b
(n = 10)
3.96 ± 0.3d
(n = 6)
4.45 ± 1.58C
(n = 7)
b
(n = 20)
a Values are expressed as mean ± one standard deviation
b Incidences for individual dose groups not reported in the study. NOAEL for males identified by EPA was
123mg/m3
cp < 0.05; dp < 0.01;e p < 0.005; fp < 0.001
Adapted from Korsak et al., (2000:1997) and Korsak and Rydzynski (1996)
Endpoints from these studies that demonstrated statistically significant pair-wise
increases or decreases relative to control were considered for the derivation of the RfC for
1,2,4-TMB. These endpoints included decreased neuromuscular function and coordination,
and decreased pain sensitivity in male rats (Korsak and Rydzynski. 1996}. increased BAL
total cells and increased BAL macrophages in male rats (Korsak et al.. 1997}. and decreased
RBCs, increased WBCs, and increased pulmonary inflammatory lesions in male rats and
decreased reticulocytes and clotting time in female rats [Korsak et al.. 2000}. Increases in
BAL polymorphonuclear leukocytes and lymphocytes were not considered for RfC
derivation due to a lack of reporting at which doses statistically significant increases
occurred. Changes in BAL protein and enzyme activity level were not considered due to
non-monotonically increasing dose-responses, and increases in sorbitol dehydrogenase
were not further considered due to the lack of accompanying hepatocellular
histopathological alterations in exposed animals. Endpoints carried forward for derivation
of an RfC for 1,2,4-TMB, along with their NOAEL and LOAEL values, are graphically
represented in Figure 1-1.
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:
c
4
Doses
ONOAEL
DLOAEL
1 4
> [
> C
I • • 4
1 [
!> C
I 4
) C
> [
: c
> n n n <
] c
3 4
) C
I 4
) C
) 4
) [
> d
<
{
(
•
] <
3
» 1
] [
) (
» 1
>
]
)
>
neuro- pain BAL BAL pulmonary RBCs WBCs reticulo- clotting maternal fetal
muscular sensitivity macro- total cells lesions (c) (c) cytes time weight gain weight
function (a) (a) phages (b) (b) (c) (c) (d) (d)
(b)
Solid lines represent range of exposure concentrations, (a) Korsak and Rydzynski [1996]: (b) Korsak et al.
(1997); (c) Korsak et al. (2000): (d) Saillenfait et al. (2005)
Figure 1-1. Exposure response array for inhalation exposure to 1,2,4-TMB.
Although the Saillenfait et al. (2005) study was a well conducted developmental
toxicity study, data from this study were not considered for identification of candidate
critical effects for 1,2,4-TMB due to the fact that maternal and developmental toxicities
were observed at doses 6- to 24-fold higher than the doses that resulted neurological,
hematological, and pulmonary effects observed in the subchronic Korsak studies.
1.2.2. Inhalation Exposure - Effects Other Than Cancer - 1,3,5-TMB
No human studies are available that would allow for the quantification of subchronic or
chronic noncancer effects resulting from inhalation exposure to 1,3,5-TMB. The available
human studies evaluated TMB exposures occurring as complex solvent or VOC mixtures,
and this consideration along with similar uncertainties as discussed for 1,2,4-TMB limit
their utility in derivation of quantitative human health toxicity values. As for 1,2,4-TMB,
the human studies do provide supportive evidence for the neurological toxicity of 1,3,5-
TMB in humans and strengthen the determination of consistency and coherency of effect in
humans and laboratory animals.
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No suitable chronic or subchronic inhalation studies investigating 1,3,5-TMB
noncancer effects in experimental animal models were identified in the literature that
would support the derivation of the RfC. Two short-term inhalation studies (Wiaderna et
al.. 2002: Gralewicz and Wiaderna. 2001} investigating neurotoxicity outcomes were
identified in the literature and data from these studies were considered as candidate
critical effects for the purpose of derivation of an RfC for 1,3,5-TMB in the absence of a
suitable chronic or subchronic study. Additionally, one developmental toxicity study was
identified in the literature; Saillenfait et al. (2005}. Data from these studies were
considered as candidate critical effects for the purpose of determining the POD for
derivation of the inhalation RfC for 1,3,5-TMB. Based on the noncancer database for 1,3,5-
TMB, these studies were determined to be adequate as evaluated using study quality
characteristics related to study populations (studies used rats as an appropriate laboratory
animal species and utilized appropriate sham-exposed controls}, exposure (the purity of
1,3,5-TMB was reported as 99% pure, the studies utilized an appropriate route [inhaled
air] duration [short-term and gestational] of exposure (although the duration for short-
term studies was not optimal}, and the studies used a reasonable range of appropriately-
spaced dose levels to facilitate dose-response analysis}, and data (appropriate latency
between exposure and development of toxicological outcomes was used, the persistence of
some outcomes after termination of exposure was investigated, adequate numbers of
animals per dose group were used, and appropriate pair-wise statistical tests were
performed}.
When considered together, these short-term and developmental studies examined
1,3,5-TMB-induced toxicity in multiple organ systems in adult, pregnant, and developing
organism. Endpoints from these studies that demonstrated statistically significant pair-
wise increases or decreases relative to control were considered for the derivation of the
RfC for 1,3,5-TMB. Gralewicz and Wiaderna (2001} and Wiaderna et al. (2002} both
indicate altered cognitive function, decreased pain sensitivity, and decreased anxiety
and/or increased motor function following inhalation exposure to 1,3,5-TMB (see Table 1-
1}. Wiaderna et al. (2002} reported that 123 mg/m3 was the LOAEL for altered cognitive
function the NOAEL for decreased pain sensitivity. As altered cognitive function was
observed at a lower exposure concentration than decreased pain sensitivity, only altered
cognitive function was further considered for derivation of an RfC for 1,3,5-TMB from the
Wiaderna et al. (2002} study. All three neurotoxic effects (altered cognitive function,
decreased pain sensitivity, and decreased anxiety and/or increased motor function} were
observed at the only exposure concentration utilized in the Gralewicz and Wiaderna (2001}
(i.e., 492 mg/m3}; these LOAELs were further considered for derivation of an RfC for 1,3,5-
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TMB. From the Saillenfait et al. (2005) study, decreased male and female fetal weights and
decreased corrected maternal weight gain were considered for derivation of the RfC (Table
1-7}. Changes in serum chemistry parameters in rats exposed sub chronically to 1,3,5-TMB
were not considered for derivation of the RfC due to inconsistent temporal patterns of
effect and the lack of accompanying histopathology.
Table 1-7: Non-cancer endpoints resulting from gestational inhalation exposure (GD
6-20) to 1,3,5-TMB considered for the derivation of the RfC
Endpoint
Species/
Sex
Exposure Concentration (mg/m3)
0
fn = 211"
492
fn = 221
1,476
fn = 211
2,952
fn = 171
5,904
fn = 181
Developmental Endpoints
Decreased fetal weight
GO
Rat, male
Rat, female
5.80 ±
0.41b
5.50 ±0.32
5.76 ±0.27
5.74 ±0.21
5.50 ±0.31
5.27 ±0.47
5.39 ±
0.55C
5.18 ±0.68
5.10 ±
0.57d
4.81 ±
0.45d
Maternal Endpoints
Decreased maternal
weight gain (g)
Rat, female
29 ±14
30 ±9
20 ±12
7±20C
-12±19d
a Number of dams with live litters, numbers of live fetuses not explicitly reported
b Values are expressed as mean ± one standard deviation
cp < 0.05; dp < 0.01
Adapted from Saillenfait et al. [2005]
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10000
1000
100
Doses
ONOAEL
DLOAEL
D
Altered
cognitive
function (a)
t ?
I 1
<
I
C
T T
o
i
> D D • •
:
Altered 4. Pain 4. Anxiety f f
cognitive sensitivity (a) and/or T" maternal fetal
function (b) motor activity (a) weight gain... weight...
Solid lines represent range of exposure concentrations, (a) Gralewicz and Wiaderna [2001]: (b) Gralewicz et
al. (19973); (c] Saillenfait et al. [2005]
Figure 1-2. Exposure response array for inhalation exposure to 1,3,5-TMB.
1.2.3. Oral Exposure - Effects Other Than Cancer - 1,2,4-TMB and 1,3,5-TMB
No human studies are available that would allow for the quantification of subchronic or
chronic noncancer effects resulting from oral exposure to either 1,2,4-TMB or 1,3,5-TMB.
Additionally, no suitable chronic or subchronic oral studies investigating 1,2,4-TMB or
1,3,5-TMB noncancer effects in experimental animal models were identified in the
literature that would support the derivation of an RfD. Although the oral database for
1,2,4-TMB and 1,3,5-TMB are inadequate to support the derivation of an RfD, a PBPK model
is available to perform a route-to-route extrapolation [Hissink et al.. 2007). The Hissink
model was chosen as an appropriate model because it was the only published 1,2,4-TMB
model that included parameterization for both rats and humans, the model code was
available, and the model adequately predicted experimental data in the dose range of
concern. The use of inhalation toxicity data to derive an oral RfD is supported by the
1,2,4-TMB and 1,3,5-TMB database: 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.
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Further, no evidence exists that would suggest toxicity profiles would differ to a substantial
degree between oral and inhalation exposures.
1.3. Carcinogenicity Analysis
Synthesis and Overall Weight of Evidence
Under the Guidelines for Carcinogen Risk Assessment (2005a}. the database for
1,2,4-TMB and 1,3,5- TMB provides "inadequate information to assess the carcinogenic
potential" of these isomers. This characterization is based on the fact that there is no
information regarding the carcinogenicity of TMB in humans and that the only animal
study on the carcinogenicity of 1,2,4-TMB observed no statistically significant carcinogenic
effects. No studies regarding the carcinogenicity of 1,3,5-TMB were identified in the
available scientific literature.
Only one animal carcinogenicity study was identified [Maltoni etal.. 1997). involving
exposure to 1,2,4-TMB by oral gavage. Although an increased incidence of total malignant
tumors in both sexes and head cancers (predominately neuroethesioepithelioma] in males
was observed in exposed rats, no statistical analyses were reported. When EPA
independently performed the Fisher's exact test on the reported data, no statistically
significant effects were observed.
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2. DOSE-RESPONSE ANALYSIS
2.1. Inhalation Reference Concentration for Effects other than Cancer
2.1.1. Dose-Response Assessment for RfC derivation for 1,2,4-TMB
As discussed in Section 1.2.1, endpoints observed in Korsak et al., (2000:1997} and
Korsak and Rydzynski (1996} that demonstrated statistically significant (at p < 0.05 or
greater} pair-wise increases or decreases relative to control for at least one dose group
were considered for the derivation of the RfC for 1,2,4-TMB; these effects are listed in Table
1-6 above. This assessment used the benchmark dose (BMD} approach, when possible, to
estimate a point of departure (POD} for the derivation of an RfC for 1,2,4-TMB (Table 2-1;
see Section C.I of Appendix B (U.S. EPA. 2011cj for detailed methodology}. The BMD
approach involves fitting a suite of mathematical models to the observed dose-response
data using EPA's Benchmark Dose Software (BMDS, version 2.2}. Each fitted model
estimates aBMD and its associated 95% lower confidence limit (BMDL} corresponding to a
selected benchmark response (BMR}. For dichotomous data (i.e., impaired neuromuscular
function and coordination, measured as % failure on rotarod} from Korsak and Rydzynski
(1996}, no information is available regarding the change in this response that would be
considered biologically significant, and thus a BMR of 10% extra risk was used to model
this endpoint, consistent with the Benchmark Dose Technical Guidance (U.S. EPA. 2000aj.
