1 

JANUARY 2012 

2 

3 

4 

5 

6 

7 

8 

9 

Toxicological Review of

1,2,4- and 1,3,5-

Trimethylbenzene
DRAFT - DO NOT CITE OR QUOTE 2 

1 EPA/635/R-11/012

2 www.epa.gov/iris

3 

4 

5 

6 

7 

8 

9 

10 

11 Toxicological review of 1,2,4- and 1,3,5-Trimethylbenzene

12 

13 (CAS No. 95-63-6 and 108-67-8) 

14 

15 In Support of Summary Information on the

16 Integrated Risk Information System (IRIS)

17 

18 

19 JANUARY 2012

20 

21 

22 

23 NOTICE

24 

25 This document is an Interagency Science Consultation draft. This information is distributed 

26 solely for the purpose of pre-dissemination peer review under applicable information quality 

27 guidelines. It has not been formally disseminated by EPA. It does not represent and should not 

28 be construed to represent any Agency determination or policy. It is being circulated for review 

29 of its technical accuracy and science policy implications.

30 

31 

32 

33 

34 

35 National Center for Environmental Assessment 

36 Office of Research and Development 

37 U.S. Environmental Protection Agency 

38 Washington, DC 

39 
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1 DISCLAIMER

2 

3 

4 This document is a preliminary draft for review purposes only. This information is 

5 distributed solely for the purpose of pre-dissemination peer review under applicable 

6 information quality guidelines. It has not been formally disseminated by EPA. It does not 

7 represent and should not be construed to represent any Agency determination or policy. 

8 Mention of trade names or commercial products does not constitute endorsement of 

9 recommendation for use.
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1 

2 CONTENTS 

3 

4 CONTENTS................................................................................................................................................. 4

5 LIST OF TABLES AND FIGURES .......................................................................................................... 5

6 ABBREVIATIONS AND ACRONYMS ................................................................................................... 6

7 PREAMBLE ................................................................................................................................................ 7

8 AUTHORS | CONTRIBUTORS | REVIEWERS .................................................................................. 17

9 PREFACE .................................................................................................................................................. 19

10 EXECUTIVE SUMMARY ...................................................................................................................... 22

11 LITERATURE SEARCH STRATEGY AND STUDY EVALUATION ............................................. 29

12 1.1. Synthesis of Major Toxicological Effects ............................................................................... 32

13 1.2. Selection of Candidate Principal Studies and Critical Effects for Derivation of Reference 

14 Values ............................................................................................................................................ 59

15 1.3. Carcinogenicity Analysis ........................................................................................................ 66

16 2. DOSE-RESPONSE ANALYSIS ......................................................................................................... 67

17 2.1. Inhalation Reference Concentration for Effects other than Cancer ........................................ 67

18 2.2. Oral Reference Dose for Effects other than Cancer ................................................................ 84

19 2.3. Cancer Assessment ................................................................................................................. 87

20 3. REFERENCES ..................................................................................................................................... 88

21 APPENDICES ........................................................................................................................................... 94

22 Appendix A: Toxicological Information in Support of Hazard Identification and Dose-Response 

23 Analysis for 1,2,4- and 1,3,5-Trimethylbenzene ........................................................................... 94

24 Appendix B: Benchmark Dose Modeling Results for the Derivation of Reference Values for 

25 1,2,4- and 1,3,5-Trimethylbenzene ................................................................................................ 94

26 Appendix C: Summary of External Peer Review and Public Comments and Disposition ............ 94

27 

28 
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1 LIST OF TABLES AND FIGURES 

2 Table I. Other Agency and International Assessments ......................................................................... 21

3 Table II. Reference concentration (RfC) for 1,2,4-TMB ....................................................................... 23

4 Table III. Reference dose (RfD) for 1,2,4-TMB ......................................................................................... 26

5 Table IV: Details of the search strategy employed for 1,2,4-TMB and 1,3,5-TMB ....................... 29

6 Table 1-1: Summary of observed in vivo neurotoxicity in subchronic and short-term studies 

7 of male Wistar rats following inhalation exposure to 1,2,4-TMB or 1,3,5-TMB .......................... 38

8 Tables 1-2: Summary of observed in vivo neurotoxicity for 1,2,4-TMB and 1,3,5-TMB — oral 

9 exposures .............................................................................................................................................................. 41

10 Tables 1-3: Summary of observed in vivo respiratory toxicity for 1,2,4-TMB and 1,3,5-TMB — 

11 inhalation exposures ......................................................................................................................................... 48

12 Table 1-4: Summary of observed developmental toxicity for 1,2,4-TMB and 1,3,5-TMB — 

13 inhalation exposures ......................................................................................................................................... 52

14 Tables 1-5: Summary of observed in vivo hematological toxicity and clinical chemistry 

15 effects for 1,2,4-TMB and 1,3,5-TMB — inhalation exposures ........................................................... 54

16 Table 1-6: Non-cancer endpoints resulting from subchronic inhalation exposure to 

17 1,2,4-TMB considered for the derivation of the RfC............................................................................... 60

18 Figure 1-1. Exposure response array for inhalation exposure to 1,2,4-TMB. .............................. 62

19 Table 1-7: Non-cancer endpoints resulting from gestational inhalation exposure (GD 6-20) to 

20 1,3,5-TMB considered for the derivation of the RfC............................................................................... 64

21 Figure 1-2. Exposure response array for inhalation exposure to 1,3,5-TMB. .............................. 65

22 Table 2-1: Summary of dose-response analysis and point of departure estimation for 

23 endpoints resulting from subchronic inhalation exposure to 1,2,4-TMB ...................................... 68

24 Table 2-2: Candidate PODADJ values, human equivalent concentrations (HECs), and applied 

25 uncertainty factors used in the derivation of RfCs for 1,2,4-TMB ..................................................... 69

26 Figure 2-1: Array of candidate PODHEC values with applied UFs and candidate RfCs for CNS,

27 hematological, and pulmonary effects resulting from inhalation exposure to 1,2,4-TMB ...... 73

28 Table 2-4: Duration adjusted point of departure (PODADJ) estimates from short-term and 

29 gestational inhalation exposures to 1,3,5-TMB ....................................................................................... 76

30 Table 2-5: Candidate PODADJ values, human equivalent concentrations (HECs), and applied 

31 uncertainty factors used in the derivation of RfCs for 1,3,5-TMB ..................................................... 78

32 
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1 ABBREVIATIONS AND ACRONYMS 

