&EPA
EPA/635/R-13/171a
Revised External Review Draft
www.epa.gov/iris
United States
Environmental Protection
Agency
lexicological Review of Trimethylbenzenes
(CASRN 25551-13-7, 95-63-6, 526-73-8, and 108-67-8}
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
August 2013
NOTICE
This document is a Revised External Review draft. This information is distributed solely
for the purpose of pre-dissemination peer review under applicable information quality
guidelines. It has not been formally disseminated by EPA. It does not represent and should
not be construed to represent any Agency determination or policy. It is being circulated for
review of its technical accuracy and science policy implications.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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Toxicological Review of Trimethylbenzene
DISCLAIMER
This document is a preliminary draft for review purposes only. This information is
distributed solely for the purpose of pre-dissemination peer review under applicable information
quality guidelines. It has not been formally disseminated by EPA. It does not represent and should
not be construed to represent any Agency determination or policy. Mention of trade names or
commercial products does not constitute endorsement of recommendation for use.
This document is a draft for review purposes only and does not constitute Agency policy.
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CONTENTS
CONTENTS iii
TABLES v
FIGURES vi
ABBREVIATIONS AND ACRONYMS vii
AUTHORS | CONTRIBUTORS | REVIEWERS viii
PREFACE xi
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS xv
EXECUTIVE SUMMARY xxxiv
LITERATURE SEARCH STRATEGY | STUDY SELECTION AND EVALUATION xlvi
1. HAZARD IDENTIFICATION 1-1
1.1. Synthesis of Evidence 1-1
1.1.1. Neurological Effects 1-1
1.1.2. Respiratory Effects 1-25
1.1.3. Reproductive and Developmental Effects 1-31
1.1.4. Hematological and Clinical Chemistry Effects 1-35
1.1.5. Carcinogenicity 1-45
1.1.6. Similarities Among TMB Isomers Regarding Observed Inhalation and Oral Toxicity 1-47
1.1.7. Similarities Among TMB Isomers Regarding Toxicokinetics 1-49
1.2. Summary and Evaluation 1-51
1.2.1. Weight of Evidence for Effects Other Than Cancer 1-51
1.2.2. Weight of Evidence for Carcinogenicity 1-54
1.2.3. Susceptible Populations and Lifestages 1-54
2. DOSE-RESPONSE ANALYSIS 2-1
2.1. Inhalation Reference Concentration for Effects Other Than Cancer for
1,2,4-TMB 2-1
2.1.1. Identification of Studies and Effects for Dose-Response Analysis for 1,2,4-TMB 2-1
2.1.2. Methods of Analysis for 1,2,4-TMB 2-7
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2.1.3. Derivation of Candidate RfC Values for 1,2,4-TMB 2-12
2.1.4. Derivation of Organ/System Specific Reference Concentrations for 1,2,4-TMB 2-15
2.1.5. Selection of the Proposed Overall Reference Concentration for 1,2,4-TMB 2-16
2.1.6. Uncertainties in the Derivation of the Reference Concentration for 1,2,4-TMB 2-19
2.1.7. Confidence Statement for 1,2,4-TMB 2-19
2.2. Inhalation Reference Concentration for Effects Other Than Cancer for
1,2,3-TMB 2-20
2.2.1. Identification of Studies and Effects for Dose-Response Analysis for 1,2,3-TMB 2-20
2.2.2. Methods of Analysis for 1,2,3-TMB 2-24
2.2.3. Derivation of Candidate RfC Values for 1,2,3-TMB 2-26
2.2.4. Derivation of Organ/System Specific Reference Concentrations for 1,2,3-TMB 2-30
2.2.5. Selection of the Proposed Overall Reference Concentration for 1,2,3-TMB 2-31
2.2.6. Uncertainties in the Derivation of the Reference Concentration for 1,2,3-TMB 2-32
2.2.7. Confidence Statement for 1,2,3-TMB 2-33
2.3. Inhalation Reference Concentration for Effects Other Than Cancer for
1,3,5-TMB 2-34
2.3.1. Identification of Studies and Effects for Dose-Response Analysis for 1,3,5-TMB 2-34
2.3.2. Methods of Analysis for 1,3,5-TMB 2-36
2.3.3. Derivation of Candidate RfC Values for 1,3,5-TMB 2-38
2.3.4. Derivation of Organ/System Specific Reference Concentrations for 1,3,5-TMB 2-41
2.3.5. Selection of the Proposed Overall Reference Concentration for 1,3,5-TMB 2-42
2.3.6. Uncertainties in the Derivation of the Reference Concentration for 1,3,5-TMB 2-43
2.3.7. Confidence Statement for 1,3,5-TMB 2-43
2.4. Oral Reference Dose for Effects Other Than Cancer for 1,2,4-TMB 2-44
2.4.1. Identification of Studies and Effects for Dose-Response Analysis for 1,2,4-TMB 2-44
2.4.2. Methods of Analysis for 1,2,4-TMB 2-44
2.4.3. Derivation of the Reference Dose for 1,2,4-TMB 2-45
2.4.4. Uncertainties in the Derivation of the Reference Dose for 1,2,4-TMB 2-46
2.4.5. Confidence Statement for 1,2,4-TMB 2-46
2.5. Oral Reference Dose for Effects Other Than Cancer for 1,2,3-TMB 2-46
2.5.1. Identification of Studies and Effects for Dose-Response Analysis for 1,2,3-TMB 2-46
2.5.2. Methods of Analysis and Derivation of the Reference Dose for 1,2,3-TMB 2-46
2.5.3. Uncertainties in the Derivation of the Reference Dose for 1,2,3-TMB 2-47
2.5.4. Confidence Statement for 1,2,3-TMB 2-47
This document is a draft for review purposes only and does not constitute Agency policy.
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2.6. Oral Reference Dose for Effects Other Than Cancer for 1,3,5-TMB 2-48
2.6.1. Identification of Studies and Effects for Dose-Response Analysis for 1,3,5-TMB 2-48
2.6.2. Methods of Analysis and Derivation of the Reference Dose for 1,3,5-TMB 2-48
2.6.3. Uncertainties in the Derivation of the Reference Dose for 1,3,5-TMB 2-49
2.6.4. Confidence Statement for 1,3,5-TMB 2-49
2.7. Cancer Risk Estimates for 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB 2-49
REFERENCES R-l
TABLES
Table ES-1. Summary of inhalation reference concentrations (RfCs) xxxv
Table ES-2. Summary of reference concentration (RfC) derivation for 1,2,4-TMB xxxvi
Table ES-3. Summary of reference concentration (RfC) derivation for 1,2,3-TMB xxxviii
Table ES-4. Summary of reference doses (RfDs) forTMB isomers xli
Table ES-5. Summary of reference dose (RfD) derivation for 1,2,4-TMB xli
Table LS-1: Details of the search strategy employed forTMBs xlvii
Table 1-1. Evidence pertaining to neurological effects of TMBs in animals — inhalation exposures 1-10
Table 1-2. Evidence pertaining to neurological effects of 1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB in animals —
oral exposures 1-16
Table 1-3. Evidence pertaining to respiratory effects of TMBs in animals — inhalation exposures 1-27
Table 1-4. Evidence pertaining to reproductive and developmental effects of 1,2,4-TMB and 1,3,5-TMB in
animals — inhalation exposures 1-33
Table 1-5. Evidence pertaining to hematological and clinical chemistry effects of 1,2,3-TMB, 1,2,4-TMB, or
1,3,5-TMB in animals — inhalation exposures 1-38
Table 1-6. Evidence pertaining to hematological and clinical chemistry effects of 1,3,5-TMB in animals —
oral exposures 1-41
Table 1-7. Similarities between 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB regarding observed inhalation and
oral toxicity 1-49
Table 2-1. Target and actual inhalation concentrations, and internal blood dose metrics of 1,2,4-TMB
calculated using the available rat PBPK model (Hissink et al., 2007) 2-3
Table 2-2. Endpoints considered for the derivation of the RfC for 1,2,4-TMB 2-5
Table 2-3. Summary of derivation of points of departure for 1,2,4-TMB 2-11
Table 2-4. Effects and corresponding derivation of candidate RfC values for 1,2,4-TMB 2-14
Table 2-5. Organ/system-specific RfCs and proposed overall RfC for 1,2,4-TMB 2-16
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Table 2-6. Target and actual exposure concentrations used in BMD modeling of 1,2,3-TMB endpoints
considered for the derivation of the RfC 2-22
Table 2-7. Endpoints considered for the derivation of the RfC for 1,2,3-TMB 2-23
Table 2-8. Summary of derivation of points of departure for 1,2,3-TMB 2-26
Table 2-9. Effects and corresponding derivation of candidate RfC values for 1,2,3-TMB 2-29
Table 2-10. Organ/system-specific RfCs and proposed overall RfC for 1,2,3-TMB 2-31
Table 2-11. Target and actual exposure concentrations used in BMD modeling of 1,3,5-TMB endpoints
considered for the derivation of the RfC 2-35
Table 2-12. Endpoints considered for the derivation of the RfC for 1,3,5-TMB 2-36
Table 2-13. Summary of derivation of points of departure for 1,3,5-TMB 2-38
Table 2-14. Effects and corresponding derivation of candidate RfC values for 1,3,5-TMB 2-40
Table 2-15. Organ/system-specific RfCs and proposed overall RfC for 1,3,5-TMB 2-42
FIGURES
Figure LS-1. Literature search and study selection strategy for TMBs xlviii
Figure 1-1. Exposure response array of neurological effects following inhalation exposure to 1,2,4-TMB 1-18
Figure 1-2. Exposure response array of neurological effects following inhalation exposure to 1,2,3-TMB 1-19
Figure 1-3. Exposure response array of neurological effects following inhalation exposure to 1,3,5-TMB 1-20
Figure 1-4. Exposure response array of neurological effects following oral exposure to 1,2,3-TMB,
1,2,4-TMB, or 1,3,5-TMB 1-21
Figure 1-5. Exposure response array of respiratory effects following inhalation exposure to 1,2,3-TMB,
1,2,4-TMB, or 1,3,5-TMB 1-29
Figure 1-6. Exposure response array of reproductive and developmental effects following inhalation
exposure to 1,2,4-TMB or 1,3,5-TMB 1-34
Figure 1-7. Exposure response array of hematological and clinical chemistry effects following inhalation
exposure to 1,2,4-TMB or 1,3,5-TMB 1-42
Figure 1-8. Exposure response array of hematological and clinical chemistry effects following inhalation
exposure to 1,2,3-TMB 1-43
Figure 1-9. Exposure response array of hematological and clinical chemistry effects following oral
exposure to 1,3,5-TMB. Mode of Action Analysis- Hematological and Clinical Chemistry
Effects 1-44
Figure 2-1. Candidate RfC values with corresponding POD and composite UF for 1,2,4-TMB 2-15
Figure 2-2. Candidate RfC values with corresponding POD and composite UF for 1,2,3-TMB 2-30
Figure 2-3. Candidate RfC values with corresponding POD and composite UF for 1,3,5-TMB 2-41
This document is a draft for review purposes only and does not constitute Agency policy.
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ABBREVIATIONS AND ACRONYMS
AAQC Ambient air quality criterion OMOE
ACGIH American Conference of OSHA
Governmental Industrial Hygienists
ADME absorption, distribution, metabolism, p
and excretion PBPK
AEGL Acute Exposure Guideline Levels
AIC Akaike Information Criterion PEL
BAL bronchoalveolar lavage POD
BMD benchmark dose PODADj
BMDL lower confidence limit on the POI
benchmark dose ppm
BMDS benchmark dose software RBC
BMR benchmark response RDso
BW body weight REL
CAS Chemical Abstracts Service RfC
CASRN Chemical Abstracts Service Registry RfD
Number RGDR
CI confidence interval ROS
CNS central nervous system SCE
CYP450 cytochrome P450 SD
DAF dosimetric adjustment factor SOA
DMBA dimethylbenzoic acid TLV
DMHA dimethylhippuric acid TMB
DNA deoxyribonucleic acid TSCA
ECso half maximal effective concentration TWA
EEC electroencephalogram UF
EPA U.S. Environmental Protection UFA
Agency UFH
GD gestational day UFs
Hb/g-A animal blood:gas partition coefficient
Hb/g-H human blood:gas partition coefficient UFi
HEC human equivalent concentration UFD
HER human epidermal keratinocytes
HERO Health and Environmental Research UV
Online VOC
HEV human epithelial keratinocytes WBC
HSDB Hazardous Substances Data Bank WS
IL-8 interleukin-8 %2
i.p. intraperitoneal
IRIS Integrated Risk Information System
JP-8 jet propulsion fuel 8
Km Michaelis-Menten constant
LDH lactate dehydrogenase
LOAEL lowest-observed-adverse-effect level
NCEA National Center for Environmental
Assessment
NIOSH National Institute for Occupational
Safety and Health
NLM National Library of Medicine
NO AEL no-observed-adverse-effect level
Ontario Ministry of the Environment
Occupational Safety and Health
Administration
probability value
physiologically based
pharmacokinetic (model)
permissible exposure limit
point of departure
duration adjusted POD
point of impingement
parts per million
red blood cell
50% respiratory rate decrease
recommended exposure limit
reference concentration
reference dose
regional gas dose ratio
reactive oxygen species
sister chromatid exchange
standard deviation
secondary organic aerosol
threshold limit value
trimethylbenzene
Toxic Substances Control Act
time-weighted average
uncertainty factor
interspecies uncertainty factor
intraspecies uncertainty factor
subchronic-to-chronic uncertainty
factor
LOAEL-to-NOAEL uncertainty factor
database deficiency uncertainty
factor
ultraviolet
volatile organic compound
white blood cell
white spirit
chi-squared
This document is a draft for review purposes only and does not constitute Agency policy.
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AUTHORS | CONTRIBUTORS | REVIEWERS
Assessment Team
J. Allen Davis, M.S.P.H. (Chemical Manager)
Eva McLanahan, Ph.D. (LCDR, USPHS)
Paul Schlosser, Ph.D.
John Cowden1, Ph.D.
Gary Foureman, Ph.D. (Currently ICF Int.)
Andrew Kraft1, Ph.D.
Ray Antonelli, B.S.
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
Research Triangle Park, NC
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
Washington, DC
Oak Ridge Institute for Scientific Education
Research Triangle Park, NC
Scientific Support Team
Reeder Sams, Ph.D.
John Stanek, Ph.D.
Rob Dewoskin, Ph.D.
George Woodall, Ph.D.
Geniece Lehmann, Ph.D.
Connie Meacham, M.S.
Martin Gehlhaus1, MHS
Nina Wang1, Ph.D.
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
Research Triangle Park, NC
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
Washington, DC
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
Cincinnati, OH
1 Chemical Assessment Support Team (CAST) Member
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Production Team
Ellen Lorang, M.S.
Deborah Wales
Gerald Gurevich
J. Sawyer Lucy
Maureen Johnson
Vicki Soto
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
Research Triangle Park, NC
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
Washington, DC
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.
Executive Direction
Kenneth Olden, Ph.D., Sc.D., L.H.D.
(Center Director)
Lynn Flowers, Ph.D., DABT
(Associate Director for Health)
Vincent Cogliano, Ph.D.2
(IRIS Program Director—Acting)
Samantha Jones, Ph.D.
(IRIS Associate Director for Science)
Jamie Strong, Ph.D.
Ted Berner, MS
Reeder Sams, Ph.D.
Lyle Burgoon, 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
Washington, DC
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
Research Triangle Park, NC
2 Chemical Assessment Support Team (CAST) Lead
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Reviewers
This assessment was provided for review to scientists in EPA's Program and Regional Offices.
Comments were submitted by:
Region/Office
Region 8, Denver, CO
Region 2, New York City, NY
Office of Children's Health Protection, Washington, DC
Office of Air and Radiation, Washington, DC
Office of Environmental Information, Washington, DC
Office of Solid Waste and Emergency Response, Washington, DC
Office of Pesticides, Washington, DC
Office of Water, Washington, DC
This assessment was provided for review to other federal agencies and the Executive Offices of the
President Comments were submitted by:
Agency/ Office / Program
Chemical Material Risk Management Program, Department of Defense
Agency for Toxic Substances and Disease Registry, Centers for Disease Control and Prevention,
Department of Health & Human Services
The National Institute for Occupational Safety and Health, Centers for Disease Control and
Prevention, Department of Health & Human Services
National Toxicology Program, National Institute of Environmental Health Sciences, National
Institutes of Health, Department of Health & Human Services
Council on Environmental Quality, Executive Office of the President
This assessment was released for public comment on June 26th, 2012 and comments were due on
August 28th, 2012. Comments were received from the following entities:
Non-Government
Leslie Berry . . „ . ,_ „ .,
.... „ J American Chemistry Council
Allison Starmann
A public listening session was held by EPA on August 1st, 2012. Attendees external to the EPA are
listed below.
Listening Session Attendees (Non-EPA)
Leslia Berry . . „ . ,_ „ .,
„. , , „ , American Chemistry Council
Richard Becker
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PREFACE
1 This Toxicological Review critically reviews the publicly available studies on the three
2 isomers of trimethylbenzene (i.e., 1,2,3-trimethylbenzene [1,2,3-TMB], 1,2,4-trimethylbenzene
3 [1,2,4-TMB], and 1,3,5-trimethylbenzene [1,3,5-TMB]) in order to identify their adverse health
4 effects and to characterize exposure-response relationships. Because more types of studies are
5 available for the 1,2,4-TMB isomer, it generally appears first when the individual isomers are listed.
6 This assessment was prepared under the auspices of EPA's Integrated Risk Information System
7 (IRIS) program.
8 This assessment was prepared because of the presence of trimethylbenzenes (TMB) at
9 Superfund sites. Of sites on EPA's National Priorities List that report TMB isomer contamination (38
10 sites), 93% report 1,3,5-TMB contamination, 85% report 1,2,4-TMB contamination, 12% report
11 1,2,3-TMB contamination, and 17% report contamination by unspecified TMB isomers.
12 The Toxicological Review of Trimethylbenzenes is a new assessment; there is no previous
13 entry on the IRIS Database for 1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB. This assessment reviews
14 information on all health effects by all exposure routes.
15 This assessment was conducted in accordance with EPA guidance, which is cited and
16 summarized in the Preamble to IRIS Toxicological Reviews. The findings of this assessment and
17 related documents produced during its development are available on the IRIS website
18 (http://www.epa.gov/iris). Appendices for chemical and physical properties, toxicokinetic
19 information, summaries of toxicity studies, and other supporting materials are provided as
20 Supplemental Information (See Appendix A to C).
Implementation of the 2011 National Research Council Recommendations
21 On December 23, 2011, The Consolidated Appropriations Act, 2012, was signed into law
22 (U.S. Congress, 2011). The report language included direction to EPA for the IRIS Program related
23 to recommendations provided by the National Research Council (NRC) in their review of EPA's
24 draft IRIS assessment of formaldehyde (NRC. 2011). The NRC's recommendations, provided in
25 Chapter 7 of the review report, offered suggestions to EPA for improving the development of IRIS
26 assessments. The report language included the following:
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1 The Agency shall incorporate, as appropriate, based on chemical-specific datasets and
2 biological effects, the recommendations of Chapter 7 of the National Research Council's
3 Review of the Environmental Protection Agency's Draft IRIS Assessment of Formaldehyde
4 into the IRIS process .... For draft assessments released in fiscal year 2012, the Agency shall
5 include documentation describing how the Chapter 7 recommendations of the National
6 Academy of Sciences (NAS) have been implemented or addressed, including an explanation
7 for why certain recommendations were not incorporated.
8 Consistent with the direction provided by Congress, documentation of how the
9 recommendations from Chapter 7 of the NRC report have been implemented in this assessment is
10 provided in Appendix D. This documentation also includes an explanation for why certain
11 recommendations were not incorporated.
12 The IRIS Program's implementation of the NRC recommendations is following a phased
13 approach that is consistent with the NRC's "Roadmap for Revision" as described in Chapter 7 of the
14 formaldehyde review report. The NRC stated that, "the committee recognizes that the changes
15 suggested would involve a multi-year process and extensive effort by the staff at the National
16 Center for Environmental Assessment and input and review by the EPA Science Advisory Board and
17 others."
18 Phase 1 of implementation has focused on a subset of the short-term recommendations,
19 such as editing and streamlining documents, increasing transparency and clarity, and using more
20 tables, figures, and appendices to present information and data in assessments. Phase 1 also
21 focused on assessments near the end of the development process and close to final posting. The
22 IRIS TMBs assessment is one of the first assessments in Phase 2 of implementation, which
23 addresses all of the short-term NRC recommendations (see Appendix D, Table D-l). The IRIS
24 Program is implementing all of these recommendations but recognizes that achieving full and
25 robust implementation of certain recommendations will be an evolving process with input and
26 feedback from the public, stakeholders, and external peer review committees. Phase 3 of
27 implementation will incorporate the longer-term recommendations made by the NRC as outlined in
28 Table D-2, including the development of a standardized approach to describe the strength of
29 evidence for noncancer effects. On May 16, 2012, EPA announced [U.S. EPA. 2012c] that as a part of
30 a review of the IRIS Program's assessment development process, the NRC will also review current
31 methods for weight-of-evidence analyses and recommend approaches for weighing scientific
32 evidence for chemical hazard identification. This effort is included in Phase 3 of EPA's
33 implementation plan.
Assessments by Other National and International Health Agencies
34 Toxicity information on 1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB has been evaluated by the
35 National Institute for Occupational Safety and Health (NIOSH), the American Conference of
This document is a draft for review purposes only and does not constitute Agency policy.
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1 Governmental Industrial Hygienists (ACGIH), the National Advisory Committee for Acute Exposure
2 Guideline Levels for Hazardous Substances, and the Ontario Ministry of the Environment (MOE).
3 The results of these assessments are summarized in Appendix A (Table A-l). It is important to
4 recognize that these assessments may have been prepared for different purposes and may utilize
5 different methods, and that newer studies may be included in the IRIS assessment.
Chemical Properties and Uses
6 TMBs are aromatic hydrocarbons with three methyl groups attached to a benzene ring and
7 the chemical formula C9Hi2. The chemical and physical properties of the TMB isomers are similar to
8 one another. TMBs are colorless, flammable liquids with a strong aromatic odor; an odor threshold
9 of 0.4 parts per million (ppm) of air has been reported [U.S. EPA. 1994a). They are insoluble in
10 water but miscible with organic solvents such as ethyl alcohol, benzene, and ethyl ether [OSHA.
11 1996]. Production and use of TMBs may result in their release to the environment through various
12 waste streams. If released to the atmosphere, 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB will exist solely
13 in the vapor phase in the atmosphere under ambient conditions, based on measured vapor
14 pressures of 1.69, 2.10, and 2.48 mm Hg at 25°C, respectively [HSDB. 2011a. b, c). All three isomers
15 are expected to have limited mobility through soil based on their Log Koc values, but are expected to
16 volatilize from both moist and dry soil surfaces and surface waters based on their respective
17 Henry's law constants and vapor pressures (see Appendix B, Table B-l). Degradation of both
18 isomers in the atmosphere occurs by reaction with hydroxyl radicals, the half-life of which is 11-12
19 hours (HSDB. 2011a. b, c). Non-volatilized TMBs may be subject to biodegradation under aerobic
20 conditions (HSDB. 2011a. b, c). The estimated bio-concentration factors (133-439) and high
21 volatility of TMBs suggest that bioaccumulation of these chemicals will not be significant (U.S. EPA,
22 1987). Additional information on the chemical identities and physicochemical properties of TMBs
23 are listed in Table B-l in Appendix B.
24 The commercially available substance known as trimethylbenzene, CAS No. 25551-13-7, is a
25 mixture of three isomers in various proportions, namely CAS No. 526-73-8 (1,2,3-TMB or
26 hemimellitene), CAS No. 95-63-6 (1,2,4-TMB or pseudocumene), and CAS No. 108-67-8 (1,3,5-TMB
27 or mesitylene). Production of TMB isomers occurs during petroleum refining, and 1,2,4-TMB
28 individually makes up approximately 40% of the C9 aromatic fraction (i.e., aromatic hydrocarbons
29 with nine carbons) (U.S. EPA. 1994a). The domestic production of the C9 fraction in 1991 was
30 estimated to be approximately 80 billion pounds (40 million tons) (U.S. EPA. 1994a). Vehicle
31 emissions are a major anthropogenic source of TMBs, due to the widespread use of the C9 fraction
32 as a component of gasoline (U.S. EPA. 1994a). Other uses of TMBs include solvents in research and
33 industry, dyestuff intermediate, paint thinner, and as a UV oxidation stabilizer for plastics (HSDB.
34 2011b. c).
This document is a draft for review purposes only and does not constitute Agency policy.
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1 Occupational levels of exposure for TMBs have been measured between 20-8,540 |ig/m3
2 [HSDB, 2011a, b, c; Tiun-Horngetal., 2008], whereas residential exposures are generally much
3 lower: 0.29-7.8 [ig/m3 [Martins etal.. 2010: Choi etal.. 2009: Guo etal.. 2009]. Total atmospheric
4 releases of 1,2,4-TMB to the environment in 2008 equaled 5.8 million pounds (2,900 tons], 265,000
5 pounds (132.5 tons] were released to surface waters, underground injection sites, or land (TRI.
6 2008]. No information is currently available regarding 1,2,3-TMB or 1,3,5-TMB releases.
7 For additional information about this assessment or for general questions regarding IRIS,
8 please contact EPA's IRIS Hotline at 202-566-1676 (phone], 202-566-1749 (fax], or
9 hotline.iris@epa.gov.
This document is a draft for review purposes only and does not constitute Agency policy.
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PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS
1. Scope of the IRIS Program
1 Soon after the EPA was established in
2 1970, it was at the forefront of developing
3 risk assessment as a science and applying it
4 in decisions to protect human health and the
5 environment. The Clean Air Act, for example,
6 mandates that the EPA provide "an ample
7 margin of safety to protect public health";
8 the Safe Drinking Water Act, that "no
9 adverse effects on the health of persons may
10 reasonably be anticipated to occur, allowing
11 an adequate margin of safety." Accordingly,
12 the EPA uses information on the adverse
13 effects of chemicals and on exposure levels
14 below which these effects are not
15 anticipated to occur.
16 IRIS assessments critically review the
17 publicly available studies to identify adverse
18 health effects from exposure to chemicals
19 and to characterize exposure-response
20 relationships. In terms set forth by the
21 National Research Council [NRC. 1983). IRIS
22 assessments cover the hazard identification
23 and dose-response assessment steps of risk
24 assessment, not the exposure assessment or
25 risk characterization steps that are
26 conducted by the EPA's program and
27 regional offices and by other federal, state,
28 and local health agencies that evaluate risk
29 in specific populations and exposure
30 scenarios. IRIS assessments are distinct from
31 and do not address political, economic, and
32 technical considerations that influence the
33 design and selection of risk management
34 alternatives.
35 An IRIS assessment may cover a single
36 chemical, a group of structurally or
37 toxicologically related chemicals, or a
38 complex mixture. These agents may be found
39 in air, water, soil, or sediment Exceptions
40 are chemicals currently used exclusively as
41 pesticides, ionizing and non-ionizing
42 radiation, and criteria air pollutants listed
43 under Section 108 of the Clean Air Act
44 (carbon monoxide, lead, nitrogen oxides,
45 ozone, particulate matter, and sulfur oxides).
46 Periodically, the IRIS Program asks other
47 EPA programs and regions, other federal
48 agencies, state health agencies, and the
49 general public to nominate chemicals and
50 mixtures for future assessment or
51 reassessment Agents may be considered for
52 reassessment as significant new studies are
53 published. Selection is based on program
54 and regional office priorities and on
55 availability of adequate information to
56 evaluate the potential for adverse effects.
57 Other agents may also be assessed in
58 response to an urgent public health need.
2. Process for developing and peer-
reviewing IRIS assessments
59 The process for developing IRIS
60 assessments (revised in May 2009 and
61 enhanced in July 2013) involves critical
62 analysis of the pertinent studies,
63 opportunities for public input, and multiple
64 levels of scientific review. The EPA revises
65 draft assessments after each review, and
66 external drafts and comments become part
67 of the public record (U.S. EPA. 2009).
68 Before beginning an assessment, the IRIS
69 Program discusses the scope with other EPA
70 programs and regions to ensure that the
71 assessment will meet their needs. Then a
72 public meeting on problem formulation
73 invites discussion of the key issues and the
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1 studies and analytical approaches that might
2 contribute to their resolution.
3 Step 1. Development of a draft
4 Toxicological Review. The draft
5 assessment considers all pertinent
6 publicly available studies and applies
7 consistent criteria to evaluate study
8 quality, identify health effects, identify
9 mechanistic events and pathways,
10 integrate the evidence of causation for
11 each effect, and derive toxicity values. A
12 public meeting prior to the integration of
13 evidence and derivation of toxicity
14 values promotes public discussion of the
15 literature search, evidence, and key
16 issues.
17 Step 2. Internal review by scientists in
18 EPA programs and regions. The draft
19 assessment is revised to address the
20 comments from within the EPA.
21 Step 3. Interagency science consultation
22 with other federal agencies and the
23 Executive Offices of the President. The
24 draft assessment is revised to address
25 the interagency comments. The science
26 consultation draft, interagency
27 comments, and the EPA's response to
28 major comments become part of the
29 public record.
30 Step 4. Public review and comment,
31 followed by external peer review. The
32 EPA releases the draft assessment for
33 public review and comment A public
34 meeting provides an opportunity to
35 discuss the assessment prior to peer
36 review. Then the EPA releases a draft for
37 external peer review. The peer review
38 meeting is open to the public and
39 includes time for oral public comments.
40 The peer reviewers assess whether the
41 evidence has been assembled and
42 evaluated according to guidelines and
43 whether the conclusions are justified by
44 the evidence. The peer review draft,
45 written public comments, and peer
46 review report become part of the public
47 record.
48 Step 5. Revision of draft Toxicological
49 Review and development of draft IRIS
50 summary. The draft assessment is
51 revised to reflect the peer review
52 comments, public comments, and newly
53 published studies that are critical to the
54 conclusions of the assessment The
55 disposition of peer review comments
56 and public comments becomes part of
57 the public record.
58 Step 6. Final EPA review and interagency
59 science discussion with other federal
60 agencies and the Executive Offices of
61 the President The draft assessment and
62 summary are revised to address the EPA
63 and interagency comments. The science
64 discussion draft, written interagency
65 comments, and EPA's response to major
66 comments become part of the public
67 record.
68 Step 7. Completion and posting. The
69 Toxicological Review and IRIS summary
70 are posted on the IRIS website
71 [http://www.epa.gov/iris/].
72 The remainder of this Preamble addresses
73 step 1, the development of a draft
74 Toxicological Review. IRIS assessments
75 follow standard practices of evidence
76 evaluation and peer review, many of
77 which are discussed in EPA guidelines
78 fU.S. EPA. 2005a. b, 2000. 1998. 1996.
79 1991. 1986a. b) and other methods [U.S.
80 EPA. 2012a. b, 2011. 2006a. b, 2002.
81 1994b). Transparent application of
82 scientific judgment is of paramount
83 importance. To provide a harmonized
84 approach across IRIS assessments, this
85 Preamble summarizes concepts from
86 these guidelines and emphasizes
87 principles of general applicability.
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3. Identifying and selecting
pertinent studies
1 3.1. Identifying studies
2 Before beginning an assessment, the EPA
3 conducts a comprehensive search of the
4 primary scientific literature. The literature
5 search follows standard practices and
6 includes the PubMed and ToxNet databases
7 of the National Library of Medicine, Web of
8 Science, and other databases listed in the
9 EPA's HERO system (Health and
10 Environmental Research Online,
11 http://hero.epa.gov/]. Searches for
12 information on mechanisms of toxicity are
13 inherently specialized and may include
14 studies on other agents that act through
15 related mechanisms.
16 Each assessment specifies the search
17 strategies, keywords, and cut-off dates of its
18 literature searches. The EPA posts the
19 results of the literature search on the IRIS
20 web site and requests information from the
21 public on additional studies and ongoing
22 research.
23 The EPA also considers studies received
24 through the IRIS Submission Desk and
25 studies (typically unpublished) submitted
26 under the Toxic Substances Control Act or
27 the Federal Insecticide, Fungicide, and
28 Rodenticide Act. Material submitted as
29 Confidential Business Information is
30 considered only if it includes health and
31 safety data that can be publicly released. If a
32 study that may be critical to the conclusions
33 of the assessment has not been peer-
34 reviewed, the EPA will have it peer-
35 reviewed.
36 The EPA also examines the toxicokinetics
37 of the agent to identify other chemicals (for
38 example, major metabolites of the agent) to
39 include in the assessment if adequate
40 information is available, in order to more
41 fully explain the toxicity of the agent and to
42 suggest dose metrics for subsequent
43 modeling.
44 In assessments of chemical mixtures,
45 mixture studies are preferred for their
46 ability to reflect interactions among
47 components.
48 The literature search seeks, in
49 decreasing order of preference (U.S. EPA.
50 2000. 32.2: 1986b. 32.1)]:
51 - Studies of the mixture being
52 assessed.
53 - Studies of a sufficiently similar
54 mixture. In evaluating similarity, the
55 assessment considers the alteration
56 of mixtures in the environment
57 through partitioning and
58 transformation.
59 - Studies of individual chemical
60 components of the mixture, if there
61 are not adequate studies of
62 sufficiently similar mixtures.
63 3.2. Selecting pertinent epidemiologic
64 studies
65 Study design is the key consideration for
66 selecting pertinent epidemiologic studies
67 from the results of the literature search.
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
Cohort studies, case-control studies,
and some population-based surveys
(for example, NHANES) provide the
strongest epidemiologic evidence,
especially if they collect information
about individual exposures and
effects.
Ecological studies (geographic
correlation studies) relate exposures
and effects 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.
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1 - Case reports of high or accidental
2 exposure lack definition of the
3 population at risk and the expected
4 number of cases. They can provide
5 information about a rare effect or
6 about the relevance of analogous
7 results in animals.
8 The assessment briefly reviews
9 ecological studies and case reports but
10 reports details only if they suggest effects
11 not identified by other studies.
12 3.3. Selecting pertinent experimental
13 studies
14 Exposure route is a key design
15 consideration for selecting pertinent
16 experimental animal studies or human
17 clinical studies.
18 - Studies of oral, inhalation, or dermal
19 exposure involve passage through an
20 absorption barrier and are
21 considered most pertinent to human
22 environmental exposure.
23 - Injection or implantation studies are
24 often considered less pertinent but may
25 provide valuable toxicokinetic or
26 mechanistic information. They also may
27 be useful for identifying effects in
28 animals if deposition or absorption is
29 problematic (for example, for particles
30 and fibers).
31 Exposure duration is also a key design
32 consideration for selecting pertinent
33 experimental animal studies.
34 - Studies of effects from chronic
35 exposure are most pertinent to
36 lifetime human exposure.
37 - Studies of effects from less-than-
38 chronic exposure are pertinent but
39 less preferred for identifying effects
40 from lifetime human exposure. Such
41 studies may be indicative of effects
42 from less-than-lifetime human
43 exposure.
44 Short-duration studies involving animals
45 or humans may provide toxicokinetic or
46 mechanistic information.
47 For developmental toxicity and
48 reproductive toxicity, irreversible effects
49 may result from a brief exposure during a
50 critical period of development Accordingly,
51 specialized study designs are used for these
52 effects [U.S. EPA. 2006b. 1998. 1996. 19911.
4. Evaluating the quality of
individual studies
53 After the subsets of pertinent
54 epidemiologic and experimental studies
55 have been selected from the literature
56 searches, the assessment evaluates the
57 quality of each individual study. This
58 evaluation considers the design, methods,
59 conduct, and documentation of each study,
60 but not whether the results are positive,
61 negative, or null. The objective is to identify
62 the stronger, more informative studies based
63 on a uniform evaluation of quality
64 characteristics across studies of similar
65 design.
66 4.1. Evaluating the quality of
67 epidemiologic studies
68 The assessment evaluates design and
69 methodological aspects that can increase or
70 decrease the weight given to each
71 epidemiologic study in the overall evaluation
72 [U.S. EPA. 2005a. 1998.1996.1994b. 19911:
73 - Documentation of study design,
74 methods, population characteristics,
75 and results.
76 - Definition and selection of the study
77 group and comparison group.
78 - Ascertainment of exposure to the
79 chemical or mixture.
80 - Ascertainment of disease or health
81 effect.
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1 - Duration of exposure and follow-up
2 and adequacy for assessing the
3 occurrence of effects.
4 - Characterization of exposure during
5 critical periods.
6 - Sample size and statistical power to
7 detect anticipated effects.
8 - Participation rates and potential for
9 selection bias as a result of the
10 achieved participation rates.
11 - Measurement error (can lead to
12 misclassification of exposure, health
13 outcomes, and other factors) and
14 other types of information bias.
15 - Potential confounding and other
16 sources of bias addressed in the
17 study design or in the analysis of
18 results. The basis for consideration
19 of confounding is a reasonable
20 expectation that the confounder is
21 related to both exposure and
22 outcome and is sufficiently prevalent
23 to result in bias.
24 For developmental toxicity, reproductive
25 toxicity, neurotoxicity, and cancer there is
26 further guidance on the nuances of
27 evaluating epidemiologic studies of these
28 effects fU.S. EPA. 2005a. 1998. 1996. 19911
29 4.2. Evaluating the quality of
30 experimental studies
31 The assessment evaluates design and
32 methodological aspects that can increase or
33 decrease the weight given to each
34 experimental animal study, in-vitro study, or
35 human clinical study [U.S. EPA. 2005a. 1998.
36 1996. 1991). Research involving human
37 subjects is considered only if conducted
38 according to ethical principles.
39 - Documentation of study design,
40 animals or study population,
41 methods, basic data, and results.
42 - Nature of the assay and validity for
43 its intended purpose.
44 - Characterization of the nature and
45 extent of impurities and
46 contaminants of the administered
47 chemical or mixture.
48 - Characterization of dose and dosing
49 regimen (including age at exposure)
50 and their adequacy to elicit adverse
51 effects, including latent effects.
52 - Sample sizes and statistical power to
53 detect dose-related differences or
54 trends.
55 - Ascertainment of survival, vital signs,
56 disease or effects, and cause of death.
57 - Control of other variables that could
58 influence the occurrence of effects.
59 The assessment uses statistical tests to
60 evaluate whether the observations may be
61 due to chance. The standard for determining
62 statistical significance of a response is a
63 trend test or comparison of outcomes in the
64 exposed groups against those of concurrent
65 controls. In some situations, examination of
66 historical control data from the same
67 laboratory within a few years of the study
68 may improve the analysis. For an uncommon
69 effect that is not statistically significant
70 compared with concurrent controls,
71 historical controls may show that the effect
72 is unlikely to be due to chance. For a
73 response that appears significant against a
74 concurrent control response that is unusual,
75 historical controls may offer a different
76 interpretation fU.S. EPA. 2005a. §2.2.2.1.3).
77 For developmental toxicity, reproductive
78 toxicity, neurotoxicity, and cancer there is
79 further guidance on the nuances of
80 evaluating experimental studies of these
81 effects (U.S. EPA. 2005a. 1998. 1996. 1991).
82 In multi-generation studies, agents that
83 produce developmental effects at doses that
84 are not toxic to the maternal animal are of
85 special concern. Effects that occur at doses
86 associated with mild maternal toxicity are
87 not assumed to result only from maternal
88 toxicity. Moreover, maternal effects may be
89 reversible, while effects on the offspring may
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1 be permanent [U.S. EPA. 1998.33.1.2.4.5.4:
2 1991 §3.1.1.4),.
3 4.3. Reporting study results
4 The assessment uses evidence tables to
5 present the design and key results of
6 pertinent studies. There may be separate
7 tables for each site of toxicity or type of
8 study.
9 If a large number of studies observe the
10 same effect, the assessment considers the
11 study quality characteristics in this section
12 to identify the strongest studies or types of
13 study. The tables present details from these
14 studies, and the assessment explains the
15 reasons for not reporting details of other
16 studies or groups of studies that do not add
17 new information. Supplemental information
18 provides references to all studies
19 considered, including those not summarized
20 in the tables.
21 The assessment discusses strengths and
22 limitations that affect the interpretation of
23 each study. If the interpretation of a study in
24 the assessment differs from that of the study
25 authors, the assessment discusses the basis
26 for the difference.
27 As a check on the selection and
28 evaluation of pertinent studies, the EPA asks
29 peer reviewers to identify studies that were
30 not adequately considered.
5. Evaluating the overall evidence
of each effect
31 5.1. Concepts of causal inference
32 For each health effect, the assessment
33 evaluates the evidence as a whole to
34 determine whether it is reasonable to infer a
35 causal association between exposure to the
36 agent and the occurrence of the effect. This
37 inference is based on information from
38 pertinent human studies, animal studies, and
39 mechanistic studies of adequate quality.
40 Positive, negative, and null results are given
41 weight according to study quality.
42 Causal inference involves scientific
43 judgment, and the considerations are
44 nuanced and complex. Several health
45 agencies have developed frameworks for
46 causal inference, among them the U.S.
47 Surgeon General fCDC. 2004: HEW. 19641
48 the International Agency for Research on
49 Cancer [IARC. 2006], the Institute of
50 Medicine [IOM. 2008], and the EPA
51 f2010.31.6: 2005a.§2.51 Although
52 developed for different purposes, the
53 frameworks are similar in nature and
54 provide an established structure and
55 language for causal inference. Each
56 considers aspects of an association that
57 suggest causation, discussed by Hill [1965]
58 and elaborated by Rothman and Greenland
59 (1998), and U.S. EPA (2005a, §2.2.1.7;
60 1994b, Appendix C).
61 Strength of association: The finding of a
62 large relative risk with narrow
63 confidence intervals strongly suggests
64 that an association is not due to chance,
65 bias, or other factors. Modest relative
66 risks, however, may reflect a small range
67 of exposures, an agent of low potency, an
68 increase in an effect that is common,
69 exposure misclassification, or other
70 sources of bias.
71 Consistency of association: An inference of
72 causation is strengthened if elevated
73 risks are observed in independent
74 studies of different populations and
75 exposure scenarios. Reproducibility of
76 findings constitutes one of the strongest
77 arguments for causation. Discordant
78 results sometimes reflect differences in
79 study design, exposure, or confounding
80 factors.
81 Specificity of association: As originally
82 intended, this refers to one cause
83 associated with one effect. Current
84 understanding that many agents cause
85 multiple effects and many effects have
86 multiple causes make this a less
87 informative aspect of causation, unless
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1 the effect is rare or unlikely to have
2 multiple causes.
3 Temporal relationship: A causal
4 interpretation requires that exposure
5 precede development of the effect.
6 Biologic gradient (exposure-response
7 relationship): Exposure-response
8 relationships strongly suggest causation.
9 A monotonic increase is not the only
10 pattern consistent with causation. The
11 presence of an exposure-response
12 gradient also weighs against bias and
13 confounding as the source of an
14 association.
15 Biologic plausibility: An inference of
16 causation is strengthened by data
17 demonstrating plausible biologic
18 mechanisms, if available. Plausibility
19 may reflect subjective prior beliefs if
20 there is insufficient understanding of the
21 biologic process involved.
22 Coherence: An inference of causation is
23 strengthened by supportive results from
24 animal experiments, toxicokinetic
25 studies, and short-term tests. Coherence
26 may also be found in other lines of
27 evidence, such as changing disease
28 patterns in the population.
29 "Natural experiments": A change in
30 exposure that brings about a change in
31 disease frequency provides strong
32 evidence, as it tests the hypothesis of
33 causation. An example would be an
34 intervention to reduce exposure in the
35 workplace or environment that is
36 followed by a reduction of an adverse
37 effect.
38 Analogy: Information on structural
39 analogues or on chemicals that induce
40 similar mechanistic events can provide
41 insight into causation.
42 These considerations are consistent with
43 guidelines for systematic reviews that
44 evaluate the quality and weight of evidence.
45 Confidence is increased if the magnitude of
46 effect is large, if there is evidence of an
47 exposure-response relationship, or if an
48 association was observed and the plausible
49 biases would tend to decrease the magnitude
50 of the reported effect. Confidence is
51 decreased for study limitations,
52 inconsistency of results, indirectness of
53 evidence, imprecision, or reporting bias
54 [Guyattetal.. 2008b: Guyattetal.. 2008a).
55 5.2. Evaluating evidence in humans
56 For each effect, the assessment evaluates
57 the evidence from the epidemiologic studies
58 as a whole. The objective is to determine
59 whether a credible association has been
60 observed and, if so, whether that association
61 is consistent with causation. In doing this,
62 the assessment explores alternative
63 explanations (such as chance, bias, and
64 confounding) and draws a conclusion about
65 whether these alternatives can satisfactorily
66 explain any observed association.
67 To make clear how much the
68 epidemiologic evidence contributes to the
69 overall weight of the evidence, the
70 assessment may select a standard descriptor
71 to characterize the epidemiologic evidence
72 of association between exposure to the agent
73 and occurrence of a health effect.
74 Sufficient epidemiologic evidence of an
75 association consistent with causation:
76 The evidence establishes a causal
77 association for which alternative
78 explanations such as chance, bias, and
79 confounding can be ruled out with
80 reasonable confidence.
81 Suggestive epidemiologic evidence of an
82 association consistent with causation:
83 The evidence suggests a causal
84 association but chance, bias, or
85 confounding cannot be ruled out as
86 explaining the association.
87 Inadequate epidemiologic evidence to
88 infer a causal association: The available
89 studies do not permit a conclusion
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1 regarding the presence or absence of an
2 association.
3 Epidemiologic evidence consistent with no
4 causal association: Several adequate
5 studies covering the full range of human
6 exposures and considering susceptible
7 populations, and for which alternative
8 explanations such as bias and
9 confounding can be ruled out, are
10 mutually consistent in not finding an
11 association.
12 5.3. Evaluating evidence in animals
13 For each effect, the assessment evaluates
14 the evidence from the animal experiments as
15 a whole to determine the extent to which
16 they indicate a potential for effects in
17 humans. Consistent results across various
18 species and strains increase confidence that
19 similar results would occur in humans.
20 Several concepts discussed by Hill [1965]
21 are pertinent to the weight of experimental
22 results: consistency of response, dose-
23 response relationships, strength of response,
24 biologic plausibility, and coherence [U.S.
25 EPA. 2005a. §2.2.1.7: 1994b. Appendix Cj.
26 In weighing evidence
27 experiments, U.S. EPA
28 distinguishes:
from multiple
[2005a. §2.5)
29 Conflicting evidence (that is, mixed positive
30 and negative results in the same sex and
31 strain using a similar study protocol)
32 from
33 Differing results (that is, positive results
34 and negative results are in different
35 sexes or strains or use different study
36 protocols).
37 Negative or null results do not invalidate
38 positive results in a different experimental
39 system. The EPA regards all as valid
40 observations and looks to explain differing
41 results using mechanistic information (for
42 example, physiologic or metabolic
43 differences across test systems) or
44 methodological differences (for example,
45 relative sensitivity of the tests, differences in
46 dose levels, insufficient sample size, or
47 timing of dosing or data collection).
48 It is well established that there are
49 critical periods for some developmental and
50 reproductive effects (U.S. EPA. 2006b.