For continuous data (i.e., decreased pain sensitivity, increased BAL macrophages,
decreased RBCs, decreased reticulocytes, and decreased clotting time} from the Korsak and
Rydzynski (1996} and Korsak et al. (2000:1997} studies, no information is available
regarding the change in these responses that would be considered biologically significance,
and thus a BMR equal to a change in the mean equal to 1 standard deviation of the model
estimated control mean was used in modeling the endpoints, consistent with the
Benchmark Dose Technical Guidance (U.S. EPA. 2000aj. The estimated BMDL is then used
as the POD for deriving the RfC.
The suitability of the above methods to determine a POD is dependent on the nature of
the toxicity database for a specific chemical. Some endpoints for 1,2,4-TMB were not
amenable to BMD modeling for a variety of reasons, including equal responses at all
exposure groups (e.g., increased BAL total cells}, responses only in the high dose group
with no significant changes in responses in lower dose groups (e.g., increased WBCs}, and
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absence of incidence data (e.g., increased inflammatory lung lesions}. Additionally, some
datasets were deemed adequate for BMD modeling, but no model provided an adequate fit
to the data (e.g., increased BAL macrophages}, estimated BMDs were greater than the
highest exposure concentration (e.g., decreased reticulocytes}, or estimated BMDLs were
determined to be biologically implausibly low (e.g., decreased clotting time}. In these
cases, the NOAEL/LOAEL approach was used to identify a POD. Detailed modeling results
are provided in Section C.2 of Appendix B (U.S. EPA. 2011c) (detailed modeling results for
maternal and fetal endpoints observed in Saillenfait et al. (2005} are provided in Appendix
B (U.S. EPA. 2011cj for comparison to endpoints observed in the Korsak et al., (2000: 1997}
and Korsak and Rydzynski (1996} studies}.
Table 2-1: Summary of dose-response analysis and point of departure estimation for
endpoints resulting from subchronic inhalation exposure to 1,2,4-TMB
Reference
Endpoint
Sex/
Species
POD
Basis
Best-fit
Model; BMR
Candidate
POD
(mg/m3)
BW
(kg)
PODAD,b
(mg/L)
Neurotoxicological Endpoints
Korsak and
Rydzynski
(1996)
Decreased pain
sensitivity
Impaired
neuromuscular
function and
coordination
Male, rat
Male, rat
BMDL
BMDL
Exponential
4;1SD
Log-logistic;
10% ER
84.0
93.9
0.387
0.387
0.085
0.096
Hematological Endpoints
Korsak et
al. (2000)
Decreased RBCs
Increased WBCs
Decreased
reticulocytes
Decreased clotting
time
Male, rat
Male, rat
Female, rat
Female, rat
BMDL
NOAEL
NOAEL
NOAEL
Exponential
4;1SD
n/a
n/a
n/a
174.1
492
492
123
0.390
0.399
0.230
0.243
0.187
0.867
0.890
0.127
Pulmonary Endpoints
Korsak et
al. (1997)
Korsak et
al. (2000)
Increased BAL
macrophages
Increased BAL total
cells
Increased
inflammatory lung
lesions
Male, rat
Male, rat
Male and
female, rat
NOAEL
LOAEL
NOAEL
n/a
n/a
n/a
123
123
123
0.383
0.383
0.390
0.127
0.127
0.127
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aGroup specific mean body weight [BW] reported in Korsak et al. (2000: Korsak et al.. 19971. For endpoints
from these studies using a NOAEL or LOAEL for the POD, the reported group specific mean BW for that dose
group was used in PBPK PODADj calculations. For decreased RBCs from Korsak et al. [2000]. the group
specific mean BW for the dose group closest to the BMDL was used. For decreased pain sensitivity and
coordination, balance, and neuromuscular function from Korsak and Rydzynski [1996]. the average of the
group specific mean BWs from Korsak et al. [2000: Korsak etal.. 1997] for the dose group closest to the
BMDL was used.
HVeekly average venous blood TMB concentration [mg/L] estimated for a rat exposed to the corresponding
candidate POD for 6 h/day, 5 d/wk. See Appendix A [U.S. EPA, 2011d] for details on PBPK modeling.
Because an RfC is a toxicity value that assumes continuous human inhalation exposure
over a lifetime, data derived from inhalation studies in animals need to be adjusted to
account for the noncontinuous exposures used in these studies. For 1,2,4-TMB, a PBPK
model (Hissink et al.. 2007} was employed to make this adjustment. This PBPK model
(described in Appendix A (U.S. EPA. 2011d}: Section A.I} was used to estimate the steady-
state weekly average venous blood concentration (mg/L} of 1,2,4-TMB for rats exposed to
1,2,4-TMB for 6 h/d, 5 d/wk. For each exposure concentration, once the model reached
steady-state, the resulting weekly average venous blood concentration (mg/L} of 1,2,4-
TMB was employed as the dose metric for these endpoints. This dose metric was
considered the duration-adjusted POD (PODADj} for each candidate critical effect (Table 2-
1).
2.1.2. RfC Derivation for 1,2,4-TMB
For the derivation of an RfC based upon an animal study, the calculated PODADj values
are further adjusted to reflect the human equivalent concentration (HEC} (Table 2-2}.
Table 2-2: Candidate PODADJ values, human equivalent concentrations (HECs), and
applied uncertainty factors used in the derivation of RfCs for 1,2,4-TMB
Reference
Endpoint
PODADJ
(mg/L)
HEC
(mg/m3
)a
Uncertainty Factors (UF)
UFA
UFH
UFL
UFS
UFD
UFTOTAL
Candidate
RfC
(mg/m3)b
Neurotoxicological Endpoints
Korsak and
Rydzynski
[1996]
Decreased pain
sensitivity
Impaired
neuromuscular
function and
coordination
0.085
0.096
15.6
17.6
3
3
10
10
1
1
10
10
3
3
1,000
1,000
1.56xlO-2
1.76 x ID'2
Hematological Endpoints
Korsak et
al. [2000]
Decreased RBCs
Increased WBCs
Decreased
reticulocytes
0.187
0.867
0.890
33.7
131.5
134.0
3
3
3
10
10
10
1
1
1
10
10
10
3
3
3
1,000
1,000
1,000
3.37x10-2
1.31 x 10-1
1.34x10-1
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Decreased
clotting time
0.127
23.2
3
10
1
10
3
1,000
2.32 x lO-2
Pulmonary Endpoints
Korsak et
al. [1997]
Korsak et
al. (2000)
Increased BAL
macrophages
Increased BAL
total cells
Increased
inflammatory
lung lesions
0.127
0.127
0.127
23.2
23.2
23.2
3
3
3
10
10
10
1
10
1
10
10
10
3
3
3
1,000
10,000
1,000
2.32 x lO-2
n/ac
2.32 x lO-2
a Human equivalent concentration
b As calculated by application of uncertainty factors, not rounded.
c Endpoint excluded for further consideration due to a UP-TOTAL of 10,000. The 2002 report, "A Review of the
Reference Dose and Reference Concentration Processes" [U.S. EPA, 20021 recommends a maximum total UF
of 3000 for derivation of an RfC.
The HEC was derived using a human PBPK model (Hissinketal.. 2007} to account for
interspecies differences in toxicokinetics. The human PBPK model was run (as described in
Appendix A [U.S. EPA. 2011d}}. assuming a continuous (24 h/day, 7 day/week] exposure,
to estimate a human PODHEC that would result from the same weekly average venous
blood concentration reflected in the PODADJ in animals (Table 2-2}.
Neurotoxicity is the most consistently observed endpoint in the toxicological database
for 1,2,4-TMB. According to EPA's Guidelines for Neurotoxicity Risk Assessment (1998},
many neurobehavioral changes are regarded as adverse, and the observation of correlated
and replicated measures of neurotoxicity strengthen the evidence for a hazard. Decreased
pain sensitivity, as measured as latency to paw-lick, is a measure of nociception (i.e.,
decreased pain sensitivity} and therefore this endpoint represents an alteration in
neurobehavioral function (U.S. EPA. 1998}. The observation of decreased pain sensitivity
was observed in multiple studies across multiple exposure durations (Gralewicz and
Wiaderna. 2001: Gralewicz etal.. 1997a: Korsak and Rydzynski. 1996: Korsak etal.. 1995}.
and in the presence of other metrics of altered neurobehavior, including impaired
neuromuscular function and coordination and altered cognitive function. Additionally,
neurotoxicological endpoints (hand tremble, weakness} are observed in human worker
populations exposed to complex VOC mixtures containing 1,2,4-TMB. In consideration of
the recommendations in the U.S. EPA's Guidelines for Neurotoxicity Risk Assessment (1998}
and given the consistency of effect across independent studies, multiple durations of
exposure in animal studies, the consistency of observed neurotoxicity in animals and
humans, the EPA concluded that neurotoxicity represents strong evidence of toxicity
hazard and that decreased pain sensitivity is an adverse effect, and the most appropriate
effect on which to base the RfC. Therefore, decreased pain sensitivity was selected as the
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critical effect and Korsak and Rydzynski (1996) as the principal study for the 1,2,4-TMB
RfC.
A PODHEC of 15.6 mg/m3 for decreased pain sensitivity (Korsak and Rydzynski. 1996}
was used as the POD to derive the chronic RfC for 1,2,4-TMB. The uncertainty factors,
selected based on EPA's A Review of the Reference Dose and Reference Concentration
Processes (2002) (Section 4.4.5 of the report], address five areas of uncertainty resulting in
a total UF of 1,000. This composite uncertainty factor was applied to the selected POD to
derive an RfC.
An interspecies uncertainty factor, UFA, of 3 (W1/2 = 3.16, rounded to 3} was applied to
account for uncertainty in characterizing the toxicokinetic and toxicodynamic differences
between rats and humans following inhalation exposure to 1,2,4-TMB. In this assessment,
the use of a PBPK model to convert internal doses in rats to administered doses in humans
reduces toxicokinetic uncertainty in extrapolating from the rat to humans, but does not
account for interspecies differences due to toxicodynamics. A default UFA of 3 was thus
applied to account for this remaining toxicodynamic uncertainty.
An intraspecies uncertainty factor, UFn, of 10 was applied to account for potentially
susceptible individuals in the absence of data evaluating variability of response in the
human population following inhalation of 1,2,4-TMB. No information is currently available
to predict potential variability in human susceptibility, including variability in the
expression of enzymes involved in 1,2,4-TMB metabolism. Due to this lack of data on
variability within the human population, a default 10-fold UFn was applied.