2 

1,2,4-TMB 1,2,4-trimethylbenzene 

1,3,5-TMB 1,3,5-trimethylbenzene 

AAQC Ambient air quality criterion 

ACGIH American Conference of 

Governmental Industrial Hygienists 

ADME Absorption, distribution, metabolism 

and excretion 

AEGL Acute exposure guideline limit 

AIC Akaike Information Criterion 

BAL bronchoalveolar lavage 

BMD benchmark dose 

BMDL lower confidence limit on the 

benchmark dose 

BMDS benchmark dose software 

BMR benchmark response 

BW body weight 

C Celsius 

CAS Chemical Abstracts Service 

CASRN Chemical Abstracts Service Registry 

Number 

CI confidence interval 

CNS central nervous system 

CYP450 cytochrome P450 

DAF dosimetric adjustment factor 

DMBA dimethylbenzoic acid 

DMHA dimethylhippuric acid 

DNA deoxyribonucleic acid 

EC50 half maximal effective concentration 

EEG Electroencephalogram 

EPA U.S. Environmental Protection 

Agency 

g gram 

GD gestational day 

Hb/g-A animal blood:gas partition coefficient 

Hb/g-H human blood:gas partition coefficient 

HEC human equivalent concentration 

HEK Human epidermal keratinocytes 

HERO Health Effects Research Online 

HEV human epithelial keratinocytes 

HSDB Hazardous Substance Database 

IL-8 interleukin-8 

i.p. intraperitoneal 

IRIS Integrated Risk Information System 

JP-8 jet propulsion fuel 8 

k kilogram 

Km Michaelis-Menten constant 

L liter 

LDH lactate dehydrogenase 

LOAEL lowest-observed-adverse-effect level 

m3 meter cubed 

mg milligram 

n nanogram 

NCEA National Center for Environmental 

Assessment 

NIOSH National Institute for Occupational 

Safety and Health 

NLM National Library of Medicine 

NOAEL No-observed-adverse-effect level 

OMOE Ontario Ministry of the Environment 

OSHA Occupational Safety and Health 

Administration 

p probability value 

p-RfC Provisional RfC 

PBPK physiologically based 

pharmacokinetic (model) 

PEL permissible exposure limit 

POD point of departure 

PODADJ duration adjusted POD 

POI Point of impingement 

ppm parts per million 

RBC red blood cell 

RD50 50% respiratory rate decrease 

REL Recommended exposure limit 

RfC reference concentration 

RfD reference dose 

RGDR regional gas dose ratio 

ROS reactive oxygen species 

SCE sister chromatid exchange 

SD standard deviation 

SOA Secondary organic aerosol 

t time 

TLV threshold limit value 

TMB trimethylbenzene 

TSCA Toxic Substances Control Act 

TWA time-weighted average 

UF uncertainty factor 

UFA interspecies uncertainty factor 

UFH intraspecies uncertainty factor 

UFS subchronic-to-chronic uncertainty 

factor 

UFL LOAEL-to-NOAEL uncertainty factor 

UFD database deficiency uncertainty 

factor 

μg microgram 

μl microliter 

μmol micromol 

UV ultraviolet 

V volt 

VOC volatile organic compound 

W watt 

WBC white blood cell 

WS white spirit 

χ2 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 exposureresponse 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 toxicologically 

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 peerreviewing 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. 

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 
DRAFT - DO NOT CITE OR QUOTE 8 

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. 

– Studies of oral, inhalation, or dermal exposure 

involve passage through an absorption 
DRAFT - DO NOT CITE OR QUOTE 9 

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 

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 
DRAFT - DO NOT CITE OR QUOTE 10 

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. 

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 
DRAFT - DO NOT CITE OR QUOTE 11 

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

et al 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 

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 lifestages. 
DRAFT - DO NOT CITE OR QUOTE 12 

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 

(Guyatt et al 2008b). 

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

(EPA 2005a). 

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. 

– 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 
DRAFT - DO NOT CITE OR QUOTE 13 

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 exposureresponse 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 exposureresponse 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 doseresponse data from inferences about lower doses 

(EPA 2005a). 

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 speciesspecific physiologic and anatomic factors 

and consider whether the effect occurs at 

the site of first contact or after systemic 

circulation (EPA 1994). 

It can be informative to convert doses across 

exposure routes. If this is done, the assessment 

describes the underlying data, algorithms, and 

assumptions (EPA 2005a). 
DRAFT - DO NOT CITE OR QUOTE 14 

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 

(EPA 2005a). 

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 doseresponse curve is expected to have a linear 

component below the point of departure. This 

includes: 

– Agents or their metabolites that are DNAreactive 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. 

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 
DRAFT - DO NOT CITE OR QUOTE 15 

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 earlylife susceptibility has been observed for 

several carcinogens that are not mutagenic 

(EPA 2005b). 

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 lowestobserved-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-tohuman 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-adverseeffect 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 

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 
DRAFT - DO NOT CITE OR QUOTE 16 

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 twogeneration reproduction study are missing, 

and a factor of 101/2 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).

 
DRAFT - DO NOT CITE OR QUOTE 17 

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.) 

U.S. Environmental Protection Agency 

Office of Research and Development 

National Center for Environmental Assessment 

Research Triangle Park, NC 

Andrew Kraft, Ph.D 

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 

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. 
DRAFT - DO NOT CITE OR QUOTE 18 

Executive Direction

Reeder Sams, Ph.D. 

John Vandenberg, Ph.D. 

Debra Walsh, M.S. 

U.S. Environmental Protection Agency 

Office of Research and Development 

National Center for Environmental Assessment 

Research Triangle Park, NC 

Vincent Cogliano, Ph.D. 

Samantha Jones, Ph.D. 

Lynn Flowers, Ph.D. 

Jamie Strong Ph.D. 

U.S. Environmental Protection Agency 

Office of Research and Development 

National Center for Environmental Assessment 

Washington, DC 

Reviewers 

This document has been provided for review to EPA scientists. 
DRAFT - DO NOT CITE OR QUOTE 19 

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 25oC, 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 

waters based on their respective Henry’s Law constants and vapor pressures (see Table 

A.1-1). Degradation of both isomers in the atmosphere occurs by reaction with hydroxyl 
DRAFT - DO NOT CITE OR QUOTE 20 

radicals, the half-life of which is 11-12 hours (HSDB, 2011a, b). Non-volatilized 1,2,4-TMB 

and 1,3,5-TMB may be subject to biodegradation under aerobic conditions (HSDB, 2011a, 

b). 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, 1987). 