51 2005a. b, 1998. 1996. 1991). Accordingly,
52 the assessment determines whether critical
53 periods have been adequately investigated.
54 Similarly, the assessment determines
55 whether the database is adequate to
56 evaluate other critical sites and effects.
57 In evaluating evidence of genetic
58 toxicity:
59 - Demonstration of gene mutations,
60 chromosome aberrations, or
61 aneuploidy in humans or
62 experimental mammals [in vivo]
63 provides the strongest evidence.
64 - This is followed by positive results in
65 lower organisms or in cultured cells
66 [in vitro] or for other genetic events.
67 - Negative results carry less weight,
68 partly because they cannot exclude
69 the possibility of effects in other
70 tissues [IARC. 2006).
71 For germ-cell mutagenicity, The EPA has
72 defined categories of evidence, ranging from
73 positive results of human germ-cell
74 mutagenicity to negative results for all
75 effects of concern [U.S. EPA. 1986a. §2.3).
76 5.4. Evaluating mechanistic data
77 Mechanistic data can be useful in
78 answering several questions.
79
80
81
82
83
84
The biologic plausibility of a causal
interpretation of human studies.
- The generalizability
studies to humans.
of animal
- The susceptibility of particular
populations or lifestages.
85 The focus of the analysis is to describe, if
86 possible, mechanistic pathways that lead to a
87 health effect. These pathways encompass:
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1 - Toxicokinetic processes of absorption,
2 distribution, metabolism, and
3 elimination that lead to the
4 formation of an active agent and its
5 presence at the site of initial biologic
6 interaction.
7 - Toxicodynamic processes that lead to
8 a health effect at this or another site
9 (also known as a mode of action}.
10 For each effect, the assessment discusses
11 the available information on its modes of
12 action and associated key events (key events
13 being empirically observable, necessary
14 precursor steps or biologic markers of such
15 steps; mode of action being a series of key
16 events involving interaction with cells,
17 operational and anatomic changes, and
18 resulting in disease). Pertinent information
19 may also come from studies of metabolites
20 or of compounds that are structurally similar
21 or that act through similar mechanisms.
22 Information on mode of action is not
23 required for a conclusion that the agent is
24 causally related to an effect [U.S. EPA. 2005a.
25 §2.5).
26 The assessment addresses several
27 questions about each hypothesized mode of
28 actionfU.S. EPA. 2005a. §2.4.3.4).
29 1) Is the hypothesized mode of action
30 sufficiently supported in test animals?
31 Strong support for a key event being
32 necessary to a mode of action can come
33 from experimental challenge to the
34 hypothesized mode of action, in which
35 studies that suppress a key event
36 observe suppression of the effect.
37 Support for a mode of action is
38 meaningfully strengthened by consistent
39 results in different experimental models,
40 much more so than by replicate
41 experiments in the same model. The
42 assessment may consider various
43 aspects of causation in addressing this
44 question.
45 2) Is the hypothesized mode of action
46 relevant to humans? The assessment
47 reviews the key events to identify critical
48 similarities and differences between the
49 test animals and humans. Site
50 concordance is not assumed between
51 animals and humans, though it may hold
52 for certain effects or modes of action.
53 Information suggesting quantitative
54 differences in doses where effects would
55 occur in animals or humans is
56 considered in the dose-response
57 analysis. Current levels of human
58 exposure are not used to rule out human
59 relevance, as IRIS assessments may be
60 used in evaluating new or unforeseen
61 circumstances that may entail higher
62 exposures.
63 3) Which populations or lifestages can
64 be particularly susceptible to the
65 hypothesized mode of action? The
66 assessment reviews the key events to
67 identify populations and lifestages that
68 might be susceptible to their occurrence.
69 Quantitative differences may result in
70 separate toxicity values for susceptible
71 populations or lifestages.
72 The assessment discusses the likelihood
73 that an agent operates through multiple
74 modes of action. An uneven level of support
75 for different modes of action can reflect
76 disproportionate resources spent
77 investigating them [U.S. EPA.
78 2005a. §2.4.3.3). It should be noted that in
79 clinical reviews, the credibility of a series of
80 studies is reduced if evidence is limited to
81 studies funded by one interested sector
82 fGuyattetal.. 2008a).
83 For cancer, the assessment evaluates
84 evidence of a mutagenic mode of action to
85 guide extrapolation to lower doses and
86 consideration of susceptible lifestages. Key
87 data include the ability of the agent or a
88 metabolite to react with or bind to DNA,
89 positive results in multiple test systems, or
90 similar properties and structure-activity
91 relationships to mutagenic carcinogens [U.S.
92 EPA. 2005a.§2.3.5).
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1 5.5. Characterizing the overall weight
2 of the evidence
3 After evaluating the human, animal, and
4 mechanistic evidence pertinent to an effect,
5 the assessment answers the question: Does
6 the agent cause the adverse effect? [NRG.
7 2009. 19831. In doing this, the assessment
8 develops a narrative that integrates the
9 evidence pertinent to causation. To provide
10 clarity and consistency, the narrative
11 includes a standard hazard descriptor. For
12 example, the following standard descriptors
13 combine epidemiologic, experimental, and
14 mechanistic evidence of carcinogenicity
15 fU.S. EPA. 2005a. 32.51.
16 Carcinogenic to humans: There is
17 convincing epidemiologic evidence of a
18 causal association (that is, there is
19 reasonable confidence that the
20 association cannot be fully explained by
21 chance, bias, or confounding); or there is
22 strong human evidence of cancer or its
23 precursors, extensive animal evidence,
24 identification of key precursor events in
25 animals, and strong evidence that they
26 are anticipated to occur in humans.
27 Likely to be carcinogenic to humans: The
28 evidence demonstrates a potential
29 hazard to humans but does not meet the
30 criteria for carcinogenic. There may be a
31 plausible association in humans,
32 multiple positive results in animals, or a
33 combination of human, animal, or other
34 experimental evidence.
35 Suggestive evidence of carcinogenic
36 potential: The evidence raises concern
37 for effects in humans but is not sufficient
38 for a stronger conclusion. This
39 descriptor covers a range of evidence,
40 from a positive result in the only
41 available study to a single positive result
42 in an extensive database that includes
43 negative results in other species.
44 Inadequate information to assess
45 carcinogenic potential: No other
46 descriptors apply. Conflicting evidence
47 can be classified as inadequate
48 information if all positive results are
49 opposed by negative studies of equal
50 quality in the same sex and strain.
51 Differing results, however, can be
52 classified as suggestive evidence or as
53 likely to be carcinogenic.
54 Not likely to be carcinogenic to humans:
55 There is robust evidence for concluding
56 that there is no basis for concern. There
57 may be no effects in both sexes of at least
58 two appropriate animal species; positive
59 animal results and strong, consistent
60 evidence that each mode of action in
61 animals does not operate in humans; or
62 convincing evidence that effects are not
63 likely by a particular exposure route or
64 below a defined dose.
65 Multiple descriptors may be used if there
66 is evidence that carcinogenic effects differ by
67 dose range or exposure route [U.S. EPA.
68 2005a.32.5).
69 Another example of standard descriptors
70 comes from the EPA's Integrated Science
71 Assessments, which evaluate causation for
72 the effects of the criteria pollutants in
73 ambient air [U.S. EPA. 2010. §1.6).
74 Causal relationship: Sufficient evidence to
75 conclude that there is a causal
76 relationship. Observational studies
77 cannot be explained by plausible
78 alternatives, or they are supported by
79 other lines of evidence, for example,
80 animal studies or mechanistic
81 information.
82 Likely to be a causal relationship:
83 Sufficient evidence that a causal
84 relationship is likely, but important
85 uncertainties remain. For example,
86 observational studies show an
87 association but co-exposures are difficult
88 to address or other lines of evidence are
89 limited or inconsistent; or multiple
90 animal studies from different
91 laboratories demonstrate effects and
92 there are limited or no human data.
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1 Suggestive of a causal relationship: At
2 least one high-quality epidemiologic
3 study shows an association but other
4 studies are inconsistent.
5 Inadequate to infer a causal relationship:
6 The studies do not permit a conclusion
7 regarding the presence or absence of an
8 association.
9 Not likely to be a causal relationship:
10 Several adequate studies, covering the
11 full range of human exposure and
12 considering susceptible populations, are
13 mutually consistent in not showing an
14 effect at any level of exposure.
15 The EPA is investigating and may on a
16 trial basis use these or other standard
17 descriptors to characterize the overall
18 weight of the evidence for effects other than
19 cancer.
6. Selecting studies for derivation
of toxicity values
20 For each effect where there is credible
21 evidence of an association with the agent,
22 the assessment derives toxicity values if
23 there are suitable epidemiologic or
24 experimental data. The decision to derive
25 toxicity values may be linked to the hazard
26 descriptor.
27 Dose-response analysis requires
28 quantitative measures of dose and response.
29 Then, other factors being equal:
30
31
32
33
34
35
36
37
38
39
40
41
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
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
toxicokinetic model can be used to
extrapolate across exposure routes.
Studies of longer exposure duration
and follow-up are preferred, to
minimize uncertainty about whether
effects are representative of lifetime
exposure.
Studies with multiple exposure levels
are preferred for their ability to
provide information about the shape
of the exposure-response curve.
Studies with adequate power to
detect effects at lower exposure
levels are preferred, to minimize the
extent of extrapolation to levels
found in the environment.
58 Studies with non-monotonic exposure-
59 response relationships are not necessarily
60 excluded from the analysis. A diminished
61 effect at higher exposure levels may be
62 satisfactorily explained by factors such as
63 competing toxicity, saturation of absorption
64 or metabolism, exposure misclassification,
65 or selection bias.
66 If a large number of studies are suitable
67 for dose-response analysis, the assessment
68 considers the study characteristics in this
69 section to focus on the most informative
70 data. The assessment explains the reasons
71 for not analyzing other groups of studies. As
72 a check on the selection of studies for dose-
73 response analysis, the EPA asks peer
74 reviewers to identify studies that were not
75 adequately considered.
7. Deriving toxicity values
76 7.1. General framework for dose-
77 response analysis
78 The EPA uses a two-step approach that
79 distinguishes analysis of the observed dose-
80 response data from inferences about lower
81 doses [U.S. EPA. 2005a. 331.
82 Within the observed range, the preferred
83 approach is to use modeling to incorporate a
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1 wide range of data into the analysis. The
2 modeling yields a point of departure (an
3 exposure level near the lower end of the
4 observed range, without significant
5 extrapolation to lower doses) (Sections 7.2-
6 7.3).
7 Extrapolation to lower doses considers
8 what is known about the modes of action for
9 each effect (Sections 7.4-7.5). If response
10 estimates at lower doses are not required, an
11 alternative is to derive reference values,
12 which are calculated by applying factors to
13 the point of departure in order to account
14 for sources of uncertainty and variability
15 (Section 7.6).
16 For a group of agents that induce an
17 effect through a common mode of action, the
18 dose-response analysis may derive a relative
19 potency factor for each agent. A full dose-
20 response analysis is conducted for one well-
21 studied index chemical in the group, then the
22 potencies of other members are expressed in
23 relative terms based on relative toxic effects,
24 relative absorption or metabolic rates,
25 quantitative structure-activity relationships,
26 or receptor binding characteristics (U.S. EPA.
27 2005_a, §3.2.6; 2000, §4.4).
28 Increasingly, the EPA is basing toxicity
29 values on combined analyses of multiple
30 data sets or multiple responses. The EPA
31 also considers multiple dose-response
32 approaches if they can be supported by
33 robust data.
34 7.2. Modeling dose to sites of biologic
35 effects
36 The preferred approach for analysis of
37 dose is toxicokinetic modeling because of its
38 ability to incorporate a wide range of data.
39 The preferred dose metric would refer to the
40 active agent at the site of its biologic effect or
41 to a close, reliable surrogate measure. The
42 active agent may be the administered
43 chemical or a metabolite. Confidence in the
44 use of a toxicokinetic model depends on the
45 robustness of its validation process and on
46 the results of sensitivity analyses (U.S. EPA,
47 2006a: 2005a. §3.1: 1994b. §4.31
48 Because toxicokinetic modeling can
49 require many parameters and more data
50 than are typically available, the EPA has
51 developed standard approaches that can be
52 applied to typical data sets. These standard
53 approaches also facilitate comparison across
54 exposure patterns and species.
55 - Intermittent study exposures are
56 standardized to a daily average over
57 the duration of exposure. For chronic
58 effects, daily exposures are averaged
59 over the lifespan. Exposures during a
60 critical period, however, are not
61 averaged over a longer duration (U.S.
62 EPA. 2005a. 33.1.1: 1991. §3.2).
63 - Doses are standardized to equivalent
64 human terms to facilitate
65 comparison of results from different
66 species.
67 - Oral doses are scaled allometrically
68 using mg/kg3/4-day as the equivalent
69 dose metric across species.
70 Allometric scaling pertains to
71 equivalence across species, not
72 across lifestages, and is not used to
73 scale doses from adult humans or
74 mature animals to infants or children
75 fU.S. EPA. 2011: 2005a. §3.1.31
76 - Inhalation exposures are scaled
77 using dosimetry models that apply
78 species-specific physiologic and
79 anatomic factors and consider
80 whether the effect occurs at the site
81 of first contact or after systemic
82 circulation (U.S. EPA. 2012a:
83 1994b. §3).
84 It can be informative to convert doses
85 across exposure routes. If this is done, the
86 assessment describes the underlying data,
87 algorithms, and assumptions (U.S. EPA.
88 2005a. 33.1.4).
89 In the absence of study-specific data on,
90 for example, intake rates or body weight, the
91 EPA has developed recommended values for
92 use in dose-response analysis (U.S. EPA.
93 1988).
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1 7.3. Modeling response in the range
2 of observation
3 Toxicodynamic ("biologically based")
4 modeling can incorporate data on biologic
5 processes leading to an effect. Such models
6 require sufficient data to ascertain a mode of
7 action and to quantitatively support model
8 parameters associated with its key events.
9 Because different models may provide
10 equivalent fits to the observed data but
11 diverge substantially at lower doses, critical
12 biologic parameters should be measured
13 from laboratory studies, not by model fitting.
14 Confidence in the use of a toxicodynamic
15 model depends on the robustness of its
16 validation process and on the results of
17 sensitivity analyses. Peer review of the
18 scientific basis and performance of a model
19 is essential [U.S. EPA. 2005a. §3.2.2).
20 Because toxicodynamic modeling can
21 require many parameters and more
22 knowledge and data than are typically
23 available, the EPA has developed a standard
24 set of empirical ("curve-fitting") models
25 (http://www.epa.gov/ncea/bmds/) that can
26 be applied to typical data sets, including
27 those that are nonlinear. The EPA has also
28 developed guidance on modeling dose-
29 response data, assessing model fit, selecting
30 suitable models, and reporting modeling
31 results (U.S. EPA. 2012bl Additional
32 judgment or alternative analyses are used if
33 the procedure fails to yield reliable results,
34 for example, if the fit is poor, modeling may
35 be restricted to the lower doses, especially if
36 there is competing toxicity at higher doses
37 (U.S. EPA. 2005a. §3.2.3).
38 Modeling is used to derive a point of
39 departure fU.S. EPA. 2012b: 2005a. §3.2.4).
40 (See Section 7.6 for alternatives if a point of
41 departure cannot be derived by modeling.):
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
If linear extrapolation is used,
selection of a response level
corresponding to the point of
departure is not highly influential, so
standard values near the low end of
the observable range are generally
used (for example, 10% extra risk for
cancer bioassay data, 1% for
epidemiologic data, lower for rare
cancers).
For nonlinear approaches, both
statistical and biologic
considerations are taken into
account
For dichotomous data, a response
level of 10% extra risk is generally
used for minimally adverse effects,
5% or lower for more severe effects.
For continuous data, a response level
is ideally based on an established
definition of biologic significance. In
the absence of such definition, one
control standard deviation from the
control mean is often used for
minimally adverse effects, one-half
standard deviation for more severe
effects.
69 The point of departure is the 95% lower
70 bound on the dose associated with the
71 selected response level.
72 7.4. Extrapolating to lower doses and
73 response levels
74 The purpose of extrapolating to lower
75 doses is to estimate responses at exposures
76 below the observed data. Low-dose
77 extrapolation, typically used for cancer data,
78 considers what is known about modes of
79 action fU.S. EPA. 2005a. §3.3.1 and §3.3.21
80 1) If a biologically based model has been
81 developed and validated for the agent,
82 extrapolation may use the fitted model
83 below the observed range if significant
84 model uncertainty can be ruled out with
85 reasonable confidence.
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1 2) Linear extrapolation is used if the dose-
2 response curve is expected to have a
3 linear component below the point of
4 departure. This includes:
5 - Agents or their metabolites that are
6 DNA-reactive and have direct
7 mutagenic activity.
8 - Agents or their metabolites for which
9 human exposures or body burdens
10 are near doses associated with key
11 events leading to an effect
12 Linear extrapolation is also used when
13 data are insufficient to establish mode
14 of action and when scientifically
15 plausible.
16 The result of linear extrapolation is
17 described by an oral slope factor or an
18 inhalation unit risk, which is the slope
19 of the dose-response curve at lower
20 doses or concentrations, respectively.
21 3) Nonlinear models are used for
22 extrapolation if there are sufficient data
23 to ascertain the mode of action and to
24 conclude that it is not linear at lower
25 doses, and the agent does not
26 demonstrate mutagenic or other activity
27 consistent with linearity at lower doses.
28 Nonlinear approaches generally should
29 not be used in cases where mode of
30 action has not ascertained. If nonlinear
31 extrapolation is appropriate but no
32 model is developed, an alternative is to
33 calculate reference values.
34 4) Both linear and nonlinear approaches
35 may be used if there a multiple modes of
36 action. For example, modeling to a low
37 response level can be useful for
38 estimating the response at doses where a
39 high-dose mode of action would be less
40 important
41 If linear extrapolation is used, the
42 assessment develops a candidate slope
43 factor or unit risk for each suitable data set
44 These results are arrayed, using common
45 dose metrics, to show the distribution of
46 relative potency across various effects and
47 experimental systems. The assessment then
48 derives or selects an overall slope factor and
49 an overall unit risk for the agent, considering
50 the various dose-response analyses, the
51 study preferences discussed in Section 6,
52 and the possibility of basing a more robust
53 result on multiple data sets.
54 7.5. Considering susceptible
55 populations and lifestages
56 The assessment analyzes the available
57 information on populations and lifestages
58 that may be particularly susceptible to each
59 effect. A tiered approach is used [U.S. EPA.
60 2005a. 33.51.
61 1) If an epidemiologic or experimental
62 study reports quantitative results for a
63 susceptible population or lifestage, these
64 data are analyzed to derive separate
65 toxicity values for susceptible
66 individuals.
67 2) If data on risk-related parameters allow
68 comparison of the general population
69 and susceptible individuals, these data
70 are used to adjust the general-population
71 toxicity values for application to
72 susceptible individuals.
73 3) In the absence of chemical-specific data,
74 the EPA has developed age-dependent
75 adjustment factors for early-life exposure
76 to potential carcinogens that have a
77 mutagenic mode of action. There is
78 evidence of early-life susceptibility to
79 various carcinogenic agents, but most
80 epidemiologic studies and cancer
81 bioassays do not include early-life
82 exposure. To address the potential for
83 early-life susceptibility, the EPA
84 recommends [U.S. EPA. 2005b. §5):
85 - 10-fold adjustment for exposures
86 before age 2 years.
87 - 3-fold adjustment for exposures
88 between ages 2 and 16 years.
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1 7.6. Reference values and uncertainty
2 factors
3 An oral reference dose or an inhalation
4 reference concentration is an estimate of an
5 exposure (including in susceptible
6 subgroups) that is likely to be without an
7 appreciable risk of adverse health effects
8 over a lifetime (U.S. EPA. 2002.54.21
9 Reference values are typically calculated for
10 effects other than cancer and for suspected
11 carcinogens if a well characterized mode of
12 action indicates that a necessary key event
13 does not occur below a specific dose.
14 Reference values provide no information
15 about risks at higher exposure levels.
16 The assessment characterizes effects
17 that form the basis for reference values as
18 adverse, considered to be adverse, or a
19 precursor to an adverse effect For
20 developmental toxicity, reproductive
21 toxicity, and neurotoxicity there is guidance
22 on adverse effects and their biologic markers
23 fU.S. EPA. 1998.1996.19911
24 To account for uncertainty and
25 variability in the derivation of a lifetime
26 human exposure where adverse effects are
27 not anticipated to occur, reference values are
28 calculated by applying a series of uncertainty
29 factors to the point of departure. If a point of
30 departure cannot be derived by modeling, a
31 no-observed-adverse-effect level or a
32 lowest-observed-adverse-effect level is used
33 instead. The assessment discusses scientific
34 considerations involving several areas of
35 variability or uncertainty.
36 Human variation. The assessment accounts
37 for variation in susceptibility across the
38 human population and the possibility
39 that the available data may not be
40 representative of individuals who are
41 most susceptible to the effect A factor of
42 10 is generally used to account for this
43 variation. This factor is reduced only if
44 the point of departure is derived or
45 adjusted specifically for susceptible
46 individuals (not for a general population
47 that includes both susceptible and non-
48 susceptible individuals) (U.S. EPA,
49 2002.54.4.5: 1998.54.2: 1996.54:
50 1994b. 54.3.9.1: 1991.53.41
51 Animal-to-human extrapolation. If animal
52 results are used to make inferences
53 about humans, the assessment adjusts
54 for cross-species differences. These may
55 arise from differences in toxicokinetics
56 or toxicodynamics. Accordingly, if the
57 point of departure is standardized to
58 equivalent human terms or is based on
59 toxicokinetic or dosimetry modeling, a
60 factor of 101/2 (rounded to 3) is applied
61 to account for the remaining uncertainty
62 involving toxicokinetic and
63 toxicodynamic differences. If a
64 biologically based model adjusts fully for
65 toxicokinetic and toxicodynamic
66 differences across species, this factor is
67 not used. In most other cases, a factor of
68 10 is applied (U.S. EPA. 2011:
69 2002.54.4.5: 1998.54.2: 1996.54:
70 1994b, §4.3.9.1; 1991, §3.4).
71 Adverse-effect level to no-observed-
72 adverse-effect level. If a point of
73 departure is based on a lowest-
74 observed-adverse-effect level, the
75 assessment must infer a dose where
76 such effects are not expected. This can be
77 a matter of great uncertainty, especially
78 if there is no evidence available at lower
79 doses. A factor of 10 is applied to
80 account for the uncertainty in making
81 this inference. A factor other than 10
82 may be used, depending on the
83 magnitude and nature of the response
84 and the shape of the dose-response
85 curve (U.S. EPA. 2002. §4.4.5; 1998. §4.2;
86 1996. 54: 1994b. 54.3.9.1: 1991. 53.4).
87 Subchronic-to-chronic exposure. If a point
88 of departure is based on subchronic
89 studies, the assessment considers
90 whether lifetime exposure could have
91 effects at lower levels of exposure. A
92 factor of 10 is applied to account for the
93 uncertainty in using subchronic studies
94 to make inferences about lifetime
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1 exposure. This factor may also be
2 applied for developmental or
3 reproductive effects if exposure covered
4 less than the full critical period. A factor
5 other than 10 may be used, depending
6 on the duration of the studies and the
7 nature of the response (U.S. EPA. 2002.
8 §4.4.5; 1998. §4.2; 1994b. §4.3.9.1).
9 Incomplete database. If an incomplete
10 database raises concern that further
11 studies might identify a more sensitive
12 effect, organ system, or lifestage, the
13 assessment may apply a database
14 uncertainty factor (U.S. EPA.
15 2002. §4.4.5: 1998. §4.2: 1996. §4:
16 1994b. §4.3.9.1; 1991. §3.4). The size of
17 the factor depends on the nature of the
18 database deficiency. For example, the
19 EPA typically follows the suggestion that
20 a factor of 10 be applied if both a
21 prenatal toxicity study and a two-
22 generation reproduction study are
23 missing and a factor of 101/2 if either is
24 missing (U.S. EPA. 2002. §4.4.5).
25 In this way, the assessment derives
26 candidate values for each suitable data set
27 and effect that is credibly associated with the
28 agent These results are arrayed, using
29 common dose metrics, to show where effects
30 occur across a range of exposures (U.S. EPA.
31 1994b, §4.3.9).
32 The assessment derives or selects an
33 organ- or system-specific reference value for
34 each organ or system affected by the agent
35 The assessment explains the rationale for
36 each organ/system-specific reference value
37 (based on, for example, the highest quality
38 studies, the most sensitive outcome, or a
39 clustering of values). By providing these
40 organ/system-specific reference values, IRIS
41 assessments facilitate subsequent
42 cumulative risk assessments that consider
43 the combined effect of multiple agents acting
44 at a common site or through common
45 mechanisms (NRC. 2009).
46 The assessment then selects an overall
47 reference dose and an overall reference
48 concentration for the agent to represent
49 lifetime human exposure levels where
50 effects are not anticipated to occur. This is
51 generally the most sensitive organ/system-
52 specific reference value, though
53 consideration of study quality and
54 confidence in each value may lead to a
55 different selection.
56 7.7. Confidence and uncertainty in the
57 reference values
58 The assessment selects a standard
59 descriptor to characterize the level of
60 confidence in each reference value, based on
61 the likelihood that the value would change
62 with further testing. Confidence in reference
63 values is based on quality of the studies used
64 and completeness of the database, with more
65 weight given to the latter. The level of
66 confidence is increased for reference values
67 based on human data supported by animal
68 data fU.S. EPA. 1994b. §4.3.9.21
69 High confidence: The reference value is not
70 likely to change with further testing,
71 except for mechanistic studies that might
72 affect the interpretation of prior test
73 results.
74 Medium confidence: This is a matter of
75 judgment, between high and low
76 confidence.
77 Low confidence: The reference value is
78 especially vulnerable to change with
79 further testing.
80 These criteria are consistent with
81 guidelines for systematic reviews that
82 evaluate the quality of evidence. These also
83 focus on whether further research would be
84 likely to change confidence in the estimate of
85 effect (Guyattetal.. 2008b).
86 All assessments discuss the significant
87 uncertainties encountered in the analysis.
88 The EPA provides guidance on
89 characterization of uncertainty [U.S. EPA.
90 2005a. §3.6). For example, the discussion
91 distinguishes model uncertainty (lack of
92 knowledge about the most appropriate
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1 experimental or analytic model) and
2 parameter uncertainty (lack of knowledge
3 about the parameters of a model).
4 Assessments also discuss human variation
5 (interpersonal differences in biologic
6 susceptibility or in exposures that modify
7 the effects of the agent).
8 Note: The Preamble reflects methods
9 that will be employed once all the 2011
10 MAS recommendations have been fully
11 implemented. As this Toxicological
12 Review was created during a period in
13 which the NAS recommendations were
14 being incorporated into the IRIS
15 process, the methods utilized in the
16 assessment may not completely reflect
17 those detailed in the Preamble. For
18 further information on which specific
19 NAS recommendations have been
20 implemented in this document, please
21 refer to Appendix D (Documentation of
22 Implementation of the 2011 National
23 Research Council Recommendations) in
24 the Supplemental Information.
Preamble References
CDC (Centers for Disease Control and
Prevention). (2004). The health
consequences of smoking: A report of the
Surgeon General. Washington, DC: U.S.
Department of Health and Human Services.
http://www.surgeongeneral.gov/librarv/smok
ingconsequences/
Guvatt GH: Oxman. AD: Kunz. R: Vist GE:
Falck-Ytter. Y: Schiinemann. HJ. (2008a).
GRADE: What is "quality of evidence" and
why is it important to clinicians? [Review].
BMJ 336: 995-998.
http://dx.doi.org/10.1136/bmi.39490.551019.
BE
Guvatt GH: Oxman. AD: Vist GE: Kunz. R:
Falck-Ytter. Y: Alonso-Coello. P:
Schiinemann. HJ. (2008b). GRADE: An
emerging consensus on rating quality of
evidence and strength of recommendations.
BMJ 336: 924-926.
http://dx.doi.org/10.1136/bmi.39489.470347.
AD
HEW (U.S. Department of Health, Education
and Welfare). (1964). Smoking and health:
Report of the advisory committee to the
surgeon general of the public health service.
Washington, DC: U.S. Department of Health,
Education, and Welfare.
http ://profiles .nlm. nih. gov/ps/retrieve/Resour
ceMetadata/NNBBMQ
Hill. AB. (1965). The environment and disease:
Association or causation? Proc R Soc Med
58: 295-300.
IARC (International Agency for Research on
Cancer). (2006). Preamble to the IARC
monographs. Lyon, France.
http://monographs.iarc.fr/ENG/Preamble/
IOM (Institute of Medicine). (2008). Improving
the presumptive disability decision-making
process for veterans. In JM Samet; CC
Bodurow (Eds.). Washington, DC: National
Academies Press.
http://www.nap.edu/openbook.php7record id
=11908
NRC (National Research Council). (1983). Risk
assessment in the federal government:
Managing the process. Washington, DC:
National Academies Press.
http://www.nap.edu/openbook.php7record id
=366&page=Rl
NRC (National Research Council). (2009).
Science and decisions: Advancing risk
assessment. Washington, DC: National
Academies Press.
http://www.nap.edu/catalog/12209.html
Rothman. KJ: Greenland. S. (1998). Modern
epidemiology (2nd ed.). Philadelphia, PA:
Lippincott, Williams, & Wilkins.
U.S. EPA (U.S. Environmental Protection
Agency). (1986a). Guidelines for
mutagenicity risk assessment [EPA Report].
(EPA/630/R-98/003). Washington, DC.
http://www.epa.gov/iris/backgrd.html
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzene
U.S. EPA (U.S. Environmental Protection
Agency). (1986b). Guidelines for the health
risk assessment of chemical mixtures. Fed
Reg 51: 34014-34025.
U.S. EPA (U.S. Environmental Protection
Agency). (1988). Recommendations for and
documentation of biological values for use in
risk assessment [EPA Report]. (EPA/600/6-
87/008). Cincinnati, OH.
http ://cfpub. epa. gov/ncea/cfm/recordisplav. cf
m?deid=34855
U.S. EPA (U.S. Environmental Protection
Agency). (1991). Guidelines for
developmental toxicity risk assessment [EPA
Report]. (EPA/600/FR-91/001). Washington,
DC: U.S. Environmental Protection Agency,
Risk Assessment Forum.
http://www.epa.gov/raf/publications/guidelin
es-dev-toxicitv-risk-assessment, htm
U.S. EPA (U.S. Environmental Protection
Agency). (1994b). Methods for derivation of
inhalation reference concentrations and
application of inhalation dosimetry [EPA
Report]. (EPA/600/8-90/066F). Research
Triangle Park, NC.
http ://cfpub. epa. gov/ncea/cfm/recordisplav. cf
m?deid=71993
U.S. EPA (U.S. Environmental Protection
Agency). (1996). Guidelines for reproductive
toxicity risk assessment [EPA Report].
(EPA/630/R-96/009). Washington, DC.
http://www.epa.gov/raf/publications/pdfs/RE
PRO51.PDF
U.S. EPA (U.S. Environmental Protection
Agency). (1998). Guidelines for
neurotoxicity risk assessment [EPA Report].
(EPA/630/R-95/001F). Washington, DC.
http://www.epa.gov/raf/publications/pdfs/NE
UROTOX.PDF
U.S. EPA (U.S. Environmental Protection
Agency). (2000). Supplementary guidance
for conducting health risk assessment of
chemical mixtures [EPA Report].
(EPA/630/R-00/002). Washington, DC.
http ://cfpub. epa. gov/ncea/cfm/recordisplav. cf
m?deid=20533
U.S. EPA (U.S. Environmental Protection
Agency). (2002). A review of the reference
dose and reference concentration processes
[EPA Report]. (EPA/630/P-02/002F).
Washington, DC: Risk Assessment Forum,
U.S. Environmental Protection Agency.
http ://cfpub. epa. gov/ncea/cfm/recordisplav .cf
m?deid=51717
U.S. EPA (U.S. Environmental Protection
Agency). (2005a). Guidelines for carcinogen
risk assessment [EPA Report]. (EPA/630/P-
03/00IF). Washington, DC: Risk Assessment
Forum.
http://www.epa.gov/cancerguidelines/
U.S. EPA (U.S. Environmental Protection
Agency). (2005b). Supplemental guidance
for assessing susceptibility from early-life
exposure to carcinogens [EPA Report] (pp.
1125-1133). (EPA/630/R-03/003F).
Washington, DC.
http://www.epa.gov/cancerguidelines/guideli
nes-carcinogen-supplement.htm
U.S. EPA (U.S. Environmental Protection
Agency). (2006a). Approaches for the
application of physiologically based
pharmacokinetic (PBPK) models and
supporting data in risk assessment (Final
Report) [EPAReport]. (EPA/600/R-
05/043F). Washington, DC.
http ://cfpub. epa. gov/ncea/cfm/recordisplav. cf
m?deid= 157668
U.S. EPA (U.S. Environmental Protection
Agency). (2006b). A framework for
assessing health risk of environmental
exposures to children [EPA Report].
(EPA/600/R-05/093F). Washington, DC.
http ://cfpub. epa. gov/ncea/cfm/recordisplav .cf
m?deid=158363
U.S. EPA (U.S. Environmental Protection
Agency). (2009). EPAs Integrated Risk
Information System: Assessment
development process [EPA Report].
Washington, DC.
http ://epa. gov/iris/process. htm
U.S. EPA (U.S. Environmental Protection
Agency). (2010). Integrated science
assessment for carbon monoxide [EPA
Report]. (EPA/600/R-09/019F). Research
Triangle Park, NC.
http ://cfpub. epa. gov/ncea/cfm/recordisplav .cf
m?deid=218686
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U.S. EPA (U.S. Environmental Protection
Agency). (2011). Recommended use of body
weight 3/4 as the default method in
derivation of the oral reference dose [EPA
Report]. (EPA/100/R11/0001). Washington,
DC.
http://www.epa.gov/raf/publications/interspe
cies-extrapolation.htm
U.S. EPA (U.S. Environmental Protection
Agency). (2012a). Advances in inhalation
gas dosimetry for derivation of a reference
concentration (rfc) and use in risk assessment
[EPA Report]. (EPA/600/R-12/044).
Washington, DC.
http ://cfpub. epa. gov/ncea/cfm/recordisplav. cf
m?deid=244650
U.S. EPA (U.S. Environmental Protection
Agency). (2012b). Benchmark dose technical
guidance. (EPA/100/R-12/001). Washington,
DC: Risk Assessment Forum.
http ://www .epa. gov/raf/publications/pdfs/ben
chmark dose guidance.pdf
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EXECUTIVE SUMMARY
Occurrence and Health Effects
1 Trimethylbenzenes are a commercially available mixture of three individual
2 isomers: 1,2,3-, 1,2,4-, and 1,3,5-trimethylbenzene (TMBs). 1MB isomers are
3 produced during petroleum refining and production of aromatic hydrocarbons with
4 nine carbons (i.e., C9 aromatic fraction). As the vast majority of the C9 fraction is
5 used as a component of gasoline, vehicle emissions are expected to be the major
6 anthropogenic source of TMBs. TMBs are volatile hydrocarbons, and thus humans
7 are exposed to these isomers primarily through breathing air containing TMB
8 vapors, although ingestion through food or drinking water is also possible.
9 Effects on the nervous system, respiratory system, and hematological
10 system (i.e., blood) have been reported in occupationally- and residentially-exposed
11 humans, but these effects were observed following exposure to complex mixtures
12 containing TMB isomers, thus making it difficult to determine the contribution of
13 each TMB isomer to the observed health effects. Health effects that are roughly
14 analogous to those seen in humans have been observed in animals exposed to the
15 individual isomers. Effects on the nervous system, including cognitive effects and
16 decreased pain sensitivity, are the most widely observed effects in animals. Effects
17 on other organ systems, including the respiratory and hematological systems, have
18 also been observed in animals. Both 1,2,4-TMB and 1,3,5-TMB have been observed
19 to elicit effects on pregnant animals and developing fetuses, but at exposure levels
20 greater than those that cause effects on the nervous system. There is inadequate
21 information to evaluate the carcinogenicity of TMBs.
1. Effects Other Than Cancer Following Inhalation Exposure
22 The relationship between exposure to 1,2,3-TMB, 1,2,4-TMB, 1,3,5-TMB and health effects
23 has been evaluated in studies of (1) exposed human adults, (2) animals exposed via inhalation for
24 acute, short-term, and subchronic durations, and (3) animals exposed gestationally via inhalation.
25 Human studies included occupational exposure to various solvent mixtures containing
26 TMBs. Health effects noted in these studies were eye irritation, neurological (hand tremble,
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1 abnormal fatigue, lack of coordination), and hematological effects [Chenetal.. 1999: Norsethetal..
2 1991: Battigetal.. 1958: Battigetal.. 1956]. Also, residential exposure to mixtures containing
3 1,2,4-TMB were observed to result in asthma [Billionnetetal.. 2011]. However, as these studies
4 involved exposures to mixtures containing multiple TMB isomers and other volatile organic
5 compounds (VOCs], it is difficult to ascertain the specific contribution of each TMB isomer to the
6 specific health effects reported. Controlled human exposures to individual isomers also exist,
7 although these studies generally report little or no effect on respiratory or sensory irritation [Tones
8 etal.. 2006: Tarnbergetal.. 1997a: Tarnbergetal.. 1997b: Kostrzewskietal.. 1997: Tarnbergetal..
9 1996: Kostrewski and Wiaderna-Brycht. 1995]. One controlled human exposure study reported
10 some deficits in attention following exposure to white spirit (WS], a complex mixture containing
11 1,2,4-TMB fLammers etal.. 20071.
12 Animal inhalation studies [Wiadernaetal., 2002: Gralewicz and Wiaderna, 2001: Wiaderna
13 etal.. 1998: Gralewicz etal.. 1997b: Gralewicz etal.. 1997a: Korsak etal.. 19951 included acute and
14 short-term studies of TMBs that reported respiratory irritation (decreased respiration rates] and
15 neurological effects (decreased pain sensitivity, altered cognitive function, and decreased anxiety
16 and/or increased motor function] that are consistent with effects seen in human studies. Four
17 subchronic inhalation studies for 1,2,3-TMB and 1,2,4-TMB observed exposure-response effects in
18 multiple organ systems, including the nervous, hematological, and respiratory systems (Korsak et
19 al., 2000a, bj Korsak etal., 1997: Korsak and Rydzynski, 1996]. In these studies, disturbances in
20 central nervous system (CNS] function, including decreased pain sensitivity and decreased
21 neuromuscular function and coordination, appear to be the most sensitive endpoints following
22 exposure to 1,2,3-TMB or 1,2,4-TMB. No subchronic studies were found that investigated exposure
23 to 1,3,5-TMB. One developmental toxicity study (Saillenfaitetal.. 2005] observed similar levels of
24 maternal and fetal toxicity (i.e., decreased maternal weight gain and fetal weight] following
25 exposure to either 1,2,4-TMB or 1,3,5-TMB; other indices of fetal toxicity (i.e., fetal death and
26 malformations] were not affected by exposure.
27 Table ES-1 summarizes the RfCs derived for all three TMB isomers, and the sections that
28 follow provide details on the RfC derivation for each isomer.
Table ES-1. Summary of inhalation reference concentrations (RfCs)
Isomer
1,2,4-TMB
1,2,3-TMB
1,3,5-TMB
Source
Decreased pain sensitivity
Decreased pain sensitivity
Adopted from 1,2,4-TMB based on
sufficient similarity of these isomers
Reference value
(mg/m3)
5 x 10"2
5 x 10"2
5 x 10"2
Confidence
Low-to-medium
Low-to-medium
Low
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2. Inhalation Reference Concentration (RfC) for 1,2,4-TMB for Effects Other
Than Cancer
Table ES-2. Summary of reference concentration (RfC) derivation for
1,2,4-TMB
Critical effect
Decreased pain sensitivity
90 day male rat study
Korsak and Rydzyriski (1996)
Point of departure
PODHEc(mg/m3) = 15.8
Uncertainty
factor
300
Chronic RfC
(mg/m3)
5 x 10"2
1 Decreased pain sensitivity was observed in multiple studies of acute, short-term, and
2 subchronic durations [Gralewicz and Wiaderna. 2001: Gralewicz etal.. 1997b: Korsak and
3 Rydzynski. 1996: Korsak etal.. 1995). Given the consistency of this effect and the determination
4 that decreased pain sensitivity is an appropriate adverse effect with which to derive reference
5 values (see Section 2.1.5 of this Toxicological Review), in accordance with the U.S. EPA's Guidelines
6 for Neurotoxicity Risk Assessment [1998], decreased pain sensitivity was selected as the critical
7 effect and Korsak and Rydzynski [1996] was selected as the principal study for derivation of the
8 RfC for 1,2,4-TMB.
9 The RfC calculation is summarized in Table ES-2. The available rat PBPK model fHissink et
10 al.. 2007] was used to convert the external concentrations (in mg/m3] from the animal study to the
11 internal blood metric of weekly average venous 1,2,4-TMB concentration (in mg/L]. These internal
12 blood metrics were then used as the dose inputs for benchmark dose (BMD] modeling.
13 A benchmark response [BMR] equal to a 1 standard deviation change in the control mean for
14 decreased pain sensitivity was used. A BMDLiso of 0.086 mg/L was estimated for decreased pain
15 sensitivity in male rats exposed to 1,2,4-TMB via inhalation for 90 days (6 hours/day, 5 days/week]
16 [data used in model: [Korsak and Rydzynski. 1996]].
17 The available human PBPK model [Hissink et al.. 2007] was then used to estimate a human
18 equivalent concentration [HEC] of 15.8 mg/m3 from the BMDLiso of 0.086 mg/L. This HEC was used
19 as the PODnEc with which to derive the RfC. A composite uncertainty factor (UF] of 300 was applied:
20 3 to account for uncertainty in extrapolating from laboratory animals to humans (interspecies
21 variability], 10 to account for variation in susceptibility among members of the human population
22 (interindividual variability], 3 to account for subchronic-to-chronic extrapolation due to the use of a
23 subchronic study with effects observed to recover within weeks of exposure termination, and 3 to
24 account for deficiencies in the database (no two-generation reproductive/developmental toxicity or
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1 developmental neurotoxicity studies were available). Dividing the PODHEc by the composite UF of
2 300 yielded a chronic RfC of 5 x 1Q-2 mg/m3 for 1,2,4-TMB.
3. Confidence in the Chronic Inhalation RfC for 1,2,4-TMB
3 A confidence level of high, medium, or low is assigned to the study used to derive the RfC,
4 the overall database, and the RfC itself, as described in Section 4.3.9.2 of EPA's Methods for
5 Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry [U.S. EPA.
6 1994b].
7 Confidence in the study from which the critical effect was identified, Korsak and Rydzyhski
8 [1996], is low to medium. This peer-reviewed study was well designed, using three dose groups
9 plus untreated controls and a typical number of animals per dose group for evaluating
10 neurotoxicity following subchronic exposure.
11 One area of uncertainty regarding this study is the lack of reported actual concentrations.
12 However, as the methods by which the test atmosphere was generated and analyzed were reported
13 in sufficient detail, and given the fact that this laboratory has used this methodology in subsequent
14 studies [Korsak et al.. 2000a. b] and achieved appropriate actual concentrations (i.e., within 10% of
15 target concentrations), the concern regarding the lack of reported actual concentrations is minimal.
16 Another source of uncertainty is the fact that Korsak and Rydzyhski [1996] does not explicitly state
17 that the reported measures of variance in Table 1 of that reference are standard deviations.
18 However, careful analysis of the reported levels of variance and magnitude of statistical significance
19 reported indicate that the measures of variance are standard deviations. Supporting this
20 conclusions is the observation that all other papers by Korsak et al. [2000a. b; 1997: 1995] report
21 variance as standard deviations. The critical effect on which the RfC is based is well-supported as
22 the evidence for 1,2,4-TMB-induced neurotoxicity is coherent across multiple animals species (i.e.,
23 human, mouse, and rat] and consistent across multiple exposure durations (i.e., acute, short-term,
24 and subchronic] [Gralewicz and Wiaderna, 2001: Chenetal., 1999: Wiaderna et al., 1998: Gralewicz
25 etal.. 1997b: Gralewicz etal.. 1997a: Korsak and Rydzyhski. 1996: Norseth etal.. 19911
26 The database for 1,2,4-TMB includes acute, short-term, subchronic, and developmental
27 toxicity studies in rats and mice. However, confidence in the database is low to medium because it
28 lacks chronic, multi-gene ration reproductive/developmental, and developmental neurotoxicity
29 studies, and the studies supporting the critical effect predominantly come from the same research
30 institute. Consequently, the overall confidence in the RfC for 1,2,4-TMB is low to medium.
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4. Inhalation Reference Concentration (RfC) for 1,2,3-TMB for Effects Other Than
Cancer
Table ES-3. Summary of reference concentration (RfC) derivation for
1,2,3-TMB
Critical effect
Decreased pain sensitivity
90 day male rat study
Korsak and Rydzyriski
(1996)
Point of departure
PODHEc(mg/m3) = 16.3
Uncertainty
factor
300
Chronic RfC
(mg/m3)
5 x 10"2
1 Decreased pain sensitivity was observed in multiple studies of acute, short-term, and
2 subchronic durations [Lutz etal.. 2010: Wiaderna et al.. 1998: Korsak and Rydzynski. 1996). Given
3 the consistency of this effect and the determination that decreased pain sensitivity is an adverse
4 effect, in accordance with the U.S. EPA's Guidelines for Neurotoxicity Risk Assessment [U.S. EPA.
5 1998]. decreased pain sensitivity was selected as the critical effect and Korsak and Rydzynski
6 [1996] was selected as the principal study for derivation of the RfC for 1,2,3-TMB.
7 The RfC calculation is summarized in Table ES-3. BMD modeling was used in order to
8 identify the POD for decreased pain sensitivity. A BMR equal to a 1 standard deviation change in the
9 control mean was used. A BMDLiso of 17.36 mg/m3 was estimated for decreased pain sensitivity in
10 male rats exposed to 1,2,3-TMB via inhalation for 90 days (6 hours/day, 5 days/week] [Korsak and
11 Rydzynski. 19961
12 As no PBPK model was available for 1,2,3-TMB, default dosimetry methodologies were used
13 to estimate the HEC of 16.3 mg/m3, based on the ratio of the human and animal blood:air partition
14 coefficients [U.S. EPA, 1994b]. This PODHEc was used to derive the RfC. A composite uncertainty
15 factor (UF] of 300 was applied: 3 to account for uncertainty in extrapolating from laboratory
16 animals to humans (interspecies variability], 10 to account for variation in susceptibility among
17 members of the human population (interindividual variability], 3 to account for subchronic-to-
18 chronic extrapolation due to the use of a subchronic study, and 3 to account for deficiencies in the
19 database (no two-generation reproductive/developmental toxicity, developmental toxicity, or
20 developmental neurotoxicity studies were available]. Dividing the PODHEc by the composite UF of
21 300 yielded a chronic RfC of 5 x 1Q-2 mg/m3 for 1,2,3-TMB.