A LOAEL to NOAEL uncertainty factor, UFi of 1 was applied because the current
approach is to address this factor as one of the considerations in selecting a BMR for BMD
modeling. In this case, a BMR equal to a change in the mean equal to 1 standard deviation
of the model estimated control mean for decreased pain sensitivity was selected under the
assumption that this BMR represents a minimally, biologically significant change for this
endpoint.
A subchronic to chronic uncertainty factor, UFs, of 10 was applied to account for
extrapolation from a subchronic exposure duration study to derive a chronic RfC.
A database uncertainty factor, UFo, of 3 (W1/2 = 3.16, rounded to 3} was applied to
account for database deficiencies due to the lack of a multi-generation reproductive toxicity
study. The database contains three subchronic studies that are well-designed and observe
exposure-response effects in multiple organ systems in rats exposed to 1,2,4-TMB via
inhalation (nervous, hematological, and pulmonary systems}. The database additionally
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contains a well-designed developmental toxicity study that investigated standard measures
of maternal and fetal toxicity in a different strain of rat. Although there is no information
regarding the potential for developmental neurotoxicity and the critical effect for the RfC is
altered CNS function, this raises concern regarding possible neurodevelopmental effects of
1,2,4-TMB exposure. However, in the absence of information regarding the magnitude of
transfer of 1,2,4-TMB or its metabolites across the placenta, and given the observation that
other developmental effects occur at concentrations 6- to 24-fold greater than those
eliciting neurotoxicity in adult animals, it may be unlikely that neurodevelopmental data
would result in a lower RfC.
Application of this 1000-fold composite UF to the PODHEC yields the following chronic
RfC for 1,2,4-TMB:
RfC = PODHEC * UF = 15.6 mg/m3 * 1,000 = 0.02 mg/m3 = 2 x 10 2 mg/m3 (rounded to
one significant digit)
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. 1994). Confidence in the study from which the critical effect was
identified, Korsak and Rydzynski (1996} is medium. The study is a well-conducted peer-
reviewed study that utilized three dose groups plus untreated controls, an appropriate
number of animals per dose group, and performed statistical analyses. The critical effect
on which the RfC is based is well-supported as the weight of evidence for 1,2,4-TMB-
induced neurotoxicity is coherent across multiple animals species (i.e., human, mouse, and
rat] and consistent across multiple exposure durations (i.e., acute, short-term, and sub-
chronic] (Gralewicz and Wiaderna. 2001: Chenetal.. 1999: Wiaderna etal.. 1998:
Gralewicz etal.. 1997a: Gralewicz etal.. 1997b: Korsak and Rydzynski. 1996: Norseth et al..
1991). Confidence in the database for 1,2,4-TMB is low to medium as the database includes
acute, short-term, subchronic, and developmental toxicity studies in rats and mice. The
database lacks a chronic and multigenerational reproductive study, and the studies
supporting the critical effect predominately come from the same research institute. Overall
confidence in the RfC for 1,2,4-TMB is low to medium.
2.1.3. Comparison of Candidate RfCs for 1,2,4-TMB
The predominant noncancer effect observed following acute, short-term, and
subchronic inhalation exposures to 1,2,4-TMB is neurotoxicity, although respiratory
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toxicity is also observed following acute and subchronic exposures, while hematological
effects are observed after subchronic exposures. Figure 2-1 provides a graphical display of
all of the candidate PODs and RfCs derived from the three subchronic studies considered in
the selection of the final POD for the inhalation RfC.
Decreased
pain
sensitivity
Decreased Increased BAL Increased Decreased
neuro- macrophages inflammatory RBCs
muscular lung lesions
function
Neurotoxicological
Endpoints
Pulmonary Endpoints
ncreased Decreased Decreased
WBCs reticulocytes clotting time
Respiratory Endpoints
D Data base Uncertainty • Subchronic to Chronic Q Intraspecies Variability Qlnterspecies Extrapolation ORfC O POD
Figure 2-1: Array of candidate PODHEC values with applied UFs and candidate RfCs for
CNS, hematological, and pulmonary effects resulting from inhalation exposure to
1,2,4-TMB
2.1.4. Uncertainties in the Derivation of the RfC for 1,2,4-TMB
As presented above, the UF approach following EPA practices and RfC guidance (U.S.
EPA. 2002.19941 was applied to the PODHEC in order to derive the chronic RfC for
1,2,4-TMB. Factors accounting for uncertainties associated with a number of steps in the
analyses were adopted to account for extrapolation from animals to humans, a diverse
human population of varying susceptibilities, POD determination methodologies (NOAEL,
LOAEL, or BMDL), and database deficiencies.
The critical effect selected, decreased pain sensitivity, does not introduce substantial
uncertainty into the RfC calculation as selection of alternative CNS, hematological, or
pulmonary effects would result in an equivalent RfCs (i.e., 2 x 10-2 mg/m3, see Figure 2-1}.
Some uncertainty does exist regarding the selection of the BMRs for use in BMD modeling,
but selection of 10% extra risk for dichotomous endpoints and 1 SD for continuous
endpoints is supported by current EPA guidance (U.S. EPA. 2000a). Uncertainty regarding
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the selection of particular models for individual endpoints does exist as selection of
alternative models could decrease or increase the RfC. However, the best-fit model is the
most appropriate for RfC derivation based on current EPA guidance (U.S. EPA. 2000a}.
Uncertainty may exist in the PBPK model estimates of internal blood dose metrics for the
rat, and subsequent HEC calculations for the human, including parameter uncertainly, but
such uncertainties would apply equally to all endpoints. Lastly, the extent of inter-
individual variation of 1,2,4-TMB metabolism in humans and potential susceptible
subpopulations have not been well characterized, and thus these two considerations
remain sources of some uncertainty in the current assessment.
2.1.5. Dose-Response Assessment for RfC derivation for 1,3,5-TMB
As discussed above in Section 1.2.2, endpoints observed in Saillenfait et al. (2005) that
demonstrated statistically significant (at p < 0.05 or greater] pair-wise increases or
decreases relative to control for at least one dose group were considered for the derivation
of the RfC for 1,3,5-TMB; these effects are listed in Table 1-7. Additionally, altered
cognitive function, decreased pain sensitivity, and decreased anxiety and/or increased
motor function observed in Gralewicz et al. (2001) and Wiaderna et al. (2002) were also
considered as the basis for the derivation of the RfC for 1,3,5-TMB. This assessment used
the BMD approach, when possible, to estimate a POD for the derivation of an RfC for
1,3,5-TMB (Table 2-4; see Section C.I of Appendix B (U.S. EPA. 2011c} for detailed
methodology}. The BMD approach involves fitting a suite of mathematical models to the
observed dose-response data using EPA's BMDS (version 2.2}. Each fitted model estimates
a BMD and its associated BMDL corresponding to a selected BMR. For continuous data (i.e.,
decreased male and female fetal weight} from the Saillenfait et al. (2005} study, a BMR
equal to 5% relative deviance from the control mean was used. A decrease in body weight
of 10% is generally assumed to be a minimally biologically significant response in adult
animals. Because the developing organism may be more sensitive to decreases in body
weight, a 5% decrease in fetal body weight relative to control was assumed to be a
minimally biologically significant response for the fetuses in the Saillenfait et al. (2005}
study. As recommended by EPA's Benchmark Dose Technical Guidance (2000aj. a BMR
equal to a change in the mean of 1 standard deviation of the model estimated control mean
was also used in modeling the fetal endpoints for comparison purposes. No information is
available regarding the magnitude of response that would be considered biologically
significant for decreased maternal weight gain. Thus, a BMR equal to a change in the mean
equal to 1 standard deviation of the model estimated control mean was used in modeling
this endpoint. The estimated BMDL is then used as the POD for deriving the RfC.
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The suitability of the above methods to determine a POD is dependent on the nature of
the toxicity database for a specific chemical. The data for neurotoxicity (i.e., altered
cognitive function, decreased pain sensitivity, and decreased anxiety and/or increased
motor function] for 1,3,5-TMB were not amenable to BMD modeling. Gralewicz and
Wiaderna (2001} only employed one exposure concentration when investigating the
neurotoxic effects of 1,3,5-TMB following short-term inhalation exposures. Thus, the
observed neurotoxic effects in this study were not amenable to modeling according to EPA
guidance (2000a}. For altered cognitive function (as measured as decreased passive and
active avoidance] reported in Wiaderna et al. (2002], responses were observed to be equal
in all exposure groups. Therefore, for the neurotoxic effects observed in Gralewicz and
Wiaderna (2001] and Wiaderna et al. (2002] the NOAEL/LOAEL approach was used to
determine a POD. In the Saillenfait et al. (2005] study, although decreased fetal body
weight in females was considered appropriate for BMD modeling, BMDS was unable to
adequately model the variance in response for this endpoint. Therefore, the
NOAEL/LOAEL approach was also used in this case to identify a POD. Detailed modeling
results are provided in Section B.2 of Appendix B (U.S. EPA. 2011c].
Because an RfC is a toxicity value that assumes continuous human inhalation exposure
over a lifetime, data derived from inhalation studies in animals need to be adjusted to
account for the noncontinuous exposures used in these studies. In the Gralewicz and
Wiaderna (2001] and Wiaderna et al. (2002] studies, rats were exposed to 1,3,5-TMB for 6
hours/day, 5 days/week for 4 weeks. Because no PBPK model exists for 1,3,5-TMB, the
duration-adjusted PODs for neurobehavioral effects in rats were calculated as follows:
PODADJ (mg/m3) = POD (mg/m3) x hours exposed per day/24 hours x days
exposed per week/7 days
Therefore, for altered cognitive function from Gralewicz and Wiaderna (2001], the
PODADJ would be calculated as follows:
PODADJ (mg/m3) = 492 mg/m3x 6 hours/24 hours x 5 days/7 days
PODADJ (mg/m3) = 87.9 mg/m3
In the Saillenfait et al. (2005] study, rats were exposed to 1,3,5-TMB for 6 hours/day for
15 consecutive days (CDs 6-20]. Therefore, the duration-adjusted PODs for
developmental/ maternal effects were calculated as follows:
PODADJ (mg/m3) = POD (mg/m3) x hours exposed per day/24 hours
For example, for decreased fetal weight in males, the PODADJ would be calculated as
follows:
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(mg/m3) = 1649 mg/m3 x 6 hours/24 hours
PODADj (mg/m3) =412.0 mg/m3
The calculated PODADJ (mg/m3} values for all neurotoxicity and developmental
endpoints considered for RfC derivation are presented in Table 2-4.
Table 2-4: Duration adjusted point of departure (PODADj) estimates from short-term
and gestational inhalation exposures to 1,3,5-TMB
Reference
Endpoint
Sex/
Species
POD
Basis
Best-fit
Model; BMR
Candidate
POD
(mg/m3)
PODADJ
(mg/L)»
Neurotoxicological Endpoints
Gralewicz and
Wiaderna
[2001]
Wiaderna et al.
[20021
Altered cognitive
function
Decreased pain
sensitivity
Decreased anxiety
and/or increased
motor function
Altered cognitive
function
Male, rat
Male, rat
Male, rat
Male, rat
LOAEL
LOAEL
LOAEL
LOAEL
n/a
n/a
n/a
n/a
492
492
492
123
87.9
87.9
87.9
22.0
Developmental Endpoints
Saillenfait et al.