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 95-63-6 108-67-8 

Synonym(s) 

1,2,4-

Trimethylbenzene, 

pseudocumene, 

asymmetrical 

trimethylbenzene 

1,3,5-Trimethylbenzene, 

mesitylene, symmetrical 

trimethylbenzene 

Molecular formula C9H12

Molecular weight 120.19 

Chemical structure 

CH3

CH3

CH3

CH3

H3C CH3

Melting point, °C -43.8 -44.8 

Boiling point, °C @ 760 mm Hg 168.9 164.7 

Vapor pressure, mm Hg @ 25°C 2.10 2.48 

Density, g/mL at 20 °C relative to 

the density of H2O at 4 °C 0.8758 0.8637 

Flashpoint, °C 44 50 

Water solubility, mg/L at 25 °C 57 48.2 

Other solubilities 

Miscible with ethanol, 

benzene, ethyl ether, 

acetone, carbon 

tetrachloride, petroleum 

ether 

Miscible with alcohol, 

ether, benzene, acetone, 

and oxygenated and 

aromatic solvents 

Henry’s Law Constant, atmm3/mole 6.16 × 10-3 8.77 × 10-3

Log KOW 3.78 3.42 

Log KOC 2.73 2.70-3.13 

Bioconcentration Factor 439 234 

Conversion factors 1 ppm = 4.92 mg/m3

1 mg/m3 = 0.2 ppm 
DRAFT - DO NOT CITE OR QUOTE 21 

CAS Registry Number 95-63-6 108-67-8 

Synonym(s) 

1,2,4-

Trimethylbenzene, 

pseudocumene, 

asymmetrical 

trimethylbenzene 

1,3,5-Trimethylbenzene, 

mesitylene, symmetrical 

trimethylbenzene 

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 Inhalation value

National Institue of 

Occupational Safety and 

Health (NIOSH, 1992) 

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) 

American Conference of 

Governmental Industrial 

Hygienists (ACGIH, 2002) 

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) 

National Advisory Committee 

for Acute Exposure Guideline 

Levels for Hazardous 

Substances (U.S. EPA, 2007) 

For TMBs: AEGL-1 (nondisabling) – 180 ppm (890 mg/m

3

) to 45 ppm 

(220 mg/m3) (10 minutes to 8 hours, respectively) (Korsak and 

Rydzyński, 1996) 

AEGL-2 (disabling) – 460 ppm (2300 mg/m3) to 150 ppm (740 mg/m3) 

(10 minutes to 8 hours, respectively) (Gage, 1970) 

Ontario Ministry of the 

Environment (MOE, 2006) 

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 Rydzyński, 1996) 

 
DRAFT - DO NOT CITE OR QUOTE 22 

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; Chen et al., 1999; Norseth et al., 1991; Battig et al., 1956). 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 et al., 1997a; Gralewicz et al., 1997b; Korsak et al., 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 (Korsak et al., 2000; Korsak et al., 1997; Korsak and Rydzyński, 1996). 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 × 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 
DRAFT - DO NOT CITE OR QUOTE 23 

male rats exposed to 1,2,4-TMB via inhalation for 90 days (6 hours/day, 5 days/week) 

(Korsak and Rydzyński, 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 Point of Departure (mg/m3) UF Chronic RfC (mg/m3) 

Decreased pain sensitivity

90 day rat study 

Korsak and Rydzyński (1996)

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. 

1,000 2 × 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 Rydzyński (1996) is medium. The study is a wellconducted 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 et al., 1999; Wiaderna et al., 1998; 

Gralewicz et al., 1997a; Gralewicz et al., 1997b; Korsak and Rydzyński, 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 
DRAFT - DO NOT CITE OR QUOTE 24 

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 × 10-1 vs. 2 

× 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 Rydzyński, 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 et al., 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; 

Järnberg et al., 1996; Dahl et al., 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 × 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. 
DRAFT - DO NOT CITE OR QUOTE 25 

Confidence Levels for the Derivation of the RfC 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 RfC, the overall database, and the RfC itself, as described in EPA (1994), 

Section 4.3.9.2. The chronic RfC of 2 × 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 Rydzyński (1996) is medium (see 

above). Confidence in the database is low to medium as the database includes acute, shortterm, 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 (Tomas et al., 1999a; Tomas et al., 1999b; Tomas et al., 1999c). In 

these studies exposed rats demonstrated changes in electrocortical arousal, altered EEG 

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 
DRAFT - DO NOT CITE OR QUOTE 26 

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 

Point of Departure (mg/kgday) 

UF 

Chronic RfD (mg/kgday) 

Decreased pain sensitivity

90 day rat study 

Korsak and Rydzyński (1996) 

Route to route 

extrapolation using Korsak 

and Rydzyński (1996) 

subchronic inhalation study 

in Wistar rats 

1000 6 × 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 Rydzyński (1996) inhalation study 

and corresponding critical effect. The confidence in the study from which the critical effect 

was identified, Korsak and Rydzyński (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 
DRAFT - DO NOT CITE OR QUOTE 27 

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 firstpass 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 × 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 × 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 Rydzyński (1996) is medium (see 

above). Confidence in the database is low to medium as the database includes acute, shortterm, 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 et al., 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, 

2005a), 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 
DRAFT - DO NOT CITE OR QUOTE 28 

database was deemed inadequate to assess carcinogenic potential; no cancer values were 

derived for 1,3,5-TMB. 
DRAFT - DO NOT CITE OR QUOTE 29 

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 TMBinduced 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-

trimethylphenol OR 697-82-5; 2,3,6-trimethylphenol OR 2416-

94-6; 2,4,6-trimethylphenol OR 527-60-6; 3,5-dimethylbenzoic 

Search 

constraints: 

none 

Last search: 

December 

2011 
DRAFT - DO NOT CITE OR QUOTE 30 

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, 

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, 
DRAFT - DO NOT CITE OR QUOTE 31 

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. 
DRAFT - DO NOT CITE OR QUOTE 32 

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 et al., 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 et al., 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 et al., 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/m3 1,2,4-TMB, or 123 mg/ m3 1,3,5-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 (Järnberg et al., 1996). The Järnberg 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. 

Decreases in pain sensitivity have been observed at concentrations ≥ 492 mg/m3 following 

subchronic and short-term exposure to 1,2,4-TMB (Wiaderna et al., 2002; Gralewicz and 
DRAFT - DO NOT CITE OR QUOTE 33 

Wiaderna, 2001; Korsak and Rydzyński, 1996) and short-term exposure to 1,3,5-TMB 

(Wiaderna et al., 2002; Gralewicz and Wiaderna, 2001). In the subchronic study (Korsak 

and Rydzyński, 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 et al., 2002; Gralewicz et al., 1997a) 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 shortterm studies, in contrast to the observations in Korsak and Rydzyńsky, (1996). Similar to 

the subchronic study, acute exposures to 1,2,4-TMB or 1,3,5-TMB induced concentrationdependent decreases in pain sensitivity, with EC50 values of 5,682 and 5,963 mg/m3, 

respectively (Korsak and Rydzyński, 1996; Korsak et al., 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 et al., 1997a). In these studies, treatmentrelated, statistically significant changes in pain sensitivity at ≥ 492 mg/m3 1,2,4-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 Rydzyński, 1996; Korsak et al., 1995). In contrast, 

performance in the hot plate test was significantly impaired following short-term exposure 