5. Confidence in the Chronic Inhalation RfC for 1,2,3-TMB
22 Confidence in the study from which the critical effect was identified, Korsak and Rydzynski
23 [1996] is low to medium. This peer-reviewed study was well designed, using three dose groups
24 plus untreated controls and a typical number of animals per dose group for evaluating
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1 neurotoxicity following subchronic exposure. One area of uncertainty regarding this study is the
2 lack of reported actual concentrations. However, as the methods by which the test atmosphere was
3 generated and analyzed were reported in sufficient detail, and given the fact that this laboratory
4 has used this methodology in subsequent studies [Korsak et al., 2000a, b] and achieved appropriate
5 actual concentrations (i.e., within 10% of target concentrations), the concern regarding the lack of
6 reported actual concentrations is minimal. Another source of uncertainty is the fact that Korsak and
7 Rydzyhski [1996] does not explicitly state that the reported measures of variance in Table 1 of that
8 reference are standard deviations. However, careful analysis of the reported levels of variance and
9 magnitude of statistical significance reported indicate that the measures of variance are standard
10 deviations. Supporting this conclusions is the observation that all other papers by Korsak et al.
11 [2000a, b; 1997: 1995] report variance as standard deviations.The critical effect on which the RfC is
12 based is well-supported as the evidence for 1,2,3-TMB-induced neurotoxicity is coherent across
13 multiple animals species (i.e., mouse, and rat] and consistent across multiple exposure durations
14 (i.e., acute, short-term, and subchronic] (Lutz etal.. 2010: Wiaderna et al.. 1998: Korsak and
15 Rydzynski. 19961
16 The database for 1,2,3-TMB includes acute, short-term, and subchronic toxicity studies in
17 rats and mice. However, confidence in the database is low to medium because it lacks chronic,
18 multi-generation reproductive/developmental, developmental toxicity, or developmental
19 neurotoxicity studies, and the studies supporting the critical effect predominantly come from the
20 same research institute. Consequently, the overall confidence in the RfC for 1,2,3-TMB is low to
21 medium.
6. Inhalation Reference Concentration (RfC) for 1,3,5-TMB for Effects Other Than
Cancer
22 No chronic or subchronic studies exist that would support the derivation of an RfC for
23 1,3,5-TMB, however one developmental toxicity study (Saillenfaitetal.. 2005] was identified as a
24 potential study from which to identify a critical effect for RfC derivation.
25 The use of decreased maternal weight gain observed in Saillenfait et al. (2005] as the critical
26 effect for RfC derivation would result in an RfC 20-fold greater than that derived for 1,2,4-TMB (1
27 mg/m3 vs. 5 x 1Q-2 mg/m3]. This large difference is not consistent with the rest of the toxicological
28 database for 1,2,4-TMB and 1,3,5-TMB, which demonstrates that the two isomers are similar to one
29 another with regard to respiratory and developmental toxicity in acute and developmental studies
30 fSaillenfaitetal..2005: Korsak and Rydzynski. 1996: Korsak etal.. 19951 The 1,3,5-TMB isomer
31 was observed to induce some measures of neurotoxicity (e.g., passive and active avoidance] at
32 lower doses than 1,2,4-TMB, in short-term studies (Wiaderna etal.. 2002: Gralewicz and Wiaderna.
33 2001: Gralewicz etal., 1997b]. Additionally, available toxicokinetic data regarding blood:air
34 partition coefficients, respiratory uptake, and absorption into the bloodstream in humans and rats
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1 do not suggest any appreciable differences can be expected between the two isomers [Meulenberg
2 and Vijverberg. 2000: TarnbergetaL 1996: Dahletal.. 1988).
3 Therefore, the chronic RfC of 5 x 10 2 mg/m3 derived for 1,2,4-TMB was adopted as the
4 RfC for 1,3,5-TMB. This is based on the determination of sufficient similarity with regard to
5 chemical properties, kinetics, and toxicity between the two isomers (see Section 2.3.5).
7. Confidence in the Chronic Inhalation RfC for 1,3,5-TMB
6 The chronic RfC for 1,2,4-TMB was adopted as the RfC for 1,3,5-TMB; thus, confidence in the
7 study from which the critical effect was identified, Korsak and Rydzyhski [1996). is low to medium
8 (see above). The database for 1,3,5-TMB includes acute, short-term, and developmental toxicity
9 studies in rats and mice. However, confidence in the database is low to medium because it lacks
10 chronic, subchronic, multi-generation reproductive/developmental toxicity, and developmental
11 neurotoxicity studies and most of the studies supporting the critical effect come from the same
12 research institute.
13 Reflecting the confidence in the study and the database and the uncertainty surrounding the
14 adoption of the RfC derived for 1,2,4-TMB as the RfC for 1,3,5-TMB, the overall confidence in the
15 RfC for 1,3,5-TMB is low.
8. Effects Other Than Cancer Observed Following Oral Exposure
16 Only one subchronic study was identified that examined the effects of oral exposure to
17 1,3,5-TMB. Effects in the hematological system, including changes in clinical chemistry parameters
18 and differential white blood cell numbers, were observed following exposure to 1,3,5-TMB via oral
19 gavage. Ultimately, the Koch Industries [1995b) study was determined to not be suitable for RfD
20 derivation following an external peer review of the study (see Appendix F). No other subchronic
21 studies were found that investigated the effects of oral exposure to 1,2,4-TMB or 1,2,3-TMB, and no
22 chronic oral studies were found that investigated noncancer effects of any of the TMB isomers.
23 A series of studies utilizing single exposures (oral gavage or i.p. injection) were identified
24 that investigated the acute neurotoxic effects of TMBs (Tomasetal.. 1999a: Tomasetal.. 1999b:
25 Tomas etal.. 1999c). In these studies, exposed rats demonstrated changes in electrocortical arousal,
26 altered EEC activity in the cortical and hippocampal regions of the brain, and altered locomotor
27 activity in open field tests. As these effects were only observed in studies investigating acute
28 exposures, they were considered insufficient for derivation of oral toxicity reference values.
29 Therefore, given that Koch Industries study was not suitable for RfD derivation and effects
30 from acute studies generally are not suitable for derivation of chronic health values, RfDs were
31 derived for 1,2,4-TMB using route-to-route extrapolation and for 1,2,3-TMB and 1,3,5-TMB based
32 on sufficient similarity.
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1 Table ES-4 below summarizes the RfDs derived for all three 1MB isomers, and the sections
2 that follow provide details on the derivation of the RfD for each isomer.
Table ES-4. Summary of reference doses (RfDs) for TMB isomers
Isomer
1,2,4-TMB
1,2,3-TMB
1,3,5-TMB
Source
Route-to-route extrapolation from
RfC for 1,2,4-TMB
Adopted from 1,2,4-TMB based on
sufficient similarity of these isomers
Adopted from 1,2,4-TMB based on
sufficient similarity of these isomers
Reference value
2 x 10"2
2 x 10"2
2 x 10"2
Confidence
Low
Low
Low
9. Oral Reference Dose (RfD) for 1,2,4-TMB for Effects Other Than Cancer
Table ES-5. Summary of reference dose (RfD) derivation for 1,2,4-TMB
Critical effect
Decreased pain sensitivity
90 day male rat study
Korsak and Rydzyriski
(1996)
Point of departure
Route-to-route
extrapolation using Korsak
and Rydzyriski (1996)
subchronic inhalation study
in Wistar rats
PODHED (mg/kg-day) = 6.3
Uncertainty
factor
300
Chronic RfD
(mg/kg-day)
2xl02
3 A human PBPK model [Hissinketal.. 2007]. modified by EPA to include an oral
4 compartment, was available for estimating the oral dose that would yield a blood concentration
5 equal to the blood concentration at the POD used in the derivation of the RfC for 1,2,4-TMB (Section
6 B.3.3.5, Appendix B). The RfD calculation is summarized in Table ES-5. Under the assumption of
7 constant oral ingestion and 100% absorption of 1,2,4-TMB via constant infusion rate into the liver,
8 a PODHED of 6.3 mg/kg-day was derived. Hepatic first-pass metabolism was also evaluated in
9 humans using the modified PBPK model: following 50 days of low daily doses, inhalation doses
10 were estimated to result in steady state venous blood concentrations 4-fold higher than blood
11 concentrations resulting from equivalent oral doses due to hepatic first pass metabolism (see
12 Figure B-18, Appendix B). The same composite UF of 300 used for the RfC derivation was applied: 3
13 to account for uncertainty in extrapolating from laboratory animals to humans (interspecies
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1 variability), 10 to account for variation in susceptibility among members of the human population
2 (interindividual variability), 3 to account for subchronic-to-chronic extrapolation due to the use of a
3 subchronic study, and 3 to account for deficiencies in the database (no multi-generation
4 reproductive/developmental toxicity or developmental neurotoxicity studies). Dividing the PODHED
5 by the composite UF of 300 yielded a chronic RfD of 2 x 10-2 mg/kg-day for 1,2,4-TMB.
10. Confidence in the Chronic Oral RfD for 1,2,4-TMB
6 A PBPK model was utilized to perform a route-to-route extrapolation to determine a POD
7 for the derivation of the RfD from the Korsak and Rydzyhski [1996) inhalation study and
8 corresponding critical effect. The confidence in the study from which the critical effect was
9 identified, Korsak and Rydzyhski [1996), is low to medium (see above). The database for 1,2,4-TMB
10 includes acute, short-term, subchronic, and developmental toxicity studies in rats and mice.
11 However, confidence in the database for 1,2,4-TMB is low to medium because it lacks chronic,
12 multi-generation reproductive/developmental and developmental neurotoxicity studies, and the
13 studies supporting the critical effect predominantly come from the same research institute.
14 Reflecting the confidence in the study and the database and the uncertainty surrounding the
15 application of the available PBPK model for the purposes of a route-to-route extrapolation, the
16 overall confidence in the RfD for 1,2,4-TMB is low.
11. Oral Reference Dose (RfD) for 1,2,3-TMB for Effects Other Than Cancer
17 The oral database is inadequate to derive an RfD for 1,2,3-TMB. No chronic, subchronic, or
18 short-term oral exposure studies were found in the literature. However, as discussed in Sections
19 1.1.6 and B.2, the toxicokinetic and toxicity similarities between 1,2,3-TMB and 1,2,4-TMB support
20 adopting the RfD for 1,2,4-TMB as the RfD for 1,2,3-TMB. 1,2,3-TMB is observed to elicit the same
21 neurotoxic effects in rats (decreased pain sensitivity) as 1,2,4-TMB following subchronic inhalation
22 exposures, and the calculated RfCs for these two isomers are equal: 5 x 1Q-2 mg/m3. In addition to
23 the outlined similarities in toxicokinetics, the qualitative metabolic profiles for the two isomers are
24 similar such that first-pass metabolism through the liver is not expected to differ greatly between
25 1,2,4-TMB and 1,2,3-TMB. Therefore, the chronic RfC of 2 x 10-2 mg/kg-day derived for
26 1,2,4-TMB was adopted as the RfD for 1,2,3-TMB based on the determination of sufficient
27 similarity between the two isomers with regard to chemical properties, toxicokinetics, and toxicity.
12. Confidence in the Chronic Oral RfD for 1,2,3-TMB
28 The chronic oral RfD for 1,2,4-TMB was adopted as the chronic oral RfD for 1,2,3-TMB; thus,
29 confidence in the study from which the critical effect was identified, Korsak and Rydzyhski (1996).
30 is low to medium (see above). The database for 1,2,3-TMB includes acute, short-term, and
31 subchronic studies in rats and mice. However, confidence in the database is low to medium because
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1 it lacks chronic, multi-generation reproductive/developmental, developmental toxicity, or
2 developmental neurotoxicity studies, and the studies supporting the critical effect predominantly
3 come from the same research institute. Reflecting the confidence in the study and the database and
4 the uncertainty surrounding the adoption of the RfD derived for 1,2,4-TMB as the RfD for
5 1,2,3-TMB, the overall confidence in the RfD for 1,2,3-TMB is low.
13. Oral Reference Dose (RfD) for 1,3,5-TMB for Effects Other Than Cancer
6 The oral database is inadequate to derive an RfD for 1,3,5-TMB. No chronic, oral exposure
7 study was found in the literature. However, one subchronic oral gavage study was identified that
8 observed effects on multiple clinical chemistry parameters and monocyte levels [Koch Industries,
9 1995b]. However, following an external peer review of this study (see Appendix F), it was
10 concluded that the Koch Industries [1995b] study was not suitable as the basis for quantifying the
11 noncancer human health risk following oral exposure. The most critical shortcoming of this study
12 was its failure to investigate the neurotoxicity of 1,3,5-TMB.
13 However, as determined for the RfC derivation for 1,3,5-TMB, the toxicokinetic and
14 toxicological similarities between 1,3,5-TMB and 1,2,4-TMB demonstrate sufficient similarity
15 between the two isomers to support adopting the RfD for 1,2,4-TMB for the RfD for 1,3,5-TMB. In
16 addition to the previously discussed similarities in toxicokinetics, the qualitative metabolic profiles
17 for the two isomers are similar to such a degree that first-pass metabolism through the liver is not
18 expected to differ greatly between 1,2,4-TMB and 1,3,5-TMB. Therefore, the chronic RfD of 2 x 10
19 2 mg/kg-day derived for 1,2,4-TMB was adopted as the RfD for 1,3,5-TMB based on the
20 determination of sufficient similarity between the two isomers with regard to chemical properties,
21 toxicokinetics, and toxicity.
Confidence in the Chronic Oral RfD for 1,3,5-TMB
22 The chronic oral RfD for 1,2,4-TMB was adopted as the chronic oral RfD for 1,3,5-TMB; thus
23 confidence in the study from which the critical effect was identified, Korsak and Rydzyhski [1996].
24 is low to medium (see above). The database for 1,3,5-TMB includes acute, short-term, and
25 developmental toxicity studies in rats and mice. However, confidence in the database is low to
26 medium because it lacks chronic, multi-gene ration reproductive/developmental and
27 developmental neurotoxicity studies, and the studies supporting the critical effect predominately
28 come from the same research institute. Reflecting the confidence in the study and the database and
29 the uncertainty surrounding the adoption of the RfD derived for 1,2,4-TMB as the RfD for
30 1,3,5-TMB, the overall confidence in the RfD for 1,3,5-TMB is low.
14. Evidence of Carcinogenicity
31 Under EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a). there is
32 "inadequate information to assess carcinogenic potential" of TMBs. No chronic inhalation studies
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1 that investigated cancer outcomes were identified in the literature for 1,2,3-TMB, 1,2,4-TMB, or
2 1,3,5-TMB. One cancer study in which rats were exposed to 1,2,4-TMB via oral gavage at one
3 experimental dose of 800 mg/kg-day observed marginal increases in total malignant tumors and
4 head tumors (e.g., neuroesthesioepitheliomas), but provided no statistical analyses of the results
5 [Maltoni et al., 1997]. A number of methodological issues limit the utility of this study (e.g., only one
6 dose group and no discussion of histopathological analyses). When Fisher's exact test was
7 performed by EPA on the incidences calculated from the reported percentages of animals bearing
8 tumors in the control and 800 mg/kg dose groups, no statistically significant elevations were
9 observed. Therefore, a quantitative cancer assessment for TMBs was not conducted.
15. Susceptible Populations and Lifestages
10 No chemical-specific data that would allow for the identification of populations or lifestages
11 with increased susceptibility to TMB exposure exist. However, some inferences can be made based
12 on the toxicokinetics of TMB isomers. TMB isomers are metabolized via side-chain oxidation to
13 form alcohols and aromatic carboxylic/mercapturic acids or by hydroxylation to form phenols,
14 which are then conjugated with glucuronic acid, glycine, or sulfates for urinary excretion. The
15 activities of multiple cytochrome P450 (GYP P450) mono-oxygenase isozymes and rates of
16 glucuronidation and sulfation conjugation are reduced in children up to 1 year in age, and renal
17 clearance is reduced in infants up to 2 months of age (Ginsberg et al., 2004]. Therefore, as GYP P450
18 mono-oxygenase activities, the rate of glucuronidation and sulfation, and renal clearance appear to
19 be decreased in early life, newborns and young infants may experience higher and more persistent
20 blood concentrations of 1,2,3-TMB, 1,2,4-TMB, 1,3,5-TMB, and/or their respective metabolites
21 compared with adults at similar exposure levels. Additionally, those with pre-existing respiratory
22 diseases (e.g., asthma] may be more sensitive to the respiratory irritative and inflammatory effects
23 resulting from exposure to TMB isomers.
16. Key Issues Addressed in the Assessment: Adoption of 1,2,4-TMB Toxicity Values
for the 1,3,5- and 1,2,3-TMB Isomers
24 The toxicity database for 1,3,5-TMB was inadequate for derivation of either a reference
25 concentration or a reference dose. The chemical, toxicokinetic, and toxicological properties of the
26 individual isomers are sufficiently similar to one another to support adoption of 1,2,4-TMB's
27 reference values for 1,3,5-TMB (see Section 2.3.5]. Both isomers are similar in their (1] chemical
28 properties (e.g., blood:tissue partition coefficients], (2] toxicokinetic properties (i.e., absorption,
29 metabolism, and excretion profiles], and (3] toxicity profiles across studies utilizing multiple
30 durations of exposure and multiple endpoints (i.e., neurological, respiratory, maternal, and fetal
31 effects]. Therefore, given these similarities, the RfC and RfD derived for 1,2,4-TMB were adopted as
32 the RfC and RfD for 1,3,5-TMB.
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1 The toxicity database for 1,2,3-TMB was inadequate for derivation of a reference dose. No
2 chemical-specific PBPK model is available for 1,2,3-TMB, and therefore, no route-to-route
3 extrapolation can be performed on which to derive an RfD from the RfC for 1,2,3-TMB. The
4 chemical, toxicokinetic, and toxicological properties of the individual isomers are sufficiently
5 similar to one another to support adoption of 1,2,4-TMB's reference dose for 1,2,3-TMB (see
6 Section 2.5.2). Both isomers are similar in their (1) chemical properties (e.g., blood:air and
7 tissue:air partition coefficients), (2) toxicokinetic properties (i.e., the degree of absorption into the
8 bloodstream between the two isomers indicates the internal blood dose metrics for 1,2,3-TMB
9 would be similar to those calculated for 1,2,4-TMB by that isomer's available PBPK model), and (3)
10 toxicity profiles (i.e., the observation that both isomers affected pain sensitivity to an equal degree
11 and that the two isomer's RfCs for this effect were equal). Therefore, given these similarities, the
12 deficiencies in the 1,2,3-TMB oral database, and the lack of a 1,2,3-TMB PBPK model with which to
13 perform a route-to-route extrapolation, the RfD derived for 1,2,4-TMB was adopted as the RfD for
14 1,2,3-TMB.
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LITERATURE SEARCH STRATEGY | STUDY
SELECTION AND EVALUATION
1 The literature search strategy used to identify primary, peer-reviewed literature pertaining
2 to TMBs was conducted using the databases and keywords listed in Table LS-1. References from
3 health assessments developed by other national and international health agencies were also
4 examined. Other peer-reviewed information, including review articles, literature necessary for the
5 interpretation of TMB-induced health effects, and independent analyses of the health effects data
6 were retrieved and included in the assessment where appropriate. EPA requested public
7 submissions of additional information in April 2008; no submissions in response to the data call-in
8 were received. A comprehensive literature search was last conducted in December 2011.
9 Figure LS-1 depicts the literature search and study selection strategy and the number of
10 references obtained at each stage of the literature screening. Approximately 4,300 references were
11 obtained from the chemical name, keyword, and metabolite searches for 1,2,4-TMB, 1,2,3-TMB, and
12 1,3,5-TMB including references retrieved from specific literature searches necessary for the
13 interpretation of TMB-induced health effects (e.g., literature on specific modes of action, PBPK
14 analysis). From this full list of references, there were 218 references that were considered for
15 inclusion in the Toxicological Review.
16 Selection of studies for inclusion in the Toxicological Review was based on consideration of
17 the extent to which the study was informative and relevant to the assessment and general study
18 quality considerations. In general, the relevance of health effect studies was evaluated as outlined in
19 the Preamble and EPA Guidance (/I Review of the Reference Dose and Reference Concentration
20 Processes [U.S. EPA, 2002] and Methods for Derivation of Inhalation Reference Concentrations and
21 Application of Inhalation Dosimetry [U.S. EPA. 1994b]]. From the list of "considered" references,
22 161 full text publications were identified as providing relevant information for use in the
23 development of this document, and included 30 studies in humans (e.g., occupational epidemiologic
24 studies, workplace exposure studies, and controlled human exposures), 63 inhalation or oral
25 animal studies, and 68 other studies (e.g., studies that provided supporting information on mode of
26 action, chemical properties, and susceptible subpopulations).
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1 The references that are cited in the document, as well as those that were considered but not
2 included in the Toxicological Review of TMBs, can be found within the Health and Environmental
3 Research Online (HERO) website3. This site contains HERO links to lists of references, including
4 bibliographic information and abstracts, which were considered for inclusion in the Toxicological
5 Review of TMBs.
Table LS-1: Details of the search strategy employed for TMBs
Databases
EBSCO
DISCOVERY
SERVICE:
HERO
SCI
NLM
TOXLINE
WOS
Keywords3'
Chemical name, CASRN, and synonym search:
1,2,4-trimethylbenzene, OR pseudocumene, OR 95-63-6;
1,2,3-trimethylbenzene, OR hemimellitene, OR 526-73-8;
1,3,5-trimethylbenzene, OR mesitylene, OR 108-67-8
Keyword search: neurotoxicity, genotoxicity, developmental toxicity, inflammation,
irritation, toxicokinetics, pbpk, mode of action, white spirit, C9, C9 fraction, JP-8
Additional search on specific metabolites:
2,3-dimethylbenzoic acid, OR 26998-80-1;
2,3-dimethylhippuric acid, OR 187980-99-0;
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;
2,6-dimethylbenzoic acid OR 632-46-2;
2,6-dimethylhippuric acid OR 187980-98-9;
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 acid OR 499-06-9;
3,5-dimethylhippuric acid OR 23082-14-6
Potentially relevant publications on TMBs were identified through a literature search conducted with the EBSCO
Discovery Service feature of Health and Environmental Research Online (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).
bLiterature search was performed using related words (i.e., lemmatization) of included search terms. Search terms
were entered into the EBSCO Discovery Service portal with no qualifiers and the results from individual search
engines were returned and exported to HERO.
HERO is a database of scientific studies and other references used to develop EPA's risk assessments aimed at
understanding the health and environmental effects of pollutants and chemicals. It is developed and managed in
EPA's Office of Research and Development (ORD) by the National Center for Environmental Assessment (NCEA).
The database includes more than 600,000 scientific articles from the peer-reviewed literature. New studies are added
continuously to HERO.
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References identified based on initial keyword search (see Table LS-1): "4300
V L
Reference excluded based on preliminary manual screen of
titles/abstracts: "4100
Reasons for excluding references included the following:
• Published in non-relevant journals (i.e., Journal of Chemical
Catalysis
Topics not relevant to TMB toxicity
Reference considered for inclusion in the Toxicological Review: 226
Human Studies: 39
Animal Studies: 78
Other supporting studies: 144
Including:
• Reviews
• Background and physical/chemical properties
« Studies related to mode-of-action
• Studies related to toxicokinetics or PBPK analyses
References excluded based on manual review of
papers/abstracts: 65
Types of papers evaluated and not considered further for
Inclusion in the Toxicological Review:
* Studies of exposure assessment
• Studies of chemicals other than TMBs (including studies of
complex solvent mixtures containing TMBs)
* Non-relevant human and animal toxicity studies
• C ase reports
• Ecotoxicity studies
• In vitro studies
• Not available in English
References cited in the Toxicological Review
Human studies/reports: 30
•Controlled Human Exposures: 10
• Epidemiological Studies: 11
•Hematatological: 3
• Neurotoxicity: 9
• Respiratory: 5
•Toxicokinetic Studies: 14
•Workplace Exposure studies: 10
Animal Studies: 63
• Cancer/Genotoxicity:
• Developmental: 5
•Hematological: 8
• Nephro toxicity: 1
• Neurotoxicity: 24
• Respiratory: 6
• Toxicokinetic: 20
Other supporting studies: 68
• Chemical properties: 6
•BlvlE) modeling: 6
• Exposure assessment: 4
• Mode of action: 22
•PBPIC27
•Susceptible populations: 3
Guidance documents: 41
•EPA: 23
• Non-EPA: 18
Note: Some references may provide information on more than one topic, and therefore, may be included in more than one
study type. Accordingly, the sum of the references for subcategories of studies is not expected to equal the number of
references for the larger category.
Figure LS-1. Literature search and study selection strategy for TMBs.
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l.HAZARD IDENTIFICATION
1.1. Synthesis of Evidence
1.1.1. Neurological Effects
1 There is evidence in humans and animals that inhalation exposure to trimethylbenzenes
2 (TMBs) induces neurotoxic effects. The human evidence comes from occupational studies involving
3 complex volatile organic compound (VOC) mixtures that include TMBs; thus, effects cannot be
4 attributed to any TMB isomer specifically. Prevalence rates of neuropsychological symptoms
5 increased with exposure duration in dockyard painters, with symptoms related to motor
6 coordination exhibiting the strongest association [Chen etal.. 1999): similarly, a significant
7 association between exposure and impaired performance in short term memory (symbol digit
8 substitution) and motor speed/ coordination (finger tapping) tests was observed in shipyard
9 painters exposed to TMBs (isomers were not specified) and other solvents (Lee etal., 2005). A
10 significant, positive association between exposure symptoms (e.g., abnormal fatigue) and
11 1,2,4-TMB exposure, but not exposure to lower levels of 1,2,3-TMB or 1,3,5-TMB, was reported in
12 asphalt workers (Norsethetal.. 1991). Nervousness, tension, headaches, vertigo, and anxiety were
13 reported in paint shop workers exposed to 49-295 mg/m3 of a solvent mixture containing 50%
14 1,2,4-TMB, 30% 1,3,5-TMB, and unspecified amounts of 1,2,3-TMB (listed as possibly present)
15 (Battig et al. (1956). as reviewed by MOE (2006) and Baettig et al. (1958)).
16 Additional evidence suggests damage or dysfunction of the inner ear and increased
17 occurrence of vertigo following exposure to TMBs and other organic solvents in paint and varnish
18 factories (Sulkowski etal.. 2002). Increased reaction time was significantly and consistently
19 associated with exposure in controlled, acute volunteer studies in which humans were exposed to
20 mixtures containing 1,2,4-TMB (Lammers etal.. 2007). although it is unclear whether 1,2,4-TMB or
21 other constituents within the mixtures were responsible for the observed effects. Uptake of TMBs
22 was reported in human volunteers exposed for 2 hours to either: 300 mg/m3 white spirit (WS,
23 corresponding to 11 mg/m31,2,4-TMB); 11 or 123 mg/m31,2,4-TMB; 123 mg/m31,2,3-TMB; or
24 123 mg/m31,3,5-TMB. However, effects on the central nervous system (CNS) were based on
25 measures of overt CNS depression (heart rate and pulmonary ventilation) and a subjective rating of
26 CNS symptoms (i.e., headache, fatigue, nausea, dizziness, and intoxication) (Tarnbergetal.. 1997a:
27 Jarnbergetal.. 1996). For full details of the epidemiologic and controlled human exposures studies
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Toxicological Review of Trimethylbenzene
1 (including human subjects research ethics procedures), see individual study summary tables in
2 Appendix B.
3 In two studies examining the toxicokinetics of TMBs following controlled human exposures
4 to 5-150 mg/m31,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB, no neurological abnormalities in routine
5 clinical examinations were reported following exposure, although results data or details regarding
6 the specific tests performed were not provided [Kostrzewskietal.. 1997: Kostrewski and
7 Wiaderna-Brycht. 1995). Studies identifying an association between occupational exposure to TMB
8 isomers and neurological effects are limited due to an inability to attribute effects due to 1,2,3-TMB,
9 1,2,4-TMB, or 1,3,5-TMB individually versus those due to the other isomers or additional
10 constituents within the mixture. The studies detailing controlled exposures to human volunteers
11 are also limited for evaluating neurotoxicity to TMBs due to a lack of methods to adequately assess
12 CNS function and a lack of no-exposure controls, short exposure duration, and exposure of
13 individual subjects to different concentrations of TMB isomers.
14 In animals, there is consistent evidence of neurotoxicity following inhalation exposure, and
15 to a lesser extent following oral exposure, to either 1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB; a summary
16 of the evidence pertaining to neurotoxic effects for TMBs is shown in Tables 1-1 and 1-2 for
17 inhalation and oral exposures, respectively. This information is presented graphically in Figures 1-1
18 to 1-4.
Pain sensitivity
19 Decreased pain sensitivity has been observed following inhalation exposure to TMBs in
20 multiple studies conducted in male Wistar rats (Table 1-1; Figures 1-1 - 1-3). To test pain
21 responses following TMB exposure, animal studies have employed the hot plate test In this test, a
22 thermal stimulus is applied to determine pain sensitivity, as indicated by the animals' latency to
23 paw-lick following introduction of the stimulus. In short-term exposure studies, the animals were
24 subjected to an additional environmental challenge, namely foot shock, which itself decreases pain
25 sensitivity. Short-term TMBs exposure without the foot shock challenge did not result in
26 statistically significant effects on pain sensitivity in the hot plate test several weeks after exposures
27 had ended. Decreases in pain sensitivity have been observed at concentrations > 123 mg/m3 or >
28 492 mg/m3 following subchronic exposure to 1,2,4-TMB or 1,2,3-TMB, respectively (Wiaderna et
29 al., 2002: Gralewicz and Wiaderna, 2001: Korsak and Rydzynski, 1996). Decreased pain sensitivity
30 after a foot shock challenge was observed at concentrations > 492 mg/m3 following short-term
31 exposure to 1,2,4-TMB (Gralewicz and Wiaderna. 2001: Gralewicz etal.. 1997b). 1,3,5-TMB
32 (Wiaderna etal.. 2002: Gralewicz and Wiaderna. 2001). or 1,2,3-TMB (Wiaderna etal.. 1998:
33 Korsak and Rydzynski. 1996). although changes were not observed at 492 mg/m31,2,3-TMB
34 (latencies 75% longer than controls were not statistically significant) in another short-term
35 exposure study (Gralewicz and Wiaderna, 2001).
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1 In the subchronic study [Korsak and Rydzynski. 1996}. inhalation of 1,2,4-TMB or
2 1,2,3-TMB resulted in reduced pain sensitivity which occurred in a concentration-dependent
3 manner. In short-term studies that examined a range of concentrations [Wiaderna et al.. 2002.
4 1998: Gralewicz etal., 1997b], decreases in pain sensitivity after foot shock challenge following
5 exposure to TMB isomers were non-monotonic. Differences in experimental design (discussed
6 below) may account for the lack of monotonicity in these short-term studies, in contrast to the
7 observations in Korsak and Rydzynski [1996]. Similar to the subchronic study, acute exposures to
8 1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB induced concentration-dependent decreases in pain sensitivity,
9 with ECso values of 4,172, 5,682,and 5,963 mg/m3 for increased latency to paw-lick compared to
10 controls, respectively [Korsak and Rydzynski. 1996: Korsak et al.. 1995].
11 The decreases in pain sensitivity measured in the subchronic and acute studies were
12 observed immediately after exposure [Korsak and Rydzynski, 1996: Korsak etal., 1995], with no
13 significant effects persisting 2 weeks after subchronic exposures were terminated (i.e., increases in
14 latency were reduced from 95 to 12% or from 78 to 13% of controls at 1,230 mg/m31,2,4- or
15 1,2,3-TMB, respectively] fKorsak and Rydzynski. 1996: Korsak etal.. 19951 In contrast,
16 performance in the hot plate test after foot shock challenge was significantly impaired following
17 short-term exposure to the TMB isomers when tested 51 days after exposure (Wiaderna etal..
18 1998] (Wiaderna etal.. 2 002: Gralewicz and Wiaderna. 2001: Gralewicz et al.. 1997b], indicating a
19 persistence of these pain sensitivity- related effects.
20 The addition of a foot shock challenge to the hot plate tests following short-term (i.e., 4-
21 week], inhalation exposure to TMB isomers makes these experiments somewhat distinct from
22 those performed following subchronic exposure, as the foot shock challenge can elicit a cognitive
23 response from the animals in later hot plate test trials (see below] (Wiaderna etal.. 2002: Gralewicz
24 and Wiaderna. 2001: Wiaderna etal.. 1998: Gralewicz etal.. 1997b]. In the short-term studies
25 (Wiaderna etal.. 2 002: Gralewicz and Wiaderna. 2001: Wiaderna etal.. 1998: Gralewicz etal..
26 1997b], treatment-related, statistically significant changes at > 492 mg/m31,2,3-TMB, 1,2,4-TMB,
27 or 1,3,5-TMB were observed 24 hours after rats were given a foot shock; no consistent, significant
28 effects at any concentration were observed immediately following foot shock. Additionally, no
29 statistically significant effects were observed prior to foot shock at 50 days post-exposure; studies
30 did tend to observe increases in latency in non-shocked rats that were not statistically significant at
31 > 492 mg/m31,2,4-TMB (up to 206% longer than controls], 1,3,5-TMB (up to 215% longer than
32 controls], or 1,2,3-TMB (up to 95% longer than controls], but these responses were highly variable
33 and not consistently observed across studies. As foot shock alone is known to cause transient
34 reductions in pain sensitivity, these findings suggest that inhalation exposure to TMBs prolongs
35 foot shock-induced reductions in pain sensitivity. However, although a lengthening of the foot
36 shock-induced decrease in pain sensitivity by TMBs exposure is the most likely reason for the
37 observed effects, and, accordingly, these responses are discussed in this context herein, this is not
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1 the only possible explanation. It is also plausible that cognitive effects resulting from TMBs
2 exposure could contribute to the responses observed 24 hours after foot shock. Specifically, control
3 groups may better associate the hot plate environment with the previously-applied aversive
4 stimulus and more quickly withdraw their paws than their TMB-exposed counterparts, who may
5 exhibit a decreased fear response or shorter retention of that fear-associated memory.
6 Alternatively, since this test paradigm can cause the hot plate test apparatus to become associated
7 with the effects of foot shock, inducing stress-related responses in the shocked animal such that
8 subsequent exposure to the hot plate test apparatus alone can reduce sensitivity to pain (possibly
9 via the release of endogenous opioids), prior TMBs exposure could amplify this effect. From the
10 data available, the relative contribution(s) of these behaviors to the observed effects cannot be
11 easily distinguished. Despite the possible overlap between contributing neurological processes in
12 this test paradigm, these observations are still regarded as significant and adverse, and clearly
13 indicate a persistence of neurological effects long after TMBs exposures have ceased.
14 Substantial differences in study design between short-term and subchronic studies make it
15 impossible to distinguish the particular aspects of the pain sensitivity phenotype that appear to be
16 latent and only manifest with an environmental challenge from those that appear to be reversible.
17 Regardless, the ability of male Wistar rats to respond to a thermal stimulus in the hot plate test was
18 consistently impaired following inhalation exposure to TMBs. The overall database indicates that
19 TMB isomers are similar in their capacity to decrease pain sensitivity following inhalation exposure
20 (Table 1-1; Figures 1-1 - 1-3). Pain sensitivity was not examined following oral exposure.
Neuromuscular function and coordination
21 Human exposures to solvent mixtures containing 1,2,4-TMB (Lammers etal.. 2007] or
22 multiple TMB isomers [(Battig etal.. 19561. as reviewed by MOE (20061 and (Lee etal.. 2005:
23 Sulkowskietal..20Q2: Battig etal.. 19581] result in effects that suggest alterations to
24 neuromuscular function and balance, including increased reaction time and vertigo. Animal studies
25 using rotarod performance, which tests motor coordination, balance, and overall neuromuscular
26 function, indicate that inhalation of TMB isomers can affect neuromuscular system function (Table
27 1-1; Figures 1-1 and 1-2). Significant decreases in rotarod performance were observed at 1,230
28 mg/m31,2,4-TMB and > 493 mg/m31,2,3-TMB when tested immediately after exposure for 13
29 weeks (Korsak and Rydzynski, 1996]: significant decreases in performance were also observed at
30 1,230 mg/m3after 4 or 8 weeks of exposure to 1,2,3-TMB or 1,2,4-TMB, respectively. This impaired
31 function was still evident at 2 weeks post-exposure and, while not statistically significant for
32 1,2,4-TMB, may indicate long-lasting neuromuscular effects of subchronic exposures to 1,2,4-TMB
33 and 1,2,3-TMB. Acute inhalation exposure studies support this observation. Effects such as loss of
34 reflexes and righting responses, have been observed following acute inhalation exposure to 1,250-
35 45,000 mg/m31,2,4-TMB (MOE. 2006: Henderson. 20011. Similarly, acute exposure to 1,2,3-TMB,
36 1,2,4-TMB, or 1,3,5-TMB resulted in decreased performance in rotarod tests immediately following
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1 exposure, with ECso values of 3,779 mg/m3,4,693 mg/m3, and 4,738 mg/m3, respectively [Korsak
2 and Rydzynski. 1996: Korsak etal.. 1995). These results indicate that 1,2,4-TMB and 1,3,5-TMB are
3 similar in their ability to impair neuromuscular function, balance, and coordination while
4 1,2,3-TMB exposure may elicit effects at lower concentrations compared to the other two isomers.
5 No studies evaluating oral exposure to TMB isomers address this endpoint.
6 The neurobehavioral tests administered (i.e., hot plate and rotarod) in the subchronic and
7 acute studies by Korsak and Rydzynski, [1996) and Korsak et al. [1995) appear to have been
8 conducted on the same days; however, it is unclear whether the tests were performed sequentially
9 in the same cohorts of animals. Performing the hot plate test immediately following the rotarod test
10 could introduce a potential confounder, as shock alone (such as that used as negative reinforcement
11 following rotarod failure, see Table B-30, Appendix B) can cause reductions in pain sensitivity.
12 Thus, if the tests were performed sequentially in the same animals, TMB-exposed animals failing
13 more often in the rotarod test may exhibit increases in paw-lick latency unrelated to treatment, as
14 compared to controls receiving less shock reinforcement However, the observations by Korsak and
15 Rydzynski, (1996) and Korsak et al. (1995) are supported by 2- to 3-fold increases in latency to
16 paw-lick that, although not statistically significant, were observed 50 days after termination of
17 short-term exposures to 492 mg/m31,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB (Gralewicz and Wiaderna.
18 2001): increases of this magnitude were not present in the studies evaluating multiple
19 concentrations of the isomers (Wiaderna et al., 2002,1998: Gralewicz etal., 1997b).
Motor function and/or anxiety
20 Effects in open field testing have been consistently reported in oral and inhalation studies of
21 exposure to 1,2,4-TMB and 1,3,5-TMB, but not 1,2,3-TMB, in male rats (Table 1-1; Figures 1-1-1-3);
22 however, open field locomotion following injections with the stimulant, amphetamine, were
23 amplified by prior 1,2,3-TMB exposure, but not by prior 1,2,4-TMB exposure (Lutz etal., 2010)
24 Altered behaviors and locomotion in open field tests can be attributed to anxiety responses due to
25 open spaces and bright light, as well as changes to motor system function. Factors other than
26 anxiety and motor function (e.g., interpretation of olfactory or visual cues) may also contribute to
27 alterations in open field behavior, but these are unlikely to be drivers of the responses. As all but
28 one of the studies (Lutz etal.. 2010) observed animals for only 5 or 10 minutes, these experimental
29 tests are insufficient to identify the relative contribution(s) of the various neurological responses to
30 the observed increases in open field behaviors. Thus, EPA has concluded that decreased anxiety
31 and/or increased motor function are the most likely explanations for the TMB-induced effects.
32 Decreased anxiety and/or increased motor function at > 492 mg/m31,2,4-TMB or
33 1,3,5-TMB has been reported in short-term studies, as evidenced by increases in horizontal
34 locomotion or grooming activities (Lutz etal.. 2010: Gralewicz and Wiaderna. 2001: Gralewicz etal..
35 1997b). Statistically significant increases in horizontal locomotion were observed in short-term
36 studies assessing open field behavior following inhalation exposure to 1,2,4-TMB or 1,3,5-TMB
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1 [Lutz etal.. 2010: Gralewicz and Wiaderna. 2001). Non-monotonic increases in grooming were
2 reported following short-term exposure to 1,2,4-TMB, although changes in horizontal locomotion
3 were not statistically significant (increases of 3-35% were also non-monotonic) [Gralewicz etal..
4 1997b]. No statistically significant effects on open field activity have been observed following short-
5 term exposure of male rats to 1,2,3-TMB [Lutz etal., 2010: Gralewicz and Wiaderna, 2001:
6 Wiaderna et al.. 1998). Open field locomotion following injections with the stimulant amphetamine
7 was amplified by previous short-term exposure to 1,2,3-TMB, but not 1,2,4-TMB (which actually
8 tended to inhibit amphetamine-induced increases in activity at 492 mg/m3), suggesting possible
9 effects of 1,2,3-TMB on sensitization-type responses. As open field testing was conducted 14 or 25
10 days after termination of exposure in these studies and TMB isomers are cleared rapidly from the
11 body following the end of inhalation exposures (Section B.2, Appendix B), the results suggest
12 persistence of the effects of 1,2,4-TMB and 1,3,5-TMB on anxiety and/or motor function following
13 clearance of the toxic moiety from the nervous system.
14 Slight, transient increases in locomotor activity were also observed in open field tests
15 immediately following acute, oral exposure to the TMB isomers (Table 1-2; Figure 1-4). Significant
16 increases in locomotor activity—measured as number of squares crossed after exposure compared
17 with prior to exposure—were observed at 3,850 mg/kg for 1,2,4-TMB and 1,2,3-TMB, and at >
18 1,920 mg/kg for 1,3,5-TMB, with minimal concentration-effect or time-effect relationships and
19 negligible differences in the magnitude of the change in activity between isomers (Tomas etal.,
20 1999b]. Increases in locomotor activity were biphasic in nature. At early timepoints following
21 exposure, increased locomotor activity was associated with perturbed motor coordination and
22 tremor, whereas after 90 minutes, this apparent motor ataxia progressed to hind limb paralysis, full
23 immobility, and respiratory distress (e.g., tachypnea), leading to several deaths by 24 hours (Tomas
24 etal.. 1999b].
25 As mentioned previously, open field tests cannot easily distinguish between anxiety-related
26 responses and changes in motor activity. However, effects on motor activity were observed
27 following inhalation exposure to elevated concentrations of TMBs in several acute studies, although
28 the results are inconsistent with observations in open field tests. Decreased motor activity was
29 observed in male rats immediately after exposure to 5,000 mg/m31,2,4-TMB (McKee etal.. 2010].
30 Decreased motor activity was also reported in rats acutely exposed via inhalation to a mixture
31 containing TMB isomers (Lammers etal., 2007], but the use of a mixture precludes a determination
32 of the toxicity specifically associated with individual isomers. As biphasic changes in activity are
33 frequently observed following exposures to solvents, it is likely that the timing of the evaluations
34 conducted in the short-term versus acute studies, as well as the differing isomer concentrations,
35 may influence the consistency of these results.
36 Overall, exposure to 1,2,4-TMB and 1,3,5-TMB affects anxiety and/or motor function at
37 concentrations above 492 mg/m3, although the exact, potentially biphasic, concentration-response
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1 relationship remains unclear. The results for 1,2,3-TMB are difficult to interpret, as no effects were
2 observed following short term inhalation exposure while acute oral exposure elicited responses
3 consistent with 1,2,4-TMB and 1,3,5-TMB. Although an explanation for this disparity is lacking,
4 these data highlight a potential difference between 1,2,3-TMB and the other isomers, regarding
5 altered motor function and/or anxiety.
Cognitive function
6 Cognitive function following exposure to TMB isomers alone has not been evaluated in
7 humans or following oral exposure in animals; controlled exposure of human volunteers to
8 mixtures containing TMBs did not indicate any effects on short-term learning and memory tests
9 [Lammers etal., 2007]. Similarly, short-term spatial memory (radial maze performance) was
10 unaffected by exposure to either 1,2,4-TMB or 1,3,5-TMB via inhalation in animal studies
11 fWiaderna etal.. 2002: Gralewicz and Wiaderna. 2001: GralewlczetaL 1997bl Similarly, although
12 one study indicates a significant decrement in radial maze performance following exposure to
13 123 mg/m31,2,3-TMB t(Wiaderna etal.. 1998). higher concentrations had no effect[Wiadernaetal..
14 1998). preventing interpretations regarding the significance of this finding. In contrast, effects on
15 cognitive function in passive and active avoidance tests of conditioning behaviors were consistently
16 observed across multiple studies in male rats 6-8 weeks following short-term inhalation exposure
17 to the TMB isomers, although clear concentration-effect relationships were not observed (Table 1-
18 1; Figures 1-1-1-3). Comparing the results of the behavioral tests reveals that there are differences
19 in cognitive effects reported for each TMB isomer, as well as differences in the concentrations at
20 which the cognitive effects were observed.
21 In the passive avoidance tests, rats were conditioned to avoid stepping down from a small,
22 elevated platform (the impulse of rats is to step down in order to escape the bright light and
23 constrained, elevated space of the platform) through the use of a brief series of foot shocks applied
24 on the lower level. It is important to clarify that these tests are distinct from tests of pain sensitivity
25 and that observations of decreased step down latency in these tests do not contrast with the
26 increases in paw lick latency observed in hot plate tests; in fact, they may be complementary (see
27 below; note: the foot shocks used are of a much shorter duration than those used to induce
28 decreased pain sensitivity in the hot plate tests). Decreases in step-down latency in passive
29 avoidance tests, particularly at 7 days following foot shock conditioning, were observed 6-7 weeks
30 after short-term inhalation exposure to > 123 mg/m31,2,3-TMB and 1,3,5-TMB or > 492 mg/m3
31 1,2,4-TMB (Wiaderna etal.. 2002: Gralewicz and Wiaderna. 2001: Wiaderna etal.. 1998: Gralewicz
32 etal.. 1997b). Differences in latency prior to foot shock were not observed. Decreases in latency
33 were consistently observed and similar in magnitude across all studies at 7 days post foot shock,
34 although the decreases were not statistically significant for 1,2,4-TMB or 1,2,3-TMB in the study by
35 Gralewicz and Wiaderna (2001). At 3 days post-foot shock, decreases in latency were less
36 consistent (i.e., statistically significant decreases were observed at 123 mg/m31,2,3-TMB and at
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1 492 mg/m31,2,4-TMB, but not at other concentrations, and were not observed following exposure
2 to 1,3,5-TMB), and only 123 mg/m31,2,3-TMB was shown to have an effect at 1 day post-foot shock.