[2005]
Decreased fetal
body weight
Male, rat
Female, rat
BMDLC
NOAELC
Exponential
2; 5% RD
n/a
1,649
2,952
412.0
738.0
Maternal Endpoints
Saillenfait et al.
[2005]
Decreased
maternal weight
body gain
Female, rat
BMDL
Power, 1 SD
1,303
326.0
a Duration adjusted PODADj [mg/m3] = POD x [6 hours/24 hours] for developmental/maternal endpoints, or
POD x [6 hours/24 hours] x [5 days/week] in accordance with EPA policy [U.S. EPA. 2002]
2.1.6. RfC Derivation for 1,3,5-TMB
Because the selected endpoints for consideration as the critical effect (altered cognitive
function, decreased pain sensitivity, decreased anxiety and/or increased motor function,
decreased fetal body weight, and maternal body weight gain] are assumed to result
primarily from systemic distribution of 1,3,5-TMB, and no available PBPK model exists for
1,3,5-TMB, the human equivalent concentration for 1,3,5-TMB was calculated by the
application of the appropriate dosimetric adjustment factor (DAF) for systemically acting
gases (i.e., Category 3 gases], in accordance with the U.S. EPA RfC Methodology (U.S. EPA.
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1994). DAFs are ratios of animal and human physiologic parameters, and are dependent on
the nature of the contaminant (particle or gas] and the target site (e.g., respiratory tract or
remote to the portal-of-entry) (U.S. EPA. 1994}. For gases with systemic effects, the DAF is
expressed as the ratio between the animal and human blood:air partition coefficients:
DAF = (Hb/g)A/(Hb/b)H
DAF = 55.7/43
DAF = 1.3
where:
(Hb/g)A = the animal blood:air partition coefficient
(Hb/g)H = the human blood:air partition coefficient
In cases where the animal blood:air partition coefficient is higher than the human value
(Meulenberg and Vijverberg. 2000: larnbergandlohanson. 1995). resulting in a DAF > 1, a
default value of 1 is substituted (U.S. EPA. 1994). For example, the HEC for altered CNS
function (reported in Wiaderna et al. (2002) is calculated as follows:
PODHEc = PODADj (mg/m3) x DAF
PODHEc = PODADj (mg/m3) x 1.0
PODHEC = 22 mg/m3 x 1.0
PODHEC = 22 mg/m3
Table 2-5 presents the derivation of candidate RfCs from the selected short-term and
developmental toxicity studies (Saillenfait et al.. 2005: Wiaderna etal.. 2002: Gralewicz and
Wiaderna. 20011
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Table 2-5: Candidate PODADJ values, human equivalent concentrations (HECs), and
applied uncertainty factors used in the derivation of RfCs for 1,3,5-TMB
Referenc
e
Endpoint
PODADJ
(mg/L)
HEC
(mg/m3
)a
Uncertainty Factors (UF)
UFA
UFH
UFL
UFS
UFD
UFTOTAL
Candidate
RfC
(mg/m3)b
Neurotoxicological Endpoints
Gralewicz
and
Wiaderna
C20Q1)
Wiaderna
etal.
[2002]
Altered cognitive
function
Decreased pain
sensitivity
Decreased anxiety
and/or increased
motor function
Altered cognitive
function
87.9
87.9
87.9
22.0
87.9
87.9
87.9
22.0
3
3
3
3
10
10
10
10
10
10
10
10
10
10
10
10
3
3
3
3
10,000
10,000
10,000
10,000
n/ac
n/ac
n/ac
n/ac
Developmental Endpoints
Saillenfait
etal.
[2005]
Decreased fetal
body weight, male
Decreased fetal
body weight,
female
412.0
738.0
412.0
738.0
3
3
10
10
1
1
1
1
3
3
100
100
4.12
7.38
Maternal Endpoints
Saillenfait
etal.
T20051
Decreased
maternal weight
body gain
326.0
326.0
3
10
1
10
3
1,000
3.26x10-1
a Human equivalent concentration
b As calculated by application of uncertainty factors, not rounded.
c Endpoint excluded for further consideration due to a UFTOTAL of 10,000. The 2002 report "A Review of the
Reference Dose and Reference Concentration Processes" [U.S. EPA. 2002] recommends a maximum total UF
of 3000 for derivation of an RfC.
The magnitude of the total uncertainty factors associated with the neurotoxicological
endpoints from Gralewicz and Wiaderna (2001} and Wiaderna et al. (2002} indicate that
these endpoints cannot support the derivation of an RfC for 1,3,5-TMB. The composite UF
for 1,3,5-TMB for the neurotoxicological endpoints from Gralewicz and Wiaderna (2001}
and Wiaderna et al. (2002} would be 10,000. In the report, A Review of the Reference Dose
and Reference Concentration Processes fU.S. EPA. 20021 the RfD/RfC Technical Panel
concluded that, in cases where maximum uncertainly exists in four or more areas of
uncertainly, or when the total uncertainly factor is 10,000 or more, it is unlikely that the
database is sufficient to derive a reference value. Therefore, consistent with the
recommendations in U.S. EPA (2002J. the available neurotoxicity data following short-term
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inhalation exposure to 1,3,5-TMB were considered insufficient to support reference value
derivation and an RfC for 1,3,5-TMB was not derived based on these data.
Of the remaining effects considered for derivation of the RfC, decreased maternal
weight gain (PODHEC = 326.0 mg/m3} was identified as the most sensitive endpoint. A
PODHEC of 326.0 mg/m3 for decreased maternal weight gain by Saillenfait et al. (2005}
was used as the PODHEC to derive a candidate chronic RfC for 1,3,5-TMB as shown in Table
2-5. The uncertainty factors, selected based on EPA's A Review of the Reference Dose and
Reference Concentration Processes (2002}. address five areas of uncertainty resulting in a
total UF of 1000. This composite uncertainty factor was applied to the selected POD to
derive an RfC.
An interspecies uncertainty factor, UFA, of 3 (W1/2 = 3.16, rounded to 3} was applied to
account for uncertainty in characterizing the toxicokinetic and toxicodynamic differences
between rats and humans following inhalation exposure to 1,3,5-TMB. In this assessment,
the use of a DAF to extrapolate external exposure concentrations from rats to humans
reduces toxicokinetic uncertainty in extrapolating from the rat data, but does not account
for the possibility that humans may be more sensitive to 1,3,5-TMB than rats due to
toxicodynamic differences. A default UFA of 3 was thus applied to account for this
remaining toxicodynamic uncertainty.
An intraspecies uncertainty factor, UFn, of 10 was applied to account for potentially
susceptible individuals in the absence of data evaluating variability of response in the
human population following inhalation of 1,3,5-TMB. No information is currently available
to predict potential variability in human susceptibility, including variability in the
expression of enzymes involved in 1,3,5-TMB metabolism. Due to this lack of data on
variability within the human population, a default 10-fold UFn is applied.
A LOAEL to NOAEL uncertainty factor, UFi of 1 was applied because the current
approach is to address this factor as one of the considerations in selecting a BMR for BMD
modeling. In this case, a BMR equal to a change in the mean of 1 standard deviation of the
model estimated control mean for decreased maternal body weight gain was selected
under an assumption that this BMR level represents a minimally, biologically significant
change for this endpoint.
A subchronic to chronic uncertainty factor, UFs, of 10 was applied to account for
extrapolation from a subchronic exposure duration study to derive a chronic RfC.
A database uncertainty factor, UFo, of 3 (101/2 = 3.16, rounded to 3} was applied to
account for database deficiencies due to the lack of a multi-generation reproductive toxicity
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study. The database contains two short-term studies that are well-designed and observe
exposure-response effects in the central nervous system of exposed rats. The database
additionally contains a well-designed developmental toxicity study that investigated
standard measures of maternal and fetal toxicity in a different strain of rat. A limitation of
the database is the lack of any information regarding the potential for developmental
neurotoxicity. As altered neurobehavioral function is observed in rats exposed to
1,3,5-TMB (manifested as decreased ability to learn passive and active avoidance and
decreased pain sensitivity], this raises concern for neurodevelopmental effects of
1,3,5-TMB exposure. However, in the absence of information regarding the magnitude of
transfer of 1,3,5-TMB or its metabolites across the placenta, it may be unlikely that
neurodevelopmental data would result in a lower RfC.
Application of this 1000-fold composite UF yields the calculation of the chronic RfC for
1,3,5-TMB as follows:
RfC = PODHEc * UF = 326 mg/m3 * 1000 = 0.326 mg/m3 = 3 x 10 1 mg/m3 (rounded to
one significant digit)
While Saillenfait et al. (2005) is a well-conducted developmental toxicity study that
utilizes appropriate study design, group sizes, and statistics, and investigates a wide range
of fetal and maternal endpoints resulting from 1,3,5-TMB inhalation exposure, a number of
additional factors lessens its suitability with which to derive the RfC for 1,3,5-TMB. First,
although maternal and fetal toxicities were observed in this study, it is important to note
that the candidate RfC for 1,3,5-TMB derived based on the critical effect of decreased
corrected (for gravid uterine weight] maternal body weight gain is 15-fold higher than the
RfC derived for 1,2,4-TMB (based on altered CNS function measured as decreased pain
sensitivity]. As discussed in Section 1.1, the available toxicological database for 1,2,4-TMB
and 1,3,5-TMB, across all exposure durations, indicates there are important similarities in
the two isomer's toxicity that are supportive of not deriving an RfC for 1,3,5-TMB that is
substantially greater than the RfC value derived for 1,2,4-TMB.
In acute studies investigating the respiratory irritative effects of the two isomers, the
RD50 of 1,2,4-TMB and 1,3,5-TMB were observed to be very similar: 2,844 and 2,553
mg/m3, respectively (Korsak et al.. 1997]. This similarity regarding toxicity was also
observed in acute neurotoxicity studies: the EC50 for decreased coordination, balance, and
neuromuscular function (i.e., performance on the rotarod] was 4,694 mg/m3 for 1,2,4-TMB
and 4,738 mg/m3 for 1,3,5-TMB. The ECso for decreased pain sensitivity (i.e., latency to
paw-lick measured on the hot plate apparatus] were also similar for both isomers: 5,683
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mg/m3 for 1,2,4-TMB and 5,963 mg/m3 for 1,3,5-TMB fKorsakand Rydzynski. 19961
Other neurotoxic endpoints similarly affected by either isomer (albeit from oral exposures
or i.p. injections] included increased electrocortical arousal and altered EEC function
(Tomas etal.. 1999a: Tomas etal.. 1999c}. However, the doses eliciting these effects were
LOAELs, and therefore it is unclear whether this represents true similarity in toxic potency
or whether testing at lower doses would reveal differences between the two isomers.
Additionally, the magnitude of effect differed between isomers, with 1,2,4-TMB and
1,3,5-TMB inducing greater changes in brain EEGs and electrocortical arousal, respectively.