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 et al., 1997a), indicating a persistence of 
DRAFT - DO NOT CITE OR QUOTE 34 

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 et al., 2007) or 

1,2,4-TMB and 1,3,5-TMB (Battig et al., 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/m3 1,2,4-TMB when tested immediately 

after exposure for either 8 or 13 weeks (Korsak and Rydzyński, 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 EC50 values of 4693 mg/m3 and 4738 mg/m3, respectively 

(Korsak and Rydzyński, 1996; Korsak et al., 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 Rydzyńsky, (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 Rydzyńsky, (1996) and Korsak et 

al. (1995) are supported by 2.8 and 2.9-fold increases in latency to paw-lick that, although 

not statistically significant, were observed weeks subsequent to short-term exposures to 

492 mg/m3 1,2,4-TMB or 1,3,5-TMB (Gralewicz and Wiaderna, 2001). 
DRAFT - DO NOT CITE OR QUOTE 35 

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/m3 1,2,4-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 et al., 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 et al., 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 et al., 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/m3 1,2,4-TMB (McKee et al., 2010). Decreased 

motor activity was also reported in rats acutely exposed to a mixture containing TMB 

isomers (Lammers et al., 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 

consistency of these results. Overall, exposure to 1,2,4-TMB and 1,3,5-TMB affects anxiety 
DRAFT - DO NOT CITE OR QUOTE 36 

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 et al., 2007). Similarly, short-term spatial memory (radial maze 

performance) was unaffected by exposure to either TMB isomer in animal studies 

(Wiaderna et al., 2002; Gralewicz and Wiaderna, 2001; Gralewicz et al., 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/m3 1,2,4-TMB or ≥ 123 

mg/m3 1,3,5-TMB was observed in treated male rats (Wiaderna et al., 2002; Gralewicz and 

Wiaderna, 2001; Gralewicz et al., 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/m3 1,2,4-TMB 

(Wiaderna et al., 2002; Gralewicz and Wiaderna, 2001); however, these changes were not 

observed in the other 1,2,4-TMB short-term study (Gralewicz et al., 1997a). Decreased 

performance in active avoidance tests was consistently observed following short-term 

exposure to ≥ 492 mg/m3 1,3,5-TMB(Wiaderna et al., 2002; Gralewicz and Wiaderna, 

2001). Similar to 1,2,4-TMB (Wiaderna et al., 2002; Gralewicz and Wiaderna, 2001), 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 

psychomotor tasks, observed immediately after exposure (effects did not persist to 24 
DRAFT - DO NOT CITE OR QUOTE 37 

hours later), were reported in male rats following acute exposure to 5000 mg/m3 1,2,4-

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/m3 1,2,4-TMB 

or 1,3,5-TMB do not cause overt CNS depression (Järnberg 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/m3 1,3,5-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/m3 1,2,4-TMB, suggesting persistent functional 

changes in the rat CNS (Gralewicz et al., 1997b). In recordings from rats that were awake, 

but immobile (not exhibiting pronounced exploratory activity, as determined by EEG 

morphology), statistically significant decreases in spike wave discharge activity were 

observed at 24 hours following short-term exposure to 492 mg/m3 1,2,4-TMB (Gralewicz 

et al., 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 et al., 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 et al., 1999a). Similar effects 

were observed following i.p. injection of 1,2,4-TMB and 1,3,5-TMB (Tomas et al., 1999c). 

The observed EEG 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 et al., 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.
DRAFT - DO NOT CITE OR QUOTE 38 

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)

a

1,2,4-TMB 

Decreased Pain 

Sensitivity 

0, 123, 492, 1,230 mg/m3 

recovery (1,230 mg/ m3 at 2 

weeks post-exposure) 

90 days; 10/group 

Korsak & Rydzynsky (1996) 

Exposure-dependent increases in paw-lick 

latency which recover by two weeks postexposure. 

Response relative to control: 0, 18, 79*, 95*% 

(recovery= 12%) 

492 

0, 492 mg/m3

4 weeks; 11/group 

Gralewicz & Wiaderna (2001) 

Increased paw-lick latency 24 hours after 

intermittent footshockb

. 

Response relative to control: 0, 191*%

492

0, 123, 492, 1,230 mg/m3

4 weeks; 15/group 

Gralewicz et al. (1997a) 

Non-monotonic increases in paw-lick latency 

24 hours after intermittent footshockb

. 

Response relative to control: 0, 9, 61*, 46*% 

492

Impaired 

Neuromuscular 

Function and 

Coordination 

0, 123, 492, 1,230 mg/m3 

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/m3

4 weeks; 11/group 

Gralewicz & Wiaderna (2001) 

Increased horizontal locomotion in open field 

activity tests. 

Response relative to control: 0, 62*% 

492 

0, 123, 492, 1,230 mg/m3

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 

Altered Cognitive 

Function

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, 43*%c ; 0, 

60*%d

492 

0, 123, 492, or 1,230 mg/m3

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 ; 

0, 30, 27, 34% e

492 
DRAFT - DO NOT CITE OR QUOTE 39 

Decreased CorticoHippocampal 

Activity 

0, 123, 492, 1,230 mg/m3

4 weeks; 9/group 

Gralewicz et al. (1997b) 

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 

 
DRAFT - DO NOT CITE OR QUOTE 40 

1,3,5-TMB 

Decreased Pain 

Sensitivity 

0, 492 mg/m3

4 weeks; 11/group 

Gralewicz & Wiaderna (2001) 

Increased paw-lick latency 24 hours after 

intermittent footshockb. 

Response relative to control: 0, 250*% 

492

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 

concentrationb. 

Response relative to control: 0, 4, 70*, 17% 

492 

Decreased Anxiety 

and/or Increased 

Motor Function 

0, 492 mg/m3

4 weeks; 11/group 

Gralewicz & Wiaderna (2001) 

Increased horizontal locomotion in open field 

activity tests. 