3 In these tests, the effects occurring several days following conditioning with foot shock are most
4 likely attributable to a reduced ability to inhibit motor reactions (or a lowered motor threshold) in
5 response to the fear-inducing environment. Alternative explanations involve possible contributions
6 of the following in the TMBs exposed rats: a diminished fear response to the foot shock; decreased
7 pain sensitivity leading to a less effective negative reinforcement by the (less painful) foot shock; or
8 diminished retention of the fear-associated memory (i.e., from the foot shock). However, as
9 statistically significant changes were observed < 24 hours following foot shock only after exposure
10 to 123 mg/m31,2,3-TMB, neither diminished fear responses to the foot shock nor decreases in pain
11 sensitivity are likely to be the sole driver (s) of these effects. This suggests that, in this particular
12 test paradigm, TMBs exposure causes latent effects on neurological functions associated with the
13 persistence of adaptive behaviors to a fear-inducing stimulus. Despite the consistency of the results
14 at 7 days post-foot shock, these tests are insufficient to pinpoint whether the effects of TMBs
15 exposure are specific to diminished memory retention, increased impulsivity, and/ or decreased
16 motor control.
17 Reduced performance in two-way active avoidance tests was observed in male rats
18 following short-term inhalation exposure to > 492 mg/m31,2,4-TMB (Gralewicz and Wiaderna,
19 2001: Gralewicz etal.. 1997b). > 123 mg/m31.3.5-TMB (Wiaderna etal.. 2002: Gralewicz and
20 Wiaderna. 2001). and at 492 mg/m31,2,3-TMB (Wiaderna etal.. 1998):. The effects of TMBs were
21 particular to the learning component of the test (acquisition/ training session), rather than the
22 memory component (retention session 7 days later) (Wiaderna etal.. 2002: Gralewicz and
23 Wiaderna. 2001: Wiaderna etal.. 1998). The conditioning or training of active avoidance behaviors
24 was based on avoiding a painful foot shock (the unconditioned stimulus) upon presentation of a
25 tone (conditioned stimulus). Similar to the interpretation of results from passive avoidance tests, it
26 is unclear whether and to what extent potential alterations in locomotor activity (rats had to shuttle
27 between compartments) and/ or pain sensitivity following exposure to TMB isomers could
28 contribute to learning deficits in these tests.
29 Acute inhalation exposure studies provide some support for the observed effects of TMB
30 isomers on learned behaviors. Significant increases in response latency in psychomotor tasks,
31 observed immediately after exposure (effects did not persist to 24 hours later), were reported in
32 male rats following acute exposure to 5,000 mg/m31,2,4-TMB (McKee etal.. 2010) or to 4,800
33 mg/m3 of a mixture containing TMBs (Lammers etal., 2007). The effects on active and passive
34 avoidance behaviors indicate that learning and/or long-term memory processes are affected by
35 exposure to the TMB isomers. The data suggest that 1,3,5-TMB may be a more potent inducer of
36 toxic effects on cognitive function than 1,2,4-TMB and 1,2,3-TMB, as the effects following exposure
37 to 1,3,5-TMB were more consistent and sometimes occurred at lower concentrations than those
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1 reported following exposure to the other two isomers. Overall, however, these differences were
2 slight
3 Controlled human exposure studies suggest that exposures of < 123 mg/m3 of the 1MB
4 isomers do not cause overt CNS depression (measured as heart rate and respiration) (Tarnberg et
5 al., 1996], although symptoms related to this effect (e.g., lightheadedness, fatigue) have been
6 reported in workers occupationally exposed to mixtures containing TMBs. In mice, CNS depression
7 has been observed following acute inhalation exposure to > 25,000 mg/m31,3,5-TMB, with similar
8 effect levels for 1,2,4-TMB fACGIH. 20021
Electrocortical activity
9 Neurophysiological evidence from short-term inhalation studies in animals, as well as
10 supportive evidence from acute oral and injection studies, suggests that exposures to 1MB isomers
11 at lower concentrations (at least for 1,2,4-TMB) may affect parameters associated with brain
12 excitability. Decreases in a particular component of electrocortical arousal (i.e., spike-wave
13 discharge, SWD, bursts in recordings from cortical-hippocampal electroencephalograms, EEGs)
14 were observed in male rats 120 days after short-term exposure to > 492 mg/m31,2,4-TMB
15 (statistically significant at 1,230 mg/m3), suggesting persistent functional changes in the rat CNS
16 (Gralewicz etal., 1997a). Altered EEC patterns can be induced by anesthetics as well as stimuli that
17 produce arousal, and may precede other measures of neurotoxicity (U.S. EPA. 1998). In recordings
18 from rats that were awake, but immobile (not exhibiting pronounced exploratory activity, as
19 determined by EEC morphology), statistically significant decreases in the frequency of SWD
20 episodes were observed at 24 hours following short-term exposure to 492 mg/m31,2,4-TMB
21 (decreases that were not statistically significant were also observed at >492 mg/m31,2,4-TMB at
22 30 and 120 days after exposure) (Gralewicz et al.. 1997a).
23 Complementing these findings, dose-related decreases in the duration and number of SWD
24 bursts (termed high-voltage spindles) were observed at > 240 mg/kg of the TMB isomers
25 subsequent to acute oral exposure (Tomas etal.. 1999a] (Table 1-2; Figure 1-4). The stronger and
26 more persistent effects on electrocortical activity followed a pattern of 1,2,3-TMB > 1,3,5-TMB >
27 1,2,4-TMB (Tomas etal.. 1999a). Similarly, electrophysiological alterations in cortical and
28 hippocampal EEGs were more pronounced following i.p. injection of 1,2,3-TMB, with 1,2,4-TMB and
29 1,3,5-TMB exerting lesser effects (Tomas etal., 1999c). Although it is unclear whether these
30 changes affect related processes such as memory and seizure initiation/propagation, the observed
31 EEC abnormalities following inhalation (Gralewicz etal.. 1997a). oral (Tomas etal.. 1999a). and i.p.
32 (Tomas etal.. 1999c] exposure to TMB isomers provide supportive evidence of possible acute CNS
33 depression by TMB isomers (Tomas etal.. 1999a: Tomas etal.. 1999c] and indicate persistent (up
34 to 120 days post-exposure) (Gralewicz etal.. 1997a] alterations in CNS activity that may reflect an
35 adaptive response to TMB exposure.
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Neurological effects: Inhalation
Table 1-1. Evidence pertaining to neurological effects of TMBs in animals —
inhalation exposures
Study Designa'b and Reference
Assay and Results (as response relative to control)
1,2,4-TMB
Pain sensitivity
0, 123, 492, 1,230 mg/m3, (recovery:
1,230 mg/m3 at 2 wks post-exposure)
90 day; Rat, Wistar, male, N = 10
Korsak and Rydzyriski (1996) Table B-30C
0, 492 mg/m3
4 wks; Rat, Wistar, male, N = 11
Gralewicz and Wiaderna (2001), Table B-26
0, 123, 492, 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 15
Gralewicz et al. (1997b), Table B-24
Hot plate- exposure-dependent increase in paw-lick latency which
recovers by 2 weeks post-exposure:
Response after exposure: 0, 18, 79*, 95*%
Response at 2 weeks post-exposure: 0, ND, ND, 12%
Hot plate- increased paw-lick latency 24 hr after foot shock:
Response at 50 days post-exposure: 0, 206%
Response at 50 days post-exposure seconds after foot shock: 0, 25%
Response at 51 days post-exposure 24hr after foot shock: 0, 191*%
Hot plate- increased paw-lick latency 24 hr after foot shockd:
Response at 50 days post-exposure: 0, -6, 7, -9%
Response at 50 days post-exposure seconds after foot shock:
0,-8, 17, -11%
Response at 51 days post-exposure 24 hr after foot shock:
0, 2, 74*, 33*%
Neuromuscular function and coordination
0, 123, 492, 1,230 mg/m3, (recovery:
1,230 mg/m3 at 2 wks post-exposure)
90 day; Rat, Wistar, male, N = 10
Korsak and Rydzyriski (1996), Table B-30
Rotarod- exposure-dependent increase in failures at 13 weeks which
does not recover by 2 weeks post-exposure:
Response after 13 weeks of exposure: 0, 10, 20, 40*%
Response at 2 weeks post-exposure: 0, ND, ND, 30%
Motor function and/or anxiety
0, 123, 492, 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 15
Lutzetal. (2010), Table B-35
0, 492 mg/m3
4 wks; Rat, Wistar, male, N = 11
Gralewicz and Wiaderna (2001), Table B-26
Open field- increased horizontal locomotion (distance traveled); no
overall effects with amphetamine challenge6:
Response at 2 weeks post-exposure with no challenge: 0, 100, 84, 154*%
Response to single amphetamine injection challenge: 0, 90, -25, 69%
Response to challenge after conditioning: 0, 43, -50, 31%
Open field- increased horizontal locomotion (number of crossings):
Response at 25 days post-exposure: 0, 61*%
Wo change in exploration (rearings) or grooming episodes
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Table 1-1. (Continued): Evidence pertaining to neurological effects of TMBs in
animals — inhalation exposures
Study Design ' and Reference
Assay and Results (as response relative to control)
0, 123, 492, 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 15
Gralewicz et al. (1997b), Table B-24
Open field- increased grooming at middle concentration:
Response at 25 days post-exposure: 0, 82,147*, 76%
Wo change in horizontal locomotion (number of crossings) or exploration
Cognitive function
0, 492 mg/m3
4 wks; Rat, Wistar, male, N = 1
Gralewicz and Wiaderna (2001), Table B-26
Passive avoidance- decreased step-down latency 7 days post-foot shockf:
Response at 39 days post-exposure prior to foot shock: 0, 34%
Response at 42 days post-exposure 1 day after foot shock: 0, -23%
Response at 44 days post-exposure 3 days after foot shock: 0,-51 %
Response at 48 days post-exposure 7 days after foot shock: 0, -43%
[Note: statistical significance 7 days after foot shock was noted after the
highest and lowest responder from each group was excluded]
Active avoidance- decreased performance during training (learning):
Trials to reach avoidance criteria at 54-60 days post-exposure: 0, 58*%
Wo differences were noted during retraining (retention)
Radial maze- no notable change in performance 14-18 days
post-exposure
0,123, 492, or 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 15
Gralewicz et al. (1997b), Table B-24
Passive avoidance- decreased step-down latency 3-7 days post-foot
shock:
Response at 39 days post-exposure prior to foot shock:
0, 26, 41, -31%
Response at 42 days post-exposure 1 day after foot shock:
0, 95, -28, -87%
Response at 44 days post-exposure 3 days after foot shock:
0, 7, -67*, -36%
Response at 48 days post-exposure 7 days after foot shock:
0, -20, -79*, -47*%
Active avoidance- decreased performance during acquisition (learning)8:
Slower increases in avoidance performance across trials: p < 0.003
Non-significant decrease in total avoidance responses: p = 0.08
Radial maze- no notable change in performance 14-18 days
post-exposure
Electrocortical activity
0, 123, 492, 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 9
Gralewicz et al. (1997a), Table B-25
EEG recordingsh- decreased spike wave discharge (SWD) bursts/ hr:
Response at 120 days post-exposure: 0,13, -35, -55*%
Wo change in global arousal level or in SWD/hr at 1 or 30 days
post-exposure
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Table 1-1. (Continued): Evidence pertaining to neurological effects of TMBs in
animals — inhalation exposures
Study Designa'b and Reference
Assay and Results (as response relative to control)
1,2,3-TMB
Pain sensitivity
0, 123, 492, 1,230 mg/m3, (recovery:
1,230 mg/m3 at 2 wks post-exposure)
90 days; Rat, Wistar, male, N = 10
Korsak & Rydzyriski (1996), Table B-30
0, 492 mg/m3
4 weeks; Rat, Wistar, male, N = 11
Gralewicz and Wiaderna (2001), Table B-26
0, 123, 492, 1,230 mg/m3
4 weeks; Rat, Wistar, male, N = 15
Wiaderna et al. (1998), Table B-42
Hot plate- exposure-dependent increase in paw-lick latency which
recovers by 2 weeks post-exposure:
Response after exposure: 0, 22*, 68, 78*%
Response at 2 weeks post-exposure: 0, ND, ND, 13%
Hot plate- no statistically significant change in paw-lick latency:
Response at 50 days post-exposure: 0, 95%
Response at 50 days post-exposure seconds after foot shock: 0, -1%
Response at 51 days post-exposure 24 hr after foot shock: 0, 75%
Hot plate- increased paw-lick latency 24 hr after foot shock at middle
concentration:
Response at 50 days post-exposure: 0, -28, -13, -12%
Response at 50 days post-exposure seconds after foot shock: 0, -9, -16, -
15%
Response at 51 days post-exposure 24 hr after foot shock: 0, -19, 45*, 8%
Neuromuscular function and coordination
0, 123, 492, 1,230 mg/m3, (recovery:
1,230 mg/m3 at 2 weeks post-exposure)
90 days; Rat, Wistar, male, N = 10
Korsak and Rydzyriski (1996), Table B-30
Rotarod- exposure-dependent increase in failures at 13 weeks which
does not recover by 2 weeks post-exposure:
Response after 13 weeks of exposure: 0, 20, 40*, 70*%
Response at 2 weeks post-exposure: 0, ND, ND, 50*%
Motor function and/or anxiety
0, 123, 492, 1,230 mg/m3
4 weeks; Rat, Wistar, male, N = 15
Lutzetal. (2010), Table B-35
0, 492 mg/m3
4 wks; Rat, Wistar, male, N = 11
Gralewicz and Wiaderna (2001), Table B-26
Open field- statistically significant increase in horizontal locomotion
(distance traveled) only after amphetamine challenge6:
Response at 2 weeks post-exposure with no challenge: 0, 96, 85, 115%
Response to single amphetamine injection challenge: 0, 15, 198*, 111%
Response to challenge after conditioning: 0, -21, 103*, 41%
Open field- no change in horizontal locomotion (crossings):
Response at 25 days post-exposure: 0, -9%
Wo change in exploration (rearings), or grooming
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Table 1-1. (Continued): Evidence pertaining to neurological effects of TMBs in
animals — inhalation exposures
Study Design ' and Reference
Assay and Results (as response relative to control)
0, 123, 492, 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 15
Wiaderna et al. (1998), Table B-42
Open field- no significant change in horizontal locomotion (crossings):
Response at 25 days post-exposure: 0,19, 51, 37%
Wo statistically significant change' in exploration (rearings) or grooming
Cognitive function
0, 492 mg/m3
4 wks; Rat, Wistar, male, N = 11
Gralewicz and Wiaderna (2001), Table B-26
Active avoidance- decreased performance during training (learning):
Trials to reach avoidance criteria at 54-60 days post-exposure: 0, 53*%
Wo differences were noted during retraining (retention)
Passive avoidance- no significant change in step down latency':
Response at 39 days post-exposure prior to foot shock: 0, -39%
Response at 42 days post-exposure 1 day after foot shock: 0, -40%
Response at 44 days post-exposure 3 days after foot shock: 0,-23 %
Response at 48 days post-exposure 7 days after foot shock: 0, -28%
Radial maze- no notable change in performance 14-18 days
post-exposure
0,123, 492, or 1,230 mg/m
4 wks; Rat, Wistar, male, N = 15
Wiaderna et al. (1998), Table B-42
Passive avoidance- decreased step-down latency after foot shock:
Response at 39 days post-exposure prior to foot shock: 0, -41, -37,19%
Response at 42 days post-exposure 1 day after foot shock: 0, -74*, -52, -
43%
Response at 44 days post-exposure 3 days after foot shock: 0, -54*, -49, -
14%
Response at 48 days post-exposure 7 days after foot shock: 0, -50*, -62*,
-37%
Active avoidance- decreased performance during training (learning):
Trials to reach avoidance criteria at 54-60 days post-exposure: 0, 3, 41*,
14%
Wo statistically significant differences noted during retraining (retention)
Radial maze- decreased performance at low concentration':
Increased errors on trial day 3: 0, 32*, -28, -4% & day 5: 0, 30*, -16,1%
Wo notable change in trial duration at any day (14-18 days
post-exposure)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzene
Table 1-1. (Continued): Evidence pertaining to neurological effects of TMBs in
animals — inhalation exposures
Study Design ' and Reference
Assay and Results (as response relative to control)
1,3,5-TMB
Pain sensitivity
0, 492 mg/m3
4 wks; Rat, Wistar, male, N = 11
Gralewicz and Wiaderna (2001), Table B-26
Hot plate- increased paw-
Response at 50 days post-
Response at 50 days post-
Response at 51 days post-
lick latency 24 hr after foot shock:
exposure: 0, 215%
exposure seconds after foot shock: 0, 26%
exposure 24 hr after foot shock: 0, 246*%
0, 123, 492, 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 12
Wiaderna et al. (2002), Table B-43
Hot plate- increased paw-lick latency 24 hr after foot shock at middle
concentration:
Response at 50 days post-
Response at 50 days post-
4%
exposure: 0, -6, 36, 24%
exposure seconds after foot shock: 0, -14, 8, -
Response at 51 days post-exposure 24 hr after foot shock: 0, -4, 68*, 18%
Motor function and/or anxiety
0, 492 mg/m3
4 wks; Rat, Wistar, male, N = 11
Gralewicz and Wiaderna (2001), Table B-26
Open field- increased horizontal locomotion (number of crossings):
Response at 25 days post-exposure: 0, 65*%
Wo change in exploration (rearings) or grooming
Cognitive function
0, 123, 492, 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 12
Wiaderna et al. (2002), Table B-43
Passive avoidance- decreased step-down latency 7 days post-foot shock:
Response at 39 days post-exposure prior to foot shock: 0, -5,146, 40%
Response at 42 days post-exposure 1 day after foot shock: 0, 99,108,
113%
Response at 44 days post-exposure 3 days after foot shock: 0, -32, -41, -
40%
Response at 48 days post-exposure 7 days after foot shock: 0, -47*, -53*,
-43*%
Active avoidance- decreased performance during training (learning):
Trials to reach avoidance criteria at 54-60 days post-exposure: 0,40*,
35*, 50*%
Radial maze- no notable change in performance 14-18 days post-
exposure
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzene
Table 1-1. (Continued): Evidence pertaining to neurological effects of TMBs in
animals — inhalation exposures
Study Design ' and Reference
Assay and Results (as response relative to control)
Cognitive function (continued)
0, 492 mg/m3
4 wks; Rat, Wistar, male, N = 11
Gralewicz & Wiaderna (2001), Table B-26
Passive avoidance- decreased step-down latency 7 days post-foot shock8:
Response at 39 days post-exposure prior to foot shock: 0, -3%
Response at 42 days post-exposure 1 day after foot shock: 0, -61%
Response at 44 days post-exposure 3 days after foot shock: 0,-65%
Response at 48 days post-exposure 7 days after foot shock: 0, -57*%
[Note: statistical significance 3 days after foot shock was noted after the
highest and lowest responder from each group was excluded]
Active avoidance- decreased performance during training (learning):
Trials to reach avoidance criteria at 54-60 days post-exposure: 0, 65*%
Radial maze- no notable change in performance 14-18 days
post-exposure
*Significantly different from controls (p< 0.05).
Notes: For studies other than Korsak and Rydzynski (1996). % change from control calculated from digitized data using Grab
It! XP software; ND= Not determined
aRotarod and hot plate tests were administered immediately after termination of exposure or following a 2 week recovery
period by Korsak and Rydzynski (1996). EEC recordings were acquired prior to exposure and one, 30, or 120 days after
exposure by Gralewicz et al. (1997a). Motor behavior in an open field (tested for 30 min) was assessed 14 days after
exposure and re-tested following single and multiple (to induce sensitization) injections with amphetamine for 120 min by
Lutz et al. (2010). For the remaining studies (Wiaderna etal.. 2002: Gralewicz and Wiaderna. 2001: Wiaderna etal.. 1998:
Gralewicz etal.. 1997b): radial maze tests were administered prior to exposure and on days 14-18 after exposure; open field
activity (tested for 5-10 minutes) was assessed prior to exposure and on day 25 after exposure; passive avoidance was tested
on days 35-48 after exposure; hot plate sensitivity was assessed on days 50 and 51 after exposure; and active avoidance tests
were administered on or after day 54 post-exposure.
bln instances where authors reported exposures in ppm, EPA converted these values to mg/m3. See Appendix B (Table B-l)
for conversion factor, and individual study summary tables for ppm values.
cTables referenced in Study Design and Reference column correspond to study summary tables in Appendix B
dObservations of hot plate latency were made prior to (LI); immediately following (L2); or 24hr after foot shock (L3). Values
for L3 in Graleiwicz et al. (1997b) were determined from reported values for LI and the ratio of L3/L1 x 100.
eNo challenge= prior to amphetamine challenge, evaluated for 30 min, and reported as Block 1: statistical significance
indicated in study text only; amphetamine challenge-induced activity was measured following a single injection or following
a single injection challenge after conditioning with 5 daily injections and evaluated for 120 min
fResults of passive avoidance tests in Gralewicz and Wiaderna (2001) may reflect adjusted data where, due to large individual
differences, 2 rats (the highest and lowest responders to foot shock) in each group were excluded. As a result, the exact
magnitude of change is assumed to be somewhat inaccurate and statistical comparisons of the modified groups are provided
in the above evidence table only as notes.
sAt 54 days post-exposure, TMB-exposed rats were slower to increase the percentage of avoidance responses across blocks
(1 block = 5 trials). This reduction in avoidance responses across blocks appeared to be lowest (although not statistically
significant) at 1,230 mg/m3. Rats were also observed to have a lower (p = 0.08) number of avoidance responses in the whole
30-trial session.
hElectroencephalograms (EEGs) were recorded at electrodes implanted in the fronto-parietal cortex and the dorsal
hippocampus (one recording from each region was analyzed for each rat).
'Dose-dependent increases in exploration and nonlinear increases in grooming were not statistically significant
iData represents % change relative to control in same trial day, but statistical significance determined by the authors based
on comparison to trial day 1 responses within the same group.
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Toxicological Review of Trimethylbenzene
Neurological effects: Oral
Table 1-2. Evidence pertaining to neurological effects of 1,2,3-TMB, 1,2,4-TMB, or
1,3,5-TMB in animals — oral exposures
,a,b
Study Design3' and Reference
Assay and Results (as response relative to control)
1,2,4-TMB
Motor function and/or anxiety
0, 960,1,920, 3,850 mg/kg single oral gavage
Rat, Wag/Rij, male, N = 10
Tomas et al. (1999b), Table B-40
Open field- transient increases in locomotor activity:
Response at 20 min after exposure relative to pre-injection controls: 0,
34.1, 57.8, 60.6*%
Wo significant changes were reported at 10, 30, 40, 50, 60, or 70 min
Electrocortical activity
0, 240, 960, 3,850 mg/kg, single oral gavage
Rat, Wag/Rij, male, N = 6
Tomas et al. (1999a), Table B-39
EEG recordings - inhibition of the duration and number of high voltage
spindle episodes (response relative to vehicle control):
Duration
Number
20 min
0, -72, -58, -83%
0, -26, -44,-62*%
40 min
0, -80*, -97*, -45%
0, -53*,-88*,-73*%
60 min
0, 11, -67, -45%
0, 7, -53*, -22%
1,2,3-TMB
Motor function and/or anxiety
0, 960,1,920, 3,850 mg/kg single oral gavage
Rat, Wag/Rij, male, N = 10
Tomas et al. (1999b), Table B-40
Open field- transient increases in locomotor activity:
Response at 20 or 30 min after exposure relative to pre-injection controls:
0, 30.9, 26.5, 56.1*% (increased 65.6*% at 30 min in at the highest
concentration
Wo significant changes were noted at 10, 40, 50, 60, or 70 min
Electrocortical activity
0, 960, 3,850 mg/kg, single oral gavage
Rat, Wag/Rij, male, N = 6
Tomas et al. (1999a), Table B-39
EEG recordings - inhibition of the duration and number of high voltage
spindle episodes (response relative to vehicle control):
Duration
Number
20 min
0, -86, -97*, -76*%
0,-71*,-86*,-48%
40 min
0, -95, -98*, -97*%
0, -84*,-93*,-86*%
60 min
0, -81, -94*, -99*%
0, -70*,-99*,-96*%
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzene
Table 1-2 (Continued): Evidence pertaining to neurological effects of 1,2,3-TMB,
1,2,4-TMB, or 1,3,5-TMB in animals — oral exposures
Study Design3' and Reference
Assay and Results (as response relative to control)
1,3,5-TMB
Motor function and/or anxiety
0, 960,1,920, 3,850 mg/kg single oral gavage
Rat, Wag/Rij, male, N = 10
Tomas et al. (1999b), Table B-40
Open field- transient increases in locomotor activity:
Response at 20 min after exposure relative to pre-injection controls: 0, 0,
46.7*, 42.4*% (increased 65-70% at 40-60 min at the highest
concentration
Wo significant changes were noted at 10, 30, or 70 min
Electrocortical activity
0, 240, 960, 3,850 mg/kg, single oral gavage
Rat, Wag/Rij, male, N = 6
Tomas et al. (1999a), Table B-39
EEG recordings - inhibition of the duration and number of high voltage
spindle episodes (response relative to vehicle control):
Duration
Number
20 min
0, -76*, -79,-86%
0, -57,- 67, -77%
40 min
0, -85*,-97*,-95*%
0,-52*,-93*,-91*%
60 min
0, -66*,-94*,-88*%
0,-49*,-91*,-89*%
*Significantly different from controls (p < 0.05).
Note: % change from control calculated from digitized data using Grab It! XP software.
aLocomotor activity in open field tests and electrocortical arousal were assessed prior to exposure and immediately after
exposure every 10 minutes for up to 70 minutes.
bln instances where authors reported exposures in ppm, EPA converted these values to mg/m3. See Appendix B (Table B-l) for
conversion factor, and individual study summary tables for ppm values.
cTables referenced in Study Design and Reference column correspond to study summary tables in Appendix B.
dElectroencephalograms (EEGs) were recorded prior to exposure and at 20,40, and 60 minutes after exposure via electrodes
implanted in the fronto-parietal cortex.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzene
Exposure Response Arrays
EffectSummary Duration
4< Pain Sensitivity Sub-chronic
4, Pain Sen sitivity (after .
t i i i i ii i Short-term
foot shock challenge,!
Impaired Neuro- . .
h Sub-chronic
muscular Function
T Motor Activity
an d/ori Anxiety Short-term
Altered Cognitive Function Short-term
4- Electro-cortical Activity Short-term
1,2,4-TMB
End point
t- Paw-lick latency
(hot pi ate) (a)
f1 Paw-lick latency
(hot plate) (d)
T Paw-lick latency
(hotplate) (b)
T Rotarod failure (a)
•T- Horizontal
Locomotion (e)
N/CAnnph,- Induced
Locomotion (e)
•T- Horizontal
Locomotion (d)
t Grooming Activity (b)
N/C Passive
Avoidance (d)
4/ Active Avoidance
W
-,J,- Passive
Avoidance (b)
4- Active
Avoidance (b)
N/C Radial Maze (b)
N/C Radial Maze (d)
4- SWD bursts (c)
i- LOW Dose -i High Dose 4 NOAEL • LOAEL
•
«|
1
[
•
• II
•
•
f
|
•
I
20 500 ;50 1,000 1,30
Concentration [mg/mSj
Note: Solid lines represent range of exposure concentrations, (a) Korsak and Rydzynski (1996): (b) Gralewicz et al.
(I997b); (c) Gralewicz et al. (I997a); (d) Gralewicz and Wiaderna (2001): (e) Lutz et al. (2010). All effects are in male
Wistar rats.
Figure 1-1. Exposure response array of neurological effects following inhalation
exposure to 1,2,4-TMB.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzene
Effect Summary Duration
•^ Pain Sensitivity Sub-chronic
\|/ Pain Sensitivity [after ,
t t ti ' Short-term
footshock challenge)
impaired Neuro- Sub.chronic
muscular Function
N/C Motor Activity
,, „ . ' Short-term
and/or Anxiety
Altered Cognitive Function Short-term
1,2,3-TMB
Endpoint
•t Paw-lick latency
(hot plate) (a)
N/C Paw- lick latency
(hot plate) (b)
•f Paw-lick latency
(hot plate) (c)
1* Rotarod failure (a)
N/C Horizontal
Locomotion (d)
1* Arnph,- Induced
Locomotion (d)
N/C Open Field
Activity(b)
N/C Open Field
Activity (c)
N/C Passive
Avoidance (b)
\|/ Passive
Avoidance (c)
J/ Active Avoidance
(b)
-J, Active
Avoidance (c)
N/C Radial Maze (b)
4, Radial Maze (c)
i- Low Dose -i High Dose * NOAEL • LOAEL
• i
•
«i
• i
i
• I
•
i
•
• i
•
• I
•
• i
250 500 750 1,000
Concentration (mg/m3)
1,250
Note: Solid lines represent range of exposure concentrations, (a) Korsak and Rydzynski (1996) ; (b) Gralewicz and
Wiaderna (2001): (c) Wiaderna et al. (1998): (d) Lutz et al. (2010). All effects are in male Wistar rats.
Figure 1-2. Exposure response array of neurological effects following inhalation
exposure to 1,2,3-TMB.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzene
EffectSummary Duration
4 Pain Sensitivity [after
, . i iii, . Short-term
foot shock challenge)
T- Motor Activity
an d/ori Anxiety Short-term
Altered Cognitive Function Short-term
1,3,5-TMB
End point
11 Paw-lick latency
(hot plate) (a)
T" Paw-lick latency
(hot plate) (b)
T Horizontal
Locomotion (a)
4, Passive
Avoidance (a)
J, Passive
Avoidance (b)
4, Active
Avoidance (a)
4r Active
Avoidance (b)
N/C Radial Maze (a)
N/C Radial Maze(b)
i- Low Dose -i High Dose * NOAEL • LOAEL
•
«i
•
•
• 1
•
*
250 500 750 1,000 1,350
Concentration (mg/m3)
Note: Solid lines represent range of exposure concentrations, (a) Gralewicz and Wiaderna (2001): (b) Wiaderna et al.
(2002). All effects are in male Wistar rats.
Figure 1-3. Exposure response array of neurological effects following inhalation
exposure to 1,3,5-TMB.
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Toxicological Review of Trimethylbenzene
1,2,4-TMB, 1,3,5-TMB,
Isomer Effect Summary
1,2,4-TMB t Motor Activity
and/ or 4/ Anxiety
•4/ Electro-
cortical Activity
•t Motor Activity
and/or \|/ Anxiety
4- Electro-
cortical Activity
•t Motor Activity
and/or \|/ Anxiety
4- Electro-
cortical Activity
or 1,2,3-TMB
Duration Endpoint
"t Horizontal
Acute . .
Locomotion (a)
4- HVS Episodes
Acute .,r.
and Duration (b)
"t Horizontal
Acute
Locomotion (a)
4- HVS Episodes
Acute , ' ...
and Duration (b)
•t Horizontal
Acute ,
Locomotion (a)
4, HVS Episodes
Acute , '
and Duration (b)
i- Low Dose H High Dose • NOAEL • LOAEL
I
• 1
I
• i
• i
• i
0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,OOC
Dose (mg/kg)
Note: Solid lines represent range of exposure concentrations, (a) Tomas et al. (I999a); (b) Tomas et al. (I999b). All effects
are in male WAG/Rij (Tomas et al. (I999a)) or Wistar (Tomas et al. (I999b)) rats.
Figure 1-4. Exposure response array of neurological effects following oral exposure
to 1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzene
1.1.1.1. Mode of Action Analysis - Neurological Effects
1 The observation of neurotoxicity following acute-, short-term-, and subchronic-duration
2 exposure to 1MB [Lutz etal.. 2010: Lammers etal.. 2007: Wiadernaetal.. 2002: Gralewicz and
3 Wiaderna. 2001: Wiadernaetal.. 1998: Gralewicz etal.. 1997b: Gralewicz etal.. 1997a: Korsakand
4 Rydzynski. 1996: Korsak etal.. 1995] may indicate thatTMB perturbs normal neurotransmission in
5 exposed animals, although the specific key events necessary for TMB-induced neurotoxicity are not
6 established. Although mechanistic and mode-of-action data is lacking for TMBs, structurally similar
7 compounds like toluene and xylene have been more thoroughly characterized and it is reasonably
8 assumed that TMBs would operate through a similar mechanism in producing the resultant
9 neurotoxicological effects. Aromatic hydrocarbons are known to interact with catecholaminergic
10 systems [Kyrklund. 1992}. Inhalation exposures to toluene and xylene have been shown to
11 significantly change concentration and turnover rate of both dopamine and norepinephrine in
12 various regions of the rat brain [Reaetal.. 1984: Anderssonetal.. 1983: Anderssonetal.. 1981:
13 Andersson et al.. 1980]. These changes have been hypothesized to be due to potential metabolites
14 with affinity to catecholamine receptors that would, in turn, influence the uptake and release of
15 neurotransmitters [Anderssonetal., 1983: Anderssonetal., 1981: Anderssonetal., 1980].
16 Catecholaminergic changes with toluene have been reported and are similar to that
17 observed with TMBs which would therefore increase the plausibility that the mechanisms of
18 neurotoxicity are similar between the two compounds. For example, subchronic inhalation
19 exposures of rats to low concentrations of toluene (as low as 80 ppm [300 mg/m3]] have been
20 shown to decrease spatial learning and memory, increase dopamine-mediated locomotor activity,
21 increase the number of dopamine D2 receptors, and increase dopamine D2 agonist receptor
22 binding (Hillefors-Berglund etal.. 1995: vonEuler etal.. 1994: vonEuler etal.. 1993]. These effects
23 were observed to persist up to four weeks after the termination of the toluene exposure.
24 Activation of the dopaminergic system may also result in an inability to inhibit locomotor
25 responses normally suppressed by punishment (Tackson and Westlind-Danielsson. 1994]. Direct
26 application of dopamine to the nucleus accumbens of rats has been observed to result in
27 retardation of the acquisition of passive avoidance learning at concentrations that also stimulated
28 locomotor activity [Braes etal.. 1984]. Increases in catecholaminergic neurotransmission (through
29 exposure to norepinephrine or dopamine agonists] result in dose-dependent reductions in the
30 duration of spike wave discharges in rats (Snead, 1995: Warter etal., 1988]. These observations
31 and findings are in concordance with those resulting from exposure to TMBs (Wiadernaetal.. 2002:
32 Gralewicz and Wiaderna. 2001: Tomas et al.. 1999a: Tomas et al.. 1999c: Gralewicz etal.. 1997b:
33 Gralewicz etal.. 1997a]. Additionally, with regards to toluene and related aromatic hydrocarbons, it
34 is known that there is direct interaction with these compounds on various ion channels (ligand and
35 voltage gated] that are present in the central nervous system (Bowenetal., 2006: Balster, 1998].
36 There is not enough information to ascertain the specific molecular sites and how the changes
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Toxicological Review of Trimethylbenzene
1 correlate to the observed neurotoxicological effects. However, it is widely believed that the
2 interactions with the neuronal receptors in the brain (e.g., ion channels, catecholaminergic
3 systems) may influence these changes.
4 Aromatic hydrocarbons may also affect the phospholipids in the nerve cell membrane
5 [Andersson et al., 1981]. Perturbation of the phospholipids on the cell membrane could indirectly
6 affect the binding of neurotransmitters to the catecholamine or other receptors and potentially lead
7 to alterations in receptor activity or uptake-release mechanisms. Uneven distribution of
8 metabolites within differing regions of the brain, or spatial variations in phospholipid composition
9 of nerve cell membranes, may explain the differential effects seen in regard to catecholamine levels
10 and turnover [Andersson etal.. 1981]. Based on effect levels with other related solvents (e.g.,
11 toluene - see Balster (1998]], it is hypothesized that with TMBs there may be an initial interaction
12 with the neuronal receptors (e.g., catecholaminergic systems, ion channels] followed by, at much
13 higher exposures, interaction with the lipid membrane when the available sites on the neuronal
14 receptors are completely occupied.
15 Additional mechanisms that may play a role in 1MB neurotoxicity include production of
16 reactive oxygen species (ROS]. Myhre et al. (2000] observed increased respiratory burst in
17 neutrophils after 1,2,4-TMB exposure demonstrated by fluorescence spectroscopy, hydroxylation of
18 4-hydroxybenzoic acid, and electron paramagnetic resonance spectroscopy. The authors suggest
19 that the observation of solvent-induced ROS production may be relevant to brain injury, as
20 microglia cells have a respiratory burst similar to neutrophils. Stronger evidence of potential ROS-
21 related mechanisms of neurotoxicity was observed in a related study by Myhre and Fonnum (2001]
22 in which rat neural synaptosomes exposed to 1,2,4-TMB produced a dose-dependent increase in
23 reactive oxygen and nitrogen species demonstrated by the formation of the fluorescence of 2'7'-
24 dichlorofluorescein. This observation of ROS production in rat synaptosomes may potentially
25 explain the observed TMB-induced neurotoxicity in acute, short-term, and subchronic inhalation
26 studies.
1.1.1.2. Summary of Neurological Effects
27 Neurotoxicity is associated with exposure to TMBs based on evidence in humans exposed to
28 mixtures containing TMBs and in animals exposed to individual TMB isomers. All three TMB
29 isomers are taken up in humans (Tarnbergetal., 1998,1997a: Tarnbergetal., 1996], and
30 occupational studies involving exposure to TMBs and other VOCs show neuropsychological effects
31 (Chen etal., 1999], deficits in short term memory and reduced motor speed/coordination (Lee et
32 al.. 2005], abnormal fatigue (Norsethetal.. 1991], and nervousness, anxiety, and/or vertigo [(Battig
33 etal.. 1956], as reviewed by MOE (2006] and (Battig etal.. 1958]]. These effects, however, cannot
34 be attributed to any specific compound. None of the available human studies have addressed the
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Toxicological Review of Trimethylbenzene
1 potential for latent neurological effects and no studies examined the potential for neurological
2 effects in sensitive populations.
3 There is strong, consistent evidence of neurotoxicity in male Wistar rats exposed to any
4 TMB isomer via inhalation across multiple concentrations and multiple durations; however, the
5 studies were all conducted at the same institute [Wiadernaetal., 2002: Gralewicz and Wiaderna,
6 2001: Wiadernaetal.. 1998: Gralewicz etal.. 1997b: Gralewicz etal.. 1997a: Korsakand Rydzynski.
7 1996: Korsak etal.. 1995). By gavage, similar effects were observed (e.g., altered EEC recordings;
8 increased locomotor activity in open field tests) [Tomas etal.. 1999a: Tomas et al.. 1999b). although
9 testing by this route was not as extensive as by inhalation.
10 The spectrum of observed effects suggests that TMBs affect multiple, possibly overlapping,
11 CNS systems rather than a single brain region or neuronal nuclei (suggested by the solvent activity
12 of the compounds). Almost all tests (including pain sensitivity) involve a contributing component of
13 motor system function. It is notable that none of the identified studies on individual TMB isomers
14 employed protocols capable of distinguishing effects on motor activity alone (e.g., the majority of
15 studies used open field tests 5-10 minutes in duration); thus, it remains to be determined whether
16 TMBs exposure specifically affects motor system function. Some endpoints exhibited clear
17 exposure-response relationships, including measures of pain sensitivity and neuromuscular
18 function, when tested immediately after exposure. Most other endpoints did not show a clear
19 concentration-effect relationship, although the direction and magnitude of responses was relatively
20 consistent across studies. In most cases, effects at 1,230 mg/m3 were less robust than those
21 observed at lower TMB concentrations (i.e., responses were nonlinear). However, nonlinear
22 relationships are not uncommon for solvents and, as they were observed across multiple studies
23 using each of the three isomers, they are considered to be biologically-relevant observations rather
24 than experimental artifacts. Latent neurological effects following TMBs exposure were consistently
25 observed, but were difficult to characterize as deficits in a single neurological function. For
26 example, latent measures of pain sensitivity following TMBs exposure, although consistent, were
27 only statistically significant when the rats were challenged with a foot shock on the prior day. The
28 most likely explanation for this observation is that TMBs exposure extends the duration of foot
29 shock-induced decreases in pain sensitivity, since the immediate response to foot shock was similar
30 across groups; yet, it cannot be ruled out that TMBs exposure could alter cognitive function,
31 resulting in the observed responses. In summary, the evidence supports a determination that TMBs
32 are neurotoxic following inhalation or oral exposure, based on consistency and coherency of effects
33 in animals and humans, biological plausibility, evidence of delayed-onset and/ or latent
34 neurological effects in animals several weeks following exposure, and observed exposure-response
35 relationships in animals tested immediately after exposure.
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Toxicological Review of Trimethylbenzene
1.1.2. Respiratory Effects
1 There is evidence in humans and animals that inhalation exposure to TMBs induces
2 respiratory toxicity. The human evidence comes from occupational and residential studies
3 involving complex VOC mixtures that include TMBs; thus, effects cannot be attributed to any TMB
4 isomer specifically. TMB isomers are associated with increased measures of respiratory irritation,
5 such as laryngeal and/or pharyngeal irritation [Norseth etal.. 1991] and asthmatic bronchitis
6 [[Battigetal.. 1956]. as reviewed in MOE [2006] and Baettig et al. [1958]] following occupational
7 exposures. Residential exposures have demonstrated significant associations between 1,2,4-TMB
8 and asthma [Billionnetetal., 2011]. Controlled human exposures [Tones etal., 2006: Tarnbergetal.,
9 1997a: Tarnbergetal., 1996] have failed to observe substantial irritative symptoms following acute
10 (less than 4 hours] inhalation exposures to TMB isomers of up to 25 ppm (123 mg/m3]. For full
11 details of the epidemiologic and controlled human exposures studies (including human subjects
12 research ethics procedures], see individual study summary tables in Appendix B.
13 In animals, there is consistent evidence of respiratory toxicity following inhalation exposure
14 of rodents to the TMB isomers (Table 1-3; Figure 1-5]. Markers of inflammation and irritation in the
15 lungs of rats have been observed following subchronic inhalation exposures of Wistar rats to
16 1,2,4-TMB or 1,2,3-TMB. Increases in immune and inflammatory cells in bronchoalveolar lavage
17 (BAL] fluid have been observed following subchronic exposures of male Wistar rats to 1,2,4-TMB at
18 concentrations > 123 mg/m3 (Korsaketal.. 1997]. Specifically, the number of cells in the BAL fluid
19 of exposed rats was increased for both total cells (> 123 mg/m3] and macrophages (> 492 mg/m3].
20 However, some attenuation of these effects was observed at high concentrations (i.e., at 1,230
21 mg/m3] compared to lower concentrations. For example, the number of macrophages was
22 increased 2.7-fold relative to control at 492 mg/m3, but only 2.2-fold at 1,230 mg/m3. This may
23 indicate either adaptation to the respiratory irritation effects of 1,2,4-TMB, saturation of metabolic
24 pathways, or immune suppression at higher doses. Subchronic exposure of male Wistar rats also
25 significantly increased the BAL fluid content of polymorphonuclear leukocytes and lymphocytes;
26 however the specific concentrations eliciting these significant increases were not reported by study
27 authors. A small, but not significant, decrease in cell viability (all cells] was observed following
28 subchronic exposure to 1,2,4-TMB at > 123 mg/m3 (Korsaketal.. 1997].
29 In addition to increases in immune and inflammatory cells in BAL fluid following exposure
30 to 1,2,4-TMB, histopathological alterations characterized by increases in lymphatic tissue in the
31 lower respiratory tract have also been observed following subchronic exposures of male and female
32 Wistar rats to 1,2,4-TMB or 1,2,3-TMB (Korsak etal.. 2000a. b]. Significant proliferation of
33 peribronchial lymphatic tissue was observed in male rats exposed to 123 mg/m31,2,3-TMB or 492
34 mg/m31,2,4-TMB and female rats exposed to 123 and 492 mg/m31,2,3-TMB, although trend
35 analysis demonstrated that these increases were not concentration-dependent. Non-concentration
36 dependent increases in interstitial lymphocytic infiltrations were also observed in male rats
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Toxicological Review of Trimethylbenzene
1 exposed to 492 mg/m31,2,4-TMB. However, statistically significant increases in interstitial
2 lymphocytic infiltrations observed in male and female rats exposed to 1,230 mg/m31,2,3-TMB or
3 1,2,4-TMB, respectively, were concentration-dependent based on trend analysis.
4 In some 1,2,4-TMB or 1,2,3-TMB-exposed rats exhibiting peribronchial lymphatic
5 proliferation, the bronchial epithelium lost its cuboidal shape and formed lymphoepithelium.
6 However, this formation of lymphoepithelium was apparently non-monotonic and not dependent
7 on concentration. Alveolar macrophages were increased in both sexes exposed to 1,230 mg/m3
8 1,2,4-TMB (significant only for males), with trend analysis demonstrating concentration-
9 dependence across the entire concentration range. Goblet cells were statistically significantly
10 increased in a concentration-dependent manner in female rats exposed to > 492 mg/m31,2,3-TMB.
11 When the incidences of all pulmonary lesions were analyzed in aggregate, lesions were significantly
12 increased in males at 492 mg/m31,2,4-TMB, but not at any concentration in females. However,
13 trend-analysis demonstrated significant increases in aggregate pulmonary lesions in both sexes
14 across the entire concentration range. In rats exposed to 1,2,3-TMB, the aggregate incidences of
15 pulmonary lesions were not statistically significantly increased at any single concentration in males
16 or females. Male rats, however, did exhibit a concentration-dependent increase in aggregate lesions
17 according to trend analysis. Studies on the respiratory effects of subchronic exposures to 1,3,5-TMB
18 were not available.
19 Additional effects on clinical chemistry including increased total protein (37% increase at
20 exposures of both 123 and 492 mg/m3), decreased mucoprotein (13% decrease at 123 mg/m3
21 exposure), increased lactate dehydrogenase (170% and 79% increase at 123 and 492 mg/m3,
22 respectively) and increased acid phosphatase activity (47-75% increase at > 123 mg/m3) were
23 observed in animals exposed to 1,2,4-TMB, suggesting pulmonary irritation or inflammation. All of
24 these effects also exhibited either some attenuation of effect at high concentrations compared to
25 lower concentrations. Therefore, some adaptation to the respiratory irritation effects of 1,2,4-TMB
26 may be occurring.
27 Decreased respiration, a symptom of sensory irritation, has been observed in male BALB/C
28 mice during acute inhalation exposures to the TMB isomers for 6 minutes. These acute exposures
29 were observed to result in dose-dependent depression of respiratory rates, with the maximum
30 decrease in respiration occurring in the first 1 or 2 minutes of exposure (Korsaketal.. 1997: Korsak
31 etal.. 1995). The concentration of 1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB that was observed to result
32 in a 50% depression in the respiratory rate (RDso) was similar between the three isomers: 578, 541,
33 or 519 ppm (2,844, 2,662, or 2,553 mg/m3), respectively.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzene
Table 1-3. Evidence pertaining to respiratory effects of TMBs in animals —
inhalation exposures
Study design3 and reference
Results
1,2,4-TMB
Pulmonary inflammation/irritation
0, 123, 492, 1,230 mg/m3, 90 days
(6 hr/day, 5 days/week)
Rat, Wistar, male, N = 6-7
Korsak et al. (1997), Table B-31
0, 123, 492, 1,230 mg/m3, 90 days
(6 hr/day, 5 days/week)
Rat, Wistar, male, N = 6-7
Korsak et al. (1997), Table B-31
0, 123, 492, 1,230 mg/m3, 90 days
(6 hr/day, 5 days/week)
Rat, Wistar, male and female, N = 10
Korsak et al. (2000a), Table B-32
Increased total bronchoalveolar cell count with evidence of attenuation
at high exposure.
Response relative to control: 0, 202***, 208**, 131*%
Increased macrophage count with evidence of attenuation at high
exposure.