In short-term neurotoxicity studies, a similar pattern of effects (inability to learn
passive or active avoidance, decreased pain sensitivity, increased spontaneous motor
activity] indicating altered neurobehavioral function was observed in rats exposed to
either isomer (Wiaderna etal.. 2002: Gralewicz and Wiaderna. 2001: Gralewicz et al..
1997a]. In these studies, 1,3,5-TMB was shown to be more toxic that 1,2,4-TMB, with
neurobehavioral effects occurring at lower exposures (123 vs. 492 mg/m3] in animals
exposed to 1,3,5-TMB vs. those exposed to 1,2,4-TMB. When comparing the magnitude of
TMB isomer-induced neurotoxicity, exposure to 492 mg/m3 1,3,5-TMB induced greater
decrements in avoidance acquisition (50% vs. 40%] compared to exposure to the same
concentration of 1,2,4-TMB. Lastly, manifestation of neurotoxicity occurred at earlier time
points (three vs. seven days] in rats exposed to 1,3,5-TMB compared to those exposed to
1,2,4-TMB.
Finally, the observed developmental effects observed in Saillenfait et al. (2005] were
shown to be similar between isomers. Exposure to 1,2,4-TMB and 1,3,5-TMB significantly
decreased male fetal body weights to a similar degree (5% and 7%, respectively] at 2952
mg/m3. 1,2,4-TMB and 1,3,5-TMB also decreased female body weights to a similar degree
(5% and 6%, respectively] at the same exposure concentration. This body weight decrease
was significant in animals exposed to 1,2,4-TMB, but was not significant in those females
exposed to 1,3,5-TMB. 1,3,5-TMB was observed to be more toxic with regard to maternal
toxicity, inducing a 75% reduction in maternal weight gain at 2952 mg/m3 compared to a
50% reduction in animals exposed to the same concentration of 1,2,4-TMB.
The two isomers are similar to one another in their chemical and toxicokinetic
properties, although important differences do also exist. Both isomers have very similar
Log KOW values, and blood:air partition coefficients reported for humans and rats in the
literature are similar between isomers: 43.0 for 1,2,4-TMB and 59.1 for 1,3,5-TMB. This
gives the strong indication that the two isomers would partition into the blood in a similar
fashion. Supporting this is the observation that 1,2,4-TMB and 1,3,5-TMB absorb equally
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into the bloodstream of exposed humans (6.5 and 6.2 |iM, respectively] Qarnberg et al..
1996). Also, the net respiratory uptake of 1,2,4-TMB and 1,3,5-TMB was similar in humans,
and the respiratory uptake for 1,2,4-TMB was similar between humans and rats Qarnberg
etal.. 1996: Dahl etal.. 1988}. Distribution of the two isomers throughout the body is
qualitatively similar, although it appears that liver and kidney concentrations for
1,2,4-TMB were greater than those for 1,3,5-TMB in both acute and short-term exposures
fSwiercz etal.. 2006: Swiercz etal.. 2003: Swiercz etal.. 20021 Although 1,2,4-TMB was
observed to distribute to the brain (Swiercz et al.. 2003: Eide and Zahlsen. 1996}.
distribution of 1,3,5-TMB to the brain was not experimentally measured in any study.
However, the predicted brain:air partition coefficient was similar between 1,2,4-TMB and
1,3,5-TMB for both humans (206 vs. 199} and rats (552 vs. 535} fMeulenberg and
Viiverberg. 20001 This strongly suggests that 1,2,4-TMB and 1,3,5-TMB can be expected to
distribute similarly to the brain in both humans and rats. Both isomers were observed to
primarily metabolize to benzoic and hippuric acids in humans and rats Qarnberg etal..
1996: Huoetal.. 1989: Mikulski and Wiglusz. 19751 although the amount of inhaled TMB
recovered as hippuric acid metabolites following exposure to 1,2,4-TMB or 1,3,5-TMB was
somewhat dissimilar in humans (22% vs. 3%, respectively} and rats (24-38% vs. 59%,
respectively} Qarnberg et al.. 1996: Mikulski and Wiglusz. 1975}. Other terminal
metabolites included mercapturic acids (-14-19% total dose}, phenols (-12% total dose},
and glucuronides and sulphuric acid conjugates (4-9% total dose} for 1,2,4-TMB and
phenols (-4-8% total dose} and glucuronides and sulphuric acid conjugates (-5-9% total
dose} for 1,3,5-TMB fTsuiimoto etal.. 2005: Tsuiimoto etal.. 2000: Huoetal.. 1989:
Wiglusz. 1979: Mikulski and Wiglusz. 1975}. In humans, the half-lives of elimination from
blood were observed to be greater for 1,3,5-TMB (1.7 minutes, 29 minutes, 4.9 hours, and
120 hours} than for 1,2,4-TMB (1.3 minutes, 21 minutes, 3.6 hours, and 87 hours}
Qarnberg etal.. 1997a: Tarnbergetal.. 1997b: Tarnbergetal.. 19961 although this
difference may be due to small sample sizes and difficulties in measuring slow elimination
phases rather than a true difference in half-lives. At low exposure concentrations, half-lives
in elimination from the blood were somewhat similar for 1,2,4-TMB and 1,3,5-TMB (3.6 vs.
2.7 hours}, but this difference became much greater with increasing doses (17.3 hours for
1,2,4-TMB and 4 hours for 1,3,5-TMB following exposure to 1230 mg/m3 for six hours}
fSwiercz etal.. 2003: Swiercz etal.. 20021.
Given the above information regarding the observed toxicity following 1,2,4-TMB and
1,3,5-TMB exposures across acute, short-term, and developmental studies, the use of
1,3,5-TMB-specific data for derivation of an RfC was not considered by EPA to be
scientifically supported. Derivation of an RfC for 1,3,5-TMB using the only adequate
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toxicity data available (i.e., Saillenfait et al. (2005)) would result in an RfC 15-fold higher
than the RfC derived for 1,2,4-TMB based on altered CNS function (i.e., decreased pain
sensitivity}. The available toxicity data indicates that 1,2,4-TMB and 1,3,5-TMB are similar
in acute respiratory and neurological toxicity and developmental toxicity, but that
1,3,5-TMB appears to be more potent in eliciting neurotoxicity and maternal toxicity
following short-term exposures. 1,3,5-TMB is observed to elicit neurotoxic effects in rats in
acute and short-term studies, and therefore the selected critical effect for 1,2,4-TMB,
altered CNS function, is relevant to observed 1,3,5-TMB-induced toxicity. Similarities in
blood:air partition coefficients, respiratory uptake, and absorption into the bloodstream
between the two isomers support the conclusion that internal blood dose metrics for
1,3,5-TMB would be similar as those calculated for 1,2,4-TMB using the available PBPK
model.
Thus, the chronic RfC of 2 x 10-2 mg/m3 derived for 1,2,4-TMB was adopted as
the RfC for 1,3,5-TMB based on the conclusion that the two isomers were sufficiently
similar regarding chemical properties, kinetics, and toxicity.
As noted previously, 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 EPA
(1994). Section 4.3.9.2. The chronic RfC of 2 x 10-2 mg/m3 derived for 1,2,4-TMB was
adopted as the RfC for 1,3,5-TMB based on the conclusion that the two isomers were
sufficiently similar regarding chemical properties, kinetics, and toxicity. Thus, confidence
in the study from which the critical effect was identified, Korsak and Rydzynski (1996) is
medium (see above). Confidence in the database is low to medium as the database includes
acute, short-term, and developmental toxicity studies in rats and mice. The database lacks
a chronic, subchronic, and multigenerational reproductive study. Additionally, the studies
supporting the critical effect predominately come from the same research institute.
Overall confidence in the RfC for 1,3,5-TMB is low due to uncertainties surrounding the
adoption of the RfC derived for 1,2,4-TMB as the RfC for 1,3,5-TMB.
2.1.7. Uncertainties in the Derivation of the RfC for 1,3,5-TMB
Uncertainties exist in adopting the RfC derived for 1,2,4-TMB based on altered CNS
function (i.e., decreased pain sensitivity) as the RfC for 1,3,5-TMB. The available database
for 1,3,5-TMB was considered insufficient with which to derive an RfC. If the most
sensitive endpoint from the only adequate study in the 1,3,5-TMB database (i.e., decreased
maternal weight gain; Saillenfait et al. (2005)). was used for the RfC derivation, an RfC 15-
fold higher would be derived for 1,3,5-TMB vs. that derived for 1,2,4-TMB (3 x 10-1 vs. 2 x
10-2 mg/m3, respectively). Although uncertainly exists in adopting the 1,2,4-TMB RfC
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value for 1,3,5-TMB, both isomers share multiple commonalities and similarities regarding
their toxicokinetic and toxicological properties that support the adoption of the value of
one isomer for the other. The majority of uncertainty regarding 1,3,5-TMB's database
involves the lack of a chronic, subchronic, or multi-generational reproductive study for this
isomer. Given the similarities in toxicity from the available developmental toxicity study,
and neurotoxicity and respiratory toxicity observed in the available acute and short-term
studies, there is strong evidence that the two isomer's toxicity resulting from subchronic
exposure can be expected to be similar. More so, 1,3,5-TMB may actually be expected to be
slightly more toxic than 1,2,4-TMB following subchronic exposures than 1,2,4-TMB given
the observation of greater magnitude and earlier onset of effect following 1,3,5-TMB
exposures in short-term studies. Therefore, while uncertainty does exist in the derivation
of 1,3,5-TMB's RfC, the available information regarding sufficient toxicokinetic and
toxicological similarity between the two isomers indicates this uncertainly does not
preclude adopting the RfC for 1,2,4-TMB as the RfC for 1,3,5-TMB.
2.2. Oral Reference Dose for Effects other than Cancer
2.2.1. Methods of analysis for RfD derivation for 1,2,4-TMB
No chronic or subchronic studies were identified for 1,2,4-TMB that utilized the oral
route of exposure. Therefore, the available oral database for 1,2,4-TMB is minimal as
defined by EPA guidance (i.e., there is no human data available nor any adequate oral
animal data] (U.S. EPA. 2002}. and this database is inadequate for the derivation of an RfD.
Even though the available oral database for 1,2,4-TMB is inadequate to derive an RfD, a
route-to-route extrapolation from inhalation to oral for the purposes of deriving an RfD is
possible using the existing inhalation data and the available 1,2,4-TMB PBPK model
(Hissink et al.. 2007}. Using route-to-route extrapolation via application of PBPK models is
supported by EPA guidance (U.S. EPA. 2002.1994} given enough data and ability to
interpret that data regarding differential metabolism and toxicity between different routes
of exposure. 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 (Appendix A (U.S. EPA.
2011djj. Further, no evidence exists that would suggest toxicity profiles would differ to a
substantial degree between oral and inhalation exposures.