Response relative to control: 0, 70*% 

492 

Altered Cognitive 

Function 

0, 123, 492, 1,230 mg/m3

4 weeks; 12/group 

Wiaderna et al. (2002) 

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

123 

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

492 

* Significantly different from controls 

For studies other than Korsak and Rydzynsky, 1996, % change from control calculated from digitized data using GrabIt 

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 

d

 Increased number of trials to reach avoidance criteria 

e Decreased avoidance response % in trials 25-30 of training
DRAFT - DO NOT CITE OR QUOTE 41 

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 CorticoHippocampal 

Activity 

240, 960, 3,850 mg/kg, single 

oral gavage 

Rat, Wag/Rij, male, 18/group 

Tomas et al. (1999a) 

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 

240 

Decreased Anxiety 

and/or Increased 

Motor Function 

960, 1920, 3850 mg/kg single 

oral gavage 

Rat, Wag/Rij, male, 10/group 

Tomas et al. (1999b) 

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 × time interaction) 

3,850 

Decreased CorticoHippocampal 

Activity 

790 mg/kg, single i.p injection 

Rat, Wistar, Male, 4 

Tomas et al. (1999c) 

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% 

790 

1,3,5-TMB 

Decreased CorticoHippocampal 

Activity 

240, 960, 3,850 mg/kg, single 

oral gavage 

Rat, Wag/Rij, male, 18/group 

Tomas et al. (1999a) 

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 

240 

Decreased Anxiety 

and/or Increased 

Motor Function 

960, 1920, 3850 mg/kg single 

oral gavage 

Rat, Wag/Rij, male, 10/group 

Tomas et al. (1999b) 

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 

1920 

Decreased CorticoHippocampal 

Activity 

790 mg/kg, single i.p injection 

Rat, Wistar, Male, 4 

Tomas et al. (1999c) 

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% 

790 

* Significantly different from controls 

a Lowest effect level at which statistically significant changes were observed
DRAFT - DO NOT CITE OR QUOTE 42 

 Mode of Action Analysis for neurotoxicity 

The observation of neurotoxicity following acute-, short-term-, and subchronicduration exposure to TMB (Lammers et al., 2007; Wiaderna et al., 2002; Gralewicz and 

Wiaderna, 2001; Wiaderna et al., 1998; Gralewicz et al., 1997a; Gralewicz et al., 1997b; 

Korsak and Rydzyński, 1996; Korsak et al., 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 et al., 1984; Andersson et al., 1983; Andersson et al., 1981; Andersson 

et al., 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 et al., 1983; Andersson et al., 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 dopaminemediated locomotor activity, increase in the number of dopamine D2 receptors, and 

increase dopamine D2 agonist receptor binding (Hillefors-Berglund et al., 1995; von Euler 

et al., 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 

(Bracs et al., 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 et al., 1988). These 

observations and findings are in concordance with those resulting from exposure to 
DRAFT - DO NOT CITE OR QUOTE 43 

1,2,4-TMB and 1,3,5-TMB (Wiaderna et al., 2002; Gralewicz and Wiaderna, 2001; 

Gralewicz et al., 1997a; Gralewicz et al., 1997b) (Tomas et al., 1999a; Tomas et al., 1999c). 

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 et al., 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 et al., 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 TMBinduced neurotoxicity in acute, short-term, and subchronic inhalation studies. 
DRAFT - DO NOT CITE OR QUOTE 44 

Is 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. 

Is 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., EEG; 

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 
DRAFT - DO NOT CITE OR QUOTE 45 

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 (Jones et al., 

2006; Järnberg et al., 1997a; Järnberg 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 et al., 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. 
DRAFT - DO NOT CITE OR QUOTE 46 

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, trendanalysis 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. 
DRAFT - DO NOT CITE OR QUOTE 47 

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. 
DRAFT - DO NOT CITE OR QUOTE 48 

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 

123-1,230 mg/m3, 90 days 

(6h/day, 5 days/week) 

Rat, Wistar, male, 6-7 

Korsak et al. (1997) 

Increased total bronchoalveolar cell count 

with evidence of attenuation at high 

exposure 

Response relative to control: 0, 202*, 208*, 

231*% 

123 

123-1,230 mg/m3, 90 days 

(6h/day, 5 days/week) 

Rat, Wistar, male, 6-7 

Korsak et al. (1997) 

Increased macrophage count with evidence 

of attenuation at high exposure 

Response relative to control:0, 107, 170*, 

116*% 

492 

123-1,230 mg/m3, 90 days 

(6h/day, 5 days/week) 

Rat, Wistar, male and female, 

6-7 

Korsak et al. (2000)

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 

492 

Clinical Chemistry 

Effect 

123-1,230 mg/m3, 90 days 

(6h/day, 5 days/week) 

Rat, Wistar, male, 6-7 

Korsak et al. (1997)

Increased acid phosphatase activity with 

evidence of attenuation at high exposure 

Response relative to control:0, 47*, 74*, 45*%

123 

Sensory Irritation 

(Decreased 

respiration) 

1,245-9,486 mg/m3, single 

exposure, 6 minutes 

Mouse, BALB/C, male, 8-10 

Korsak et al. (1997); Korsak 

et al. (1995) 

Decreased respiratory rate as measured 

during first minute of exposure 

Response relative to control:RD50 = 2,844 

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 

Korsak et al. (1997) 

Decreased respiratory rate as measured 

during first minute of exposure 

Response relative to control: RD50 = 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 

respiratory rates, with the maximum decrease in respiration occurring in the first one or 

two minutes of exposure (Korsak et al., 1997; Korsak et al., 1995). The concentration of 
DRAFT - DO NOT CITE OR QUOTE 49 

1,2,4-TMB and 1,3,5-TMB that was observed to result in a 50% depression in the 

respiratory rate (RD50) 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 (Korsak et al., 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 RD50 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) (Korsak et al., 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 subchronically 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 

aerosol (SOA) particles derived from 1,3,5-TMB (Gaschen et al., 2010). The observation 

that IL-8 levels increase following exposure to 1,3,5-TMB-derived SOA is noteworthy as a 
DRAFT - DO NOT CITE OR QUOTE 50 

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 et al., 2000), and the related compounds benzene and toluene have been shown to 

induce oxidative stress in cultured lung cells (Mögel et al., 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 (Chou et al., 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). 

Is 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 TMBinduced respiratory effects in exposed animals. 

Is 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 

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 
DRAFT - DO NOT CITE OR QUOTE 51 

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-

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 
DRAFT - DO NOT CITE OR QUOTE 52 

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 Study Design and Reference Results 

Lowest Level 

at which 

Significant 

Effects were 

observed 

(mg/m3)

a

1,2,4-TMB 

Developmental 

Toxicity 

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) 

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*% 

2,952 

Maternal Toxicity 

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) 

Decreased corrected maternal weight gain 

Response relative to control: 0, +7, 7, 51*, 

100*% (weight gain = 0 g) 

2,952 

1,3,5-TMB 

Developmental 

Toxicity 

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) 

Decreased fetal body weight of male and 

female 

Response relative to control: 

Male: 0, 1, 5, 7*, 12*% 

Female: 0, 1, 4, 6, 13*% 

2,952 

Maternal Toxicity 

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) 

Decreased corrected maternal weight gain 

Response relative to control: 0, +3, 31, 76*, 

159*% (weight gain = -12 g) 

2,952 

* Significantly different from controls 

a Lowest effect level at which statistically significant changes were observed

 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 
DRAFT - DO NOT CITE OR QUOTE 53 

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 concentrationdependent 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 

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. 
DRAFT - DO NOT CITE OR QUOTE 54 

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 et al., 1975a; Wiglusz et al., 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)

a

1,2,4-TMB 

Hematological 

toxicity 

123-1,230 mg/m3, 90 days 

(6h/day, 5days/week) 

Rat, Wistar, female & male, 

6-7 

Korsak et al. (2000) 

Decreased red blood cells in males only. 