Response relative to control: 0, 107, 170**, 116**%
Increase in number of pulmonary lesions.
Response relative to control: Incidences not reported, thus calculation of
response relative to control not possible; authors report statistically
significant increases at 492 and 1,230 mg/m3.
Clinical chemistry effect
0, 123, 492, 1,230 mg/m3, 90 days
(6 hr/day, 5 days/week)
Rat, Wistar, male, N = 10
Korsak et al. (1997), Table B-31
Increased acid phosphatase activity with evidence of attenuation at
exposure.
Response relative to control: 0, 47*, 74*, 45*%
high
Sensory irritation (decreased respiration)
1,245, 3,178, 5,186, 6,391, 9,486 mg/m3,
6 min
Mouse, BALB/C, male, N = 8-10
Korsak et al. (1997); Korsak et al. (1995),
Tables B-31 and B-29
Decreased respiratory rate as measured during first minute of exposure.
Response relative to control: RD50 = 2,844
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzene
Table 1-3 (Continued): Evidence pertaining to respiratory effects of TMBs in animals
— inhalation exposures
Study design3 and reference
Results
1,2,3-TMB
Pulmonary inflammation/irritation
0, 123, 492, 1,230 mg/m3, 90 days
(6 hr/day, 5 days/week)
Rat, Wistar, male and female, N = 10
Korsak et al. (2000b), Table B-33
Increase in number of pulmonary lesions.
Response relative to control: Incidences not reported, thus calculation
of response relative to control not possible; authors report statistically
significant increases at 492 and 1,230 mg/m3.
Sensory irritation (decreased respiration)
1,255, 2,514, 4,143, 7,828 mg/m3, 6 min
Mouse, BALB/C, male, N = 8-10
Korsak et al. (1997); Tables B-31
Decreased respiratory rate as measured during first minute
exposure.
Response relative to control: RD50 = 2,662
of
1,3,5-TMB
Sensory irritation (decreased respiration)
1,348, 2,160, 2,716, 3,597, 4,900 mg/m3,
6 min
Mouse, BALB/C, male, N = 8-10
Korsak et al. (1997), Table B-31
Decreased respiratory rate as measured during first minute
exposure.
Response relative to control: RD50 = 2,553
of
*, **, *** Statistically different from controls at p < 0.05, p < 0.01, and p < 0.001, respectively.
aln instances where authors reported exposures in ppm, EPA converted these values to mg/m3. See Appendix B (Table B-l) for
conversion factor, and individual study summary tables for ppm values.
Tables referenced in Study Design and Reference column correspond to study summary tables in Appendix B
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzene
1,2,4-TMB, 1,2,3-TMB,
Isomer Effect Summary
Pulmonary
1,2,4-TMB Inflammation/
Irritation
Clinical
Chemistry
Effects
Sensory
Irritation
Pulmonary
1,2,3-TMB Inflammation/
Irritation
Sensory
Irritation
1,3,5-TMB f^
Irritation
or 1,3,5-TMB
i-
Duration Endpoint
^ BAL Total
Subchronic
Cell Count (a)
1" BALMacro-
phage Count (a)
't' Pulmonary
Inflammatory
Lesions (b)
I"1 Acid
Subchronic Phosphatase
Activity (a)
d. Respiratory
Acute , i
Rate (a, c)
T1 Pulmonary
Subchronic Inflammatory
Lesions (d)
>!• Respiratory
Acute ' ,. , ,
Rate (a)
4/ Respiratory
Acute , ,
Rate (a)
Low Dose H High Dose • NOAEL • LOAEL A RD50
• i
• i
LOO 1000 1000(
Concentration (mg/m3)
Note: Solid lines represent range of exposure concentrations, (a) Korsak et al. [1997]: (b) Korsak et al. [2000a]: (c) Korsak
et al. [1995]: [d] Korsak [2000b]. Y-axis is displayed on a logarithmic scale. All subchronic effects are in male Wistar rats,
except for increased pulmonary lesions, which occur in both male and female Wistar rats; acute effects are in BALB/c
mice.
Figure 1-5. Exposure response array of respiratory effects following inhalation
exposure to 1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB.
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Toxicological Review of Trimethylbenzene
1.1.2.1. Mode of Action Analysis - Respiratory Effects
1 Data regarding the potential mode of action for the respiratory effects resulting from TMB
2 inhalation exposures are limited and the key events for TMB-induced respiratory toxicity are not
3 established. However, the available toxicity data suggest that TMB isomers act as potent acute
4 respiratory irritants and induce inflammatory responses following longer exposures (i.e.,
5 subchronic) in animals. Korsak et al. [1995] and Korsak et al. [1997] have suggested that decreased
6 respiratory rate following TMB inhalation exposure is indicative of irritation, and proposed that
7 respiratory irritants such as TMB may activate a "sensory irritant receptor" on the trigeminal nerve
8 ending in the nasal mucosa leading to an inflammatory response. Korsak et al. [1997: 1995] further
9 suggested that activation of this irritant receptor follows either adsorption of the agonist, or
10 adsorption and chemical reaction with the receptor. The authors referenced a proposed model for
11 the receptor protein that includes two main binding sites for benzene moieties and a thiol group.
12 Further, they suggested that in the case of organic solvents (i.e., toluene, xylene, and TMB], a
13 correlation between the potency of the irritating effect and the number of methyl groups is likely
14 given the observation that RDso values for depressed respiratory rates following exposure to TMB
15 isomers is approximately 8-fold lower than toluene and 4-fold lower than xylene.
16 Following subchronic inhalation exposure of rats to 1,2,4-TMB, inflammatory cell (i.e.,
17 macrophages, polymorphonuclear leukocytes, and lymphocytes] numbers were increased along
18 with markers of their activation (i.e., total lactate dehydrogenase and acid phosphatase activity in
19 BAL] (Korsak et al.. 1997]. further indicating the inflammatory nature of responses in the
20 respiratory tract of TMB-exposed animals. Inflammatory pulmonary lesions were also observed
21 following subchronic inhalation exposures in rats. However, many of these effects were not
22 observed to be concentration-dependent in repeat exposure studies (i.e., no progression of effect
23 over an order of magnitude of concentrations], suggesting that there may be adaptation to
24 respiratory irritation that occurs following extended inhalation exposure to TMB. The processes
25 responsible for the respiratory inflammatory responses observed in subchronically exposed
26 animals are unknown. However, a major inflammatory mediator, interleukin 8 (IL-8], was
27 increased following exposure of porcine and human macrophages to secondary organic aerosol
28 (SOA] particles derived from 1,3,5-TMB (Gaschenetal.. 2010]. The observation that IL-8 levels
29 increase following exposure to 1,3,5-TMB-derived SOA is noteworthy as a major function of IL-8 is
30 to recruit immune cells to sites of inflammation. Therefore, the observation of inflammatory lesions
31 involving immune cells (i.e., macrophages and leukocytes] may be partially explained by increases
32 in inflammatory cytokines following TMB exposures. Additionally, ROS-generation has been
33 observed in cultured neutrophil granulocytes and rat neural synaptosomes exposed to TMB (Myhre
34 andFonnum, 2001: Myhre etal., 2000], and the related compounds benzene and toluene have been
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzene
1 shown to induce oxidative stress in cultured lung cells [Mogeletal.. 2011). Although pulmonary
2 ROS-generation has not been observed following in vivo or in vitro 1MB exposures, there is
3 suggestive evidence that it could play a role in the irritative and inflammatory responses seen in
4 exposed animals.
5 In a study investigating jet fuel-induced cytotoxicity in human epidermal keratinocytes
6 (HEK), aromatic hydrocarbons were more potent inducers of cell death than aliphatic constituents,
7 even though the aromatic compounds only accounted for less than one-fourth of aliphatic
8 constituents [Chou etal.. 2003). Of the single aromatic ring hydrocarbons, 1,2,4-TMB and xylene
9 were the most lethal to HEK. Increased cytotoxicity may explain the small, but insignificant,
10 decrease in BAL cell viability observed in Korsak et al. [1997].
1.1.2.2. Summary of Respiratory Effects
11 Respiratory toxicity is associated with inhalation exposure to TMBs based on evidence in
12 humans and animals. All three 1MB isomers are taken up by humans [Tarnbergetal.. 1998.1997a:
13 Jarnbergetal.. 1996]. and occupational and residential studies involving exposure to TMBs and
14 other VOCs suggest an association between 1MB exposure and asthmatic symptoms [Billionnet et
15 al., 2011: Battigetal., 1956] and sensory irritation [Norseth etal., 1991]. These effects, however,
16 cannot be attributed to any specific compound.
17 There is strong, consistent evidence of respiratory toxicity in male and female Wistar rats
18 exposed to any TMB isomer via inhalation across multiple concentrations and multiple durations,
19 although the studies were conducted at the same institute [Korsak etal.. 2000a. b; Korsak etal..
20 1997: Korsak etal.. 1995]. Some endpoints (i.e., BAL macrophages and alkaline phosphatase]
21 showed concentration-dependence at low- and mid-exposures, all effects were observed to exhibit
22 some attenuation of effect at high doses, potentially indicating either adaptation to the respiratory
23 irritation effects, saturation of metabolic and/or toxicity pathways, or immune suppression at
24 higher doses. In summary, the evidence supports a determination that TMBs are respiratory
25 toxicants following inhalation exposure, based on consistency and coherency of effects observed in
26 humans and animals, biological plausibility, and observed exposure-response relationships.
1.1.3. Reproductive and Developmental Effects
27 There are no studies in humans that investigated the reproductive or maternal toxicity of
28 the TMB isomers by any route of exposure. Maternal toxicity in the form of decreased corrected
29 body weight (i.e., maternal body weight minus the weight of the gravid uterus] was observed in
30 Sprague-Dawley rat dams following inhalation exposure during gestation to 1,2,4-TMB or
31 1,3,5-TMB [Saillenfaitetal..20Q5] (Table 1-4; Figure 1-6]. Dams exposed to 2,952 mg/m3
32 1,2,4-TMB gained only 50% of the weight gained by control animals, whereas dams exposed to
33 2,952 mg/m31,3,5-TMB gained only 25% of the weight gained by controls. Decreased maternal
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Toxicological Review of Trimethylbenzene
1 food consumption (across GD6-GD21) was also observed at > 2,952 mg/m31,2,4-TMB and > 1,476
2 mg/m31,3,5-TMB, although the magnitude of the difference compared to controls (88-83% and 92-
3 75% of controls, respectively) was modest relative to the observed decreases in maternal weight
4 gain. The decrease in food consumption at 1,476 mg/m31,3,5-TMB (92% relative to controls) was
5 not considered to be a marker of adversity given no accompanying decrease in maternal weight
6 gain was observed at that concentration.
7 There are no studies in humans that investigated the developmental toxicity of either
8 1,2,4-TMB or 1,3,5-TMB by any route of exposure. Developmental toxicity (reported as decreased
9 fetal body weight) has been observed in male and female rats following gestational exposure to
10 1,2,4-TMB and 1,3,5-TMB on gestational days 6 through 20 via inhalation for 6 hours a day
11 (Saillenfaitetal., 2005) (Table 1-4). Fetal body weights were decreased (statistically significantly)
12 by 5-13% at concentrations of > 2,952 mg/m3 of 1,2,4-TMB and 1,3,5-TMB. No adverse effects were
13 noted on embryo/fetal viability and no increase in skeletal, visceral, or external morphology (i.e.,
14 teratogenesis) was observed up to the highest concentrations for either isomer. Studies on the
15 developmental or reproductive effects of 1,2,3-TMB by any route of exposure were not available.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzene
Table 1-4. Evidence pertaining to reproductive and developmental effects of
1,2,4-TMB and 1,3,5-TMB in animals — inhalation exposures
Study Design3 and Reference
1,2,4-TMB
Developmental toxicity
0, 492, 1,476, 2,952, 4,428 mg/m3,
GD6-GD20 (6 hr/day)
Rat, Sprague-Dawley, female and male0
Saillenfait et al. (2005), Table B-38
Maternal toxicity
0, 492, 1,476, 2,952, 4,428 mg/m3,
GD6-GD20 (6 hr/day)
Rat, Sprague-Dawley, female, N = 24-25
dams
Saillenfait et al. (2005), Table B-38
1,3,5-TMB
Developmental toxicity
0, 492, 1,476, 2,952, 5,904 mg/m3,
GD6-GD20 (6 hr/day)
Rat, Sprague-Dawley, female and malea'c
Saillenfait et al. (2005), Table B-38
Maternal Toxicity
0, 492, 1,476, 2,952, 5,904 mg/m3,
GD6-GD20 (6 hr/day)
Rat, Sprague-Dawley, female, N = 24-25
dams
Saillenfait et al. (2005), Table B-38
Results
Decreased fetal body weight of male and female fetuses.
Response relative to control:
Male:0, -l,-2, -5*, -11**%
Female: 0, -1, -3, -5*, -12**%
Decreased corrected maternal weight gain.
Response relative to control: 0, +7, -7, -51**, -100**%
(weight gain = 0g)
Decreased fetal body weight of male and female.
Response relative to control:
Male:0, -1, -5, -7*, -12**%
Female: 0, -1, -4, -6, -13**%
Decreased corrected maternal weight gain.
Response relative to control: 0, +3, -31,- 76**, -159**%
weight gain = -12 g)
*, ** Statistically significantly different from controls at p < 0.05 and p < 0.01, respectively.
aln instances where authors reported exposures in ppm, EPA converted these values to mg/m3. See Appendix B (Table B-l) for
conversion factor, and individual study summary tables for ppm values.
Tables referenced in Study Design and Reference column correspond to study summary tables in Appendix B.
cNumber of fetuses analyzed not reported.
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Toxicological Review of Trimethylbenzene
1,2,4-TMB, or 1,3,5-TMB
Isomer Effect Summary Endpoint Sex
1,2,4-TMB D^'°Pmental ^Fetal Male;
Toxicity Body Weight
Females
. \1/ Maternal
Maternal
Body Weight Female
T™^ Gain
1,3,5-TMB D^P™^1 4, Fetal ^
Toxicity Body Weight
Females
4/ Maternal
Maternal '...,,,,-.,
Body Weight Female
Toxicity
Gain
i- Low Dose -i High Dose • NOAEL • LOAEL
I I
i I
i i
i i
i i
i i
I • I
I * \
1,000 2,000 3,000 4,000 5,000 6,0011
Concentration (mg/m3)
Note: Solid lines represent range of exposure concentrations. All effects from Saillenfait et al. (2005).
Figure 1-6. Exposure response array of reproductive and developmental effects
following inhalation exposure to 1,2,4-TMB or 1,3,5-TMB.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzene
1.1.3.1. Summary of Reproductive and Developmental Effects
1 The database for reproductive and developmental toxicity following inhalation exposure to
2 1,2,4-TMB and 1,3,5-TMB is limited to one animal developmental study; no studies in humans are
3 available. Thus, these isomers may cause developmental toxicity, although this is based on only one
4 study that demonstrated clear, exposure-related effects on fetal and maternal body weights.
1.1.4. Hematological and Clinical Chemistry Effects
5 There is limited evidence in humans, and stronger evidence in animals, that exposure to
6 TMB isomers via inhalation induces hematological toxicity and alterations in clinical chemistry
7 parameters. Alterations in blood clotting and anemia in workers exposed to a paint solvent
8 containing 50% 1,2,4-TMB, 30% 1,3,5-TMB, and unspecified amounts of 1,2,3-TMB (listed as
9 possibly present) was reported by Battig et al. [1956]. as reviewed by MOE [2006]: effects observed
10 at 295 mg/m3. However, as workers were exposed to a solvent mixture containing multiple TMB
11 isomers and other VOCs, effects cannot be attributed to any TMB isomer specifically.
12 In animals, there is evidence of hematological toxicity following subchronic inhalation
13 exposure to 1,2,4-TMB or 1,2,3-TMB and short-term inhalation exposure to 1,3,5-TMB (Table 1-5;
14 Figures 1-7 and 1-8). Subchronic exposures to 1,2,4-TMB or 1,2,3-TMB have been shown to result
15 in hematological effects and changes in serum chemistry in rats [Korsak et al.. 2000a. b). In male
16 rats exposed to 1,230 mg/m31,2,4-TMB or 1,2,3-TMB, red blood cells (RBC) counts were
17 significantly decreased 23 and 15%, respectively. The observed alterations in RBCs were
18 concentration-dependent as determined by trend analysis. Exposure to 1,2,4-TMB or 1,2,3-TMB did
19 not significantly decrease RBCs in female rats, but trend analysis demonstrated that decreases in
20 RBC counts in female rats exposed to 1,2,3-TMB were concentration dependent, with a maximum
21 decrease of 9% at 1,230 mg/m3. RBCs in both sexes were observed to still be depressed relative to
22 controls 2 weeks following termination of exposure to both isomers, but these decreases were not
23 statistically significant.
24 White blood cell (WBC) counts were significantly increased 80% in male rats and increased
25 30% (not statistically significant) in female rats exposed to 1,230 mg/m31,2,4-TMB. After a two-
26 week follow-up after termination of exposure, WBC counts had returned to normal in female rats
27 and were slightly depressed (18%) in male rats. WBC numbers were unchanged in male rats
28 exposed to 1,2,3-TMB, but were increased (not statistically significant) 22% in female rats exposed
29 to 1,230 mg/m3. After two weeks following termination of exposure, WBC counts in male and
30 female rats had fallen to roughly 60% of controls.
31 Significant decreases in reticulocytes (71% decrease relative to controls) and clotting time
32 (37% decrease relative to controls) were observed in female rats exposed to 1,230 mg/m3 and 492
33 mg/m31,2,4-TMB, respectively. Both of these effects were concentration-dependent across the
34 entire-range of concentrations as determined by trend-analysis; animals fully recovered within 2
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1 weeks after termination of exposure. Reticulocyte numbers were statistically significantly
2 increased 60% in male rats exposed to 1,230 mg/m31,2,3-TMB, with reticulocyte numbers even
3 further increased (150%) two weeks following the termination of exposure. Reticulocyte numbers
4 in females exposed to 1,2,3-TMB were significantly increased 77% and 100% at 123 and 492
5 mg/m3, and increased 69% (not statistically significant) at 1,230 mg/m3. Reticulocyte numbers
6 were still increased in males and females 2 weeks after the termination of exposure to 1,2,3-TMB.
7 Segmented neutrophils were statistically significantly decreased 29% in male rats exposed to 1,230
8 mg/m31,2,3-TMB; statistically significant decreases of 29% and 48% were observed in female rats
9 exposed to 492 and 1,230 mg/m31,2,3-TMB. Lymphocytes were statistically increased 11% and
10 15% in male and female rats exposed to 1,230 mg/m3, respectively. Numbers of segmented
11 neutrophils and lymphocytes returned to control values 2 weeks after termination of exposure.
12 Exposure to TMB isomers was also observed to have an effect on clinical chemistry markers
13 that possibly indicate hepatic injury. Sorbitol dehydrogenase was increased at > 123 mg/m3 in male
14 rats exposed to 1,2,4-TMB (18-23% relative to controls) and at 1,230 mg/m3 in male rats exposed
15 to 1,2,3-TMB (69% relative to controls)(Korsaketal.. 2000a. b). However, the increases following
16 exposure to 1,2,4-TMB were not concentration-dependent Sorbitol dehydrogenase activity was
17 also higher in female rats exposed to 1,2,4-TMB (19-23% relative to controls) but the increases in
18 activity were not significantly higher when compared to controls. Sorbitol dehydrogenase activity
19 was not affected in female rats exposed to 1,2,3-TMB. Alanine aminotransferase was decreased
20 (23% relative to controls) and alkaline phosphatase was increased (42-45% relative to controls) at
21 1,230 mg/m3 and > 492 mg/m3 (respectively) in female rats exposed to 1,2,3-TMB. Absolute iver
22 weights were only observed to increase (9%) in male rats exposed to 1,230 mg/m31,2,3-TMB, and
23 no histopathological changes were observed in either sex exposed to 1,2,3-TMB or 1,2,4-TMB.
24 Therefore, the adversity of the observed changes in clinical chemistry parameters is unclear.
25 An increase (30% relative to controls) in aspartate aminotransferase, but no other
26 substantial hematological effects, was observed in rats 14 days following short-term exposure (6
27 hours/day, 6 days/week for 5 weeks) (Wiglusz etal.. 1975b: Wiglusz etal.. 1975a). The adversity of
28 aspartate aminotransferase is uncertain given the lack of a clear pattern in temporality (effects at
29 some days post-exposure, but not others) and the lack of accompanying liver histopathology.
30 Acute inhalation exposures of male Wistar rats to 1,500-6,000 mg/m31,3,5-TMB for 6
31 hours did not result in substantial effects on hemoglobin or RBC or WBC count (Wiglusz etal.,
32 1975b). However, the number of segmented neutrophilic granulocytes was increased in 1,3,5-TMB-
33 exposed rats up to 28 days following exposure (statistics not reported). The greatest increase in
34 granulocyte numbers (100%) was observed the day of exposure and 1 day following in rats
35 exposed to 6,000 mg/m3, although attenuation was seen 7-28 days following exposure, possibly
36 indicating induction of metabolizing enzymes or saturation of toxicity pathways. Investigation of
37 clinical chemistry parameters in rats acutely exposed to 300-3,000 mg/m3 for 6 hours did not
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Toxicological Review of Trimethylbenzene
1 reveal any consistent pattern in the levels of aspartate or alanine aminotransferases, although
2 alkaline phosphatase was statistically increased 84% in rats 7 days following exposure to 3,000
3 mg/m3 [Wigluszetal.. 1975a).
4 Slight alterations in clinical chemistry parameters and differential white blood cell counts
5 were also observed in rats following subchronic, oral exposure to 1,3,5-TMB (Table 1-6; Figure 1-9)
6 [Koch Industries. 1995b). While no hematological parameters (i.e., RBC counts, hematocrit) were
7 observed to differ between exposed rats and controls, the number of monocytes were observed to
8 increase (100-200% increase) in male rats exposed to > 200 mg/kg-day 1,3,5-TMB. Additionally, a
9 number of clinical chemistry parameters were altered in exposed rats. In female rats exposed to
10 600 mg/kg-day, sodium and chloride levels were statistically significantly decreased (2.3 and 2.7%,
11 respectively) relative to controls, and cholesterol and phosphorus were statistically significantly
12 increased (41% and 23%, respectively). In male rats, exposure to 600 mg/kg-day resulted in a
13 significant decrease (19%) in glucose levels, and significant increases in phosphorus levels and
14 alkaline phosphatase activity (17% and 46%, respectively). In a related, preliminary study (Koch
15 Industries. 1995a). hematological and clinical chemistry effects were also observed following 14
16 days of oral exposure. Female Sprague Dawley rats exposed to either 150 or 600 mg/kg-day
17 1,3,5-TMB had increased cholesterol levels, and high-dose males exhibited increased white blood
18 cell counts with corresponding increased neutrophil and lymphocyte numbers.
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Toxicological Review of Trimethylbenzene
Table 1-5. Evidence pertaining to hematological and clinical chemistry effects of
1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB in animals — inhalation exposures
Study Design3 and Reference
Results
1,2,4-TMB
Hematological toxicity
0, 123, 492, 1,230 mg/m3, 90 days (6 hr/day,
5 days/week)
Rat, Wistar, female and male, N = 10
Korsak et al. (2000a), Table B-32
0, 123, 492, 1,230 mg/m3, 90 days (6 hr/day,
5 days/week)
Rat, Wistar, female and male, N = 10
Korsak et al. (2000a), Table B-32
0, 123, 492, 1,230 mg/m3, 90 days (6 hr/day,
5 days/week)
Rat, Wistar, female and male, N = 10
Korsak et al. (2000a), Table B-32
0, 123, 492, 1,230 mg/m3, 90 days (6 hr/day,
5 days/week)
Rat, Wistar, female and male, N = 10
Korsak et al. (2000a), Table B-32
Decreased red blood cells in males only.
Response relative to control: 0, -1, -15, -23**%
(recovery = 24% decrease)
Increased white blood cells in males only.
Response relative to control: 0, 2, 4, 80**%
(recovery = 18% decrease)
Decreased reticulocytes in females only.
Response relative to control: 0, -51, -49, -71*%
(recovery = 65% increase)
Decreases in clotting time in females only.
Response relative to control: 0, -23, -37**, -27*%
(recovery = 60% increase)
Clinical chemistry effects
0, 123, 492, 1,230 mg/m3, 90 days (6 hr/day,
5 days/week)
Rat, Wistar, female and male, N = 10
Korsak et al. (2000a), Table B-32
Non-monotonic increases in sorbitol dehydrogenase in males
only.
Response relative to control: 0, 73**, 74*,73**%
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Toxicological Review of Trimethylbenzene
Table 1-5 (Continued): Evidence pertaining to hematological and clinical chemistry
effects of 1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB in animals —
inhalation exposures
Study Design3 and Reference
Results
1,2,3-TMB
Hematological toxicity
0,123, 492,1,230 mg/m3, 90 days (6 hr/day,
5 days/week)
Rat, Wistar, female and male, N = 10
Korsak et al. (2000b), Table B-33
Decreased red blood cells in males only.
Response relative to control: 0, 8, 6, -15*%
(recovery = 9% decrease)
0,123, 492,1,230 mg/m3, 90 days (6 hr/day,
5 days/week)
Rat, Wistar, female and male, N = 10
Korsak et al. (2000b), Table B-33
Decreased segmented neutrophils in males and females.
Response relative to control:
Males: 0, 2, -17, -29*% (recovery = 11% increase)
Females: 0, -15, -29*, -48*% (recovery = 15% decrease)
0,123, 492,1,230 mg/m3, 90 days (6 hr/day,
5 days/week)
Rat, Wistar, female and male, N = 10
Korsak et al. (2000b), Table B-33
Increased lymphocytes in males and females.
Response relative to control:
Males: 0,1, 6,11**% (recovery = 11% decrease)
Females: 0, 6,10,15**% (recovery = 3% increase)
0,123, 492,1,230 mg/m3, 90 days (6 hr/day,
5 days/week)
Rat, Wistar, female and male, N = 10
Korsak et al. (2000b), Table B-33
Increased reticulocytes in males and females (non-monotonic).
Response relative to control:
Males: 0, -25, 36, 61*% (recovery = 146**% increase)
Females: 0, 77*, 100**, 69% (recovery = 162**% increase)
Clinical chemistry effects
0,123, 492,1,230 mg/m , 90 days (6 hr/day,
5 days/week)
Rat, Wistar, female and male, N = 10
Korsak et al. (2000b), Table B-33
Decreased alanine aminotransferase in females only.
Response relative to control: 0, -1, -6, -23*%
0,123, 492,1,230 mg/m3, 90 days (6 hr/day,
5 days/week)
Rat, Wistar, female and male, N = 10
Korsak et al. (2000b), Table B-33
Increased alkaline phosphatase in females only.
Response relative to control: 0, 20,45*, 42*%
0,123, 492,1,230 mg/m3, 90 days (6 hr/day,
5 days/week)
Rat, Wistar, female and male, N = 10
Korsak et al. (2000b), Table B-33
Increased sorbitol dehydrogenase in males only.
Response relative to control: 0,44, 56, 69*%
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Toxicological Review of Trimethylbenzene
Table 1-5 (Continued): Evidence pertaining to hematological and clinical chemistry
effects of 1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB in animals —
inhalation exposures
Study Design3 and Reference
Results
1,3,5-TMB
Hematological toxicity
1,500, 3,000, 6,000 mg/m3, 6 hr
Samples collected 0, 1, 7, 14, and 28 days post
exposure
Rat, Wistar, male, N = 5.8
Wiglusz et al. (1975b), Table B-44
Increased segmented neutrophilic granulocytes (1-28 days
post-exposure).
Response relative to control:
Day 0: 0, 59, 118, 95%
Day 1: control response not reported
Day 7: control response not reported
Day 14: 0, 15, 184, 94%
Day 28: 0, -20, 124, 1%
Clinical chemistry effects
3,000 mg/m3, 5 weeks (6 hr/day, 6 days/week)
Samples collected 1, 3, 7, 14, and 28 days during
exposure
Rat, Wistar, male, N = 6
Wiglusz et al. (1975a), Table B-45
300-3,000 mg/m3, 6 hr, Samples collected 0, 2, 7, 14
and 28 days post exposure
Rat, Wistar, male, N = 6
Wiglusz et al. (1975a), Table B-45
Increased aspartate aminotransferase on day 14.
Response relative to control (day 14): 12*%
Increased alkaline phosphatase on day 7 post-exposure.
Response relative to control (on day 7 :0, -0.1, 0.03, 84*%
*, ** Statistically different from controls at p < 0.05 and p < 0.01, respectively.
aln instances where authors reported exposures in ppm, EPA converted these values to mg/m3. See Appendix B
(Table B-l) for conversion factor, and individual study summary tables for ppm values.
bTables referenced in Study Design and Reference column correspond to study summary tables in Appendix B.
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Toxicological Review of Trimethylbenzene
Table 1-6. Evidence pertaining to hematological and clinical chemistry effects of
1,3,5-TMB in animals — oral exposures
Study Design and Reference
Results
1,3,5-TMB
Hematological toxicity
0, 50, 200, 600 mg/kg-day, 90 days (once
5 days/week)
Rat, Sprague-Dawley, female and male, N
Koch Industries (1995b), Table B-28a
daily,
= 10
Increased monocyte levels in males only
Response relative to control:
Male: 0, 100, 200*, 100*% (recovery = 100% increase)
Clinical chemistry effects
0, 50, 200, 600 mg/kg-day, 90 days (once
5 days/week)
Rat, Sprague-Dawley, female and male, IN
Koch Industries (1995b), Table B-28
0, 50, 200, 600 mg/kg-day, 90 days (once
5 days/week)
Rat, Sprague-Dawley, female and male, IN
Koch Industries (1995b), Table B-28
0, 50, 200, 600 mg/kg-day, 90 days (once
5 days/week)
Rat, Sprague-Dawley, female and male, N
Koch Industries (1995b), Table B-28
0, 50, 200, 600 mg/kg-day, 90 days (once
5 days/week)
Rat, Sprague-Dawley, female and male, IN
Koch Industries (1995b), Table B-28
0, 50, 200, 600 mg/kg-day, 90 days (once
5 days/week)
Rat, Sprague-Dawley, female and male, N
Koch Industries (1995b) Table B-28
0, 50, 200, 600 mg/kg-day, 90 days (once
5 days/week)
Rat, Sprague-Dawley, female and male, IN
Koch Industries (1995b), Table B-28
daily,
= 10
daily,
= 10
daily,
= 10
daily,
= 10
daily,
= 10
daily,
= 10
Increased phosphorus levels in males and females
Response relative to control:
Male1 038 17*% (recovery ~~ 11% decrease)
Female: 0, 0, 5, 23*% (recovery = 13% decrease)
Decreased sodium levels in females only
Response relative to control: 0, 0, 0, -2*%
(recover = 1% decrease)
Decreased chloride levels in females only
Response relative to control: 0, 0, 0, -3*%
(recovery = 1% increase)
Increased cholesterol levels in females only
Response relative to control: 0, -3, 7, 41*%
(recovery = 21% decrease)
Decreased glucose levels in males only
Response relative to control: 0, -10, -9, -19*%
(recovery = 12% increase)
Increased alkaline phosphatase activity in males only
Response relative to control: 0, 5, 13, 46*%
(recovery = 28% decrease)
*, ** Statistically different from controls at p < 0.05 and p < 0.01, respectively.
a Tables referenced in Study Design and Reference column correspond to study summary tables in Appendix B.
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Toxicological Review of Trimethylbenzene
1,2,4-TMB and 1,3,5-TMB
Isomer Effect Summary Duration Endpoint Sex
1,2,4-TMB
1,3,5-TMB
Hematological
Toxidty
Clinical
Chemistry
Effects
Hematological
Toxidty
Clinical
Chemistry
Effects
Sub-chronic
4/Red Blood
Cells (a)
f White Blood
Cells (a)
•I- Reticulocytes
4- Clotting
Time (a)
Males
Males
Females
Females
^ Sorbitol Dehydro-
'jub-:hn:inic ' Males
genase(a)
1" Segmented
Acute Neutrophilic Males
granulocytes(b)
't'AspartateAmino-
Short-term transferaseonday Males
11 post-exposure (c)
t Alkaline
Acute Phosphataseonday Males
7 post-exposure (c)
i- Low Dose H High Dose • NOAEL • LOAEL
100 1000
Concentration (mg/m3)
10001
Note: Solid lines represent range of exposure concentrations, (a) Korsak et al. [2000a]: (b) Wiglusz et al. (1975b); (c)
Wiglusz et al. [I975a]. Y-axis is displayed on a logarithmic scale.
Figure 1-7. Exposure response array of hematological and clinical chemistry effects
following inhalation exposure to 1,2,4-TMB or 1,3,5-TMB.
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Toxicological Review of Trimethylbenzene
Effect Summary
Hematological
Toxicity
Clinical
Chemistry
Effects
1,2,3-TMB
Duration Endpoint Sex
4/ Red Blood
Subchromc .. , , Males
Cells (a)
4/ Segmented
Neutrophils (a)
\1/ Segmented
Females
Neutrophils (a)
t Lymphocytes ^
't' Lymphocytes
' Females
*}'• Reticulocytes
(a) MalS!
f- Reticulocytes
, , Females
(a)
\1/ AlanineAmino-
Subchronic Females
transferase (a)
t Alkaline
, , Females
Phosphatase(a)
t Sorbitol
. . . . . Male;
dehydrogenase(a)
i- Low Dose H High Dose • NOAEL • LOAEL
1
I
• i
I
i
1 A
• 1
1
1
«|
I
250 500 750 1,000 1,250
Concentration (mg/mD)
Note: Solid lines represent range of exposure concentrations, (a) Korsaketal. (200Qb).
Figure 1-8. Exposure response array of hematological and clinical chemistry effects
following inhalation exposure to 1,2,3-TMB.
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Toxicological Review of Trimethylbenzene
Isomer Effect Summary
Hematological
Subchromc
Toxicity
Clinical
Chemistry Subchronic
Effects
1,3,5-TMB
Duration Sex
f Monocytes(a) Males
t Phosphorus (a) Males
t Phosphorus (a) Females
4- Sodium (a) Females
4/ Chloride (a) Females
t Cholesterol (a) Females
4/ Glucose (a) Males
t Alkaline
, / v Males
Fhosphatase(a)
i- Low Do SB -i High Dose * HOAEL • LOAEL
• i
i
i
i
0 50 100 150 200 250 300 350 400 450 500 550 600 650 700
Dose (mg/kg-day)
Note: Solid lines represent range of exposure concentrations, (a) Koch Industries f!995b).
Figure 1-9. Exposure response array of hematological and clinical chemistry effects
following oral exposure to 1,3,5-TMB.
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Toxicological Review of Trimethylbenzene
1.1.4.1. Mode of Action Analysis - Hematological and Clinical Chemistry Effects.
1 The mode of action for TMB-induced hematological and clinical chemistry effects has not
2 been established. Increased sorbitol dehydrogenase activity is a marker for hepatic injury
3 [Ramaiah. 2007] and therefore, underlying hepatotoxicity could explain its increase in rats exposed
4 to 1,2,4-TMB or 1,2,3-TMB. However, absolute and relative liver weights were not observed to
5 increase with inhalation exposure to 1,2,4-TMB, and microscopic histopathological analysis of the
6 liver did not demonstrate any observable changes following exposure to either isomer. Similarly,
7 although increased cholesterol levels and alkaline phosphatase levels could indicate hepatic
8 dysfunction, no gross or histopathological lesions were observed in animals orally exposed to
9 1,3,5-TMB. The increases in WBC counts in exposed animals could be secondary to the observed
10 respiratory irritative and inflammatory effects of 1,2,4-TMB exposure in Korsak et al. [2000a:
11 19971
1.1.4.2. Summary of Hematological and Clinical Chemistry Effects
12 Hematological and clinical chemistry toxicity was observed following inhalation and oral
13 exposure to TMBs based on evidence in humans and animals. The information regarding
14 hematological toxicity in humans is limited to one study involving exposure to a complex VOC
15 mixture containing both 1,2,4-TMB and 1,3,5-TMB [Battigetal.. 1956). as reviewed in MOE [2006]
16 and Baettig et al. [1958]. Although this study reported hematological effects (alterations in clotting
17 and anemia], exposure was to a mixture of TMB isomers and other VOCs. Therefore, it is impossible
18 to attribute the effects to any TMB isomer. There is evidence of hematological effects in male and
19 female Wistar rats following inhalation exposure [Korsak etal., 2000a, b], that are roughly
20 analogous to those observed in humans. Additionally, there is some evidence of hematological and
21 clinical chemistry effects in male and female Sprague-Dawley rats following oral exposure [Koch
22 Industries. 1995bl
23 In summary, the evidence supports a determination that 1,2,4-TMB and 1,2,3-TMB result in
24 hematological toxicity following inhalation exposure, based on consistency and coherency of effects
25 across species (human and rats]. The general lack of data on hematological effects following
26 exposure to 1,3,5-TMB precludes a determination of hazard to humans for this isomer, although it
27 is reasonably anticipated given the observed effects following 1,2,4-TMB or 1,2,3-TMB exposure.
1.1.5. Carcinogenicity
28 There are no studies in humans that investigated the carcinogenic potential of the TMB
29 isomers by any route of exposure. One animal study was identified that investigated the association
30 of chronic oral exposure (via gavage] to 1,2,4-TMB and cancer endpoints (Maltoni et al., 1997]. Male
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Toxicological Review of Trimethylbenzene
1 and female Sprague-Dawley rats were exposed to a single dose of 800 mg/kg-day of 1,2,4-TMB in
2 olive oil by stomach tube for 4 days/week starting at 7 weeks of age. Exposures were terminated at
3 the end of 104 weeks (i.e., at 111 weeks of age) and the animals were keptunder observation until
4 natural death. The authors report that chronic oral exposure to 1,2,4-TMB resulted in an
5 "intermediate" reduction of survival in male rats and a "slight" reduction in females (no
6 quantitative information on survival was reported). A slight increase in total malignant tumors in
7 both sexes of rats was observed, with the incidence of head cancers being specifically increased in
8 male rats. The predominant type of head cancer identified was neuroesthesioepithelioma, which
9 arises from the olfactory neuroepithelium and is normally rare in Sprague-Dawley rats. Other head
10 cancers observed included those in the Zymbal gland, ear duct, and nasal and oral cavities. No tests
11 of statistical significance were reported for these data. When EPA performed the Fisher's exact test
12 on the incidences calculated from the reported percentages of animals bearing tumors in the
13 control and exposed animals, no statistically significant elevations in tumor incidence relative to
14 controls were observed.
15 Janik-Spiechowicz et al. [1998) investigated the genotoxicity of TMB isomers by measuring
16 three genotoxic endpoints: mutation frequency in bacteria, micronucleus formation in mice, and
17 sister chromatid exchanges in mice. Neither 1,2,4-TMB or 1,3,5-TMB induced gene mutations in any
18 Salmonella typhimurium strain tested (TA102, TA100, TA98, and TA97a). However, 1,2,3-TMB
19 induced gene mutations in all four strains in absence of rat S9 fraction. When cells were incubated
20 in the presence of S9,1,2,3-TMB did not induce gene mutation, indicating possibly that 1,2,3-TMB
21 itself is the primary mutagen. No isomer induced the formation of micronuclei in Imp:BALB/c mice
22 following i.p. injection. Males in the high-dose groups for 1,2,4-TMB and 1,3,5-TMB, but not
23 1,2,3-TMB, exhibited a statistically significant reduction in the ratio of polychromatic erythrocytes
24 to normochromatic erythrocytes, indicating bone marrow cytotoxicity. All three isomers
25 significantly increased the frequency of sister chromatid exchanges (SCEs) in Imp:BALB/c mice
26 following i.p. injection, with 1,2,4-TMB eliciting the more significant response. These results appear
27 to have occurred at doses that did not induce significant bone marrow cytotoxicity.
28 In summary, very little genotoxicity data are available on TMBs. Janik-Spiechowicz et al.
29 [1998) observed varying results in the Ames mutation assay in Salmonella, with 1,2,3-TMB, but not
30 1,2,4-TMB or 1,3,5-TMB, inducing gene mutations. Results for the in vivo assays for micronucleus
31 and SCE formation were consistent across isomers: TMB isomers were observed to induce SCEs, but
32 not micronuclei in mouse bone marrow cells. Increased frequency of SCEs indicates that DNA
33 damage has occurred as a result of exposure to these isomers, but it does not provide a specific
34 indication of mutagenic potential, as there is no known mechanistic association between SCE
35 induction and a transmissible genotoxic effect. With only one isomer (1,2,3-TMB) demonstrating a
36 positive result for gene mutation and positive SCE results for all three isomers, there is inadequate
37 evidence to conclude that any isomer is directly genotoxic.
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1.1.6. Similarities Among TMB Isomers Regarding Observed Inhalation and Oral
Toxicity
1 In the existing toxicological database for 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB, important
2 similarities have been observed in the potency and magnitude of effect resulting from exposure to
3 these three isomers in male and female Wistar rats, although some important differences also exist
4 (Table 1-7).
5 In acute studies investigating respiratory irritative effects (i.e., decreased respiratory rate),
6 the RDso for the three isomers were very similar, ranging from 2,553 to 2,844 mg/m3 (Korsaketal.,
7 1997). Measures of acute inhalation neurotoxicity, namely ECso values for decreases in rotarod
8 performance (4,694 and 4,738 mg/m3) and pain sensitivity (5,683 5,963 mg/m3), were also similar
9 for 1,2,4-TMB and 1,3,5-TMB, respectively (Korsak and Rydzynski. 1996). However, the ECso values
10 for both measures were lower following exposure to 1,2,3-TMB (3.779 and 4,172 mg/m3,
11 respectively). The observation that 1,2,3-TMB may be slightly more neurotoxic than 1,2,4-TMB or
12 1,3,5-TMB was also observed following acute oral and injection exposures. Although all three
13 isomers were observed to result in altered EEC readings, stronger and more persistent effects
14 followed a pattern of 1,2,3-TMB > 1,3,5-TMB > 1,2,4-TMB following oral exposures (Tomasetal..
15 1999al and 1,2,3-TMB > 1,2,4-TMB > 1,3,5-TMB following i.p. injections fTomas etal.. 1999cl Acute
16 exposure to both 1,2,4-TMB and 1,2,3-TMB affected motor function and/or anxiety at similar
17 exposure levels, whereas 1,3,5-TMB appeared to be slightly more potent, although the magnitude of
18 the response across isomers suggests that this difference is negligible (Tomas etal.. 1999b).
19 In short-term neurotoxicity studies, a qualitatively similar pattern of effects (inability to
20 learn passive and/or active avoidance and decreased pain sensitivity following foot shock
21 challenge) indicating altered neurobehavioral function was observed for TMBs, although some
22 quantitative differences were noted (Wiadernaetal..20Q2: Gralewicz and Wiaderna. 2001:
23 Wiaderna et al.. 1998: Gralewicz etal.. 1997b). Exposure to any isomer resulted in statistically
24 significant decreases in pain sensitivity following foot shock challenge at the same concentration,
25 although the magnitude of effect and consistency across studies was greater for 1,3,5-TMB and
26 1,2,4-TMB compared to 1,2,3-TMB (Wiaderna etal.. 2002: Gralewicz and Wiaderna. 2001:
27 Wiaderna etal.. 1998: Gralewicz etal.. 1997b). 1,2,4-TMB and 1,3,5-TMB were also observed to
28 increase motor function and/or decrease anxiety in open field tests, whereas 1,2,3-TMB was
29 observed to have no statistically significant effects fLutz etal.. 2010: Wiaderna etal.. 2002.1998:
30 Gralewicz etal.. 1997b). In contrast, increased locomotor activity elicited by amphetamine was
31 amplified following exposure to 1,2,3-TMB, but not 1,2,4-TMB (Lutz etal.. 2010). All three isomers
32 elicited effects on cognitive function, as measured by learning decrements in two-way active
33 avoidance or by decreased fear responses in a passive avoidance test paradigm(Wiaderna et al.,
34 2002: Gralewicz and Wiaderna. 2001: Wiaderna etal.. 1998: Gralewicz et al.. 1997b). 1,3,5-TMB
35 was observed to be the most potent isomer in this regard, eliciting effects on both passive and
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1 active avoidance at > 123 mg/m3.1,2,3-TMB and 1,2,4-TMB affected passive avoidance
2 performance at > 123 and > 492 mg/m3, respectively, and both 1,2,3-TMB and 1,2,4-TMB affected
3 the ability to learn active avoidance at 492 mg/m3. For all isomers, short-term exposure to 1,230
4 mg/m3 TMB was nearly always less effective (or ineffective), as compared to lower TMB
5 concentrations, at eliciting responses (i.e., responses were nonlinear).
6 Following subchronic exposure to either 1,2,4-TMB or 1,2,3-TMB, both decreased pain
7 sensitivity and decreased rotarod performance were observed. With regard to decreased pain
8 sensitivity, although 1,2,3-TMB was observed to decrease pain sensitivity at a lower concentration
9 than 1,2,4-TMB, the magnitude of effect was similar between isomers at every concentration
10 (Korsak and Rydzynski. 1996). For either isomer, effects on pain sensitivity appeared to be
11 reversible at 1,230 mg/m3 TMB; lower concentrations were nottested. 1,2,3-TMB was more potent
12 than 1,2,4-TMB in reducing rotarod performance. Specifically, 1,2,3-TMB elicited effects at a lower
13 concentration and caused a greater magnitude of effect at each concentration, as well as following a
14 period of recovery (Korsak and Rydzynski. 1996).
15 Similarities were also observed in 1,2,4-TMB- and 1,3,5-TMB-induced developmental and
16 maternal effects (Saillenfaitetal.. 2005). Male fetal weights were significantly reduced in animals
17 exposed gestationally to 2,952 mg/m31,2,4-TMB (5% decrease) or 1,3,5-TMB (7% decrease).
18 1,2,4-TMB also significantly decreased female fetal weights by approximately 5% in animals
19 exposed to the same concentration. Although, 1,3,5-TMB significantly reduced female fetal weights
20 by 13% in animals exposed to 5,904 mg/m3, female fetal weights were decreased at 2,952 mg/m3 to
21 a similar degree (6%) as animals exposed to the same concentration of 1,2,4-TMB. Maternal
22 toxicity, measured as decreased corrected maternal weight gain, was significantly decreased in
23 animals exposed to 2,952 mg/m31,2,4-TMB or 1,3,5-TMB. However, 1,3,5-TMB exposure resulted
24 in a 75% reduction of maternal weight gain compared to controls, whereas 1,2,4-TMB exposure
25 reduced maternal weight gain by 50%.
26 Lastly, 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB were observed to elicit hematological toxicity
27 in exposed animals. Although all three isomers were observed to qualitatively affect similar
28 hematological parameters, the direction and magnitude of effect often differed between isomers.