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Therefore, assuming oral exposure would result in the same systemic effect as
inhalation exposure (altered CNS function, measured as decreased pain sensitivity [Korsak
and Rydzynski. 1996}. an oral exposure component was added to the Hissink et al. (2007}
PBPK model by EPA (Appendix A (U.S. EPA. 2011dj: Section A.1.4}, assuming continuous
oral ingestion and 100% of the ingested 1,2,4-TMB is absorbed by constant infusion of the
oral dose into the liver. This is a common assumption when information about the oral
absorption of the compound is unknown. The contribution of the first-pass metabolism in
the liver for oral dosing was evaluated by simulating steady state venous blood levels (at
the end of 50 days continuous exposure} for a standard human at rest (70 kg} for a range of
concentrations and doses; at low daily doses (0.1-10 mg/kg-day}, equivalent inhalation
concentrations result in steady state blood concentrations 4-fold higher than those
resulting from oral doses, indicating the presence of first-pass metabolism following oral
exposure. This difference became insignificant for daily doses exceeding 50 mg/kg-day
(Appendix A (U.S. EPA. 2011dj: Section A.1.4}.
The human PBPK model inhalation dose metric (weekly average blood concentration,
mg/L} for the PODADJ (0.085 mg/L} was used as the target for the oral dose metric. The
human PBPK model was run to determine what oral exposure would yield an equivalent
weekly average blood concentration and the resulting value of 6.2 mg/kg-day was used as
the human equivalent dose POD (PODHED} for the RfD derivation.
2.2.2. RfD Derivation for 1,2,4-TMB
A PODHED of 6.2 mg/kg-day was derived for the oral database using route-to-route
extrapolation based on the neurotoxic effects observed by Korsak and Rydzynski (1996}
following inhalation exposure (decreased pain sensitivity}. Thus, the same uncertainty
factors applied to derive the RfC (see Section 2.1.2} were also applied to derive the RfD.
The uncertainty factors, selected based on EPA's report, A Review of the Reference Dose and
Reference Concentration Processes (2002J. address five areas of uncertainty resulting in a
composite UF of 1,000.
Application of this 1,000-fold composite UF yields the calculation of the chronic RfD for
1,2,4-TMB as follows:
RfD = PODHED •*• UF = 6.2 mg/kg-day •*• 1,000 = 0.006 mg/kg-day = 6 x 10 3 mg/kg-day
(rounded to one significant digit)
A PBPK model was utilized to perform a route-to-route extrapolation to determine a
POD for the derivation of the RfD from the Korsak and Rydzynski (1996} inhalation study
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and corresponding critical effect. The confidence in the study from which the critical effect
was identified, Korsak and Rydzynski (1996) is medium (see above}. Confidence in the
database for 1,2,4-TMB is low to medium as the database includes acute, short-term,
subchronic, and developmental toxicity studies in rats and mice. The database lacks a
multigenerational reproductive study, and the studies supporting the critical effect
predominately come from the same research institute. Overall confidence in the RfD for
1,2,4-TMB is low due to uncertainties surrounding the application of the available PBPK
model for the purposes of a route-to-route extrapolation.
2.2.3. Uncertainties in the Derivation of the RfD for 1,2,4-TMB
As the oral RfD for 1,2,4-TMB was based on a route-to-route extrapolation in order to
determine the oral dose that would result in the same effect as inhalation exposure
(decreased pain sensitivity; Korsak and Rydzynski (1996)). the uncertainties regarding this
derivation are the same as for the RfC for 1,2,4-TMB (see Section 2.1.4), with the exception
of the uncertainty surrounding the route-to-route extrapolation. The model used to
perform this route-to-route extrapolation is a well-characterized model deemed
appropriate for the purposes of the Toxicological Review. One source of uncertainty
regarding the route-to-route extrapolation is the assumption of 100% bioavailability, that
is,100% of the ingested 1,2,4-TMB would be absorbed and pass through the liver. If not all
of the compound is bioavailable, a lower blood concentration would be expected compared
to the current estimate, and thus, a higher RfD would be calculated.
2.2.4. Methods of analysis for RfD derivation for 1,3,5-TMB
The available oral database is inadequate to derive an RfD for 1,3,5-TMB. No chronic,
subchronic, or short-term oral exposure studies were found in the literature. However, as
outlined in RfC Derivation for 1,3,5-TMB, the toxicokinetic and toxicological similarities
between 1,3,5-TMB and 1,2,4-TMB support adopting the RfC for 1,2,4-TMB as the RfC
1,3,5-TMB. These considerations also apply to the oral reference value, thus the RfD for
1,2,4-TMB was adopted for 1,3,5-TMB. 1,3,5-TMB is observed to elicit neurotoxic effects in
rats in acute and short-term studies, and therefore the selected critical effect for 1,2,4-TMB,
altered CNS function, is relevant to observed 1,3,5-TMB-induced toxicity. Similarities in
blood:air and tissue:air partition coefficients and absorption into the bloodstream between
the two isomers support the conclusion that internal blood dose metrics for 1,3,5-TMB
would be similar as those calculated for 1,2,4-TMB using the available PBPK model. Also,
the qualitative metabolic profiles for the two isomers are similar, with dimethylbenzyl
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hippuric acids being the major terminal metabolite for both isomers, so that first-pass
metabolism through the liver is not expected to differ greatly between 1,2,4-TMB and
1,3,5-TMB.
Therefore, given the above similarities in toxicokinetics and toxicity, the RfD derived for
1,2,4-TMB: 6 x 10-3 mg/kg-day was adopted for the RfD for 1,3,5-TMB.
As noted previously, a confidence level of high, medium, or low is assigned to the study
used to derive the RfD, the overall database, and the RfD itself, as described in EPA (1994).
Section 4.3.9.2. The chronic RfD of 6 x 10-3 mg/kg-day derived for 1,2,4-TMB was adopted
as the RfD for 1,3,5-TMB based on the conclusion that the two isomers were sufficiently
similar regarding chemical properties, kinetics, and toxicity. Thus, confidence in the study
from which the critical effect was identified, Korsak and Rydzynski (1996) is medium (see
above}. Confidence in the database is low to medium as the database includes acute, short-
term, and developmental toxicity studies in rats and mice. The database lacks a
multigenerational reproductive study, and the studies supporting the critical effect
predominately come from the same research institute. Overall confidence in the RfD for
1,3,5-TMB is low due to uncertainties surrounding the adoption of the RfD derived for
1,2,4-TMB as the RfD for 1,3,5-TMB.
2.2.5. Uncertainties in the Derivation of the RfD for 1,3,5-TMB
The uncertainties regarding adopting the RfD for 1,2,4-TMB as the RfD for 1,3,5-TMB
encompass previous areas of uncertainty involved in the derivation of the RfC for 1,3,5-
TMB and the RfD for 1,2,4-TMB (see Sections 2.1.7 and 2.2.4}. There does exist uncertainty
regarding this adoption. However, as discussed above in Section 2.1.7, both isomers share
multiple commonalities and similarities regarding their toxicokinetic and toxicological
properties that support adopting one isomer's value for the other. Additionally, as the RfD
derivation for 1,2,4-TMB was based on a route-to-route extrapolation, the uncertainties in
that toxicity value's derivation (see Section 2.2.4} apply to the derivation of the RfD for
1,3,5-TMB.
2.3. Cancer Assessment
Under the U.S. EPA Guidelines for Carcinogen Risk Assessment (2005aj. the database for
1,2,4-TMB and 1,3,5-TMB provides "inadequate information to assess carcinogenic
potential". This characterization is based on the limited and equivocal genotoxicity
findings, and the lack of data indicating carcinogenicity in experimental animal species.
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Information available on which to base a cancer assessment is lacking, and thus, no cancer
risk value is derived.
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3. REFERENCES
ACGIH. (American Conference of Governmental Industrial Hygienists). (2002). Trimethyl benzene isomers. In
Documentation of the threshold limit values and biological exposure indices (7 ed.). Cincinnati, OH.
Andersson. K: Fuxe. K: Toftgard. R: Nilsen. OG: Eneroth. P: Gustafsson. JA. (1980). Toluene-induced activation of
certain hypothalamic and median eminence catecholamine nerve terminal systems of the male rat and its effects on
anterior pituitary hormone secretion. ToxicolLett 5: 393-398. http://dx.doi.org/10.1016/0378-4274(80)90021-1.
Andersson. K: Fuxe. K: Nilsen. OG: Toftgard. R: Eneroth. P: Gustafsson. JA. (1981). Production of discrete
changes in dopamine and noradrenaline levels and turnover in various parts of the rat brain following exposure to
xylene, ortho-, meta-, and para-xylene, and ethylbenzene. Toxicol Appl Pharmacol 60: 535-548.
Andersson. K: Nilsen. OG: Toftgard. R: Eneroth. P: Gustafsson. JA: Battistini. N: Agnati. LF. (1983). Increased
amine turnover in several hypothalamic noradrenaline nerve exposure to various concentrations of toluene.
Neurotoxicology 4: 43-55.
Balster. RL. (1998). Neural basis of inhalant abuse. Drug Alcohol Depend 51: 207-214.
Battig. K: Grandjeaa E: Turrian. V. (1956). Health damage after continuous exposure to trimethyl benzene in a
painting workshop. Soz Praventivmed 1: 389-403. http://dx.doi.org/10.1007/BF02031676.
Billionnet C: Gay. E: Kirchner. S: Leynaert B: Annesi-Maesano. I. (2011). Quantitative assessments of indoor air
pollution and respiratory health in a population-based sample of French dwellings. Environ Res 111: 425-434.
http://dx.doi.0rg/10.1016/j.envres.2011.02.008.
Bowen. SE: Batis. JC: Paez-Martinez. N: Cruz. SL. (2006). The last decade of solvent research in animal models of
abuse: Mechanistic and behavioral studies. Neurotoxicol Teratol 28: 636-647.
http://dx.doi.0rg/10.1016/j.ntt.2006.09.005.
Braes. PU: Gregory. P: Jackson. DM. (1984). Passive avoidance in rats: Disruption by dopamine applied to the
nucleus accumbens. Psychopharmacology 83: 70-75.
CDC. (Centers for Disease Control and Prevention). (2004). The health consequences of smoking: A report of the
Surgeon General. Washington, DC: U.S. Department of Health and Human Services.
http://www.surgeongeneral.gov/library/smokingconsequences/.
Chen. R: Dick. F: Seaton. A. (1999). Health effects of solvent exposure among dockyard painters: Mortality and
neuropsychological symptoms. Occup EnvironMed 56: 383-387. http://dx.doi.0rg/10.1136/oem.56.6.383.
Chou. CC: Riviere. JE: Monteiro-Riviere. NA. (2003). The cytotoxicity of jet fuel aromatic hydrocarbons and dose-
related interleukin-8 release from human epidermal keratinocytes. Arch Toxicol 77: 384-391.
http://dx.doi.org/10.1007/s00204-003-0461-z.
Dahl AR: Damon. EG: Mauderly. JL: Rothenberg. SJ: Seiler. FA: McClellan. RO. (1988). Uptake of 19
hydrocarbon vapors inhaled by F344 rats. Fundam Appl Toxicol 10: 262-269. http://dx.doi.org/10.1016/0272-
0590(88)90310-7.
Eide. I: Zahlsen. K. (1996). Inhalation experiments with mixtures of hydrocarbons. Experimental design, statistics
and interpretation of kinetics and possible interactions. Arch Toxicol 70: 397-404.
http://dx.doi.org/10.1007/s002040050291.