Response relative to control:0, 1, 15, 23*%

(recovery = 24%) 

1,230 

123-1,230 mg/m3, 90 days 

(6h/day, 5days/week) 

Rat, Wistar, female & male, 

6-7 

Korsak et al. (2000) 

Increased white blood cells in males only. 

Response relative to control:0, 2, 4, 80*%

(recovery = 18% decrease) 

1,230 

123-1,230 mg/m3, 90 days 

(6h/day, 5days/week) 

Rat, Wistar, female & male, 

6-7 

Korsak et al. (2000) 

Decreased reticulocytes in females only. 

Response relative to control:0, 51, 49,71*%

(recovery = 65% increase) 

1,230 

123-1,230 mg/m3, 90 days 

(6h/day, 5days/week) 

Rat, Wistar, female & male, 

6-7 

Korsak et al. (2000) 

Non-monotonic decreases in clotting time in 

females only. 

Response relative to control:0, 23,37*,27*% 

(recovery = 60% increase) 

492 

Clinical Chemistry 

Effects 

123-1,230 mg/m3, 90 days 

(6h/day, 5days/week) 

Rat, Wistar, female & male, 

6-7 

Korsak et al. (2000) 

Non-monotonic increases in sorbitol 

dehydrogenase in males only. 

Response relative to control:0, 73*, 74*,73*%

123 

1,3,5-TMB 
DRAFT - DO NOT CITE OR QUOTE 55 

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. (1975b) 

Increased alkaline phosphatase on day 7 postexposure 

Response relative to control (on day 7):0, -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 

et al., 1975b). 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 

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 
DRAFT - DO NOT CITE OR QUOTE 56 

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 Toxicological 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 et al., 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 

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 
DRAFT - DO NOT CITE OR QUOTE 57 

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 RD50 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 (Korsak et al., 1997). 

The similarity regarding toxicity was also observed in acute inhalation neurotoxicity 

studies: the EC50 for decreased coordination, balance, and neuromuscular function was 

4,694 mg/m3 for 1,2,4-TMB and 4,738 mg/m3 for 1,3,5-TMB. The EC50 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 (Korsak and Rydzyński, 1996). Neurotoxic endpoints were also 

similarly affected by oral exposure to individual isomers: increased electrocortical arousal 
DRAFT - DO NOT CITE OR QUOTE 58 

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 et al., 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 et al., 2002; 

Gralewicz and Wiaderna, 2001; Gralewicz et al., 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 et al., 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, 2001). 

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% 

reduction of maternal weight gain compared to controls, whereas 1,2,4-TMB exposure 

reduced maternal weight gain by 50%. 
DRAFT - DO NOT CITE OR QUOTE 59 

1.1.7. Susceptible Populations or Lifestages 

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 et al., 

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 (CYP P450) mono-oxygenase isozymes have been 

shown to be reduced in children up to 1 year of age compared to adult activities (Ginsberg 

et al., 2004). Additionally, the rate of glucuronidation and sulfation is decreased in 

children. Therefore, as both CYP 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 

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 
DRAFT - DO NOT CITE OR QUOTE 60 

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 Rydzyński (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) 

0 123 492 1,230 

Neurotoxicological Endpoints 

Decreased pain sensitivity (measured 

as latency to paw-lick in seconds)

Rat, male 

15.4 ± 5.8a 

(n = 9) 

18.2 ± 5.7 

(n = 10) 

27.6 ± 3.2d 

(n = 9) 

30.1 ± 7.9d 

(n = 10) 

Impaired neuromuscular function 

and coordination (% failures on 

0 

(n = 10) 

10 

(n = 10) 

20

(n = 10) 

40e 

(n = 10)
DRAFT - DO NOT CITE OR QUOTE 61 

rotarod) 

Hematological Endpoints 

Decreased RBCs (106/cm3) 

Rat, male 

9.98 ± 1.68

(n = 10) 

9.84 ± 1.82

(n = 10) 

8.50 ± 1.11

(n = 10) 

7.70 + 1.38d 

(n = 10) 

Increased WBCs (106/cm3)

8.68 ± 2.89

(n = 10) 

8.92 ± 3.44

(n = 10) 

8.30 ± 1.84

(n = 10) 

15.89 ± 5.74d 

(n = 10) 

Decreased reticulocytes (%)

Rat, female 

3.5 ± 2.6 

(n = 10) 

1.7 ± 2.0 

(n = 10) 

1.8 ± 0.9 

(n = 10) 

1.0 ± 0.6c 

(n = 20) 

Decreased clotting time (seconds) 30 ± 10 

(n = 10) 

23 ± 4 

(n = 10) 

19 ± 5d 

(n = 10)

22 ± 7c 

(n = 20)

Pulmonary Endpoints 

Increased BAL macrophages 

(106/cm3) 

Rat, male 

1.83 ± 0.03

(n = 6) 

3.78 ± 0.8 

(n = 6) 

4.95 ± 0.2d 

(n = 7) 

3.96 ± 0.3d 

(n = 6) 

Increased BAL total cells (106/cm3) 1.93 ± 0.79

(n = 6) 

5.82 ± 1.32f 

(n = 6) 

5.96 ± 2.80d

(n = 7) 

4.45 ± 1.58c 

(n = 7) 

Increased inflammatory lung lesions 

b 

(n = 10) 

b 

(n =10) 

b 

(n = 10) 

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 

123 mg/m3 

c p < 0.05; d p < 0.01; e

 p < 0.005; f p < 0.001 

Adapted from Korsak et al., (2000; 1997) and Korsak and Rydzyński (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 Rydzyński, 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. 
DRAFT - DO NOT CITE OR QUOTE 62 

Solid lines represent range of exposure concentrations. (a) Korsak and Rydzyński (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. 