29 Red blood cells were significantly decreased in male rats exposed to 1,230 mg/m31,2,3-TMB (23%
30 decrease) or 1,2,4-TMB (15% decrease) (Korsak et al.. 2000a. b). Reticulocyte numbers were also
31 altered in rats following exposure to these isomers, although 1,2,4-TMB was observed to
32 significantly decrease reticulocytes in male rats at 1,230 mg/m3 (71% decrease), while exposure to
33 1,2,3-TMB increased reticulocytes in male rats at 1,230 mg/m3 (61% increase) and female rats at
34 123 and 492 mg/m3 (77% and 100% increases, respectively). 1,2,3-TMB and 1,2,4-TMB were also
35 altered the numbers of white blood cells in exposed animals following subchronic exposures. In
36 male rats exposed to 1,230 mg/m31,2,4-TMB, white blood cell numbers were significantly
37 increased by 80%. Exposure to 1,230 mg/m31,2,3-TMB also increased lymphocyte numbers by
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Toxicological Review of Trimethylbenzene
1 11% and 15% in male and female rats, respectively. Exposure to 1,230 mg/m31,2,3-TMB decreased
2 segmented neutrophils by 29% in male rats, whereas exposure to 492 mg/m3 and 1,230 mg/m3
3 decreased neutrophil numbers in female rats by 29% and 48%, respectively. Acute exposure (six
4 hours) to 1,500 - 6,000 mg/m31,3,5-TMB was also reported to result in increased numbers of
5 segmented neutrophils that persisted for up to 28 days post exposure [Wiglusz etal., 1975b]. A
6 summary of these comparisons across isomers is presented below in Table 1-7.
Table 1-7. Similarities between 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB regarding
observed inhalation and oral toxicity
Health Outcome Measure
Pain Sensitivity
Pain Sensitivity following foot
shock challenge
Neuromuscular Function
Motor Function / Anxiety
Sensitization
Cognitive Function
Electrocortical activity
Respiratory Effects
Developmental Effects
Hematological Effects
Exposure Duration
acute
subchronic
short-term
acute
subchronic
short-term
short-term
short-term
acute
acute
gestational
subchronic
TMB Isomer Potency
1,2,3-TMB > 1,2,4-TMB * 1,3,5-TMB
1,2,4-TMB * 1,2,3-TMB
1,2,4-TMB * 1,3,5-TMB > 1,2,3-TMB
1,2,3-TMB > 1,2,4-TMB * 1,3,5-TMB
1,2,3-TMB > 1,2,4-TMB
1,2,4-TMB * 1,3,5-TMB » 1,2,3-TMB
1,2,3-TMB > 1,2,4-TMB
1,3,5-TMB > 1,2,4-TMB * 1,2,3-TMB
1,2,3-TMB » 1,3,5-TMB > 1,2,4-TMB
1,2,4-TMB * 1,3,5-TMB * 1,2,3-TMB
1,2,4-TMB = 1,3,5-TMB
1,2,4-TMB * 1,2,3-TMB
1.1.7. Similarities Among TMB Isomers Regarding Toxicokinetics
7 In the existing toxicokinetic database for 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB, important
8 similarities have been observed in the chemical properties and absorption, distribution,
9 metabolism, and excretion profiles for these isomers in animals and humans, although some
10 important differences also exist
11 All three isomers have very similar Log Kow values (3.42-3.78), and blood:air partition
12 coefficients reported for humans and rats in the literature are similar: 43.0 and 55.7 for 1,2,4-TMB,
13 66.5 and 62.6 for 1,2,3-TMB, and 59.1 and 57.7 for 1,3,5-TMB fMeulenberg and Viiverberg. 20001
14 This gives an indication that the three isomers would partition into the blood in a similar fashion.
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1 Supporting this is the observation that 1,2,4-TMB and 1,3,5-TMB absorb equally into the
2 bloodstream of exposed humans (6.5 and 6.2 |iM, respectively), although the absorption for 1,2,3-
3 1MB was observed to be higher (7.3 [iM) (Jarnbergetal.. 1998.1997a: Jarnbergetal.. 1996). Also,
4 the net respiratory uptake of 1,2,3-TMB, 1,2,4-TMB and 1,3,5-TMB was similar among humans (48-
5 60%), and the respiratory uptake for 1,2,4-TMB was similar across humans and rats (50-
6 60%)(Tarnbergetal.. 1996: Dahl etal.. 1988). Although no data exist regarding the distribution of
7 TMB isomers in humans, experimentally-derived tissue-specific partition coefficients were similar
8 for all three isomers across a number of organ systems (Meulenberg and Vijverberg. 2000).
9 strongly suggesting that the individual isomers can be expected to distribute similarly to these
10 various organ systems. Distribution of the 1,2,4-TMB and 1,3,5-TMB throughout the body is
11 qualitatively similar in animals, although it appears that liver and kidney concentrations for
12 1,2,4-TMB are greater than those for 1,3,5-TMB after both acute and short-term inhalation
13 exposures fSwiercz etal.. 2006: Swiercz etal.. 2003: Swiercz etal.. 20021 Although 1,2,4-TMB was
14 observed to distribute to the brain (Swiercz etal.. 2003: Eide and Zahlsen. 1996}. distribution of
15 1,3,5-TMB to the brain was not experimentally measured in any study. However, the predicted
16 brain:air partition coefficient was similar between 1,2,4-TMB and 1,3,5-TMB for both humans (206
17 vs. 199) and rats (552 vs. 535) (Meulenberg and Vijverberg. 2000). This strongly suggests that
18 1,2,4-TMB and 1,3,5-TMB can be expected to distribute similarly to the brain in both humans and
19 rats. Detailed information regarding the distribution of 1,2,3-TMB following inhalation exposure is
20 lacking. However, similar tissue-specific partition coefficients for 1,2,3-TMB compared to 1,2,4-TMB
21 and 1,3,5-TMB indicate a similar pattern of distribution can be reasonably anticipated (Meulenberg
22 and Vijverberg. 2000).
23 All three TMB isomers were observed to primarily metabolize to benzoic and hippuric acids
24 in humans and rats (Tarnbergetal., 1996: Huoetal., 1989: Mikulski and Wiglusz, 1975), although
25 the amounts of inhaled TMB recovered as hippuric acid metabolites following exposure to 1,2,3-
26 TMB, 1,2,4-TMB, or 1,3,5-TMB was dissimilar in humans (11%, 22%, and 3%, respectively) and rats
27 (10%, 24-38%, and 59%, respectively) flarnberg etal.. 1996: Mikulski and Wiglusz. 19751 Greater
28 amounts of urinary benzoic acid and hippuric acid metabolites (73%) were observed after
29 exposure to higher amounts of 1,3,5-TMB (up to 30.5 ppm) for 8 hours (Kostrzewskietal.. 1997:
30 Kostrewski and Wiaderna-Brycht. 1995). Other terminal metabolites included mercapturic acids
31 (~14-19% total dose), phenols (~12% total dose), and glucuronides and sulphuric acid conjugates
32 (4-9% total dose) for 1,2,4-TMB; mercapturic acids (-5% total dose), phenols (
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Toxicological Review of Trimethylbenzene
1 In humans, the half-lives of elimination from blood were observed to be similar for all
2 isomers in the first three phases of elimination: 1,2,4-TMB (1.3 ± 0.8 min, 21 ± 5 min, 3.6 ± 1.1 hr),
3 1,2,3-TMB (1.5 ± 0.9 min, 24 ± 9 min, 4.7 ± 1.6 hr), and 1,3,5-TMB (1.7 ± 0.8 min, 27 ± 5 min, 4.9 ±
4 1.4 hr) (Tarnbergetal., 1996). The half-life of elimination for 1,3,5-TMB in the last and longest
5 phase is much greater than those for 1,2,4-TMB or 1,2,3-TMB (120 ± 41 hr vs. 87 ± 27 and 78 ± 22
6 hr, respectively). Urinary excretion of unchanged parent compound was extremely low (<0.002%)
7 for all three isomers (Tanasik et al.. 2008: Tarnbergetal.. 1997b). The difference observed in half-
8 lives between the three isomers in the last elimination phase may be due to small sample sizes and
9 difficulties in measuring slow elimination phases rather than a true difference in half-lives. All three
10 isomers were eliminated via exhalation: 20-37% of the absorbed dose of 1,2,4-TMB, 1,2,3-TMB, or
11 1,3,5-TMB was eliminated via exhalation during exposure to 123 mg/m3 (25 ppm) for 2 hours
12 (Tarnbergetal., 1996). At low concentrations in rats, half-life of elimination from the blood was
13 greater for 1,2,4-TMB compared to 1,3,5-TMB (3.6 vs. 2.7 hours). This difference became much
14 greater with increasing doses (17.3 hours for 1,2,4-TMB and 4 hours for 1,3,5-TMB following
15 exposure to 1,230 mg/m3 for 6 hours) fSwiercz etal.. 2003: Swiercz etal.. 20021 For a full
16 discussion of the chemical properties and toxicokinetics 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB see
17 Appendices B.I and B.2.
1.2. Summary and Evaluation
1.2.1. Weight of Evidence for Effects Other Than Cancer
18 In both humans and animals, inhalation exposure to TMBs has been shown to result in
19 toxicity in multiple organ systems, including the nervous, respiratory, and hematological systems.
20 In addition, developmental toxicity has been observed in animals exposed to either 1,2,4-TMB or
21 1,3,5-TMB. Generally, the information regarding inhalation toxicity in humans is limited for a
22 number of reasons, including that the majority of human studies involved exposure to complex VOC
23 mixtures containing several TMB isomers and other VOCs, and not the individual isomers
24 themselves. Therefore, the observed health effects cannot be attributed to specific TMB isomers.
25 However, these studies observe effects in exposed human populations that are generally analogous
26 to effects observed in animal toxicity studies, and provide qualitative, supportive evidence for
27 hazard identification. Currently, no human studies exist that investigate the oral toxicity of any TMB
28 isomer. Potential limitations in the animal inhalation and oral toxicity database for TMBs include
29 the lack of a chronic study and the fact that all of the available inhalation animal studies were
30 conducted by the same research group: The Nofer Institute of Occupational Medicine, Lodz Poland.
31 The most strongly and widely supported manifestation of toxicity in humans and animals
32 following inhalation exposure to 1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB is neurotoxicity. In humans
33 exposed to TMB-containing VOC mixtures, a multitude of effects, including neuropsychological
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1 effects [Chenetal.. 1999). deficits in short-term memory and reduced motor speed/coordination
2 [Lee etal.. 2005]. abnormal fatigue [Norseth et al.. 1991]. dysfunction of the inner ear/vertigo
3 [Sulkowskietal.. 2002]. and nervousness, anxiety, and/or vertigo [Battig et al. [1956]. as reviewed
4 by MOE [2006] and Baettig et al. [1958]], have been observed. None of the available human studies
5 have addressed the potential for latent neurological effects and no studies examined the potential
6 for neurological effects in sensitive populations. Although the reported human symptoms do not
7 directly parallel the animal data, exposure of male Wistar rats to the TMB isomers has been shown
8 to consistently result in a multitude of neurotoxic effects, including decreased pain sensitivity,
9 impaired neuromuscular function and coordination, altered cognitive function, decreased anxiety
10 and/or increased motor function, and neurophysiological effects (e.g., decreased electrocortical
11 activity] across multiple concentrations and durations [Wiadernaetal., 2002: Gralewicz and
12 Wiaderna. 2001: Wiadernaetal.. 1998: Gralewicz etal.. 1997b: Gralewicz etal.. 1997a: Korsakand
13 Rydzynski. 1996: Korsak etal.. 19951
14 The effects observed in the animal neurotoxicity studies are recognized in the U.S. EPA's
15 Guidelines for Neurotoxicity Risk Assessment [U.S. EPA. 1998] as possible indicators of neurotoxicity.
16 The effects observed include concentration-dependent decrements in pain sensitivity in hot plate
17 tests and neuromuscular function in rotarod tests following subchronic exposure. Although effects
18 on pain sensitivity appeared to be reversible atthe highest concentration (i.e., 1,230 mg/m3],
19 reversible effects occurring in occupational settings may be of high concern, particularly if they
20 diminish a person's ability to survive or adapt to the environment [(U.S. EPA. 1998]. pg.8]; such is
21 the case for exposure to TMBs in occupations with dangerous surroundings and/ or heavy
22 equipment, such as dockyard painters or asphalt workers. These effects are supported by
23 additional data from short-term exposure studies that consistently identified latent effects of TMBs
24 exposure on pain sensitivity in hot plate tests following an environmental challenge (i.e., foot
25 shock], alongside reproducible learning decrements in passive and active avoidance experiments,
26 altered EEC patterns, and increased locomotor activity in open field tests. Further, the data from
27 these short-term studies clearly indicated a persistence of neurological effects several weeks after
28 TMB exposures had ended and identified a consistent nonlinearity in many of the TMB-elicited
29 responses (e.g., 1,230 mg/m3 was nearly always substantially less effective than 123 or 492
30 mg/m3]. The neurotoxic effects are biologically plausible and analogous to effects that could occur
31 in humans. Thus, the evidence for TMBs identifies neurotoxicity as a toxicity hazard based on
32 consistency and coherency of effect across multiple studies and durations of exposure.
33 Three acute oral studies (Tomas etal., 1999a: Tomas et al., 1999b: Tomas etal., 1999c]
34 observe similar effects as observed in the available inhalation neurotoxicity studies (i.e., increased
35 locomotor activity and altered brain wave activity]. However, these studies are also limited with
36 regard to the range of endpoints investigated, and as such, no weight of evidence determination can
37 be made regarding the chronic oral toxicity of the TMB isomers.
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1 In addition to neurotoxicity, both respiratory and hematological toxicity have been
2 observed in human populations and animals exposed to TMBs, or to mixtures containing the three
3 isomers. In humans, occupational and residential exposure to VOC mixtures containing 1MB
4 isomers have resulted in number of effects characterized as respiratory toxicity, including
5 asthmatic bronchitis [[Battigetal.. 1956], as reviewed in MOE [2006] and Baettig et al. [1958]].
6 asthma [Billionnetetal.. 2011]. or laryngeal/pharyngeal irritation [Norseth etal.. 1991].
7 Additionally, workers exposed to a VOC mixture containing 1,2,4-TMB and 1,3,5-TMB, and possibly
8 1,2,3-TMB, were reported to exhibit hematological effects including alterations in clotting time and
9 anemia [[Battigetal.. 1956]. as reviewed in MOE [2006] and Baettig et al. [1958]]. Again, as
10 workers were exposed to complex VOC mixtures containing TMB isomers, the observed health
11 effects cannot be attributed to any single TMB isomer.
12 The observation of respiratory irritation and inflammation in Wistar rats and BALB/C mice
13 following exposure to 1,2,4-TMB was consistent across multiple concentrations, and subchronic
14 and acute exposure durations [Korsaketal.. 2000a: Korsaketal.. 1997: Korsaketal.. 1995].
15 Respiratory toxicity was also observed in multiple studies involving exposure to 1,2,3-TMB [Korsak
16 etal.. 2000b: Korsaketal.. 1995]. Although the reported symptoms in humans (laryngeal and/or
17 pharyngeal irritation, asthmatic bronchitis, and asthma] do not directly parallel the effects
18 observed in animal studies, the observation of irritative and/or inflammatory responses in multiple
19 species (including humans] demonstrates a consistency in TMB-induced respiratory toxicity.
20 Additionally, multiple measures of hematological toxicity have been observed in rats subchronically
21 exposed to 1,2,4-TMB or 1,2,3-TMB, including decreased RBCs, increased WBCs, decreased clotting
22 time, and decreased reticulocytes (1,2,4-TMB] and decreased RBCs, decreased segmented
23 neutrophils, increased lymphocytes and increased reticulocytes (1,2,3-TMB] [Korsak et al.. 2000a.
24 b]. At least two of these effects, decreased RBCs and decreased clotting time, are roughly analogous
25 to the hematological effects (alterations in clotting and anemia] observed in occupationally exposed
26 humans, thereby demonstrating a consistency and coherency of effect across species. Therefore, the
27 respiratory and hematological effects observed in animals are biologically plausible and analogous
28 to effects that could occur in exposed human populations. The available evidence for 1,2,4-TMB and
29 1,2,3-TMB identified respiratory and hematological toxicity as a hazard.
30 Currently, no human studies exist that investigate the reproductive or developmental
31 toxicity of 1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB. However, one animal study (Saillenfait etal.. 2005]
32 observed effects on fetal body weights and maternal body weight gains due to gestational exposure
33 to 1,2,4-TMB or 1,3,5-TMB. Although the weight of evidence regarding developmental toxicity is not
34 as strong compared to other measures of toxicity in the TMB database, these effects observed in
35 animals are considered biologically plausible and potentially analogous to effects that could occur
36 in humans. The available evidence for 1,2,4-TMB and 1,3,5-TMB identifies maternal and
37 developmental toxicity as a hazard.
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1.2.2. Weight of Evidence for Carcinogenicity
1 Under the Guidelines for Carcinogen Risk Assessment [2005a], the database for the TMBs
2 provides "inadequate information to assess carcinogenic potential" of these isomers. This
3 characterization is based on the fact that there is no information regarding the carcinogenicity of
4 TMB in humans and that the only animal study available on the carcinogenicity of 1,2,4-TMB
5 observed no statistically significant carcinogenic effects. No studies regarding the carcinogenicity of
6 1,2,3-TMB or 1,3,5-TMB were identified in the available scientific literature.
7 In the animal carcinogenicity study identified [Maltoni et al.. 1997]. involving exposure to
8 1,2,4-TMB by oral gavage, an increased incidence of total malignant tumors in both sexes and head
9 cancers (predominantly neuroethesioepithelioma) in males was observed in exposed rats, no
10 statistical analyses were reported. When EPA independently performed the Fisher's exact test on
11 the reported data, no statistically significant effects were observed.
12 Additionally, in the only study investigating the genotoxicity of TMB isomers, Janik-
13 Spiechowicz et al. [1998] observed negative results in in vitro genotoxicity assays (i.e., Ames
14 mutation assay in Salmonella) involving 1,2,4-TMB and 1,3,5-TMB. However, 1,2,3-TMB was
15 observed to induce gene mutations in all Salmonella typhimurium strains tested. All three isomers
16 failed to induce micronuclei in mouse bone marrow cells. Janik-Spiechowicz et al. [1998] observed
17 an increased incidence of SCE in mice exposed to all three TMB isomers (individually); however,
18 this observation does not provide a specific indication of mutagenic potential. Given the findings
19 regarding the in vitro genotoxicity of the TMB isomers, and increased frequency SCEs does not
20 provide specific indication of mutagenic potential, the evidence is inadequate to conclude that any
21 TMB isomer is genotoxic.
1.2.3. Susceptible Populations and Lifestages
22 Although there are no chemical-specific data that would allow for the identification of
23 susceptible populations and lifestages, the reduced metabolic and elimination capacities in children
24 relative to adults may be a source of susceptibility [Ginsberg et al.. 2004). TMB isomers are
25 metabolized following inhalation and oral exposure via side-chain oxidation to form alcohols and
26 aromatic carboxylic/mercapturic acids or by hydroxylation to form phenols, which are then
27 conjugated with glucuronic acid, glycine, or sulfates for urinary excretion. The activities of multiple
28 cytochrome P450 (GYP P450) mono-oxygenase isozymes have been shown to be reduced in
29 children up to 1 year of age compared to adult activities [Ginsberg et al.. 2004). Additionally, the
30 rate of glucuronidation and sulfation is decreased in children. Therefore, as both GYP P450 mono-
31 oxygenase activities and the rate of glucuronidation and sulfation appear to be decreased in early
32 life, newborns and young infants may experience higher and more persistent blood concentrations
33 of the TMB isomers, and/or their respective metabolites compared with adults at similar exposure
34 levels. Reduced renal clearance in children may be another important source of potential
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1 susceptibility. 1MB isomers and their metabolites are excreted in the urine of exposed laboratory
2 animals and occupationally exposed humans. Data indicating reduced renal clearance for infants up
3 to 2 months of age [Ginsberg et al.. 2004] may suggest a potential to affect 1MB excretion, thus
4 possibly prolonging its toxic effects. Additionally, those with pre-existing respiratory diseases (e.g.,
5 asthma) may be more sensitive to the respiratory irritative and inflammatory effects of TMB
6 isomers.
7
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2. DOSE-RESPONSE ANALYSIS
2.1. Inhalation Reference Concentration for Effects Other Than Cancer
for 1,2,4-TMB
1 The RfC (expressed in units of mg/m3) is defined as an estimate (with uncertainty spanning
2 perhaps an order of magnitude) of a continuous inhalation exposure to the human population
3 (including sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects
4 during a lifetime. It can be derived from a NOAEL, LOAEL, or the 95% lower bound on the
5 benchmark concentration (BMCL), with UFs generally applied to reflect limitations of the data used.
2.1.1. Identification of Studies and Effects for Dose-Response Analysis for 1,2,4-TMB
6 The nervous, respiratory, hematological systems, as well as pregnant animals and the
7 developing fetus, are the primary targets of inhaled 1,2,4-TMB in humans and experimental
8 animals, and effects in these systems have been identified as hazards following inhalation exposure
9 to 1,2,4-TMB.
10 The selection of studies and general procedures for dose-response analysis are outlined in
11 Sections 6 and 7 of the Preamble. Human data are preferred over animal data for deriving reference
12 values when possible because the use of human data is more relevant in the assessment of human
13 health and avoids the uncertainty associated with interspecies extrapolation introduced when
14 animal data serve as the basis for the reference value. In this case, while literature exists on the
15 effects of 1,2,4-TMB exposure in humans, including neurological, respiratory, and hematological
16 toxicities, no human studies are available that would allow for dose-response analysis. The human
17 studies evaluated TMB exposures occurring as complex solvents or VOC mixtures, and this
18 confounding along with other uncertainties including high imprecision in effect measures due to
19 low statistical power, lack of quantitative exposure assessment, and lack of control for
20 co-exposures, limit their utility in derivation of quantitative human health toxicity values. However,
21 these studies provide supportive evidence for the neurological, respiratory, and hematological
22 toxicity of TMB isomers in humans and indicate a coherency of effects in both humans and
23 laboratory animals.
24 Several studies investigating 1,2,4-TMB effects in experimental animal models were
25 identified in the literature. No chronic studies were available, although acute, short-term,
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1 subchronic, and developmental toxicity studies were identified. 1,2,4-TMB-induced toxicity was
2 observed across several organ systems in three subchronic studies by Korsak et al., [2000a: 1997]
3 and Korsak and Rydzyhski [1996]. and in pregnant animals and developing fetuses in a
4 developmental toxicity study by Saillenfait et al. [2005]. These four studies were the only
5 subchronic or developmental studies identified in the peer-reviewed literature. Data from these
6 studies pertaining to the primary hazards observed in humans and animals identified in Chapter 1
7 (neurological, respiratory, and hematological toxicity] or in animals only (maternal and
8 developmental toxicity] were considered as candidate critical effects for the purpose of
9 determining the point of departure (POD] for derivation of the inhalation RfC for 1,2,4-TMB.
10 Neurotoxicity was also observed in both acute and short-term inhalation studies and respiratory
11 toxicity was also observed in acute studies. However, the high concentrations used in acute studies
12 and the short exposure durations employed in both acute and short-term studies limit their utility
13 for the quantitation of chronic human health effects. Nevertheless, as with the human mixture
14 studies, these studies provide qualitative information regarding hazard identification, especially the
15 observation of the consistency and coherency of these effects across the 1,2,4-TMB database.
16 The three subchronic studies by Korsak et al., (2000a: 1997] and Korsak and Rydzyhski
17 (1996]. and the developmental toxicity study by Saillenfait et al. (2005]. adequately supported dose
18 response analysis. All four studies exposed rats, a common model for human response, by
19 inhalation, to 1,2,4-TMB (reported as > 97-99% pure [impurities not reported]]. All studies used at
20 least three exposure levels, spaced approximately threefold apart All controls were exposed under
21 similar conditions to untreated air. The durations of exposure, subchronic or gestational, were
22 suitable for the effects under evaluation: neurological, developmental, and short-term general
23 toxicity. In addition, the persistence of some outcomes after termination of exposure was
24 investigated. Typical numbers of animals per exposure group for these study designs were used: at
25 least 10/group for the subchronic studies [Korsak et al., (2000a: 1997]. Korsak and Rydzyhski
26 (1996]]: and 25/group for the developmental study (Saillenfait et al. (2005]. Regarding exposure
27 characterization, Korsak et al. (2000a] and Saillenfait et al. (2005] reported actual concentrations,
28 as measured by gas chromatography, to be within 10% of target concentrations. This increases the
29 confidence in the overall adequacy of these studies. Although Korsak and Rydzyhski (1996] and
30 Korsak et al. (1997] did not report actual, measured concentrations, these studies used the same
31 exposure methodology as Korsak et al. (2000a]: suggesting that it is likely that the actual
32 concentrations in these studies were also within 10% of target concentrations. Target and actual
33 concentrations are presented in Table 2-1.
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Table 2-1. Target and actual inhalation concentrations, and internal blood dose
metrics of 1,2,4-TMB calculated using the available rat PBPK model
(Hissinketal., 2007)
Reference
Korsak and
Rydzyriski
(1996)
Korsak et al.
(1997)
Korsak et al.
(2000a)
Saillenfait et
al. (2005)
Species/
sex
Rat, male
Rat, male
Rat, male
Rat, female
Rat:
Female
(pregnant
dam); Male
and female
(fetuses)
Body
weight (kg)a
0.387
0.404
0.403
0.383
0.409
0.416
0.390
0.399
0.389
0.243
0.230
0.229
--
--
--
--
Exposure concentration
(mg/m3)b
123
492
1,230
123
492
1,230
123 (129)
492 (492)
1,230 (1,207)
123 (129)
492 (492)
1,230 (1,207)
492 (492)
1,476 (1,471)
2,952 (2,913)
4,428 (4,408)
Internal dose - average
weekly venous blood
concentration (mg/L)
0.1272
0.8666
5.4424
0.1272
0.8661
5.4274
0.1339
0.8671
5.2481
0.1335
0.8899
5.5189
n/a
n/a
n/a
n/a
aFor Korsak et al. (2000a; 1997), exposure group-specific terminal body weights from those studies were used to calculate
internal dose metrics; for Korsak and Rydzyriski (1996) the average of the exposure group-specific body weights reported in
Korsak et al. (2000a; 1997) were used in internal dose metric calculations. For Saillenfait et al. (2005), body weights were not
provided so the PBPK model was not used to derive internal dose metrics for this study
b For Korsak et al. (2000a) and Saillenfait et al. (2005), values in parentheses are actual concentrations, as measured by gas
chromatography
Rat PBPK model (Hissinket al., 2007)
1 These subchronic and developmental toxicity studies examined 1,2,4-TMB-induced toxicity
2 in multiple organ systems and neurological, respiratory, hematological, maternal, or developmental
3 toxicity endpoints that demonstrated statistically significant increases or decreases relative to
4 control were considered for the derivation of the RfC for 1,2,4-TMB (Table 2-2). The endpoints
5 included decreased pain sensitivity in male rats [Korsak and Rydzynski. 1996]. increased BAL total
6 cells in male rats [Korsak etal.. 1997]. increased inflammatory lung lesions, decreased RBCs, and
7 increased WBCs in male rats and decreased reticulocytes and clotting time in female rats [Korsak et
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1 al., 2000a], and decreased fetal weight (males and females) and decreased maternal weight gain
2 [Saillenfaitetal.. 2005). Increases in BAL polymorphonuclear leukocytes and lymphocytes
3 observed in the Korsak et al. [1997] study were not considered for RfC derivation due to a lack of
4 reporting of exposures at which statistically significant increases occurred. Additionally, Korsak et
5 al. [1997] reported that 123 mg/m3 was the LOAEL for increased BAL total cells, butthe NOAEL for
6 increased BAL macrophages. Therefore, increased BAL macrophages were not considered for RfC
7 derivation as these effects were not observed at concentrations that elicited an increase in total
8 BAL cells. Changes in BAL protein and enzyme activity level were not considered due to non-
9 monotonically increasing dose-responses, and increases in sorbitol dehydrogenase were not
10 further considered due to the lack of accompanying hepatocellular histopathological alterations in
11 exposed animals.
12 Impaired neuromuscular function and coordination, measured as performance deficits on
13 the rotarod apparatus, was also observed in rats exposed to 1,2,4-TMB. The use of rotarod data
14 from Korsak and Rydzyhski [1996] was initially considered as a candidate critical effect for
15 1,2,4-TMB. However, upon critical evaluation of the exposure-response information in the study, it
16 was determined that rotarod performance was reported in a manner that reduced the confidence in
17 the observed effect levels. The most widely used and accepted measure of rotarod performance in
18 rodents is latency to fall from the rotating rod [Brooks and Dunnett. 2009: Kaspar etal.. 2003: Bogo
19 etal., 1981], typically with an arbitrary upper limit on the maximum latency allowed to prevent
20 confounding by fatigue. The primary limitation for these data was that rotarod performance was
21 presented as percent of failures to last 2 minutes on the apparatus. Although the quantal percent
22 failures data can provide useful information, these measures require an arbitrary selection of the
23 length of time required for successful performance; there is no scientific consensus on an optimal
24 time for this parameter. In addition, when identifying effect levels based on the data presented by
25 Korsak and Rydzyhski [1996]. latencies on the rod of 1 second versus 119 seconds would be
26 treated identically as failures when, in fact, they indicate very different levels of neurological
27 dysfunction [Bogo etal.. 1981]. This adds uncertainty when trying to extrapolate to a concentration
28 associated with a minimally adverse effect Finally, this quantal presentation of data does not allow
29 for interpretations related to intra-rat and intra-group variability in performance. Due to these
30 reporting limitations, impaired neuromuscular function and coordination, measured as
31 performance deficits on the rotarod apparatus, was considered to be less informative than the data
32 supporting decreases in pain sensitivity, and thus, was excluded from consideration for derivation
33 of the RfC for 1,2,4-TMB.
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Table 2-2. Endpoints considered for the derivation of the RfC for 1,2,4-TMB
Endpoint
Neurological endpoints
Decreased pain sensitivity
(measured as latency to paw-lick,
in seconds)b
Hematological endpoints
Decreased RBCs (106/mm3)c
(10s cells per 100 ul)
Increased WBCs (103/mm3)c
(103 cells per 100 ul)
Decreased reticulocytes (%)c
Decreased clotting time (s)c
Respiratory endpoints
Increased BAL total cells
(106/cm3)d
Increased inflammatory
lung lesions0
Developmental endpoints
Decreased fetal weight (g)f'g
Species/
sex
Rat,
male
Rat,
male
Rat,
female
Rat,
male
Rat,
male
Rat,
female
Exposure concentration (mg/m3)
0
15.4 ±
5.8a
(n = 9)
0
9.98 ±
1.68
(n = 10)
8.68 ±
2.89
(n = 10)
3.5 ±2.6
(n = 10)
30 ±10
(n = 10)
0
1.93 ±
0.79
(n = 6)
e
(n = 10)
0
5.86 ±
0.34
5.57 ±
0.33
123
18.2 ±
5.7
(n = 10)
123
9.84 ±
1.82
(n = 10)
8.92 ±
3.44
(n = 10)
1.7 ±2.0
(n = 10)
23 ±4
(n = 10)
123
5.82 ±
1.32***
(n = 6)
e
(n =10)
492
5.79 ±
0.30
5.51 ±
0.31
492
27.6 ±
3.2**
(n = 9)
492
8.50 ±
1.11
(n = 10)
8.30 ±
1.84
(n = 10)
1.8 ±0.9
(n = 10)
19 ± 5**
(n = 10)
492
5.96 ±
2.80**
(n = 7)
e
(n = 10)
1,476
5.72 ±
0.49
5.40 ±
0.45
1,230
30.1 ±7.9**
n = 10)
1,230
7.70+1.38**
(n = 10)
15.89 ± 5.74**
(n = 10)
1.0 ±0.6*
n = 10)
22 ±7*
(n =10)
1,230
4.45 ± 1.58*
(n = 7)
e
n = 10)
2,952 4,428
0.48* 5-20±°-42**
C TQ _|_
0.40* 4.92 ±0.40-
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Table 2-2 (Continued): Endpoints considered for the derivation of the RfC for
1,2,4-TMB
Endpoint
Maternal endpoints
Decreased maternal weight
gain (g)f
Species/
sex
Rat,
female
Exposure concentration (mg/m3)
0
29 ±12
(n = 24)
492
31 ±14
(n = 22)
1,476
27 ±12
(n = 22)
2,952
15 ± 17**
(n=22)
4,428
0 ± 14**
(n = 24)
*p<0.05; **p<0.01; ***p< 0.001.
aValues are expressed as mean ± 1 SD. Korsak and Rydzyriski (1996) does not explicitly state that the reported measures of
variance in Table 1 of that reference are standard deviations. However, independent analysis conducted by EPA confirms that
the reported measures of variance are standard deviations.
b Adapted from Korsak and Rydzyriski (1996)
c Adapted from Korsak et al. (2000a)
d Adapted from Korsak et al. (1997)
Incidences for individual exposure groups not reported; however, based on qualitative information reported in the study (i.e.,
that female rats exhibited a statistically significant increase in inflammatory lung lesions at 492 mg/m3), a NOAEL of
123 mg/m3 was identified.
f Adapted from Saillenfait et al. (2005)
5 Numbers of fetuses not explicitly reported. See maternal weight gain for number of litters.
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2.1.2. Methods of Analysis for 1,2,4-TMB
1 This assessment uses PBPK model estimates of internal blood dose metrics coupled with
2 the benchmark dose (BMD) approach, when possible, to estimate a POD for the derivation of an RfC
3 for 1,2,4-TMB (see Section B.3 of Appendix B and Section C.I of Appendix C for details regarding
4 PBPK model estimates and BMD modeling, respectively). As dosimetry can often be non-linear due
5 to metabolic saturation, and internal dose metrics are expected to correlate more closely to toxic
6 response than external concentrations [Mclanahanetal.. 2012]. this assessment used the PBPK
7 model-estimated internal dose metrics for dose-response modeling.
8 A deterministic rat PBPK model [Hissinketal., 2007] was used to convert non-continuous
9 external inhalation concentrations (in mg/m3] of 1,2,4-TMB to the internal blood dose metric of
10 average weekly venous blood concentration (in mg/L] of 1,2,4-TMB for Korsak et al., (2000a: 1997]
11 and Korsak and Rydzyhski (1996] only (see Table 2-1]. Weekly average venous blood 1,2,4-TMB
12 concentration was chosen as the internal dose metric on which to base the POD as it is assumed
13 that the parent compound is the toxic moiety of interest and that average venous blood
14 concentration of 1,2,4-TMB is assumed to adequately represent the target tissue dose across the
15 multiple tissues of interest. The use of concentration of parent compound in venous blood as the
16 relevant dose metric in non-metabolizing, non-first pass organs is recommended by Aylward et al.
17 (2011]. Furthermore, toluene-induced neurological effects in the brain are provided by Aylward et
18 al. (2011] as an example of a chemically induced toxic endpoint for which this dose metric is
19 relevant As discussed in Section 1 (Mode of Action Analysis - Neurotoxic Effects], 1,2,4-TMB is
20 reasonably expected to have a mode of action for neurotoxic effects similar to toluene, further
21 supporting the selection of venous blood concentration as the relevant internal dose metric.
22 One consequence of using PBPK model-estimated internal dose metrics as the dose inputs
23 for BMD modeling was the necessity of dropping the high exposure group in all datasets modeled.
24 During the validation and optimization of the animal PBPK model (Hissinketal., 2007] against
25 available animal toxicokinetic datasets, the model accurately reproduced venous blood
26 concentrations of 1,2,4-TMB following repeated (6 hours/day, 5 days/week, 4 weeks] exposures to
27 123 or 492 mg/m3 (see Section B.3.3.2, Appendix B]. However, the PBPK model consistently
28 overpredicted venous blood concentrations following exposure to 1,230 mg/m3. It was concluded
29 that the optimized animal PBPK model produces acceptable simulations of venous blood 1,2,4-TMB
30 concentrations for chronic exposures of up to 100 ppm [492 mg/m3] in rats following inhalation
31 exposure to 1,2,4-TMB (Section B.3.3.2, Appendix B]. Therefore, as the model-estimated internal
32 blood dose metrics at the high concentration are not representative of empirically observed blood
33 concentrations, using the high-dose model estimates as dose inputs for BMD modeling is not
34 appropriate. The decision to drop the high concentration results in a loss of information regarding
35 dose-response characteristics at high concentrations and a reduction in the number of available
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1 dose-response models to fit to the data (due to the number of model parameters > exposure
2 groups). However, this methodology is preferred over inclusion of demonstrably inaccurate
3 internal blood dose metrics that result from high exposure concentrations. Additionally, this
4 methodology still allows for BMD modeling of these endpoints, which is preferred over use of the
5 NOAEL/LOAEL approach.
6 After calculation of internal blood dose metrics, those dose metrics were used as the dose
7 inputs for BMD modeling. As the Hissink et al. [2007] PBPK model was not parameterized for
8 pregnant animals and did not include a fetal compartment, internal dose metrics were not
9 calculated from Saillenfait et al. [2005]. Instead, actual exposure concentrations were used for these
10 endpoints.
11 The BMD approach involves fitting a suite of mathematical models to the observed dose-
12 response data using EPA's Benchmark Dose Software (BMDS, version 2.2]. Each fitted model
13 estimates a BMD and its associated 95% lower confidence limit (BMDL] corresponding to a selected
14 benchmark response (BMR]. For continuous data (i.e., decreased pain sensitivity, increased BAL
15 total cells, decreased RBCs, decreased reticulocytes, and decreased clotting time] from the Korsak
16 and Rydzyhski [1996] and Korsak et al. [2000a: 1997] studies, and maternal weight gain from
17 Saillenfait et al. [2005]. no information is available regarding the change in these responses that
18 would be considered biologically significant, thus a BMR equal to a 1 standard deviation change in
19 the control mean was used in modeling these endpoints, consistent with EPA's Benchmark Dose
20 Technical Guidance [U.S. EPA. 2012b]. For the decreased male and female fetal body weight
21 endpoints identified from the Saillenfait et al. [2005] study, a BMR of 5% relative deviation from
22 the control mean was selected. A 5% decrease in fetal body weight relative to control was
23 determined to be a minimal, biologically significant response. This determination is based on the
24 fact that decreased body weight gain in fetuses and/or pups is considered indicative of altered
25 growth, which has been identified by EPA as one of the four major manifestations of developmental
26 toxicity [U.S. EPA, 1991]. In addition, a 10% decrease in adult body weight in animals is generally
27 recognized as a biologically significant response associated with identifying a maximum tolerated
28 dose, but since fetuses and/or pups are generally recognized as a susceptible lifestage, and thus are
29 assumed to be more greatly affected by decreases in body weight than adult animals, a 5% decrease
30 in fetal body weight is considered a biologically significant response. Finally, in humans, reduced
31 birth weight is associated with a series of adverse effects including neonatal and postnatal
32 mortality, coronary heart disease, arterial hypertension, chronic renal insufficiency, and diabetes
33 mellitus [Barker, 2007: Reyes and Manalich, 2005]. For these reasons, the selection of a BMR of 5%
34 for decreased fetal body weight was considered reasonable. Additionally, a BMR equal to a
35 1 standard deviation change in the control mean was also selected for the BMD modeling of both
36 fetal body weight and maternal body weight gain to facilitate comparisons across assessments [see
37 EPA's Benchmark Dose Technical Guidance [2012b]].
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1 Some endpoints for 1,2,4-TMB were not modeled for a variety of reasons, including equal
2 responses at all exposure groups (e.g., increased BAL total cells and decreased reticulocytes),
3 responses only in the high exposure group with no changes in responses in lower exposure groups
4 (e.g., increased WBCs), and absence of incidence data (e.g., increased inflammatory lung lesions).
5 Additionally, some datasets were modeled, but no model provided estimated BMDLs that were
6 considered to be biologically plausible (e.g., decreased clotting time). In cases where BMD modeling
7 was not feasible or modeling failed to appropriately describe the dose-response characteristics, the
8 NOAEL/LOAEL approach was used to identify a POD. Detailed modeling results are provided in
9 Section C.I of Appendix C.
10 Because an RfC is a toxicity value that assumes continuous human inhalation exposure over
11 a lifetime, data derived from inhalation studies in animals dose metrics need to be adjusted to
12 account for the noncontinuous exposures used in these studies. This is addressed by calculation of
13 internal dose metrics for the Korsak et al., (2000a: 1997) and Korsak and Rydzyhski (1996) studies.
14 For the Saillenfait et al. (2005) study, rats were exposed to 1,2,4-TMB for 6 hours/day for 15
15 consecutive days (GD6-GD20). Therefore, the duration-adjusted PODs for developmental/maternal
16 effects were calculated as follows:
17 PODADj (mg/m3) = POD (mg/m3) x hours exposed per day/24 hours
18 For example, for decreased fetal weight in males, the PODADj would be calculated as follows:
19 PODADj (mg/m3) = 1,640.07 mg/m3 x 6 hours/24 hours
20 PODADj (mg/m3) = 410 mg/m3
21 For the derivation of an RfC based upon animal data, the calculated PODADj values are
22 converted to human equivalent concentrations (HECs) using the available human PBPK model
23 (Hissinketal..20Q7) for the selected endpoints from the Korsak et al., (2000a: 1997) and Korsak
24 and Rydzyhski (1996) studies. The human PBPK model was run (as described in Appendix B),
25 assuming a continuous (24 hours/day, 7 days/week) exposure, to estimate a human PODHEc that
26 would result from the same weekly average venous blood concentration reflected in the PODADj in
27 animals (Table 2-3). As the selected endpoints from Saillenfait et al. (2005) (i.e., decreased fetal
28 body weight, and maternal body weight gain) are assumed to result primarily from systemic
29 distribution of 1,2,4-TMB, and the Hissink et al. (2007) PBPK model is not parameterized for
30 pregnant animals and did not include a fetal compartment, the human equivalent concentration
31 (HEC) for 1,2,4-TMB was calculated by the application of the appropriate dosimetric adjustment
32 factor (DAF) for systemically acting gases (i.e., Category 3 gases), in accordance with the EPA's RfC
33 Methodology (U.S. EPA. 1994b). DAFs are ratios of animal and human physiologic parameters, and
34 are dependent on the nature of the contaminant (i.e., particle or gas) and the target site (i.e.,
35 respiratory tract or remote to the portal-of-entry [i.e., systemic]) (U.S. EPA. 1994b). For gases with
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1 systemic effects, the DAF is expressed as the ratio between the animal and human blood:air
2 partition coefficients:
3 DAF = (Hb/g)A/(Hb/g)H
4 where:
5 (Hb/g)A = the animal bloochair partition coefficient
6 (Hb/g)H = the human bloochair partition coefficient
7 DAF = 57.7 flarnberg and Tohanson. 19951/59.1 fMeulenberg and Viiverberg. 20001
8 DAF = 0.98
9 In cases where the animal blood:air partition coefficient is lower than the human value,
10 resulting in a DAF < 1, the calculated value is used for dosimetric adjustments [U.S. EPA, 1994b].
11 For example, the HEC for decreased female fetal body weight (reported in Saillenfait et al. [2005]]
12 is calculated as follows:
13 PODHEc = PODADj (mg/m3) x DAF
14 PODHEC = PODADj (mg/m3) x 0.98
15 PODHEC = 403.2 mg/m3 x 0.98
16 PODHEc = 395.1 mg/m3
17 The calculated PODHEc (mg/m3] values for all endpoints considered for candidate value
18 derivation are presented in Table 2-3.
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Table 2-3. Summary of derivation of points of departure for 1,2,4-TMB
Endpoint/Reference
Species/sex
Model; BMRor
NOAEL/LOAEL
PODa
Candidate
PODADJa
Candidate
PODHEc
(mg/m3)
Neurological endpoints
Decreased pain sensitivity
(Korsakand Rydzyriski, 1996)
Rat, male
Exponential M4;
ISO
0.086
0.086
15.8
Hematological endpoints
Decreased RBCs
(Korsaketal.,2000a)
Increased WBCs
(Korsaketal.,2000a)
Decreased reticulocytes
(Korsaketal.,2000a)
Decreased clotting time
(Korsaketal.,2000a)
Rat, male
Rat, male
Rat, female
Rat, female
Linear; ISO
NOAELb
NOAELb
NOAELb
0.499
0.867
0.890
0.134
0.499
0.867
0.890
0.134
83.9
131.5
134.0
24.4
Respiratory endpoints
Increased BAL total cells
(Korsaketal., 1997)
inflammatory lung lesions (Korsak et
al., 2000a)
Rat, male
Rat, male
LOAELb
NOAELb
0.127
0.134
0.127
0.134
23.2
24.4
Developmental endpoints
Decreased fetal weight
Saillenfait et al. (2005)
Rat, male
Rat, female
Linear, 5% RD
Linear, 5% RD
1,640.07
1,612.89
410
403.2
401.8
395.1
Maternal endpoints
Decreased maternal weight gain
(Saillenfait et al., 2005)
Rat, female
Exponential M3,
ISO
2,247.99
562
550.8
a Values are weekly average venous blood 1,2,4-TMB concentration (mg/L) for Korsak et al. (2000a; 1997) and
Korsak and Rydzyriski (1996). See Appendix B for details on PBPK modeling, Values are in mg/m3 for
Saillenfait etal. (2005)
b No model was able to fit data adequately, or data were not modeled. NOAEL/LOAEL method used to identify a
POD
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2.1.3. Derivation of Candidate RfC Values for 1,2,4-TMB
1 Under EPA's A Review of the Reference Dose and Reference Concentration Processes [[U.S.
2 EPA, 2002] §4.4.5], also described in the Preamble, five possible areas of uncertainty and variability
3 were considered in deriving the candidate RfC values for 1,2,4-TMB. An explanation of these five
4 possible areas of uncertainty and variability and the values assigned to each as a designated
5 uncertainty factor (UF) to be applied to the candidate PODnEc are as follows:
6 An interspecies uncertainty factor, UFA, of 3 (101/2 = 3.16, rounded to 3) was applied to
7 account for uncertainty in characterizing the toxicokinetic and toxicodynamic differences between
8 rats and humans following inhalation exposure to 1,2,4-TMB. In this assessment, the use of a PBPK
9 model to convert internal doses in rats to administered doses in humans reduces toxicokinetic
10 uncertainty in extrapolating from the rat to humans, but does not account for interspecies
11 differences due to toxicodynamics. A default UFA of 3 was thus applied to account for this remaining
12 toxicodynamic and any residual toxicokinetic uncertainty not accounted for by the PBPK model.
13 An intraspecies uncertainty factor, UFH, of 10 was applied to account for potentially
14 susceptible individuals in the absence of data evaluating variability of response in the human
15 population following inhalation of 1,2,4-TMB. No information is currently available to predict
16 potential variability in human susceptibility, including variability in the expression of enzymes
17 involved in 1,2,4-TMB metabolism.