DRAFT - DO NOT CITE OR QUOTE 91
-------
Gage. JC. (1970). The subacute inhalation toxicity of 109 industrial chemicals. Br J Ind Med 27: 1-18.
http://dx.doi.org/10.1136/oem.27.Ll.
Gaschen. A: Lang. D: Kalberer. M: Savi M: Geiser. T: Gazdhar. A: Lehr. CM: Bur. M: Dommen. J: Baltensperger.
U: Geiser. M. (2010). Cellular responses after exposure of lung cell cultures to secondary organic aerosol particles.
Environ Sci Technol 44: 1424-1430. http://dx.doi.org/10.1021/es902261m.
Ginsberg. G: W Jr. S: Bruckner. J: Sonawane. B. (2004). Incorporating children's toxicokinetics into a risk
framework. Environ Health Perspect 112: 272-283. http://dx.doi.org/10.1289/ehp.6013.
Gralewicz. S: Wiaderna. D: Tomas. T: Rydzyriski. K. (1997a). Behavioral changes following 4-week inhalation
exposure to pseudocumene (1,2,4-trimethylbenzene) in the rat. Neurotoxicol Teratol 19: 327-333.
http://dx.doi.org/10.1016/S0892-0362(97)00001-9.
Gralewicz. S: Wiaderna. D: Tomas. T. (1997b). Retardation of the age-related increase in spontaneous cortical
spike-wave discharges (SWD) in rats after a 28-day inhalation (SWD) in rats after a 28-day inhalation exposure to
an industrial solvent, pseudocumene (1,2,4-trimethylbenzene). Int J Occup Med Environ Health 10: 213-222.
Gralewicz. S: Wiaderna. D. (2001). Behavioral effects following subacute inhalation exposure to m-xylene or
trimethylbenzene in the rat: A comparative study. Neurotoxicology 22: 79-89. http://dx.doi.org/10.1016/S0161-
813X(00)00003-6.
Guvatt GH: Oxman. AD: Vist GE: Kunz. R: Falck-Ytter. Y: Alonso-Coello. P: Schiinemann. HJ. (2008a).
GRADE: An emerging consensus on rating quality of evidence and strength of recommendations. BMJ 336: 924-
926. http://dx.doi.org/10.1136/bmj.39489.470347.AD.
Guvatt. GH: Oxman. AD: Kunz. R: Vist. GE: Falck-Ytter. Y: Schiinemann. HJ. (2008b). GRADE: What is "quality
of evidence" and why is it important to clinicians. BMJ 336: 995-998.
http://dx.doi.org/10.1136/bmj.39490.551019.BE.
Henderson. RF. (2001). Aromatic hydrocarbons: Benzene and other alkylbenzenes. 4: 231-301.
Hill. AB. (1965). The environment and disease: Association or causation? Proc R Soc Med 58: 295-300.
Hillefors-Berglund. M: Liu. Y: von Euler. G. (1995). Persistent, specific and dose-dependent effects of toluene
exposure on dopamine D2 agonist binding in the rat caudate-putamen. Toxicology 100: 185-194.
http://dx.doi.org/10.1016/0300-483X(95)03084-S.
Hissink. AM: Kriise. J: Kulig. BM: Verwei M: Muijser. H: Salmon. F: Leenheers. LH: Owen. DE: Lammers. JH:
Freidig. AP: McKee. RH. (2007). Model studies for evaluating the neurobehavioral effects of complex hydrocarbon
solvents III. PBPK modeling of white spirit constituents as a tool for integrating animal and human test data.
Neurotoxicology 28: 751-760. http://dx.doi.0rg/10.1016/j.neuro.2007.03.005.
Huo. JZ: Aldous. S: Campbell. K: Davies. N. (1989). Distribution and metabolism of 1,2,4-trimethylbenzene
(pseudocumene) in the rat. Xenobiotica 19: 161-170. http://dx.doi.org/10.3109/00498258909034688.
IARC. (International Agency for Research on Cancer). (2006). Preamble to the IARC monographs. Lyon, France.
http://monographs.iarc.fr/ENG/Preamble/.
Jacksoa DM: Westlind-Danielssoa A. (1994). Dopamine receptors: Molecular biology, biochemistry and
behavioural aspects. Pharmacol Ther64: 291-370. http://dx.doi.org/10.1016/0163-7258(94)90041-8.
Janik-Spiechowicz. E: Wvszynska. K: Dziubaltowska. E. (1998). Genotoxicity evaluation of trimethylbenzenes.
Mutat Res Genet Toxicol Environ Mutagen 412: 299-305. http://dx.doi.org/10.1016/81383-5718(97)00202-7.
Jarnberg. J: Johanson. G. (1995). Liquid/air partition coefficients of the trimethylbenzenes. Toxicol Ind Health 11:
81-88. http://dx.doi.org/10.1177/074823379501100107.
DRAFT - DO NOT CITE OR QUOTE 92
-------
Jarnberg. J: Johanson. G: Lof. A. (1996). Toxicokinetics of inhaled trimethylbenzenes in man. Toxicol Appl
Pharmacol 140: 281-288. http://dx.doi.org/10.1006/taap.1996.0223.
Jarnberg. J: Johanson. G: Lof. A: Stahlbom. B. (1997a). Inhalation toxicokinetics of 1,2,4-trimethylbenzene in
volunteers: Comparison between exposure to white spirit and 1,2,4-trimethylbenzene alone. Sci Total Environ 199:
65-71. http://dx.doi.org/10.1016/S0048-9697(97)05482-X.
Jarnberg. J: Stahlbon. B: Johanson. G: Lof. A. (1997b). Urinary excretion of dimethylhippuric acids in humans after
exposure to trimethylbenzenes. Int Arch Occup Environ Health 69: 491-497.
http://dx.doi.org/10.1007/s004200050179.
Jones. K: Meldrum M: Baird. E: Cottrell. S: Kaur. P: Plant. N: Dyne. D: Cocker. J. (2006). Biological monitoring
for trimethylbenzene exposure: A human volunteer study and a practical example in the workplace. Ann Occup Hyg
50: 593-598. http://dx.doi.org/10.1093/annhyg/mel016.
Korsak. Z: Swiercz. R: Rydzyfiski. K. (1995). Toxic effects of acute inhalation exposure to 1,2,4-trimethylbenzene
(pseudocumene) in experimental animals. Int J Occup Med Environ Health 8:331-337.
Korsak. Z: Rydzyfiski. K. (1996). Neurotoxic effects of acute and subchronic inhalation exposure to
trimethylbenzene isomers (pseudocumene, mesitylene, hemimellitene) in rats. Int J Occup Med Environ Health 9:
341-349.
Korsak. Z: Rydzynski. K: Jajte. J. (1997). Respiratory irritative effects of trimethylbenzenes: An experimental
animal study. Int J Occup Med Environ Health 10: 303-311.
Korsak. Z: Stetkiewicz. J: Majcherek. W: Stetkiewicz. I: Jaite. J: Rydzynski. K. (2000). Sub-chronic inhalation
toxicity of 1,2,4-trimethylbenzene (pseudocumene) in rats. Int J Occup Med Environ Health 13: 155-164.
Kyrklund. T. (1992). The use of experimental studies to reveal suspected neurotoxic chemicals as occupational
hazards: Acute and chronic exposures to organic solvents. Am J Ind Med 21: 15-24.
http://dx.doi.org/10.1002/ajim.4700210105.
Lammers. JH: Emmea HH: Muijser. H: Hoogendijk. EM: McKee. RH: Owen. DE: Kulig. BM. (2007). Model
studies for evaluating the neurobehavioral effects of complex hydrocarbon solvents II. Neurobehavioral effects of
white spirit in rat and human. Neurotoxicology 28: 736-750. http://dx.doi.0rg/10.1016/j.neuro.2007.03.003.
Maltoni. C: Ciliberti. A: Pinto. C: Soffritti M: Belpoggi F: Menarini. L. (1997). Results of long-term experimental
carcinogenicity studies of the effects of gasoline, correlated fuels, and major gasoline aromatics on rats. Ann N Y
Acad Sci 837: 15-52. http://dx.doi.0rg/10.llll/j.1749-6632.1997.tb56863.x.
McKee. RH: Lammers. JH: Muijser. H: Owen. DE: Kulig. BM. (2010). Neurobehavioral effects of acute exposure
to aromatic hydrocarbons. Int J Toxicol 29: 277-290. http://dx.doi.org/10.1177/1091581810365089.
Meulenberg. C: Vijverberg. H. (2000). Empirical relations predicting human and rat tissue: Air partition coefficients
of volatile organic compounds. Toxicol Appl Pharmacol 165: 206-216. http://dx.doi.org/10.1006/taap.2000.8929.
Mikulski. PI: Wiglusz. R. (1975). The comparative metabolism of mesitylene, pseudocumene, and hemimellitene in
rats. Toxicol Appl Pharmacol 31: 21-31. http://dx.doi.org/10.1016/0041-008X(75)90048-4.
MOE. (Ontario Ministry of the Environment). (2006). Rationale for the development of Ontario air standards for
trimethylbenzenes: 1,2,3-Trimethylbenzene. Ontario, Canada.
Mogel. I: Baumann. S: Bohme. A: Kohaida. T: von Bergen. M: Simon. JC: Lehmann. I. (2011). The aromatic
volatile organic compounds toluene, benzene and styrene induce COX-2 and prostaglandins in human lung epithelial
cells via oxidative stress and p38 MAPK activation. Toxicology 289: 28-37.
http://dx.doi.0rg/10.1016/j.tox.2011.07.006.
DRAFT - DO NOT CITE OR QUOTE 93
-------
Myhre. O: Vestad. TA: Sagstuen. E: Aarnes. H: Fonnum. F. (2000). The effects of aliphatic (n-nonane), naphtenic
(1,2,4-trimethylcyclohexane), and aromatic (1,2,4-trimethylbenzene) hydrocarbons on respiratory burst in human
neutrophil granulocytes. Toxicol Appl Pharmacol 167: 222-230. http://dx.doi.org/10.1006/taap.2000.9008.
Myhre. O: Fonnum F. (2001). The effect of aliphatic, naphthenic, and aromatic hydrocarbons on production of
reactive oxygen species and reactive nitrogen species in rat brain synaptosome fraction: the involvement of calcium,
nitric oxide synthase, mitochondria, and phospholipase A. Biochem Pharmacol 62: 119-128.
http://dx.doi.org/10.1016/80006-2952(01)00652-9.
NIOSH. (National Institute for Occupational Safety and Health). (1992). NIOSH recommendations for occupational
safety and health: Compendium of policy documents and statements. (92-100). Cincinnati, OH: U.S. Department of
Health and Human Services, Public Health Service, Centers for Disease Control, National Institute for Occupational
Safety and Health. http://www.cdc.gov/Niosh/92-100.html.
Norseth. T: Waage. J: Dale. I. (1991). Acute effects and exposure to organic compounds in road maintenance
workers exposed to asphalt. Am J Ind Med 20: 737-744. http://dx.doi.org/10.1002/ajim.4700200604.