100

1000

10000

↓ 

neuromuscular

function (a)

↓ 

pain 

sensitivity 

(a)

↑ 

BAL 

macrophages 

(b)

↑ 

BAL

total cells

(b)

↑ 

pulmonary 

lesions

(b)

↓ 

RBCs 

(c)

↑ 

WBCs

(c)

↓ 

reticulocytes

(c)

↓ 

clotting 

time 

(c)

↑ 

maternal

weight gain

(d)

↑ 

fetal 

weight 

(d)

Doses

NOAEL

LOAEL
DRAFT - DO NOT CITE OR QUOTE 63 

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 shortterm studies was not optimal), and the studies used a reasonable range of appropriatelyspaced 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 pairwise 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-
DRAFT - DO NOT CITE OR QUOTE 64 

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 subchronically 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 

(n = 21)a 

492 

(n = 22) 

1,476 

(n = 21) 

2,952 

(n = 17) 

5,904 

(n = 18) 

Developmental Endpoints 

Decreased fetal weight 

(g) 

Rat, male 5.80 ± 

0.41b 5.76 ± 0.27 5.50 ± 0.31 5.39 ± 

0.55c 

5.10 ± 

0.57d 

Rat, female 5.50 ± 0.32 5.74 ± 0.21 5.27 ± 0.47 5.18 ± 0.68 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 

c p < 0.05; d p < 0.01 

Adapted from Saillenfait et al. (2005)
DRAFT - DO NOT CITE OR QUOTE 65 

Solid lines represent range of exposure concentrations. (a) Gralewicz and Wiaderna (2001); (b) Gralewicz et 

al. (1997a); (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. 

100

1000

10000

Altered

cognitive

function (a)

Altered

cognitive

function (b)

↓ Pain

sensitivity (a)

↓ Anxiety

and/or ↑

motor activity (a)

↑ 

maternal

weight gain...

↑ 

fetal 

weight ...

Doses

NOAEL

LOAEL
DRAFT - DO NOT CITE OR QUOTE 66 

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 et al., 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. 

 
DRAFT - DO NOT CITE OR QUOTE 67 

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 Rydzyński (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.1 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 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 Rydzyński 

(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, 2000a). 

For continuous data (i.e., decreased pain sensitivity, increased BAL macrophages, 

decreased RBCs, decreased reticulocytes, and decreased clotting time) from the Korsak and 

Rydzyński (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, 2000a). 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 

absence of incidence data (e.g., increased inflammatory lung lesions). Additionally, some 
DRAFT - DO NOT CITE OR QUOTE 68 

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, 2011c) for comparison to endpoints observed in the Korsak et al., (2000; 1997) 

and Korsak and Rydzyński (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) 

BWa 

(kg) 

PODADJb

(mg/L) 

Neurotoxicological Endpoints 

Korsak and 

Rydzyński 

(1996) 

Decreased pain 

sensitivity Male, rat BMDL Exponential 4; 1 SD 84.0 0.387 0.085 

Impaired 

neuromuscular 

function and 

coordination 

Male, rat BMDL Log-logistic; 

10% ER 93.9 0.387 0.096 

Hematological Endpoints 

Korsak et 

al. (2000) 

Decreased RBCs Male, rat BMDL Exponential 

4; 1 SD 174.1 0.390 0.187 

Increased WBCs Male, rat NOAEL n/a 492 0.399 0.867 

Decreased 

reticulocytes Female, rat NOAEL n/a 492 0.230 0.890 

Decreased clotting 

time Female, rat NOAEL n/a 123 0.243 0.127 

Pulmonary Endpoints 

Korsak et 

al. (1997) 

Increased BAL 

macrophages Male, rat NOAEL n/a 123 0.383 0.127 

Increased BAL total 

cells Male, rat LOAEL n/a 123 0.383 0.127 

Korsak et 

al. (2000) 

Increased 

inflammatory lung 

lesions 

Male and 

female, rat NOAEL n/a 123 0.390 0.127 
DRAFT - DO NOT CITE OR QUOTE 69 

a Group specific mean body weight (BW) reported in Korsak et al. (2000; Korsak et al., 1997). 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 Rydzyński (1996), the average of the 

group specific mean BWs from Korsak et al. (2000; Korsak et al., 1997) for the dose group closest to the 

BMDL was used. 

bWeekly 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.1) was used to estimate the steadystate 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) Candidate 

RfC 

(mg/m3) UFA UFH UFL UFS UFD UFTOTAL b 

Neurotoxicological Endpoints 

Korsak and 

Rydzyński 

(1996) 

Decreased pain 

sensitivity 0.085 15.6 3 10 1 10 3 1,000 1.56 × 10-2 

Impaired 

neuromuscular 

function and 

coordination 

0.096 17.6 3 10 1 10 3 1,000 1.76 × 10-2 

Hematological Endpoints 

Korsak et 

al. (2000) 

Decreased RBCs 0.187 33.7 3 10 1 10 3 1,000 3.37 × 10-2 

Increased WBCs 0.867 131.5 3 10 1 10 3 1,000 1.31 × 10-1 

Decreased 

reticulocytes 0.890 134.0 3 10 1 10 3 1,000 1.34 × 10-1 
DRAFT - DO NOT CITE OR QUOTE 70 

Decreased 

clotting time 0.127 23.2 3 10 1 10 3 1,000 2.32 × 10-2 

Pulmonary Endpoints 

Korsak et 

al. (1997) 

Increased BAL 

macrophages 0.127 23.2 3 10 1 10 3 1,000 2.32 × 10-2 

Increased BAL 

total cells 0.127 23.2 3 10 10 10 3 10,000 n/ac 

Korsak et 

al. (2000) 

Increased 

inflammatory 

lung lesions 

0.127 23.2 3 10 1 10 3 1,000 2.32 × 10-2 

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 HEC was derived using a human PBPK model (Hissink et al., 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 et al., 1997a; Korsak and Rydzyński, 1996; Korsak et al., 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 
DRAFT - DO NOT CITE OR QUOTE 71 

critical effect and Korsak and Rydzyński (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 Rydzyński, 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 (101/2 = 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, UFH, 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 UFH was applied. 

A LOAEL to NOAEL uncertainty factor, UFL 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, UFD, 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 

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 
DRAFT - DO NOT CITE OR QUOTE 72 

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 × 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 Rydzyński (1996) is medium. The study is a well-conducted peerreviewed 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-TMBinduced 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 subchronic) (Gralewicz and Wiaderna, 2001; Chen et al., 1999; Wiaderna et al., 1998; 

Gralewicz et al., 1997a; Gralewicz et al., 1997b; Korsak and Rydzyński, 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 
DRAFT - DO NOT CITE OR QUOTE 73 

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. 