18 A LOAEL to NOAEL uncertainty factor, UFi, of 1 was applied because the current approach is
19 to address this factor as one of the considerations in selecting a BMR for BMD modeling. In this
20 case, a BMR equal to a 1 standard deviation change in the control mean for modeled endpoints was
21 selected under the assumption that this BMR represents a minimal, biologically significant change
22 for these effects. For endpoints that could not be modeled, a LOAEL to NOAEL uncertainty factor of
23 1 was applied as a NOAEL was used, except for increased BAL cells to which a uncertainty factor of
24 10 was applied due to the use of a LOAEL for this endpoint
25 A subchronic to chronic uncertainty factor, UFS, of 3 (101/2 = 3.16, rounded to 3) was applied
26 to account for extrapolation from a subchronic exposure duration study to derive a chronic RfC, for
27 all endpoints except decreases in fetal weight, to which an UFS of 1 was applied. The 3-fold
28 uncertainty factor is applied to the POD identified from the subchronic study on the assumption
29 that effects observed in a similar chronic study would be observed at lower concentrations for a
30 number of possible reasons, including potential cumulative damage occurring over the duration of
31 the chronic study or an increase in the magnitude or severity of effect with increasing duration of
32 exposure. For example, in the case of neurotoxicity, chronic exposures may overwhelm the adaptive
33 responses observed after termination of subchronic exposure, potentially resulting in more severe
34 and/or irreversible changes in neurological function. A full subchronic to chronic uncertainty factor
35 of 10 was not applied in this case as there was evidence of reversibility of not only neurotoxic
36 effects, but also hematological effects in rats exposed to 1,2,4-TMB for subchronic durations. Also,
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1 the respiratory effects appeared to be inflammatory in nature. Although reversibility was not
2 investigated for these endpoints, it is possible that adaptive mechanisms may alleviate these effects
3 following the termination of exposure.
4 A database uncertainty factor, UFD, of 3 (101/2 = 3.16, rounded to 3) was applied to account
5 for database deficiencies. Strengths of the database include the three well-designed subchronic
6 studies that observe exposure-response effects in multiple organ systems (nervous, respiratory,
7 and hematological systems), in Wistar rats exposed to 1,2,4-TMB via inhalation. An additional
8 strength of the database is the well-designed developmental toxicity study that investigated
9 standard measures of maternal and fetal toxicity in a different strain of rat (Sprague-Dawley).
10 However, the lack of a multi-generation reproductive/developmental toxicity study is a weakness
11 of the database. EPA's A Review of the Reference Dose and Reference Concentration Processes [U.S.
12 EPA, 2002] recommends that the database uncertainty factor take into consideration whether there
13 is concern from the available toxicology database that the developing organism may be particularly
14 susceptible to effects in specific organ systems. TMBs (unspecified isomer) are able to cross the
15 placenta (Cooper etal.. 2001: Dowty etal.. 1976}: therefore, as neurotoxicity is observed in adult
16 animals, there is the concern that exposure to 1,2,4-TMB may result in neurotoxicity in the
17 developing organism. EPA's Guidelines for Neurotoxicity Risk Assessment (U.S. EPA. 1998] identifies
18 specific effects observed in adult animals (e.g., cognitive and motor function] that can also affect the
19 developing organism exposed in utero. The Neurotoxicity Guidelines (U.S. EPA, 1998] also indicate
20 that neurotoxicants may have greater access to the nervous system in developing organisms due to
21 an incomplete blood-brain barrier and immature metabolic detoxifying pathways. Lastly, EPA's A
22 Review of the Reference Dose and Reference Concentration Processes (U.S. EPA. 2002] also states that
23 effects that may be mild or reversible in adults may produce more robust or permanent effects in
24 offspring following developmental exposures. Therefore, there is some concern that the lack of a
25 developmental neurotoxicity study is a deficiency in the database and that inclusion of such a study
26 would potentially result in a lower POD than the POD for neurotoxicity identified from the available
27 1,2,4-TMB toxicity database. In summary, a 3-fold database UF was applied to account for the lack
28 of both a multi-generation reproductive/developmental toxicity study and a developmental
29 neurotoxicity study in the available database for 1,2,4-TMB.
30 Table 2-4 is a continuation of Table 2-3, and summarizes the application of UFs s to each
31 POD to derive a candidate RfC value for each data set The candidate RfC values presented in Table
32 2-4 are preliminary to the derivation of the organ/system-specific RfC values. These candidate RfC
33 values are considered individually in the selection of a representative inhalation reference RfC
34 value for a specific hazard and subsequent overall RfC for 1,2,4-TMB. Figure 2-1 presents
35 graphically these candidate RfC values, uncertainty factors, and points of departure, with each bar
36 corresponding to one data set described in Tables 2-3 and 2-4.
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Table 2-4. Effects and corresponding derivation of candidate RfC values for
1,2,4-TMB
Endpoint/Reference
HEC
(mg/m3)3
UFA
UFH
UFL
UFS
UFD
Composite
UF
Candidate RfC
value (mg/m3)b
Neurological endpoints
Decreased pain sensitivity
(Korsakand Rydzyhski, 1996)
15.8
3
10
1
3
3
300
5.27 x 10"2
Hematological endpoints
Decreased RBCs,
(Korsaketal.,2000a)
Increased WBCs
(Korsaketal.,2000a)
Decreased reticulocytes
(Korsaketal.,2000a)
Decreased clotting time
(Korsaketal.,2000a)
83.9
131.5
134.0
24.4
3
3
3
3
10
10
10
10
1
1
1
1
3
3
3
3
3
3
3
3
300
300
300
300
2.80 x 10"1
4.38 x 10"1
4.47 x 10"1
8.13xlO"2
Respiratory endpoints
Increased BAL total cells
(Korsaketal., 1997)
Increased inflammatory lung lesions
(Korsaketal.,2000a)
23.2
24.4
3
3
10
10
10
1
3
3
3
3
3,000
300
n/ac
8.13xlO"2
Developmental endpoints
Decreased fetal weight
(Saillenfait et al., 2005)
Maternal endpoints
rat, male
(rat, female)
401.8
395.1
3
3
10
10
1
1
1
1
3
3
100
100
4.02
3.95
Decreased maternal weight gain
(Saillenfait et al., 2005)
550.8
3
10
1
3
3
300
1.84
aHuman equivalent concentration.
As calculated by application of uncertainty factors, not rounded to 1 significant digit.
cEndpoint excluded for further consideration due to a UFCOMPOSITE of 3,000. 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 composite uncertainty factor is 10,000 or
more, it is unlikely that the database is sufficient to derive a reference value. Although, 3,000 is generally recognized as
the maximum composite uncertainty factor for RfC derivation, a candidate RfC based on the data for increased BAL total
cells was not derived due to the fact that the uncertainty surrounding this endpoint was much higher than for any other
endpoint.
This document is a draft for review purposes only and does not constitute Agency policy.
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01
D.
_O
01
>
01
Q
01
cc
E
01
3:
O fT3
= -^
d, 00
Maternal body weight gain
(Saillenfaitetal., 2005)
Fetal body weight, females
(Saillenfaitetal., 2005)
4- Fetal body weight, males
(Saillenfaitetal., 2005)
Inflammatory lung lesions
(Korsaketal, 2000a)
t BAL Total eel Is
(Korsaketal, 1997)
4- Clotting time
(Korsaketal., 2000a)
4- Reticulocytes
(Korsaketal., 2000a)
N|/WBCs
(Korsaketal., 2000a)
N|/ RBCs
(Korsaketal., 2000a)
\|/ Pa in Sensitivity
(Korsakand Rydzynski, 1996)
Composite UF
A Candidate value
• POD(HEC)
0.001 0.01 0.1 1 10
Concentrations (mg/m3)
100
1000
Figure 2-1. Candidate RfC values with corresponding POD and composite UF for
1,2,4-TMB.
2.1.4. Derivation of Organ/System Specific Reference Concentrations for 1,2,4-TMB
1 Table 2-5 distills the candidate RfC values from Table 2-4 into a single value for each organ
2 or system. The single RfC value selected for a particular organ system was preferably chosen using
3 biological and toxicological information regarding that endpoint. If no compelling biological
4 information exists with which to select the primary hazard, the lowest RfC value for that organ
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1 system was selected. These organ- or system-specific reference concentrations may be useful for
2 subsequent cumulative risk assessments that consider the combined effect of multiple agents acting
3 at a common site. The individual organs and systems for which specific RfC values were derived
4 were the neurological, hematological, and respiratory systems, along with specific RfCs derived for
5 the pregnant animal (maternal) and developing fetus (developmental). The RfC value for the
6 neurological system, based on decreased pain sensitivity, was selected for the proposed overall RfC
7 for 1,2,4-TMB (see Section 2.1.5 for details). The RfC values for the hematological and respiratory
8 systems, based on decreased clotting time and increased inflammatory lung lesions, respectively,
9 are only slightly higher than the RfC derived for neurological effects (8 x 10~2 vs. 5 x 10~2 mg/m3),
10 indicating that effects in these organ systems may also be of concern. However, effects to pregnant
11 animals and the developing fetus may be of less concern as the RfCs for these types of effects (based
12 on decreased maternal weight gain and decreased male and female fetal weight, respectively) are
13 much higher than those derived for other organ systems.
Table 2-5. Organ/system-specific RfCs and proposed overall RfC for 1,2,4-TMB
Effect
Hematological
Respiratory
Maternal
Developmental
Proposed
overall RfC
(Neurological)
Basis
Decreased clotting time
Increased inflammatory lung
lesions
Decreased maternal weight
gain
Decreased fetal weight (males
and females)
Decreased pain sensitivity
RfC
(mg/m3)
8 x 10"2
8 x 10"2
2
4
5 x 10"2
Exposure
description
Subchronic
Subchronic
Gestational
Gestational
Subchronic
Confidence
Low to medium
Low to medium
Low to medium
Low to medium
Low to medium
2.1.5. Selection of the Proposed Overall Reference Concentration for 1,2,4-TMB
14 Neurotoxicity is the most consistently observed endpoint in the toxicological database for
15 1,2,4-TMB. According to EPA's Guidelines for Neurotoxicity Risk Assessment (U.S. EPA. 19981 many
16 neurobehavioral changes are regarded as adverse, and the observation of correlated and replicated
17 measures of neurotoxicity strengthen the evidence for a hazard. Decreased pain sensitivity,
18 measured as an increased latency to paw-lick in hot plate tests, represents an alteration in
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1 neurobehavioral function [U.S. EPA. 1998). Decreased pain sensitivity or decreased pain sensitivity
2 following a foot shock challenge was observed in multiple studies across multiple exposure
3 durations [Gralewicz and Wiaderna. 2001: Gralewicz etal.. 1997b: Korsak and Rydzynski. 1996:
4 Korsak etal., 1995], and in the presence of other measures of altered neurobehavior, including
5 impaired neuromuscular function and altered cognitive function. Additionally, neurological
6 symptoms (e.g., hand tremble, weakness) were observed in worker populations exposed to
7 complex VOC mixtures containing 1,2,4-TMB (notably, pain sensitivity has not been tested in
8 humans), suggesting a consistency and coherency of neurotoxic effects in humans and animals
9 following exposure to 1,2,4-TMB.
10 EPA's Guidelines for Neurotoxicity Risk Assessment (U.S. EPA. 1998] note that effects that are
11 reversible in minutes, hours, or days after the end of exposure and appear to be associated with the
12 pharmacokinetics of the agent and its presence in the body may be of less concern than effects that
13 persist for longer periods of time after the end of exposure. Pain sensitivity was observed to return
14 to control levels 2 weeks after termination of subchronic 1,2,4-TMB exposure at 1,230 mg/m3 in
15 one study (Korsak and Rydzynski. 1996]. However, the Neurotoxicity Guidelines also indicate that
16 reversible effects occurring in occupational settings may be of high concern, particularly if they
17 diminish a person's ability to survive or adapt to the environment (U.S. EPA. 1998] (pg. 8]; such is
18 the case for exposure to 1,2,4-TMB in occupations with dangerous surroundings and/ or heavy
19 equipment, such as dockyard painters or asphalt workers.
20 In several short-term studies of TMBs, there is evidence indicating that decreased pain
21 sensitivity in the presence of an additional environmental challenge (i.e., foot shock] is not rapidly
22 reversible and is not associated with clearance of the chemical from the body. TMB isomers have
23 been observed to clear rapidly from blood and nervous tissues (Section B.2, Appendix B], and
24 decreased pain sensitivity following foot shock persisted 51 days after termination of short-term
25 exposures (Wiaderna etal., 2002: Gralewicz and Wiaderna, 2001: Gralewicz etal., 1997b]. As
26 pointed out in A Review of the Reference Dose and Reference Concentration Process (U.S. EPA, 2002],
27 "[i]t is also important to keep in mind that effects that may initially appear to be reversible may re-
28 appear later or be predictive of later adverse outcomes." (pg. 4-16]. Additionally, the Neurotoxicity
29 Guidelines (U.S. EPA. 1998] state that "latent effects (those that become evident only after an
30 environmental challenge [e.g., in this case, footshock]] have a high level of concern." The hot plate
31 test is a relatively simple assessment that may not be sensitive enough to detect subtle changes
32 (U.S. EPA, 1998], suggesting that the large changes observed immediately after 1,2,4-TMB exposure
33 may represent gross effects. It is possible that, at longer durations after exposure, an environmental
34 challenge is necessary for the more subtle perturbations that persist to become manifest at a
35 detectable level. The latent decrements in pain sensitivity following foot shock appear to reflect a
36 lengthening of the numbing effects of foot shock following exposure to 1,2,4-TMB weeks earlier, as
37 the immediate increases in latency due to foot shock were unchanged by prior 1,2,4-TMB exposure.
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1 Although these measures may be complicated by less likely, but possible, effects on cognition, the
2 results suggest that some aspect(s) of the altered pain sensitivity phenotype fail to resolve
3 following termination of exposure. No environmental challenge was applied in the subchronic study
4 by Korsak and Rydzyhski [1996]: such an experiment may have uncovered similar latent responses.
5 Conversely, the short-term 1,2,4-TMB exposure studies testing pain sensitivity failed to analyze hot
6 plate latency with a foot shock challenge shortly after exposure, as these evaluations only occurred
7 at > 50 days post-exposure.
8 Uncertainty regarding the reversibility of pain sensitivity in non-shocked rats at all tested
9 1,2,4-TMB concentrations also exists. Reversibility of the pain sensitivity phenotype following
10 subchronic exposure was only tested at the highest concentration of 1,2,4-TMB used in any study
11 (i.e., 1,230 mg/m3). In multiple other tests of neurological function (including pain sensitivity
12 following a foot shock challenge), it was clearly shown that exposure to 1,2,4-TMB elicits nonlinear
13 effects when tested some period of time after exposure, with 1,230 mg/m31,2,4-TMB usually
14 resulting in no response or a substantially reduced response as compared to lower 1,2,4-TMB
15 concentrations (e.g., 492 mg/m3). Thus, from the data available, a determination regarding the
16 reversibility of 1,2,4-TMB-induced decreases in pain sensitivity at other concentrations (i.e., 492
17 mg/m3) at two weeks post-exposure cannot be made with confidence.
18 Although it is important to consider the potential for reversibility of neurological effects,
19 "for chronic lifetime exposures, designation of an effect as irreversible or reversible is academic, as
20 exposure is presumed to be lifetime (i.e., there is no post-exposure period)" (U.S. EPA. 2002] (pg. 3-
21 27). In other words, the nature of an RfC precludes the possibility of recovery of the critical effect.
22 This supports the choice of the principal study even were all aspects of the pain sensitivity
23 phenotype identified as transient, which, notably, does not appear to be the case. Taken as a whole,
24 the database supports the characterization of decreased pain sensitivity associated with exposure
25 to 1,2,4-TMB as being an effect of high concern. Given the consistency of observations from hot
26 plate tests with or without foot shock challenge across several studies from the same research
27 group using multiple durations of exposure in male Wistar rats, as well as the evidence and
28 biological plausibility of similarities in neurological effects between rats and humans, there is
29 strong evidence that neurotoxicity is the primary hazard associated with exposure to 1,2,4-TMB.
30 Based on the above considerations, decreased pain sensitivity measured immediately after
31 subchronic exposure is identified as an adverse neurotoxic effect and thus is an appropriate effect
32 on which to base the RfC. Therefore, the candidate RfC for neurotoxicity based on decreased pain
33 sensitivity was selected as the RfC for 1,2,4-TMB.
34 A PODnEc of 15.8 mg/m3 for decreased pain sensitivity (Korsak and Rydzynski. 1996] was
35 used as the POD from which to derive the chronic RfC for 1,2,4-TMB (see Table 2-4). The
36 uncertainty factors (UFs), selected and applied in accordance with the procedures described in
37 EPA's A Review of the Reference Dose and Reference Concentration Processes [(U.S. EPA. 2002]
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1 (Section 4.4.5 of the report)], were discussed previously in Section 2.1.3. Application of the
2 composite UF of 300 to the PODHEc yields the following chronic RfC for 1,2,4-TMB:
3 RfC = PODHEC * UF = 15.8 mg/m3 4- 300 = 0.05 mg/m3 = 5 x 1Q-2 mg/m3
4 (rounded to one significant digit)
2.1.6. Uncertainties in the Derivation of the Reference Concentration for 1,2,4-TMB
5 As presented above, the UF approach, following EPA practices and RfC guidance [U.S. EPA.
6 2002.1994b). was applied to the PODHEc in order to derive the chronic RfC for 1,2,4-TMB. Factors
7 accounting for uncertainties associated with a number of steps in the analyses were adopted to
8 account for extrapolation from animals to humans, a diverse human population of varying
9 susceptibilities, duration of exposure, POD determination methodologies (NOAEL, LOAEL, or
10 BMDL), and database deficiencies.
11 The critical effect selected, decreased pain sensitivity, does not introduce substantial
12 uncertainty into the RfC calculation as selection of alternative hematological or respiratory effects
13 would result in similar RfCs that would be equivalent when rounding to one significant digit (i.e.,
14 2 x ID-2 mg/m3, see Figure 2-2). Some uncertainty exists regarding the selection of the BMRs for use
15 in BMD modeling due to the absence of information to determine the biologically significant level of
16 response associated with the endpoints. However in cases such as this, the selection of a BMR of 1
17 standard deviation for continuous endpoints is supported by EPA guidance [U.S. EPA. 2012b). In
18 addition, there is uncertainty in the estimated standard deviation for decreased pain sensitivity
19 [Korsak and Rydzynski. 1996). which was two- to threefold higher than that estimated in the
20 parallel evaluation of 1,2,3-TMB in the same publication. Given the lack of information concerning a
21 biologically significant level of response for pain sensitivity, the concurrently estimated standard
22 deviation was judged to be most relevant for characterizing this response to 1,2,4-TMB.
23 Uncertainty regarding the selection of particular models for individual endpoints exists as
24 selection of alternative models could decrease or increase the estimated POD and consequently, the
25 RfC. The selection criteria for model selection was based on a practical approach as described in
26 EPA's Benchmark Dose Technical Guidance (U.S. EPA. 2012bl Uncertainty may exist in the PBPK
27 model estimates of internal blood dose metrics for the rat, and subsequent HEC calculations for
28 humans, including parameter uncertainty, but such uncertainties would apply equally to all
29 endpoints.
2.1.7. Confidence Statement for 1,2,4-TMB
30 A confidence level of high, medium, or low is assigned to the study used to derive the RfC,
31 the overall database, and the RfC itself, as described in Section 4.3.9.2 of EPA's Methods for
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1 Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry [U.S. EPA.
2 1994b).
3 Confidence in the study from which the critical effect was identified, Korsak and Rydzyhski
4 [1996] is low to medium. The study is a peer-reviewed study that utilized three dose groups plus
5 untreated controls and employed an appropriate number of animals per dose group. However,
6 sources of uncertainty exist that reduce confidence in this study.
7 One area of uncertainty regarding this study is the lack of reported actual concentrations.
8 However, as the methods by which the test atmosphere was generated and analyzed were reported
9 in sufficient detail, and given the fact that this laboratory has used this methodology in subsequent
10 studies [Korsak et al.. 2000a. b) and achieved appropriate actual concentrations (i.e., within 10% of
11 target concentrations), the concern regarding the lack of reported actual concentrations is minimal.
12 The critical effect on which the RfC is based is well-supported as the weight of evidence for
13 1,2,4-TMB-induced neurotoxicity is coherent across species (i.e., human and rat) and consistent
14 across multiple exposure durations (i.e., acute, short-term, and subchronic) [Gralewicz and
15 Wiaderna. 2001: Chen etal.. 1999: WiadernaetaL 1998: Gralewicz etal.. 1997b: Gralewicz etal..
16 1997a: Korsak and Rydzynski. 1996: Norseth etal.. 1991).
17 The database for 1,2,4-TMB includes acute, short-term, subchronic, and developmental
18 toxicity studies in rats and mice. However, confidence in the overall database is low to medium
19 because it lacks chronic, multi-generation reproductive/developmental, and developmental
20 neurotoxicity studies, and the studies supporting the critical effect predominantly come from the
21 same research institute. The overall confidence in the RfC for 1,2,4-TMB is low to medium.
2.2. Inhalation Reference Concentration for Effects Other Than Cancer
for 1,2,3-TMB
2.2.1. Identification of Studies and Effects for Dose-Response Analysis for 1,2,3-TMB
22 The nervous, hematological, and respiratory systems are the primary targets of inhaled
23 1,2,3-TMB in humans and experimental animals, and effects in these systems have been identified
24 as hazards following inhalation exposure to 1,2,3-TMB. Although literature exists on the effects of
25 1,2,3-TMB exposure in humans, including neurological, hematological, and respiratory toxicities, no
26 human studies are available that would allow for dose-response analysis. The human studies
27 evaluated TMB exposures occurring as complex solvents or VOC mixtures, and this consideration
28 along with other uncertainties including high imprecision in effect measures due to low statistical
29 power, lack of quantitative exposure assessment, and lack of control for co-exposures, limit their
30 utility in derivation of quantitative human health toxicity values. However, these studies provide
31 supportive evidence for the neurological, hematological, and respiratory toxicity of TMB isomers in
32 humans and indicate a coherency of effects in both humans and laboratory animals.
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1 Several studies investigating 1,2,3-TMB effects in experimental animal models were
2 identified in the literature. No chronic studies were available, although several acute, short-term,
3 and subchronic studies were identified. 1,2,3-TMB-induced toxicity was observed across several
4 organ systems in two subchronic studies by Korsak et al. [2000b] and Korsak and Rydzyhski
5 [1996]. These were the only subchronic studies identified in the peer-reviewed literature. Data
6 from these studies pertaining to the primary hazards observed in humans and animals identified
7 previously in Chapter 1 (neurological, hematological, and respiratory toxicity) were considered as
8 candidate critical effects for the purpose of determining the point of departure (POD) for derivation
9 of the inhalation RfC for 1,2,3-TMB. Neurotoxicity was also observed in both acute and short-term
10 inhalation studies and respiratory toxicity was also observed in acute studies. However, the high
11 concentrations used in acute studies and the short exposure durations employed in both acute and
12 short-term studies limit their applicability for quantitation of chronic human health effects.
13 Nevertheless, as with the human in which subjects were exposed to mixtures containing 1,2,3-TMB,
14 these studies provide qualitative information regarding the consistency and coherency of these
15 effects across the 1,2,3-TMB database.
16 The two subchronic studies by Korsak et al. (2000b) and Korsak and Rydzyhski (1996) are
17 adequate for dose-response analysis. Both studies exposed rats, a common model for human
18 response, by inhalation, tol,2,3-TMB (reported as > 97% pure [impurities not reported]). The
19 studies used three exposure levels spaced two- to threefold apart, facilitating dose-response
20 analysis and utilized sham-exposed controls. The subchronic durations of exposure were suitable
21 for the effects under evaluation. In addition, the persistence of some outcomes after termination of
22 exposure was investigated. Typical numbers of animals per exposure group for subchronic studies
23 were used, at least 10/group. Regarding exposure characterization, Korsak et al. (2000b) reported
24 actual concentrations, as measured by gas chromatography, to be within 10% of target
25 concentrations. This increases the confidence in the overall evaluation and adequacy of this study.
26 Although Korsak and Rydzyhski (1996) do not report actual, measured concentrations, this study
27 uses the same exposure methodology as Korsak et al. (2000b): suggesting that it is likely that the
28 actual concentrations in this study were also within 10% of target concentrations. Target and actual
29 concentrations for these studies are presented in Table 2-6.
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Table 2-6. Target and actual exposure concentrations used in BMD modeling of
1,2,3-TMB endpoints considered for the derivation of the RfC
Reference
Korsak and
Rydzyriski (1996)
Korsak et al.
(2000b)
Species/
sex
Rat, male
Rat, male
Rat, female
Target exposure
concentration (mg/m3)
123
492
1,230
123
492
1,230
123
492
1,230
Actual exposure
concentration (mg/m3)
n/a
n/a
n/a
128
523
1,269
128
523
1,269
1 These two subchronic studies examined 1,2,3-TMB-induced toxicity in multiple organ
2 systems and the neurological, hematological, and respiratory endpoints that demonstrated
3 statistically significant increases or decreases relative to control were considered for the derivation
4 of the RfC for 1,2,3-TMB (Table 2-7). These endpoints included decreased pain sensitivity in male
5 rats [Korsak and Rydzynski, 1996], and decreased RBCs and increased reticulocytes in male rats,
6 decreased segmented neutrophils and increased lymphocytes in male and female rats, and
7 increased inflammatory lung lesions in female rats [Korsak et al.. 2000b). Changes in liver organ
8 weights and clinical chemistry parameters from Korsak et al. [2000b] were not further considered
9 due to the lack of accompanying hepatocellular histopathological alterations in exposed animals.
10 Changes in splenic organ weights were similarly not considered further due to a lack of any
11 observed histopathological changes in that organ. Increases in reticulocytes in females were not
12 further considered due to non-monotonicity in response (increases in high concentration animals
13 that were not statistically significant). Increased lymphocytes were excluded from further
14 consideration due to the unusually high standard deviations reported in the high-concentration
15 group.
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Table 2-7. Endpoints considered for the derivation of the RfC for 1,2,3-TMB
Endpoint
Species/
sex
Exposure concentration (mg/m3)3
0
123
492
1,230
Neurological endpoints
Decreased pain sensitivity (measured as
latency to paw-lick in seconds)b
Rat, male
9.7 ±2.1
(n=30)
11.8 ±3.8*
(n = 20)
16.3 ±6.3°
(n = 10)
17.3 ±3.4**
(n = 10)
Hematological endpoints
Decreased RBCs (106/mm3)d
(10s cells per 100 ul)
Decreased segmented neutrophils (%)d
Increased reticulocytes (%)d
Rat, male
Rat, male
Rat, female
Rat, male
9.49 ± 2.03
(n = 10)
24.8 ±4.5
(n = 10)
23.1 ±6.1
(n = 10)
2.8 ±1.3
(n = 10)
10.25 ± 1.29
(n = 10)
25.4 ±5.8
(n = 10)
19.7 ±3.4
(n = 10)
2.1 ±1.7
(n = 10)
10.11 ±1.27
(n = 10)
20.7 ±5.8
(n = 10)
16.4 ±4.2*
(n = 10)
3.8 ±2.1
(n = 10)
8.05 ± 1.38*
(n = 10)
17.7 ±8.3*
(n = 10)
11.9 ±7.1**
(n = 10)
4.5 ± 1.8*
(n = 10)
Respiratory Endpoints
Increased inflammatory lung lesionsd
Rat, female
e
(n = 10)
e
(n =10)
e
(n = 10)
e
(n = 10)
p<0.05; p<0.01.
aValues are expressed as mean ± 1SD. Korsak and Rydzyriski (1996) does not explicitly state that the reported
measures of variance in Table 1 of that reference are standard deviations. However, independent analysis
conducted by EPA confirms that the reported measure of variance are standard deviations.
b Adapted from Korsak and Rydzyriski (1996)
c Level of significance not reported in Table 1 from Korsak and Rydzyriski (1996), however the results of an ad-hoc t-
test (performed by EPA) indicated significance at p < 0.01.
d Adapted from Korsak et al. (2000b)
e Incidences for exposure groups not reported; however, based on qualitative information reported in the study
(i.e., that female rats exhibited a statistically significant increase in inflammatory lung lesions at 492 mg/m3),
a NOAEL of 123 mg/m3 was identified.
1 Impaired neuromuscular function and coordination, measured as performance on the
2 rotarod apparatus, was also observed in rats exposed to 1,2,3-TMB. See Section 2.1.1 for a detailed
3 discussion of the uncertainties surrounding the use of this endpoint for derivation of an RfC. Due to
4 these uncertainties, this endpoint was excluded from consideration for the derivation of the RfC for
5 1,2,3-TMB.
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2.2.2. Methods of Analysis for 1,2,3-TMB
1 As discussed above in Section 2.2.1, endpoints observed in Korsak et al. [2000b] and Korsak
2 and Rydzynski [1996] that demonstrated statistically significant (p < 0.05 level) increases or
3 decreases relative to control for at least one exposure group were considered for the derivation of
4 the RfC for 1,2,3-TMB; these effects are listed in Table 2-7. This assessment used the BMD approach,
5 when possible, to estimate a POD for the derivation of an RfC for 1,2,3-TMB (see Section C.I of
6 Appendix C for detailed methodology). The BMD approach involves fitting a suite of mathematical
7 models to the observed dose-response data using EPA's BMDS (version 2.2). Each fitted model
8 estimates a BMD and its associated BMDL corresponding to a selected BMR. For continuous data
9 (i.e., decreased pain sensitivity, decreased RBCs, decreased segmented neutrophils, increased
10 reticulocytes) from the Korsak and Rydzynski (1996) and Korsak et al. (2000b) studies, no
11 information is available regarding the change in these responses that would be considered
12 biologically significant, and thus a BMR equal to a 1 standard deviation change in control mean was
13 used in modeling the endpoints, consistent with the Benchmark Dose Technical Guidance Document
14 (U.S. EPA. 2012b). The estimated BMDL is then used as the POD for deriving the RfC (Table 2-8).
15 The suitability of the above methods to determine a POD is dependent on the nature of the
16 toxicity database for a specific chemical. Some endpoints for 1,2,3-TMB were not modeled for a
17 variety of reasons, including responses only in the high exposure group with no changes in
18 responses in lower exposure groups (e.g., decreased RBCs) and absence of incidence data (e.g.,
19 increased inflammatory lung lesions). In cases where BMD modeling was not feasible, the
20 NOAEL/LOAEL approach was used to identify a POD. Additionally, for decreased pain sensitivity,
21 the reported SD of 3.4 in the high exposure group resulted in an inability of the variance power
22 model to fit the data adequately. For this reason, the high exposure group was dropped in order to
23 facilitate model fitting. Detailed modeling results are provided in Section C.I of Appendix C.
24 Because an RfC is a toxicity value that assumes continuous human inhalation exposure over
25 a lifetime, data derived from inhalation studies in animals need to be adjusted to account for the
26 noncontinuous exposures used in these studies. In the Korsak et al. (2000b) and Korsak and
27 Rydzynski (1996) studies, rats were exposed to 1,2,3-TMB for 6 hours/day, 5 days/week for 3
28 months. Because no PBPK model exists for 1,2,3-TMB, the duration-adjusted PODs for effects in rats
29 were calculated as follows:
30 PODADj (mg/m3) = POD (mg/m3) x hours exposed per day/24 hours x days exposed
31 per week/7 days
32 Therefore, for example, for decreased pain sensitivity from Korsak and Rydzynski (1996).
33 the PODADj would be calculated as follows:
34 PODADj (mg/m3) = 97.19 mg/m3x 6 hours/24 hours x 5 days/7 days
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1 PODADj (mg/m3) = 17.36 mg/m3
2 Because the majority of the selected endpoints for consideration as the critical effect
3 (decreased pain sensitivity, decreased RBCs, decreased segmented neutrophils, increased
4 reticulocytes) result primarily from systemic distribution of 1,2,3-TMB, and no available PBPK
5 model exists for 1,2,3-TMB, the human equivalent concentration (HEC) for 1,2,3-TMB was
6 calculated by the application of the dosimetric adjustment factor (DAF) for systemically acting gases
7 (i.e., Category 3 gases), in accordance with the U.S. EPA RfC Methodology (U.S. EPA. 1994bl
8 Additionally, although the observation of lung lesions would normally indicate portal-of-entry
9 effects, the observation that the overwhelming majority of 1,2,3-TMB-induced effects are systemic
10 in nature supports the determination that 1,2,3-TMB is a Category 3 gas. Other factors also support
11 that 1,2,3-TMB is a systemically-acting toxicant, including the isomer's relatively low water-
12 solubility and non-reactivity. Gases with these properties are expected to preferentially distribute to
13 the lower regions of the respiratory tract where larger surface areas and thin alveolar-capillary
14 boundaries facilitate uptake. Respiratory absorption of 1,2,3-TMB into the bloodstream has been
15 observed to be relatively high (~60%) following inhalation exposures to humans (Tarnbergetal..
16 1996]. Therefore, increased inflammatory lung lesions are assumed to result from systemic
17 distribution of 1,2,3-TMB in the bloodstream of exposed animals. DAFs are ratios of animal and
18 human physiologic parameters, and are dependent on the nature of the contaminant (particle or
19 gas) and the target site (e.g., respiratory tract or remote to the portal-of-entry [i.e., systemic]) (U.S.
20 EPA. 1994b). For gases with systemic effects, the DAF is expressed as the ratio between the animal
21 and human blood:air partition coefficients:
22 DAF = (Hb/g)A/(Hb/g)H
23 where:
24 (Hb/g)A = the animal bloochair partition coefficient
25 (Hb/g)H = the human bloochair partition coefficient
26 DAF = 62.6 (Tarnberg and Tohanson. 19951/66.5 (Meulenberg and Viiverberg. 2000)
27 DAF = 0.94
28 In cases where the animal blood:air partition coefficient is lower than the human value,
29 resulting in a DAF < 1, the calculated value is used for dosimetric adjustments (U.S. EPA. 1994b).
30 For example, the HEC for decreased pain sensitivity reported in Korsak and Rydzyhski (1996) is
31 calculated as follows:
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1 PODHEc = PODADj (mg/m3) x DAF
2 PODHEC = PODADj (mg/m3) x 0.94
3 PODHEC = 17.36 mg/m3 x 0.94
4 PODHEc = 16.32 mg/m3
5 Table 2-8 presents the calculated HECs for the candidate critical effects, selected
6 uncertainty factors (UFs), and the resulting derivation of candidate RfCs from the two subchronic
7 toxicity studies [Korsak et al.. 2000b: Korsak and Rydzynski. 1996).
Table 2-8. Summary of derivation of points of departure for 1,2,3-TMB
Endpoint/Reference
Species/sex
Model; BMR or
NOAEL/LOAEL
POD
(mg/m3)
Candidate
PODADJ
(mg/m3)
Candidate
PODHEC
(mg/m3)
Neurological endpoints
Decreased pain sensitivity
(Korsak and Rydzyriski, 1996)
Rat, male
Linear; 1 SD
97.19
17.36
16.32
Hematological endpoints
Decreased RBCs
(Korsak etal.. 2000b)
Increased segmented
neutrophils
(Korsak etal., 2000b)
Increased reticulocytes
(Korsak etal., 2000b)
Rat, male
Rat, male
Rat, female
Rat, male
NOAELa
Exponential M2;
1SD
Hill; 1 SD
Linear; 1 SD
523
534.81
99.21
652.90
93.39
95.50
17.72
116.58
87.79
89.77
16.66
109.58
Respiratory endpoints
inflammatory lung lesions
(Korsak etal., 2000b)
Rat, male
NOAELa
128
22.86
21.49
' No model was able to fit data adequately, or data were not modeled. NOAEL/LOAEL method used to identify
a POD.
9
10
2.2.3. Derivation of Candidate RfC Values for 1,2,3-TMB
Under EPA's A Review of the Reference Dose and Reference Concentration Processes [[U.S.
EPA. 2002] §4.4.5], also described in the Preamble, five possible areas of uncertainty and variability
were considered in deriving the candidate RfC values for 1,2,4-TMB. An explanation of these five
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1 possible areas of uncertainty and variability and the values assigned to each as a designated
2 uncertainty factor (UF) to be applied to the candidate PODHEc are as follows:
3 An interspecies uncertainty factor, UFA, of 3 (101/2 = 3.16, rounded to 3) was applied to
4 account for uncertainty in characterizing the toxicokinetic and toxicodynamic differences between
5 rats and humans following inhalation exposure to 1,2,3-TMB. In this assessment, the use of a DAF to
6 extrapolate external concentrations from rats to humans reduces toxicokinetic uncertainty in
7 extrapolating from the rat data, but does not account for the possibility that humans may be more
8 sensitive to 1,2,3-TMB than rats due to toxicodynamic differences. A default UFA of 3 was thus
9 applied to account for this remaining toxicodynamic and residual toxicokinetic uncertainty not
10 accounted for in the DAF.
11 An intraspecies uncertainty factor, UFH, of 10 was applied to account for potentially
12 susceptible individuals in the absence of data evaluating variability of response in the human
13 population following inhalation of 1,2,3-TMB. No information is currently available to predict
14 potential variability in human susceptibility, including variability in the expression of enzymes
15 involved in 1,2,3-TMB metabolism.
16 A LOAEL to NOAEL uncertainty factor, UFL, of 1 was applied because the current approach is
17 to address this factor as one of the considerations in selecting a BMR for BMD modeling. In this
18 case, a BMR equal to a 1 standard deviation change in the control mean for modeled endpoints was
19 selected under the assumption that this BMR represents a minimal, biologically significant change
20 for these effects. For endpoints that could not be modeled, a LOAEL to NOAEL uncertainty factor of
21 1 was applied as a NOAEL was used.
22 A subchronic to chronic uncertainty factor, UFs, of 3 (101/2 = 3.16, rounded to 3) was applied
23 to account for extrapolation from a subchronic exposure duration study to derive a chronic RfC. The
24 3-fold uncertainty factor is applied to the POD identified from the subchronic study on the
25 assumption that effects observed in a similar chronic study would be observed at lower
26 concentrations for a number of possible reasons, including potential cumulative damage occurring
27 over the duration of the chronic study or an increase in the magnitude or severity of effect with
28 increasing duration of exposure. For example, in the case of neurotoxicity, chronic exposures may
29 overwhelm the adaptive responses observed after termination of subchronic exposure, potentially
30 resulting in more severe and/or irreversible changes in neurological function. A full subchronic to
31 chronic uncertainty factor of 10 was not applied in this case as there was evidence of reversibility
32 of not only neurotoxic effects, but also hematological effects in rats exposed to 1,2,4-TMB for
33 subchronic durations. Also, the respiratory effects appeared to be inflammatory in nature. Although
34 reversibility was not investigated for these endpoints, it is possible that adaptive mechanisms may
35 alleviate these effects following the termination of exposure.
36 A database uncertainty factor, UFo, of 3 (101/2 = 3.16, rounded to 3) was applied to account
37 for database deficiencies. Strengths of the database include the two well-designed subchronic
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1 studies that observe exposure-response effects in multiple organ systems (i.e., neurological,
2 hematological, and respiratory effects) in Wistar rats exposed to 1,2,3-TMB via inhalation.
3 However, the lack of a either a multi-generational reproductive/developmental toxicity study or a
4 developmental toxicity study investigating effects due to 1,2,3-TMB exposure is a weakness of the
5 database. Normally, the lack of both of these types of studies in a toxicity database would warrant
6 the application of a full, 10-fold UFo in accordance with EPA's A Review of the Reference Dose and
7 Reference Concentration Processes [2002]. Although there is no developmental toxicity study for
8 1,2,3-TMB, Saillenfait et al. [2005] investigates the developmental toxicity of the other two TMB
9 isomers (1,2,4-TMB and 1,3,5-TMB] and observes developmental toxicity at levels much higher
10 than those eliciting neurotoxicity, hematotoxicity, and respiratory toxicity in adult animals (Korsak
11 studies]. Given that toxic effects were observed at lower concentrations in adult animals exposed
12 1,2,4-TMB and 1,3,5-TMB compared with rats exposed in utero and the similarities in toxicity
13 profiles amongst the three isomers, it is unlikely that the inclusion of a developmental toxicity study
14 for 1,2,3-TMB would result in a POD that is lower than the POD associated with neurotoxicity for
15 this isomer. Thus, the application of an UF to account for the lack of a developmental toxicity study
16 is not warranted.
17 EPA's A Review of the Reference Dose and Reference Concentration Processes [U.S. EPA. 2002]
18 also recommends that the database uncertainty factor take into consideration whether there is
19 concern from the available toxicology database that the developing organism may be particularly
20 susceptible to effects in specific organ systems. TMBs (unspecified isomer] are able to cross the
21 placenta [Cooper et al.. 2001: Dowtyetal.. 1976]: therefore, as neurotoxicity is observed in adult
22 animals, there is concern that exposure to 1,2,3-TMB may result in neurotoxicity in the developing
23 organism. EPA's Guidelines for Neurotoxicity Risk Assessment [U.S. EPA. 1998] identifies specific
24 effects observed in adult animals (e.g., cognitive and motor function] that can also affect the
25 developing organism exposed in utero. The Neurotoxicity Guidelines [U.S. EPA, 1998] also indicate
26 that neurotoxicants may have greater access to the nervous system in developing organisms due to
27 an incomplete blood-brain barrier and immature metabolic detoxifying pathways. Lastly, EPA's A
28 Review of the Reference Dose and Reference Concentration Processes [U.S. EPA. 2002] also states that
29 effects that may be mild or reversible in adults may produce more robust or permanent in offspring
30 following developmental exposures. Therefore, there is some concern that the lack of a
31 developmental neurotoxicity study is a deficiency in the database and that the inclusion of such a
32 study would potentially result in a lower POD than the POD for neurotoxicity identified from the
33 available 1,2,3-TMB toxicity database. In summary, a 3-fold database UF was applied to account for
34 the lack of both a multi-generation reproductive/developmental toxicity study and a developmental
35 neurotoxicology study in the available database for 1,2,3-TMB.
36 Table 2-9 is a continuation of Table 2-8, and summarizes the application of UFs to each POD
37 to derive a candidate value for each data set. The candidate values presented in Table 2-9 are
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4
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Toxicological Review of Trimethylbenzene
preliminary to the derivation of the organ/system-specific values. These candidate values are
considered individually in the selection of a representative inhalation reference value for a specific
hazard and subsequent overall RfC for 1,2,3-TMB. Figure 2-2 presents graphically these candidate
values, uncertainty factors, and points of departure, with each bar corresponding to one data set
described in Tables 2-8 and 2-9.
Table 2-9. Effects and corresponding derivation of candidate RfC values for
1,2,3-TMB
Endpoint/Reference
HEC
(mg/m3)3
UFA
UFH
UFL
UFS
UFD
Composite
UF
Candidate
value (mg/m3)b
Neurological endpoints
Decreased pain sensitivity
(Korsakand Rydzyriski, 1996)
16.32
3
m
1
3
3
300
5.44 x 10"2
Hematological endpoints
Decreased RBCs
(Korsak et al., 2000b)
Decreased segmented
neutrophils,
(Korsak etal.,2000b)
Increased reticulocytes
(Korsak etal.,2000b)
male
female
87.79
89.77
16.66
109.58
3
3
3
3
m
10
10
10
1
1
1
1
3
3
3
3
3
3
3
3
300
300
300
300
2.93 x 10"1
2.99 x 10"1
5.55 x 10 2
3.65 x 10"1
Respiratory endpoints
Increased inflammatory lung lesions
(Korsak etal.,2000b)
21.49
3
10
1
3
3
300
7.16 x 10"2
aHuman equivalent concentration.
bAs calculated by application of uncertainty factors, not rounded to 1 significant digit.
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6 ro
Inflammatory lung lesions
(Korsaketal, 2000b)
T" Reticulocytes
(Korsaketal., 2000b)
— 4- Segmented neutrophils,females
.y (Korsaketal., 2000b)
4- Segmented neutrophils, males
(Korsaketal., 2000b)
v|/ RBCs
(Korsaketal., 2000b)
\|/ Pain Sensitivity
(Korsakand Rydzynski, 1996)
Composite UF
A Candidate value
• POD(HEC)
0.001 0.01 0.1 1 10
Concentrations (mg/m3)
100
1000
Figure 2-2. Candidate RfC values with corresponding POD and composite UF for
1,2,3-TMB.
2.2.4. Derivation of Organ/System Specific Reference Concentrations for 1,2,3-TMB
1 Table 2-10 distills the candidate values from Table 2-9 into a single value for each organ or
2 system. The single RfC value selected for a particular organ system was preferably chosen using
3 biological and toxicological information regarding that endpoint. If no compelling biological
4 information exists with which to select the primary hazard, the lowest RfC value for that organ
5 system was selected. These organ- or system-specific reference concentrations may be useful for
6 subsequent cumulative risk assessments that consider the combined effect of multiple agents acting
7 at a common site. The individual organs and systems for which specific RfC values were derived
8 were the neurological, hematological, and respiratory systems. The RfC value for the neurological
9 system, based on decreased pain sensitivity, was selected for the proposed overall RfC for
10 1,2,3-TMB (see Section 2.2.5 for details). The RfC values for the hematological and respiratory
11 systems, based on decreased segmented neutrophils and increased inflammatory lung lesions, were
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1 only marginally higher than the RfC derived for neurological effects (6 x 1Q-2 and 7 x 1Q-2 mg/m3 vs.
2 5 x IQ-2 mg/m3), indicating that effects in these organ systems may also be of concern.
Table 2-10. Organ/system-specific RfCs and proposed overall RfC for 1,2,3-TMB
Effect
Hematological
Respiratory
Proposed
overall RfC
(Neurological)
Basis
Decreased segmented
neutrophils
Increased inflammatory
lung lesions
Decreased pain sensitivity
Rfc (mg/m3)
6 x 10"2
7 x 10"2
5 x 10"2
Exposure
description
Subchronic
Subchronic
Subchronic
Confidence
Low to medium
Low to medium
Low to medium
2.2.5. Selection of the Proposed Overall Reference Concentration for 1,2,3-TMB
3 Neurotoxicity is the most consistently observed endpoint in the toxicological database for
4 1,2,3-TMB. According to EPA's Guidelines for Neurotoxicity Risk Assessment [U.S. EPA, 1998], many
5 neurobehavioral changes are regarded as adverse, and the observation of correlated and replicated
6 measures of neurotoxicity strengthen the evidence for a hazard. Decreased pain sensitivity, as
7 measured as an increased latency to paw-lick in hot plate tests, represents an alteration in
8 neurobehavioral function [U.S. EPA. 1998]. Decreased pain sensitivity or decreased pain sensitivity
9 following a foot shock challenge was observed in two studies investigating short-term and
10 subchronic exposure durations [Wiadernaetal.. 1998: Korsak and Rydzynski. 1996] and in the
11 presence of other metrics of altered neurobehavior, including impaired neuromuscular function
12 and altered cognitive function. Additionally, neurological symptoms (e.g., hand tremble, weakness]
13 are observed in human worker populations exposed to complex VOC mixtures containing
14 1,2,3-TMB (notably, pain sensitivity has not been tested in humans] indicating a consistency and
15 coherency of neurotoxic effects in humans and animals following exposure to 1,2,3-TMB.