Ramaiah. SK. (2007). A lexicologist guide to the diagnostic interpretation of hepatic biochemical parameters. Food
Chem Toxicol 45: 1551-1557. http://dx.doi.0rg/10.1016/j.fct.2007.06.007.
Rea. TM: Nash. JF: Zabik. JE: Born. GS: Kessler. WV. (1984). Effects of toluene inhalation on brain biogenic
amines in the rat. Toxicology 31: 143-150. http://dx.doi.org/10.1016/0300-483X(84)90006-4.
Rothman. KJ: Greenland. S. (1998). Modern epidemiology (2nd ed.). Philadelphia, PA: Lippincott, Williams, &
Wilkins.
Saillenfait AM: Gallissot F: Sabate. JP: Morel G. (2005). Developmental toxicity of two trimethylbenzene
isomers, mesitylene and pseudocumene, in rats following inhalation exposure. Food Chem Toxicol 43: 1055-1063.
http://dx.doi.0rg/10.1016/j.fct.2005.02.008.
Snead. OC. III. (1995). Basic mechanisms of generalized absence seizures. AnnNeurol 37: 146-157.
http://dx.doi.org/10.1002/ana.410370204.
Swiercz. R: Rydzyfiski. K: Wasowicz. W: Maicherek. W: Wesolowski. W. (2002). Toxicokinetics and metabolism
of pseudocumene (1,2,4-trimethylbenzene) after inhalation exposure in rats. Int J Occup Med Environ Health 15:
37-42.
Swiercz. R: Wiaderna. D: Wasowicz. W: Rydzynskj K. (2003). Pseudocumene in brain, liver, lung and blood of rats
after single and repeated inhalation exposure. Int J Occup Med Environ Health 16: 61-66.
Swiercz. R: Wasowicz. W: Maicherek. W. (2006). Mesitylene (1,3,5-trimethylbenzene) in the liver, lung, kidney,
and blood and 3,5-dimethylbenzoic acid in the liver, lung, kidney and urine of rats after single and repeated
inhalation exposure to mesitylene. Pol J Environ Stud 15: 485-492.
Tomas. T: Lutz. P: Wiaderna. D. (1999a). Changes in electrocortical arousal following acute trimethylbenzene
administration in rats. Int J Occup Med Environ Health 12: 67-78.
Tomas. T: Swiercz. R: Wiaderna. D: Gralewicz. S. (1999b). Effects of acute exposure to aromatic hydrocarbons C 9
on locomotor activity in rats. Trimethylbenzene isomers. Int J Occup Med Environ Health 12: 331-343.
Tomas. T: Wiaderna. D: Swiercz. R. (1999c). Neurotoxicity assessment of selected organic solvents based on
spontaneous and evoked cortical and hippocampal activity in rats. Int J Occup Med Environ Health 12: 73-84.
Tsuiimoto. Y: Noda. T: Shimizu. M: Moriwakj H: Tanaka. M. (2000). Identification of the dimethylbenzyl
mercapturic acid in urine of rats administered with 1,2,4-trimethylbenzene. Chemosphere 40: 893-896.
http://dx.doi.org/10.1016/S0045-6535(99)00467-l.
DRAFT - DO NOT CITE OR QUOTE 94
-------
Tsuiimoto. Y: Warashina. M: Nam. VD: Noda. T: Shimizu. M: Yamaguchi. Y: Moriwaki. H: Morimoto. T:
Kakiuchi. K: Maeda. Y: Tanaka. M. (2005). Determination of urinary phenolic metabolites from rats treated with
1,2,3-and 1,3,5-trimethylbenzenes. JOccup Health 47: 337-339.
U.S. EPA. (U.S. Environmental Protection Agency). (1986a). Guidelines for mutagenicity risk assessment [EPA
Report]. (EPA/630/R-98/003). Washington, DC. http://www.epa.gov/iris/backgrd.html.
U.S. EPA. (U.S. Environmental Protection Agency). (1986b). Guidelines for the health risk assessment of chemical
mixtures [EPA Report]. (EPA/630/R-98/002). Washington, DC.
U.S. EPA. (U.S. Environmental Protection Agency). (1991). Guidelines for developmental toxicity risk assessment
[EPA Report]. (EPA/600/FR-91/001). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment
Forum, http://www.epa.gov/iris/backgrd.html.
U.S. EPA. (U.S. Environmental Protection Agency). (1994). Methods for derivation of inhalation reference
concentrations and application of inhalation dosimetry. (EPA/600/8-90/066F). Research Triangle Park, NC: U.S.
Environmental Protection Agency, Office of Research and Development, Office of Health and Environmental
Assessment, Environmental Criteria and Assessment Office.
http://cfpub.epa. gov/ncea/cfm/recordisplay.cfm?deid=71993.
U.S. EPA. (U.S. Environmental Protection Agency). (1996). Guidelines for reproductive toxicity risk assessment
[EPA Report]. (EPA/630/R-96/009). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment
Forum. http://www.epa.gov/raf/publications/pdfs/REPRO51 .PDF.
U.S. EPA. (U.S. Environmental Protection Agency). (1998). Guidelines for neurotoxicity risk assessment.
(EPA/630/R-95/001F). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum.
http://www.epa.gov/raf/publications/pdfs/NEUROTOX.PDF.
U.S. EPA. (U.S. Environmental Protection Agency). (2000a). Benchmark dose technical guidance document
[external review draft]. (EPA/630/R-00/001). Washington, DC: U.S. Environmental Protection Agency, Risk
Assessment Forum, http://www.epa.gov/raf/publications^enchmark-dose-doc-draft.htm.
U.S. EPA. (U.S. Environmental Protection Agency). (2000b). Supplementary guidance for conducting health risk
assessment of chemical mixtures. (EPA/630/R-00/002). Washington, DC: U.S. Environmental Protection Agency,
Risk Assessment Forum. http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=20533.
U.S. EPA. (U.S. Environmental Protection Agency). (2002). A review of the reference dose and reference
concentration processes. (EPA/630/P-02/002F). Washington, DC.
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=51717.
U.S. EPA. (U.S. Environmental Protection Agency). (2005a). Guidelines for carcinogen risk assessment.
(EPA/630/P-03/001F). Washington, DC. http://www.epa.gov/cancerguidelines/.
U.S. EPA. (U.S. Environmental Protection Agency). (2005b). Supplemental guidance for assessing susceptibility
from early-life exposure to carcinogens. (EPA/630/R-03/003F). Washington, DC: U.S. Environmental Protection
Agency, Risk Assessment Forum, http://www.epa.gov/cancerguidelines/guidelines-carcinogen-supplement.htm.
U.S. EPA. (U.S. Environmental Protection Agency). (2006). Approaches for the application of physiologically
based pharmacokinetic (PBPK) models and supporting data in risk assessment (final report). (EPA/600/R-05/043F).
Washington, DC: U.S. Environmental Protection Agency, Office of Research and Development.
U.S. EPA. (U.S. Environmental Protection Agency). (2007). Acute exposure guideline levels (AEGLs) for 1,3,5-
trimethylbenzene (CAS reg. no. 108-67-8), 1,2,4-trimethylbenzene (CAS reg. no. 95-63-6), 1,2,3-trimethylbenzene
(CAS reg. no. 526-73-8). Washington, DC.
http://www.epa.gov/opptintr/aegl/pubs/123_%20124_%20135_trimethylbenzenes_%20interim_ll_2007.vl.pdf.
DRAFT - DO NOT CITE OR QUOTE 95
-------
U.S. EPA. (U.S. Environmental Protection Agency). (201 la). IRIS Process Retrieved August 24, 2011, from
http://epa.gov/iris/process.htm
U.S. EPA. (U.S. Environmental Protection Agency). (201 Ib). Recommended use of body weight 3/4 as the default
method in derivation of the oral reference dose. (EPA/100/R11/0001). Washington, DC.
http://www.epa.gov/raf/publications/interspecies-extrapolation.htm.
U.S. EPA. (U.S. Environmental Protection Agency). (201 Ic). Supplemental Material: Benchmark Dose Modeling
Results for the Derivation of Reference Values for 1,2,4- and 1,3,5-Trimethylbenzene (Science Consultation Draft
[Appendix C]). Washington, DC.
U.S. EPA. (U.S. Environmental Protection Agency). (20lid). Supplemental Material: Toxicological Information
(PBPK and TK) in Support of Hazard Identification and Dose-Response Analysis for 1,2,4- and 1,3,5-
Trimethylbenzene (Science Consulation Draft [Appendix A]). Washington, DC.
von Euler. G: Ogrea SO: Li. XM: Fuxe. K: Gustafsson. JA. (1993). Persistent effects of subchronic toluene
exposure on spatial learning and memory, dopamine-mediated locomotor activity and dopamine D2 agonist binding
in the rat. Toxicology 77: 223-232. http://dx.doi.org/10.1016/0300-483X(93)90162-L.
von Euler. G: Ogren. SO: Eneroth. P: Fuxe. K: Gustafsson. JA. (1994). Persistent effects of 80 ppm toluene on
dopamine-regulated locomotor activity and prolactin secretion in the male rat. Neurotoxicology 15: 621-624.
Warter. JM: Vergnes. M: Depaulis. A: Tranchant. C: Rumbach. L: Micheletti. G: Marescaux. C. (1988). Effects of
drugs affecting dopaminergic neurotransmission in rats with spontaneous petit mal-like seizures.
Neuropharmacology 27: 269-274. http://dx.doi.org/10.1016/0028-3908(88)90043-3.
Wiaderna. D: Gralewicz. S: Tomas. T. (1998). Behavioral changes following a four-week inhalation exposure to
hemimellitene (1,2,3-trimethylbenzene) in rats. Int J Occup Med Environ Health 11: 319-334.
Wiaderna. D: Gralewicz. S: Tomas. T. (2002). Assessment of long-term neurotoxic effects of exposure to
mesitylene (1,3,5-trimethylbenzene) based on the analysis of selected behavioral responses. Int J Occup Med
Environ Health 15: 385-392.
Wiglusz. R: Kienitz. M: Delag. G: Galuszko. E: Mikulski. P. (1975a). Peripheral blood of mesitylene vapour treated
rats. Bull Inst Marit Trop Med Gdynia 26: 315-321.
Wiglusz. R: Delag. G: Mikulski. P. (1975b). Serum enzymes activity of mesitylene vapour treated rats. Bull Inst
Marit Trop Med Gdynia 26: 303-313.
Wiglusz. R. (1979). The effect of 1, 3, 5-trimethylbenzene inhalation exposure on the glucuronic acid pathway and
activity of some xenobiotic-metabolizing enzymes. Bull Inst Marit Trop Med Gdynia 30: 189-196.
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APPENDICES
Appendix A: Toxicological Information in Support of Hazard Identification
and Dose-Response Analysis for 1,2,4- and 1,3,5-Trimethylbenzene
Appendix B: Benchmark Dose Modeling Results for the Derivation of
Reference Values for 1,2,4- and 1,3,5-Trimethylbenzene
Appendix C: Summary of External Peer Review and Public Comments and
Disposition
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