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, 1994), 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 × 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 

0.01

0.1

1

10

100

Decreased 

pain 

sensitivity

Decreased 

neuromuscular 

function

Increased BAL

macrophages

Increased 

inflammatory 

lung lesions

Decreased 

RBCs

Increased 

WBCs

Decreased 

reticulocytes

Decreased 

clotting time

Database Uncertainty Subchronic to Chronic Intraspecies Variability Interspecies Extrapolation RfC POD

Neurotoxicological 

Endpoints Pulmonary Endpoints Respiratory Endpoints
DRAFT - DO NOT CITE OR QUOTE 74 

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 uncertainty, but 

such uncertainties would apply equally to all endpoints. Lastly, the extent of interindividual 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.1 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 (2000a), 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. 
DRAFT - DO NOT CITE OR QUOTE 75 

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) × hours exposed per day/24 hours × 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/m3× 6 hours/24 hours × 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 (GDs 6-20). Therefore, the duration-adjusted PODs for 

developmental/ maternal effects were calculated as follows: 

PODADJ (mg/m3) = POD (mg/m3) × hours exposed per day/24 hours 

For example, for decreased fetal weight in males, the PODADJ would be calculated as 

follows: 
DRAFT - DO NOT CITE OR QUOTE 76 

PODADJ (mg/m3) = 1649 mg/m3 × 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) a

Neurotoxicological Endpoints 

Gralewicz and 

Wiaderna 

(2001) 

Altered cognitive 

function Male, rat LOAEL n/a 492 87.9 

Decreased pain 

sensitivity Male, rat LOAEL n/a 492 87.9 

Decreased anxiety 

and/or increased 

motor function 

Male, rat LOAEL n/a 492 87.9 

Wiaderna et al. 

(2002) 

Altered cognitive 

function Male, rat LOAEL n/a 123 22.0 

Developmental Endpoints 

Saillenfait et al. 

(2005) 

Decreased fetal 

body weight 

Male, rat BMDLc Exponential 

2; 5% RD 1,649 412.0 

Female, rat NOAELc n/a 2,952 738.0 

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 × (6 hours/24 hours) for developmental/maternal endpoints, or 

POD × (6 hours/24 hours) × (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, 
DRAFT - DO NOT CITE OR QUOTE 77 

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; Järnberg and Johanson, 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) × DAF 

PODHEC = PODADJ (mg/m3) × 1.0 

PODHEC = 22 mg/m3 × 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 et al., 2002; Gralewicz and 

Wiaderna, 2001). 
DRAFT - DO NOT CITE OR QUOTE 78 

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) Candidate

RfC 

(mg/m3) UFA UFH UFL UFS UFD UFTOTAL b 

Neurotoxicological Endpoints 

Gralewicz 

and 

Wiaderna 

(2001) 

Altered cognitive 

function 87.9 87.9 3 10 10 10 3 10,000 n/ac 

Decreased pain 

sensitivity 87.9 87.9 3 10 10 10 3 10,000 n/ac

Decreased anxiety 

and/or increased 

motor function 

87.9 87.9 3 10 10 10 3 10,000 n/ac

Wiaderna 

et al. 

(2002) 

Altered cognitive 

function 22.0 22.0 3 10 10 10 3 10,000 n/ac 

Developmental Endpoints 

Saillenfait 

et al. 

(2005) 

Decreased fetal 

body weight, male 412.0 412.0 3 10 1 1 3 100 4.12

Decreased fetal 

body weight, 

female 

738.0 738.0 3 10 1 1 3 100 7.38 

Maternal Endpoints 

Saillenfait 

et al. 

(2005) 

Decreased 

maternal weight 

body gain 

326.0 326.0 3 10 1 10 3 1,000 3.26 × 10-1 

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 (U.S. EPA, 2002) the RfD/RfC Technical Panel 

concluded that, in cases where maximum uncertainty exists in four or more areas of 

uncertainty, or when the total uncertainty 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 (2002), the available neurotoxicity data following short-term 

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. 
DRAFT - DO NOT CITE OR QUOTE 79 

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 (101/2 = 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, UFH, 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 UFH is applied. 

A LOAEL to NOAEL uncertainty factor, UFL 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, UFD, 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 

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 
DRAFT - DO NOT CITE OR QUOTE 80 

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 × 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 EC50 for decreased pain sensitivity (i.e., latency to 

paw-lick measured on the hot plate apparatus) 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 (Korsak and Rydzyński, 1996). 

Other neurotoxic endpoints similarly affected by either isomer (albeit from oral exposures 

or i.p. injections) included increased electrocortical arousal and altered EEG function 
DRAFT - DO NOT CITE OR QUOTE 81 

(Tomas et al., 1999a; Tomas et al., 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 et al., 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 

into the bloodstream of exposed humans (6.5 and 6.2 μM, respectively) (Järnberg 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 (Järnberg 
DRAFT - DO NOT CITE OR QUOTE 82 

et al., 1996; Dahl et al., 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 

(Swiercz et al., 2006; Swiercz et al., 2003; Swiercz et al., 2002). 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) (Meulenberg and 

Vijverberg, 2000). 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 (Järnberg et al., 

1996; Huo et al., 1989; Mikulski and Wiglusz, 1975), 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) (Järnberg 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 (Tsujimoto et al., 2005; Tsujimoto et al., 2000; Huo et al., 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) 

(Järnberg et al., 1997a; Järnberg et al., 1997b; Järnberg et al., 1996), 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) 

(Swiercz et al., 2003; Swiercz et al., 2002). 

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 

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 
DRAFT - DO NOT CITE OR QUOTE 83 

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 × 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 × 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 Rydzyński (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 × 10-1 vs. 2 × 

10-2 mg/m3, respectively). Although uncertainty exists in adopting the 1,2,4-TMB RfC 

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 
DRAFT - DO NOT CITE OR QUOTE 84 

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 uncertainty 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, 

2011d)). Further, no evidence exists that would suggest toxicity profiles would differ to a 

substantial degree between oral and inhalation exposures. 

Therefore, assuming oral exposure would result in the same systemic effect as 

inhalation exposure (altered CNS function, measured as decreased pain sensitivity (Korsak 

and Rydzyński, 1996), an oral exposure component was added to the Hissink et al. (2007) 

PBPK model by EPA (Appendix A (U.S. EPA, 2011d); Section A.1.4), assuming continuous 
DRAFT - DO NOT CITE OR QUOTE 85 

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, 2011d); 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 Rydzyński (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 (2002), 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 × 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 Rydzyński (1996) inhalation study 

and corresponding critical effect. The confidence in the study from which the critical effect 

was identified, Korsak and Rydzyński (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 
DRAFT - DO NOT CITE OR QUOTE 86 

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 Rydzyński (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 

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. 
DRAFT - DO NOT CITE OR QUOTE 87 

Therefore, given the above similarities in toxicokinetics and toxicity, the RfD derived for 

1,2,4-TMB: 6 × 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 × 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 Rydzyński (1996) is medium (see 

above). Confidence in the database is low to medium as the database includes acute, shortterm, 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 (2005a), 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. 

Information available on which to base a cancer assessment is lacking, and thus, no cancer 

risk value is derived. 
DRAFT - DO NOT CITE OR QUOTE 88 

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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