16 See Section 2.1.5 for a detailed discussion of U.S. EPA's Guidelines for Neurotoxicity Risk
17 Assessment (U.S. EPA. 1998] and the use of reversible and/or latent neurotoxicological endpoints in
18 the derivation of reference values. The issues pertaining to the observed 1,2,3-TMB neurotoxicity
19 are the same as those identified for 1,2,4-TMB. For example, although 1,2,3-TMB-induced pain
20 sensitivity was observed to return to control levels two weeks after termination of subchronic
21 inhalation exposure in one study (Korsak and Rydzynski. 1996]. the Neurotoxicity Guidelines note
22 that reversible effects occurring in occupational settings may be of high concern, particularly if they
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1 diminish a person's ability to survive or adapt to the environment [U.S. EPA. 1998] (pg. 8).
2 Additionally, the "designation of an effect as irreversible or reversible is academic, as exposure is
3 presumed to be lifetime (i.e., there is no post-exposure period)" [U.S. EPA. 2002] (pg. 3-27]. In other
4 words, the nature of an RfC precludes the possibility of recovery from the critical effect Lastly, the
5 issues surrounding the use of an environmental challenge (i.e., foot shock] in short-term
6 neurotoxicity studies of 1,2,3-TMB are the same as those discussed for 1,2,4-TMB in Section 2.1.5.
7 Taken as a whole, the database supports the characterization of decreased pain sensitivity
8 associated with exposure to 1,2,3-TMB as being an effect of high concern. Given the consistency of
9 observations from hot plate tests with or without foot shock challenge across several studies from
10 the same research group using multiple durations of exposure in male Wistar rats, as well as the
11 evidence and biological plausibility of similarities in neurological effects between rats and humans,
12 there is strong evidence that neurotoxicity is the primary hazard associated with exposure to
13 1,2,3-TMB. Based on these considerations, decreased pain sensitivity observed immediately after
14 subchronic exposure is identified as an adverse neurotoxic effect and thus is an appropriate effect
15 on which to base the RfC. Therefore, the candidate RfC for neurotoxicity based on decreased pain
16 sensitivity was selected as the proposed overall RfC for 1,2,3-TMB.
17 A PODHEc of 16.3 mg/m3 for decreased pain sensitivity (Korsak and Rydzynski. 1996] was
18 used as the POD to derive the chronic RfC for 1,2,3-TMB. The uncertainty factors (UFs], selected and
19 applied in accordance with the procedures described in EPA's A Review of the Reference Dose and
20 Reference Concentration Processes (U.S. EPA. 2002] (Section 4.4.5 of the report], were discussed
21 previously in Section 2.2.3. Application of this composite UF of 300 to the PODnEc yields the
22 following chronic RfC for 1,2,3-TMB:
23 RfC = PODHEC * UF = 16.3 mg/m3 4- 300 = 0.05 mg/m3 = 5 x 10 2 mg/m3 (rounded to one
24 significant digit)
2.2.6. Uncertainties in the Derivation of the Reference Concentration for 1,2,3-TMB
25 As presented above, the UF approach following EPA practices and RfC guidance (U.S. EPA.
26 2002.1994b]. was applied to the PODHEc in order to derive the chronic RfC for 1,2,3-TMB. Factors
27 accounting for uncertainties associated with a number of steps in the analyses were adopted to
28 account for extrapolation from animals to humans, a diverse human population of varying
29 susceptibilities, duration of exposure, POD determination methodologies (NOAEL, LOAEL, or
30 BMDL], and database deficiencies.
31 The critical effect selected, decreased pain sensitivity, does not introduce substantial
32 variability into the RfC calculation as selection of alternative hematological or respiratory effects
33 would result in similar RfCs that would be equivalent when rounding to one significant digit (i.e.,
34 2 x ID-2 mg/m3, see Figure 2-4]. Some uncertainty exists regarding the selection of the BMRs for use
35 in BMD modeling due to the absence of information to determine the biologically significant level of
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1 response associated with the endpoints. However in cases such as this, the selection of a BMR of
2 1 standard deviation for continuous endpoints is supported by EPA guidance [U.S. EPA. 2012b]. In
3 addition, there is uncertainty in the estimated standard deviation for decreased pain sensitivity
4 [Korsak and Rydzynski, 1996], which was two- to threefold lower than that estimated in the
5 parallel evaluation of 1,2,4-TMB in the same publication (see Section 2.1.6.). Given the lack of
6 information concerning a biologically significant level of response for pain sensitivity, the
7 concurrently estimated standard deviation was judged to be most relevant for characterizing this
8 response to 1,2,3-TMB.
9 Uncertainty regarding the selection of particular models for individual endpoints exists as
10 selection of alternative models could decrease or increase the estimated POD and consequently, the
11 RfC. The criteria for model selection was based on a practical approach as described in EPA's
12 Benchmark Dose Technical Guidance [U.S. EPA, 2012b]. Uncertainty may exist in the default
13 dosimetry methods used to calculate HEC estimates, but such uncertainties would apply equally to
14 all endpoints.
2.2.7. Confidence Statement for 1,2,3-TMB
15 Confidence in the study from which the critical effect was identified, Korsak and Rydzynski
16 [1996] is low to medium. The study is a peer-reviewed study that utilized three dose groups plus
17 untreated controls, employed an appropriate number of animals per dose group, and appropriately
18 performed statistical analyses. However, sources of uncertainty exist that reduce confidence in this
19 study.
20 One area of uncertainty regarding this study is the lack of reported actual concentrations.
21 However, as the methods by which the test atmosphere was generated and analyzed were reported
22 in sufficient detail, and given the fact that this laboratory has used this methodology in subsequent
23 studies [Korsak et al.. 2000a. b] and achieved appropriate actual concentrations (i.e., within 10% of
24 target concentrations], the concern regarding the lack of reported actual concentrations is minimal.
25 Another source of uncertainty is the fact that Korsak and Rydzynski (1996] does not explicitly state
26 that the reported measures of variance in Table 1 of that reference are standard deviations.
27 However, careful analysis of the reported levels of variance and magnitude of statistical significance
28 reported indicate that the measures of variance are standard deviations. Supporting this
29 conclusions is the observation that all other papers by Korsak et al. (2000a, b; 1997: 1995] report
30 variance as standard deviations. The critical effect on which the RfC is based is well-supported as
31 the weight of evidence for 1,2,3-TMB-induced neurotoxicity is coherent across multiple animals
32 species (i.e., mouse, and rat] and consistent across multiple exposure durations (i.e., acute, short-
33 term, and subchronic] (Lutz etal.. 2010: Wiadernaetal.. 1998: Korsak and Rydzynski. 1996].
34 The database for 1,2,3-TMB includes acute, short-term, and subchronic toxicity studies in
35 rats and mice. However, confidence in the database is low to medium because it lacks chronic,
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1 multi-generation reproductive/developmental, developmental toxicity, or developmental
2 neurotoxicity studies, and the studies supporting the critical effect predominantly come from the
3 same research institute. The overall confidence in the RfC for 1,2,3-TMB is low to medium.
2.3. Inhalation Reference Concentration for Effects Other Than Cancer
for 1,3,5-TMB
2.3.1. Identification of Studies and Effects for Dose-Response Analysis for 1,3,5-TMB
4 The nervous, hematological, and respiratory systems are the primary targets of toxicity for
5 inhaled 1,3,5-TMB in humans, whereas the nervous system in adults, pregnant females, and
6 developing organism are the primary targets of inhaled 1,3,5-TMB in experimental animals. Effects
7 in these systems have been identified as hazards following inhalation exposures to 1,3,5-TMB.
8 Although literature exists on the effects of 1,3,5-TMB exposure in humans, including neurological,
9 hematological, and respiratory toxicities, no human studies are available that would allow for dose-
10 response analysis. The human studies evaluated TMB exposures occurring as complex solvents or
11 VOC mixtures, and this consideration along with similar uncertainties as discussed for 1,2,4-TMB
12 and 1,2,3-TMB limit their utility in derivation of quantitative human health toxicity values. As for
13 the other two isomers, the human studies provide supportive evidence for the neurological toxicity
14 of 1,3,5-TMB in humans and indicate a consistency and coherency of this effect in humans and
15 laboratory animals.
16 Several studies investigating 1,3,5-TMB effects in experimental animals models were
17 identified in the literature. No chronic or subchronic inhalation studies were identified that
18 investigated effects in adult animals. One developmental toxicity study investigating maternal and
19 fetal toxicity was identified in the literature [Saillenfaitetal.. 2005). Data from this study pertaining
20 to the primary hazards observed animals (maternal/developmental effects) was considered as
21 candidate critical effects for the purpose of determining the point of departure (POD) for derivation
22 of the inhalation RfC for 1,3,5-TMB. Neurotoxicity and respiratory toxicities were also observed in
23 acute and short-term inhalation studies investigating effects in adult animals. However, the high
24 exposure concentrations used in acute studies and the short exposure durations employed in both
25 acute and short-term studies limit their utility for the quantitation of chronic human health effects.
26 Nevertheless, as with the human in which subject were exposed to mixtures containing 1,3,5-TMB,
27 these studies provide qualitative information regarding hazard identification, especially the
28 observation of the consistency and coherency of these effects across the 1,3,5-TMB database.
29 The developmental toxicity study by Saillenfait et al. [2005) is adequate for dose-response
30 analysis. This study exposed rats, a common laboratory animal for developmental studies, by
31 inhalation to 1,3,5-TMB (reported as 99% pure [impurities not reported]). The four exposure
32 groups covered just over an order of magnitude, with the higher three groups spaced about twofold
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1 apart Typical numbers of animals per exposure group were used, 25/group. Regarding exposure
2 characterization, Saillenfait et al. [2005] reported actual concentrations, as measured by gas
3 chromatography, to be within 10% of target concentrations. This increases the confidence in the
4 overall evaluation and adequacy of this study. Target and actual concentrations are provided in
5 Table 2-11.
Table 2-11. Target and actual exposure concentrations used in BMD modeling of
1,3,5-TMB endpoints considered for the derivation of the RfC
Reference
Saillenfait et al.
(2005)
Species/sex
Rat, female
(pregnant dam);
male and female
(fetuses)
Target exposure
concentration (mg/m3)
492
1,476
2,952
5,904
Actual exposure
concentration (mg/m3)
497
1,471
2,974
5,874
6 The Saillenfait et al. [2005] study examined 1,3,5-TMB-induced toxicity in both the
7 pregnant animal and the developing fetus, and the observed effects that demonstrated statistically
8 significant decreases relative to control were considered for the derivation of the RfC for 1,3,5-TMB
9 (Table 2-12]. These endpoints included decreased male and female fetal weights and decreased
10 maternal weight gain (minus gravid uterine weight]. Changes in serum chemistry parameters in
11 rats exposed to 1,3,5-TMB in a short-term (five weeks] inhalation study (Wiglusz etal.. 1975a]
12 were not considered for derivation of the RfC due to inconsistent temporal patterns of effect and
13 the lack of accompanying histopathology.
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Table 2-12. Endpoints considered for the derivation of the RfC for 1,3,5-TMB
Endpoint
Species/sex
Exposure concentration (mg/m3)
0
492
1,476
2,952
5,904
Developmental endpoints
Decreased fetal weight
(g)a
Rat, male
Rat, female
5.80 ±
0.41b'c
5.50 ±
0.32
5.76 ±
0.27
5.74 ±
0.21
5.50 ±
0.31
5.27 ±
0.47
5.39 ±
0.55*
5.18±
0.68
5.10±
0.57**
4.81
0.45**
Maternal endpoints
Decreased maternal
weight gain (g)a
Rat, female
29 ±14
(n = 21)d
30 ±9
(n = 22)
20 ±12
(n = 21)
7 ±20*
(n = 17)
-12 ± 19**
(n = 18)
p<0.05; p<0.01
"Adapted from Saillenfait et al. (2005).
bl\lumbers of live fetuses not explicitly reported.
"Values are expressed as mean ± 1SD.
dl\lumber of dams with live litters.
2.3.2. Methods of Analysis for 1,3,5-TMB
1 As discussed above in Section 2.3.1, endpoints observed in Saillenfait et al. [2005] that
2 demonstrated statistically significant (p < 0.05) increases or decreases relative to control for at
3 least one exposure group were considered for the derivation of the RfC for 1,3,5-TMB; these effects
4 are listed in Table 2-12. This assessment used the BMD approach, when possible, to estimate a POD
5 for the derivation of an RfC for 1,3,5-TMB (see Section C.I of Appendix C for detailed methodology).
6 The BMD approach involves fitting a suite of mathematical models to the observed dose-response
7 data using EPA's BMDS (version 2.2), and then selecting the best fitting model. Each best-fit model
8 estimates a BMD and its associated BMDL (i.e., a 95% lower bound on the BMD) corresponding to a
9 selected BMR.
10 For maternal weight gain identified from the Saillenfait et al. (2005) study, no information
11 is available regarding the change in these responses that would be considered biologically
12 significant, thus a BMR equal to a 1 standard deviation change in the control mean was used in
13 modeling these endpoints, consistent with EPA's Benchmark Dose Technical Guidance (U.S. EPA.
14 2012b). For the decreased male and female fetal body weight endpoints identified from the
15 Saillenfait et al. (2005) study, a BMR of 5% relative deviation from the control mean was selected
16 (see Section 2.1.2 for a detailed discussion for the rationale for this choice). Additionally, a BMR
17 equal to a 1 standard deviation change in the control mean was also selected for the BMD modeling
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1 of fetal body weight to facilitate comparisons across assessments [U.S. EPA. 2012b]. The estimated
2 BMDL is then used as the candidate POD (Table 2-13).
3 The suitability of the above methods to determine a POD is dependent on the nature of the
4 toxicity database for a specific chemical. In the Saillenfait et al. [2005] study, although decreased
5 fetal body weight in males and females was considered for BMD modeling, BMDS was unable to
6 adequately model the variance in response for this endpoint Consequently, the NOAEL/LOAEL
7 approach was used to identify a POD. Detailed modeling results are provided in Section C.I of
8 Appendix C.
9 Because an RfC is a toxicity value that assumes continuous human inhalation exposure over
10 a lifetime, data derived from inhalation studies in animals need to be adjusted to account for the
11 noncontinuous exposures used in these studies. In the Saillenfait et al. [2005] study, rats were
12 exposed to 1,3,5-TMB for 6 hours/day for 15 consecutive days (GD6-GD20). Therefore, the
13 duration-adjusted PODs for developmental/ maternal effects were calculated as follows:
14 PODADj (mg/m3) = POD (mg/m3) x hours exposed per day/24 hours
15 For example, for decreased fetal weight in males, the PODADj would be calculated as follows:
16 PODADj (mg/m3) = 2,974 mg/m3 x 6 hours/24 hours
17 PODADj (mg/m3) = 744 mg/m3
18 Because the selected endpoints for consideration as the critical effect (i.e., decreased fetal
19 body weight, and maternal body weight gain] are assumed to result primarily from systemic
20 distribution of 1,3,5-TMB, and no available PBPK model exists for 1,3,5-TMB, the human equivalent
21 concentration [HEC] for 1,3,5-TMB was calculated by the application of the appropriate dosimetric
22 adjustment factor (DAF] for systemically acting gases (i.e., Category 3 gases], in accordance with the
23 EPAs RfC Methodology [U.S. EPA. 1994b]. DAFs are ratios of animal and human physiologic
24 parameters, and are dependent on the nature of the contaminant (i.e., particle or gas] and the target
25 site (i.e., respiratory tract or remote to the portal-of-entry [i.e., systemic]] [U.S. EPA, 1994b]. For
26 gases with systemic effects, the DAF is expressed as the ratio between the animal and human
27 blood:air partition coefficients:
28 DAF = (Hb/g)A/(Hb/g)H
29 where:
30 (Hb/g)A = the animal blood:air partition coefficient
31 (Hb/g)H = the human blood:air partition coefficient
32 DAF = 55.7 [Tarnberg and Tohanson. 1995]/43 [Meulenberg and Vijverberg. 2000]
33 DAF = 1.3
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1 In cases where the animal blood:air partition coefficient is higher than the human value,
2 resulting in a DAF > 1, a default value of 1 is substituted [U.S. EPA. 1994b]. For example, the HEC for
3 decreased female fetal body weight (reported in Saillenfait et al. [2005]] is calculated as follows:
4 PODHEc = PODADj (mg/m3) x DAF
5 PODHEc = PODADj (mg/m3) x 1.0
6 PODHEc = 744 mg/m3 x 1.0
7 PODHEc = 744 mg/m3
8 Table 2-13 presents the calculated HECs for the candidate critical effects, selected
9 uncertainty factors (UFs], and the resulting derivation of candidate RfCs from the Saillenfait et al.
10 [2005] developmental toxicity study.
Table 2-13. Summary of derivation of points of departure for 1,3,5-TMB
Endpoint/Reference
Species/sex
Model; BMRor
NOAEL/LOAEL
POD
(mg/m3)
Candidate
PODADJ
(mg/m3)
Candidate
PODHEC
(mg/m3)
Developmental endpoints
Decreased fetal body weight
(Saillenfait etal., 2005)
Rat, male
Rat, female
NOAELa
NOAELa
2,974
2,974
744
744
744
744
Maternal endpoints
Decreased maternal body
weight gain
(Saillenfait etal. ,2005)
Rat, female
Power; 1 SD
1,302
326
326
' No model was able to fit data adequately, or data were not modeled.
2.3.3. Derivation of Candidate RfC Values for 1,3,5-TMB
11 Under EPA's A Review of the Reference Dose and Reference Concentration Processes [[U.S.
12 EPA. 2002]. §4.4.5], also described in the Preamble, five possible areas of uncertainty and
13 variability were considered in deriving the candidate RfC values for 1,2,4-TMB. An explanation of
14 these five possible areas of uncertainty and variability and the values assigned to each as a
15 designated uncertainty factor [UF] to be applied to the candidate PODHEc are as follows:
16 An interspecies uncertainty factor, UFA, of 3 (101/2 = 3.16, rounded to 3] was applied to
17 account for uncertainty in characterizing the toxicokinetic and toxicodynamic differences between
18 rats and humans following inhalation exposure to 1,3,5-TMB. In this assessment, the use of a DAF to
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1 extrapolate external concentrations from rats to humans reduces toxicokinetic uncertainty in
2 extrapolating from the rat data, but does not account for the possibility that humans may be more
3 sensitive to 1,3,5-TMB than rats due to toxicodynamic differences. A default UFA of 3 was thus
4 applied to account for this remaining toxicodynamic uncertainty and any residual toxicokinetic
5 uncertainty.
6 An intraspecies uncertainty factor, UFn, of 10 was applied to account for potentially
7 susceptible individuals in the absence of data evaluating variability of response in the human
8 population following inhalation of 1,3,5-TMB. No information is currently available to predict
9 potential variability in human susceptibility, including variability in the expression of enzymes
10 involved in 1,3,5-TMB metabolism.
11 A LOAEL to NOAEL uncertainty factor, UFL, of 1 was applied because the current approach is
12 to address this factor as one of the considerations in selecting a BMR for BMD modeling. In this
13 case, a BMR equal to a 1 standard deviation change in the control mean for modeled endpoints was
14 selected under the assumption that this BMR represents a minimal, biologically significant change
15 for these effects. For endpoints that could not be modeled, a LOAEL to NOAEL uncertainty factor of
16 1 was applied as a NOAEL was used.
17 A subchronic to chronic uncertainty factor, UFS, of 3 (101/2 = 3.16, rounded to 3) was applied
18 to decreased maternal weight to account for extrapolation from a subchronic (albeit gestational)
19 exposure duration study to derive a chronic RfC. The 3-fold uncertainty factor is applied to the POD
20 identified from the subchronic study on the assumption that effects observed in a similar chronic
21 study would be observed at lower concentrations for a number of possible reasons, including
22 potential cumulative damage occurring over the duration of the chronic study or an increase in the
23 magnitude or severity of effect with increasing duration of exposure. A full subchronic to chronic
24 uncertainty factor of 10 was not applied in this case as there was no observed decrease in adult
25 body weights in rats exposed to either 1,2,4-TMB or 1,2,3-TMB for longer durations (i.e., 90 days).
26 For decreases in fetal weight, a UFS of 1 was applied.
27 A database uncertainty factor, UFD, of 3 (101/2 = 3.16, rounded to 3) was applied to account
28 for database deficiencies. Strengths of the database include the well-designed developmental
29 toxicity study that investigated standard measures of maternal and fetal toxicity in Sprague-Dawley
30 rats. However, the lack of a multi-generational reproductive/developmental toxicity study
31 investigating effects due to 1,3,5-TMB exposure is a weakness of the database. EPA's^l Review of the
32 Reference Dose and Reference Concentration Processes (U.S. EPA, 2002] also recommends that the
33 database uncertainty factor take into consideration whether there is concern from the available
34 toxicology database that the developing organism may be particular susceptible to effects in
35 specific organ systems. TMBs (unspecified isomer) are able to cross the placenta (Cooper etal..
36 2001: Dowty etal.. 1976]: therefore, as neurotoxicity is observed in adult animals in the available
37 short-term 1,3,5-TMB inhalation studies, there is concern that exposure to 1,3,5-TMB may result in
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1 neurotoxicity in the developing organism. EPA's Guidelines for Neurotoxicity Risk Assessment [U.S.
2 EPA. 1998] identifies specific effects observed in adult animals (e.g., cognitive and motor function)
3 that can also affect the developing organism exposed in utero. The Neurotoxicity Guidelines [U.S.
4 EPA, 1998] also indicate that neurotoxicants may have greater access to the nervous system in
5 developing organisms due to an incomplete blood-brain barrier and immature metabolic
6 detoxifying pathways. Therefore, there is some concern that the lack of a developmental
7 neurotoxicity study is a deficiency in the database and that the inclusion of such a study would
8 potentially result in a lower POD than the POD for maternal effects identified from the available
9 1,3,5-TMB toxicity database. In summary, a 3-fold database UF was applied to account for the lack
10 of both a multi-generation reproductive/developmental toxicity study and a developmental
11 neurotoxicity study in the available database for 1,3,5-TMB.
12 Table 2-14 is a continuation of Table 2-13, and summarizes the application of UFs to each
13 POD to derive a candidate value for each data set. The candidate values presented in Table 2-14 are
14 preliminary to the derivation of the organ/system-specific values. These candidate values are
15 considered individually in the selection of a representative inhalation reference value for a specific
16 hazard and subsequent overall RfC for 1,3,5-TMB. Figure 2-3 presents graphically these candidate
17 values, uncertainty factors, and points of departure, with each bar corresponding to one data set
18 described in Tables 2-13 and 2-14. Additionally, the RfC values for 1,2,4-TMB and 1,2,3-TMB are
19 shown for comparative purposes
Table 2-14. Effects and corresponding derivation of candidate RfC values for
1,3,5-TMB
Endpoint/Reference
HEC
(mg/m3)3
UFA
UFH
UFL
UFS
UFD
Composite
UF
Candidate RfC
value (mg/m3)b
Developmental endpoints
Decreased fetal body weight, male
(Saillenfait et al., 2005)
Decreased fetal body weight, female
(Saillenfait et al., 2005)
744
744
3
3
10
10
1
1
1
1
3
3
100
100
7.44
7.44
Maternal endpoints
Decreased maternal body weight gain
(Saillenfait et al., 2005)
326
3
10
1
3
3
300
1.09
aHuman equivalent concentration.
As calculated by application of uncertainty factors, not rounded to 1 significant digit.
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Pain Sensitivity
E xl/Maternal body weight gain
•£ (Saillenfaitetal., 2005)
Fetal body weight, females
(Saillenfaitetal., 2005)
gj -53
xl/ Fetal body weight, males
(Saillenfaitetal., 2005)
Composite UF
A Candidate value
• POD(HEC)
0.001 0.01 0.1 1 10
Concentrations (mg/m3)
100
1000
Figure 2-3. Candidate RfC values with corresponding POD and composite UF for
1,3,5-TMB.
2.3 A. Derivation of Organ/System Specific Reference Concentrations for 1,3,5-TMB
1 Table 2-15 distills the candidate values from Table 2-14 into a single value for each organ or
2 system. The single RfC value selected for a particular organ system was preferably chosen using
3 biological and toxicological information regarding that endpoint. If no compelling biological
4 information exists with which to select the primary hazard, the lowest RfC value for that organ
5 system was selected. These organ- or system-specific reference concentrations may be useful for
6 subsequent cumulative risk assessments that consider the combined effect of multiple agents acting
7 at a common site. The individual organs and systems for which specific RfC values were derived
8 were the pregnant animal (maternal) and developing fetus (developmental). The RfC value for
9 maternal effects was the lowest of the derived specific RfCs using 1,3,5-TMB data. The RfC value for
10 developmental effects was greater than that for maternal effects, indicating this effect may be of
11 less concern. However, effects to pregnant animals and the developing fetus may be of less concern
12 in general as the RfC values for these types of effects (based on decreased maternal weight gain and
13 decreased male and female fetal weight, respectively) are much greater than the RfC value derived
14 for 1,2,4-TMB based on decreased pain sensitivity (see Section 2.3.5 for details).
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Table 2-15. Organ/system-specific RfCs and proposed overall RfC for 1,3,5-TMB
Effect
Developmental
Maternal
Proposed
overall RfC
(Neurological)
Basis
Decreased fetal weight (male
and female
Decreased maternal weight
gain
Decreased pain sensitivity
(based on RfC derived for
1,2,4-TMB)
RfC
(mg/m3)
7
1
5 x 10"2
Exposure
description
Gestational
Gestational
Subchronic
Confidence
Low to medium
Low to medium
Low to medium
2.3.5. Selection of the Proposed Overall Reference Concentration for 1,3,5-TMB
1 Decreased maternal weight gain was identified as the most sensitive endpoint in the
2 1,3,5-TMB database. A PODHEc of 326.0 mg/m3 for decreased maternal weight gain from Saillenfait
3 et al. [2005] was used to derive a candidate chronic RfC for 1,3,5-TMB as shown in Table 2-14.
4 Uncertainty factors, selected and applied in accordance with the procedures described in EPA's A
5 Review of the Reference Dose and Reference Concentration Processes [U.S. EPA, 2002], were
6 previously discussed in Section 2.3.2. Application of this 300-fold composite UF yields the
7 calculation of the chronic RfC for 1,3,5-TMB as follows:
8 RfC = PODHEC * UF = 326 mg/m3 4- 300 = 1.09 mg/m3 = 1 mg/m3 (rounded to one
9 significant digit)
10 However, while Saillenfait et al. [2005] is a well-conducted developmental toxicity study
11 that evaluates a wide range of fetal and maternal endpoints resulting from 1,3,5-TMB inhalation
12 exposure, a number of other factors lessens its suitability for use in deriving an RfC for 1,3,5-TMB.
13 First, although maternal and fetal toxicities were observed in this study, it is important to note that
14 the candidate RfC for 1,3,5-TMB, derived based on the critical effect of decreased maternal body
15 weight gain (corrected for gravid uterine weight], is 20-fold higher than the RfC derived for
16 1,2,4-TMB, which is based on altered CNS function measured as decreased pain sensitivity. As
17 discussed in Section 1.1.6, the available toxicological database for 1,2,4-TMB and 1,3,5-TMB, across
18 all exposure durations, indicates there are important similarities in the two isomers' neurotoxicity
19 that are supportive of an RfC for 1,3,5-TMB that is not substantially different than the RfC derived
20 for 1,2,4-TMB. Also supporting this conclusion is the observation that 1,2,4-TMB and 1,3,5-TMB
21 display important similarities in regard to chemical properties and toxicokinetics, including
22 similarities in blood:air partition coefficients, respiratory uptake, and absorption into the
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1 bloodstream (see Section 1.1.7 and Appendices B.I and B.2). These similarities support the
2 conclusion that internal blood dose metrics for 1,3,5-TMB would be comparable to those calculated
3 for 1,2,4-TMB using the available PBPK model.
4 Given these considerations, the use of 1,3,5-TMB-specific data for derivation of an RfC was
5 not considered to be scientifically supported. Thus, the chronic RfC of 5 x 1Q-2 mg/m3 derived
6 for 1,2,4-TMB was adopted as the RfC for 1,3,5-TMB based on the conclusion that the two
7 isomers were sufficiently similar based on chemical properties, toxicokinetics, and toxicity.
2.3.6. Uncertainties in the Derivation of the Reference Concentration for 1,3,5-TMB
8 Uncertainties exist in adopting the RfC derived for 1,2,4-TMB based on altered CNS function
9 (i.e., decreased pain sensitivity) as the RfC for 1,3,5-TMB. While the available database for
10 1,3,5-TMB was considered sufficient to derive an RfC, if the most sensitive endpoint from the only
11 adequate study in the 1,3,5-TMB database [i.e., decreased maternal weight gain; Saillenfait et al.
12 (2005)] was used for the RfC derivation, an RfC 20-fold higher would be derived for 1,3,5-TMB vs.
13 that derived for 1,2,4-TMB (1 vs. 5 x 1Q-2 mg/m3, respectively). Although uncertainty exists in
14 adopting the 1,2,4-TMB RfC for 1,3,5-TMB RfC, both isomers share multiple commonalities and
15 similarities regarding their chemical, toxicokinetic, and toxicological properties that support the
16 adoption of the value of one isomer for the other. The majority of uncertainty regarding 1,3,5-TMB's
17 database involves the lack of a chronic, subchronic, or multi-generational reproductive study for
18 this isomer. Given the similarities in toxicity from the developmental toxicity study, and
19 neurotoxicity and respiratory toxicity observed in the available acute and short-term studies, there
20 is strong evidence that the two isomer's toxicity resulting from subchronic exposure can be
21 expected to be similar. Therefore, while uncertainty exists in the derivation of 1,3,5-TMB's RfC, the
22 available information regarding sufficient chemical, toxicokinetic, and toxicological similarity
23 between the two isomers supports adopting the RfC for 1,2,4-TMB as the RfC for 1,3,5-TMB.
2.3.7. Confidence Statement for 1,3,5-TMB
24 The chronic RfC for 1,2,4-TMB was adopted as the RfC for 1,3,5-TMB; thus, confidence in the
25 study from which the critical effect was identified, Korsak and Rydzynski (1996). is low to
26 medium (see above). The database for 1,3,5-TMB includes acute, short-term, and developmental
27 toxicity studies in rats and mice. However, confidence in the overall database is low to medium
28 because it lacks chronic, subchronic, multi-generation reproductive/developmental toxicity, and
29 developmental neurotoxicity studies and most of the studies supporting the critical effect come
30 from the same research institute. Reflecting the confidence in the study and the database and the
31 uncertainty surrounding the adoption of the RfC derived for 1,2,4-TMB as the RfC for 1,3,5-TMB, the
32 overall confidence in the RfC for 1,3,5-TMB is low.
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2.4. Oral Reference Dose for Effects Other Than Cancer for 1,2,4-TMB
1 The RfD (expressed in units of mg/kg-day) is defined as an estimate (with uncertainty
2 spanning perhaps an order of magnitude) of a daily exposure to the human population (including
3 sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a
4 lifetime. It can be derived from a NOAEL, a LOAEL, or a 95% lower bound on the benchmark dose
5 (BMDL), with uncertainty factors (UFs) generally applied to reflect limitations of the data used.
2.4.1. Identification of Studies and Effects for Dose-Response Analysis for 1,2,4-TMB
6 No chronic or subchronic studies were identified for 1,2,4-TMB that utilized the oral route
7 of exposure. Therefore, the available oral database for 1,2,4-TMB is minimal as defined by EPA
8 guidance (i.e., there is no human data available nor any adequate oral animal data) (U.S. EPA. 2002).
9 and thus this database is inadequate for the derivation of an RfD.
2.4.2. Methods of Analysis for 1,2,4-TMB
10 Even though the available oral database for 1,2,4-TMB is inadequate to derive an RfD, a
11 route-to-route extrapolation from inhalation to oral for the purposes of deriving an RfD is possible
12 using the existing inhalation data and the available 1,2,4-TMB PBPK model (Hissinketal.. 2007).
13 The Hissink model was chosen as an appropriate model because it was the only published
14 1,2,4-TMB model that included parameterization for both rats and humans, the model code was
15 available, and the model adequately predicted experimental data in the dose range of interest.
16 Using route-to-route extrapolation via application of PBPK models is supported by EPA guidance
17 (U.S. EPA, 2002,1994b) given enough data and the ability to interpret that data with regard to
18 differential metabolism and toxicity between different routes of exposure. The available database
19 for 1,2,4-TMB supports the use of route-to-route extrapolation; sufficient evidence exists that
20 demonstrates similar qualitative profiles of metabolism (i.e., observation of dimethylbenzoic and
21 hippuric acid metabolites) and patterns of parent compound distribution across exposure routes
22 (Section B.2, Appendix B). Further, no evidence exists that would suggest toxicity profiles would
23 differ to a substantial degree between oral and inhalation exposures. In fact, in acute oral studies in
24 rats (Tomas etal., 1999a: Tomas etal., 1999b), the observed neurotoxic effects of exposure to 1,2,4-
25 TMB (i.e., alterations in motor function and electrocortical activity) are similar to effects observed
26 following short-term exposures to 1,2,4-TMB via inhalation.
27 Therefore, assuming oral exposure would result in the same systemic effect as inhalation
28 exposure (i.e., altered CNS function, measured as decreased pain sensitivity (Korsak and Rydzynski.
29 1996)). an oral exposure component was added to the Hissink et al. (2007) PBPK model by EPA
30 (Section B.3.3.5, Appendix B), assuming continuous oral ingestion and 100% absorption of the
31 ingested 1,2,4-TMB by constant infusion of the oral dose into the liver. This is a common
32 assumption when information about the oral absorption of the compound is unknown. The
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1 contribution of the first-pass metabolism in the liver for oral dosing was evaluated by simulating
2 steady-state venous blood levels (at the end of 5 0 days continuous exposure) for a standard human
3 at rest (70 kg) for a range of concentrations and doses; at low daily doses (0.1-10 mg/kg-day),
4 equivalent inhalation concentrations result in steady state blood concentrations 4-fold higher than
5 those resulting from oral doses, indicating the presence of first-pass metabolism following oral
6 exposure (see Figure B-18, Appendix B). This difference became insignificant for daily doses
7 exceeding 50 mg/kg-day.
8 The human PBPK model inhalation dose metric (weekly average blood concentration,
9 mg/L) for the PODADj (0.086 mg/L) for decreased pain sensitivity was used as the target for the oral
10 dose metric. The human PBPK model was run to determine what oral exposure would yield an
11 equivalent weekly average blood concentration, and then the resulting value of 6.3 mg/kg-day was
12 used as the human equivalent dose POD (PODHED) for the RfD derivation.
2.4.3. Derivation of the Reference Dose for 1,2,4-TMB
13 A PODHED of 6.3 mg/kg-day was derived for the oral database using route-to-route
14 extrapolation based on the neurotoxic effects (i.e., decreased pain sensitivity) observed by Korsak
15 and Rydzyhski (1996) following inhalation exposure to 1,2,4-TMB. Thus, the same uncertainty
16 factors applied to derive the RfC (see Section 2.1.5) were also applied to derive the RfD. The
17 uncertainty factors, selected and applied in accordance with the procedures described in EPA's A
18 Review of the Reference Dose and Reference Concentration Processes (U.S. EPA. 2002) (Section 4.4.5
19 of the report), address five areas of uncertainty resulting in a composite UF of 300.
20 Application of this 300-fold composite UF yields the calculation of the chronic RfD for
21 1,2,4-TMB as follows:
22 RfD = PODHED * UF = 6.3 mg/kg-day 4- 300 = 0.02 mg/kg-day = 2 x 10 2 mg/kg-day
23 (rounded to one significant digit)
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2.4.4. Uncertainties in the Derivation of the Reference Dose for 1,2,4-TMB
1 As the oral RfD for 1,2,4-TMB was based on a route-to-route extrapolation in order to
2 determine the oral dose that would result in the same effect (i.e., decreased pain sensitivity) as
3 inhalation exposure in Korsak and Rydzyhski [1996]. the uncertainties regarding this derivation
4 are the same as those for the RfC for 1,2,4-TMB (see Section 2.1.6), with the exception of the
5 uncertainty surrounding the route-to-route extrapolation. The model used to perform this route-to-
6 route extrapolation is a well-characterized model considered appropriate for the purposes of this
7 assessment. One source of uncertainty regarding the route-to-route extrapolation is the assumption
8 of 100% bioavailability, that is, 100% of the ingested 1,2,4-TMB would be absorbed and pass
9 through the liver. If not all of the compound is bioavailable, a lower blood concentration would be
10 expected compared to the current estimate, and thus, a higher RfD would be calculated.
2.4.5. Confidence Statement for 1,2,4-TMB
11 A PBPK model was utilized to perform a route-to-route extrapolation to determine a POD
12 for the derivation of the RfD from the Korsak and Rydzyhski (1996) inhalation study and
13 corresponding critical effect. The confidence in the study from which the critical effect was
14 identified, Korsak and Rydzyhski (1996), is low to medium (see Section 2.1.7). The inhalation
15 database for 1,2,4-TMB includes acute, short-term, subchronic, and developmental toxicity studies
16 in rats and mice. However, confidence in the database for 1,2,4-TMB is low to medium because it
17 lacks chronic, multi-generation reproductive/developmental and developmental neurotoxicity
18 studies, and the studies supporting the critical effect predominantly come from the same research
19 institute. Reflecting the confidence in the study and the database and the uncertainty surrounding
20 the application of the available PBPK model for the purposes of a route-to-route extrapolation, the
21 overall confidence in the RfD for 1,2,4-TMB is low.
2.5. Oral Reference Dose for Effects Other Than Cancer for 1,2,3-TMB
2.5.1. Identification of Studies and Effects for Dose-Response Analysis for 1,2,3-TMB
22 No chronic or subchronic studies were identified for 1,2,3-TMB that utilized the oral route
23 of exposure. Therefore, the available oral database for 1,2,3-TMB is minimal as defined by EPA
24 guidance (i.e., there is no human data available nor any adequate oral animal data) (U.S. EPA. 2002).
25 and thus this database is inadequate for the derivation of an RfD.
2.5.2. Methods of Analysis and Derivation of the Reference Dose for 1,2,3-TMB
26 The available oral database is inadequate to derive an RfD for 1,2,3-TMB. No chronic,
27 subchronic, or short-term oral exposure studies were found in the literature. However, as discussed
28 in Section 1.1.6, there are sufficient similarities between isomers regarding observed toxicological
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1 effects that support adopting the RfD for 1,2,4-TMB as the RfD for 1,2,3-TMB. Specifically, the
2 qualitative pattern of neurotoxic effects following short-term and subchronic inhalation exposures
3 is similar between 1MB isomers. Particularly important to this determination is that, although
4 1,2,3-TMB is observed to decrease pain sensitivity at lower concentrations than 1,2,4-TMB (LOAEL
5 values of 123 vs. 492 mg/m3, respectively), the magnitude of decreased pain sensitivity is similar
6 for 1,2,4-TMB and 1,2,3-TMB, especially at the low- and mid-concentrations. This similarity of effect
7 in the low-dose region of the dose-response curve is exhibited by equal RfC values derived from
8 isomer-specific data: 5 x 10~2 mg/m3. Additionally, given that similar patterns of neurotoxicity are
9 observed following acute oral and subchronic inhalation exposures to 1,2,4-TMB, it is reasonable to
10 assume that neurotoxicity profiles would not differ substantially between oral and inhalation
11 exposures to 1,2,3-TMB. Although a PBPK model exists for 1,2,4-TMB that allows for route-to-route
12 extrapolation from inhalation to oral exposure, no such model exists for 1,2,3-TMB. However,
13 similarities in blood:air and tissue:air partition coefficients and degree of absorption into the
14 bloodstream between 1,2,4-TMB and 1,2,3-TMB support the conclusion that internal blood dose
15 metrics for 1,2,3-TMB would be similar to those calculated for 1,2,4-TMB using that isomer's
16 available PBPK model. Also, the qualitative metabolic profiles for the two isomers are similar, with
17 dimethylbenzyl hippuric acids being the major terminal metabolite for both isomers, such that first-
18 pass metabolism through the liver is not expected to differ greatly between 1,2,4-TMB and
19 1,2,3-TMB. Therefore, given the similarities in chemical properties, toxicokinetics, and
20 toxicity, the RfD derived for 1,2,4-TMB, 2 x 10 2 mg/kg-day was adopted as the RfD for
21 1,2,3-TMB.
2.5.3. Uncertainties in the Derivation of the Reference Dose for 1,2,3-TMB
22 The uncertainties regarding adopting the RfD for 1,2,4-TMB as the RfD for 1,2,3-TMB
23 encompass previous areas of uncertainty involved in the derivation of the RfC for 1,2,3-TMB and
24 the RfD for 1,2,4-TMB (see Sections 2.1.6 and 2.2.6). Additionally, there is uncertainty in this
25 adoption regarding the assumptions made about the similarity in toxicokinetics and toxicity
26 between the two isomers. However, as discussed above in Sections 1.1.6 and 1.1.7 and in Appendix
27 B (Section B.2), there is strong evidence that both isomers share multiple commonalities and
28 similarities regarding their toxicokinetic and toxicological properties that support adopting one
29 isomer's value for the other.
2.5.4. Confidence Statement for 1,2,3-TMB
30 The chronic RfD for 1,2,4-TMB was adopted as the RfD for 1,2,3-TMB; thus, confidence in
31 the study from which the critical effect was identified, Korsak and Rydzyhski (1996). is low to
32 medium (see above). The inhalation database for 1,2,3-TMB includes acute, short-term, and
33 subchronic studies in rats and mice. However, confidence in the database is low to medium because
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1 it lacks chronic, multi-generation reproductive/developmental, developmental toxicity, or
2 developmental neurotoxicity studies, and the studies supporting the critical effect predominantly
3 come from the same research institute. Reflecting the confidence in the study and the database and
4 the uncertainty surrounding the adoption of the RfD derived for 1,2,4-TMB as the RfD for
5 1,2,3-TMB, the overall confidence in the RfD for 1,2,3-TMB is low.
2.6. Oral Reference Dose for Effects Other Than Cancer for 1,3,5-TMB
2.6.1. Identification of Studies and Effects for Dose-Response Analysis for 1,3,5-TMB
6 Only one subchronic study [Koch Industries, 1995b] investigating 1,3,5-TMB's toxicity was
7 located that utilized the oral route of exposure. As this study was not located in the peer-reviewed
8 literature (it was submitted to EPA under a TSCA 4(a) test rule), EPA sought an independent
9 external peer review to assess the study's reliability and suitability for use as the basis of an RfD
10 derivation [Versar. 2013). Ultimately, the results of the external peer review led EPA to conclude
11 that this study was not suitable to serve as a principal study with which to derive human health
12 reference doses (see Appendix F). The most critical shortcoming noted in the external peer review
13 of the Koch Industries (1995b] study was its lack of investigation of neurotoxicity endpoints, as
14 these effects (e.g., decreased pain sensitivity, altered cognitive ability) have been demonstrated to
15 be the most sensitive endpoints following inhalation exposure to other TMB isomers. Given the
16 conclusion that the Koch Industries (1995b) study is insufficient for use in RfD derivation, the
17 available oral database for 1,3,5-TMB is minimal as defined by EPA guidance (i.e., there is no human
18 data available nor any adequate oral animal data) (U.S. EPA. 2002). and thus this database is
19 inadequate for the derivation of an RfD.
2.6.2. Methods of Analysis and Derivation of the Reference Dose for 1,3,5-TMB
20 The available oral database is inadequate to derive an RfD for 1,3,5-TMB. The only identified
21 oral toxicity study was judged to be unsuitable for derivation of the RfD. However, as outlined in the
22 RfC Derivation for 1,3,5-TMB, the chemical, toxicokinetic, and toxicological similarities between
23 1,3,5-TMB and 1,2,4-TMB support adopting the RfC for 1,2,4-TMB as the RfC for 1,3,5-TMB. These
24 considerations also apply to the oral reference value, thus the RfD for 1,2,4-TMB was adopted for
25 1,3,5-TMB. 1,3,5-TMB and 1,2,4-TMB are observed to elicit similar neurotoxic effects in rats in acute
26 and short-term oral and inhalation studies, and therefore the selected critical effect for 1,2,4-TMB,
27 altered CNS function, is relevant to observed 1,3,5-TMB-induced toxicity. Further, no evidence
28 exists to suggest that toxicity profiles would differ substantially between oral and inhalation
29 exposures to 1,3,5-TMB. In fact, in acute oral studies in rats (Tomas etal.. 1999a: Tomas etal..
30 1999b). the observed neurotoxic effects of exposure to 1,3,5-TMB (i.e., alterations in motor
31 function) are similar to effects observed following short-term exposures via inhalation. Similarities
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1 in blood:air and tissue:air partition coefficients and absorption into the bloodstream between the
2 two isomers support the conclusion that internal blood dose metrics for 1,3,5-TMB would be
3 similar to those calculated for 1,2,4-TMB using the available PBPK model. Also, the qualitative
4 metabolic profiles for the two isomers are similar, with dimethylbenzyl hippuric acids being the
5 major terminal metabolite for both isomers, so that first-pass metabolism through the liver is not
6 expected to differ greatly between 1,2,4-TMB and 1,3,5-TMB. Therefore, given the similarities in
7 chemical properties, toxicokinetics, and toxicity, the RfD derived for 1,2,4-TMB of 2 x 1Q-2
8 mg/kg-day was adopted as the RfD for 1,3,5-TMB.
2.6.3. Uncertainties in the Derivation of the Reference Dose for 1,3,5-TMB
9 The uncertainties regarding adopting the RfD for 1,2,4-TMB as the RfD for 1,3,5-TMB
10 encompass previous areas of uncertainty involved in the derivation of the RfC for 1,3,5-TMB and
11 the RfD for 1,2,4-TMB (see Sections 2.3.6 and 2.4.4). There is uncertainty regarding this adoption.
12 However, as discussed above in Section 2.3.3, both isomers share multiple commonalities and
13 similarities regarding their chemical, toxicokinetic, and toxicological properties that support
14 adopting one isomer's value for the other. Additionally, as the RfD derivation for 1,2,4-TMB was
15 based on a route-to-route extrapolation, the uncertainties in that toxicity value's derivation (see
16 Section 2.4.3) apply to the derivation of the RfD for 1,3,5-TMB.
2.6.4. Confidence Statement for 1,3,5-TMB
17 The chronic RfD for 1,2,4-TMB was adopted as the RfD for 1,3,5-TMB; thus confidence in the
18 study from which the critical effect was identified, Korsak and Rydzyhski (1996). is low to medium
19 (see above). The inhalation database for 1,3,5-TMB includes acute, short-term, and developmental
20 toxicity studies in rats and mice. However, confidence in the database is low to medium because it
21 lacks chronic, multi-generation reproductive/developmental and developmental neurotoxicity
22 studies, and the studies supporting the critical effect predominantly come from the same research
23 institute. Reflecting the confidence in the study and the database and the uncertainty surrounding
24 the adoption of the RfD derived for 1,2,4-TMB as the RfD for 1,3,5-TMB, the overall confidence in
25 the RfD for 1,3,5-TMB is low.
2.7. Cancer Risk Estimates for 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB
26 Under the U.S. EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a). the
27 database for 1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB provides "inadequate information to assess
28 carcinogenic potential". This characterization is based on the limited and equivocal genotoxicity
29 findings, and the lack of data indicating carcinogenicity in experimental animal species via any
30 route of exposure. Information available on which to base a quantitative cancer assessment is
31 lacking, and thus, no cancer risk estimates for either oral or inhalation exposure are derived.
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