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EPA/635/R-16/001a
Final Agency/Interagency Science Discussion Draft
www.epa.gov/iris
Toxicological Review of Trimethylbenzenes
(CASRNs 25551-13-7, 95-63-6, 526-73-8, and 108-67-8]
June 2016
NOTICE
This document is a Final Agency/Interagency Science Discussion 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.
Integrated Risk Information System
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
A EPA
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Toxicological Review of Trimethylbenzenes
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
TABLES v
FIGURES vi
ABBREVIATIONS vii
AUTHORS | CONTRIBUTORS | REVIEWERS ix
PREFACE xiii
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS xvi
EXECUTIVE SUMMARY xxiii
LITERATURE SEARCH STRATEGY | STUDY SELECTION AND EVALUATION xxxi
1. HAZARD IDENTIFICATION 1-1
1.1. OVERVIEW OF THE TOXICOKINETICS OF TMBs 1-2
1.1.1. Toxicokinetics of TMB isomers 1-2
1.1.2. Description of Toxicokinetic Models 1-4
1.2. SYNTHESIS OF EVIDENCE 1-5
1.2.1. Neurological Effects 1-5
1.2.2. Respiratory Effects 1-33
1.2.3. Reproductive and Developmental Effects 1-39
1.2.4. Hematological and Clinical Chemistry Effects 1-45
1.2.5. General Toxicity 1-53
1.2.6. Carcinogenicity 1-54
1.2.7. Similarities among TMB Isomers Regarding Observed Inhalation and Oral Toxicity 1-56
1.3. SUMMARY AND EVALUATION 1-60
1.3.1. Weight of Evidence for Effects Other than Cancer 1-60
1.3.2. Weight of Evidence for Carcinogenicity 1-65
1.3.3. Susceptible Populations and Lifestages 1-66
2. DOSE-RESPONSE ANALYSIS 2-1
2.1. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER THAN CANCER FOR
TMBs 2-1
2.1.1. Identification of Studies and Effects for Dose-Response Analysis and Derivation of
Reference Concentrations for TMBs 2-1
2.1.2. Methods of Analysis for Derivation of Reference Concentrations for TMBs 2-8
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2.1.3. Derivation of Candidate Inhalation Values for TMBs 2-14
2.1.4. Derivation of Organ/System-Specific Reference Concentrations for TMBs 2-21
2.1.5. Selection of the Overall Reference Concentration for TMBs 2-22
2.1.6. Uncertainties in the Derivation of the Reference Concentration for TMBs 2-24
2.1.7. Confidence Statement for the Reference Concentration for TMBs 2-26
2.1.8. Calculation of Subchronic Reference Concentrations for TMBs 2-26
2.2. ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER FOR TMBs 2-28
2.2.1. Identification of Studies and Effects for Dose-Response Analysis and Derivation of
Reference Doses for TMBs 2-28
2.2.2. Methods of Analysis for Derivation of Reference Doses for TMBs 2-29
2.2.3. Derivation of the Reference Dose for TMBs 2-31
2.2.4. Uncertainties in the Derivation of the Reference Dose for TMBs 2-36
2.2.5. Confidence Statement for the Reference Dose for TMB 2-36
2.2.6. Calculation of Subchronic Reference Doses for TMBs 2-37
2.3. CANCER RISK ESTIMATES FOR TMBs 2-37
REFERENCES R-l
SUPPLEMENTAL INFORMATION (see Volume 2)
This document is a draft for review purposes only and does not constitute Agency policy.
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TABLES
Table P-l. Physical properties and chemical identities of TMB isomers xv
Table ES-1. Organ/system-specific RfCs for individual TMB isomers xxv
Table ES-2. Organ/system-specific subchronic RfCs for individual TMB isomers xxvi
Table LS-1. Details of the initial search strategy employed for TMBs xxxi
Table 1-1. Toxicokinetic similarities between TMB isomers 1-3
Table 1-2. Evidence pertaining to neurological effects of TMBs in animals—inhalation exposures 1-17
Table 1-3. Evidence pertaining to neurological effects of TMBs in animals—oral exposures 1-22
Table 1-4. Evidence pertaining to respiratory effects of TMBs in animals—inhalation exposures 1-35
Table 1-5. Evidence pertaining to reproductive and developmental effects of TMBs in animals-
inhalation exposures 1-41
Table 1-6. Evidence pertaining to hematological and clinical chemistry effects of TMBs in
animals—inhalation exposures 1-48
Table 1-7. Evidence pertaining to hematological and clinical chemistry effects of TMBs in
animals—oral exposures 1-50
Table 1-8. Similarities between 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB regarding observed
inhalation and oral toxicity 1-60
Table 2-1. Target and actual exposure concentrations used in BMD modeling of 1,2,4-TMB,
1,2,3-TMB, and 1,3,5-TMB endpoints considered for the derivation of the RfC 2-4
Table 2-2. Endpoints observed in rats in the Korsak studies (Korsak et al., 2000a, b; Korsak et
al., 1997; Korsak and Rydzynski, 1996) considered for the derivation of the RfC for
TMBs 2-5
Table 2-3. Endpoints observed in rats in the Saillenfait et al. (2005) study considered for the
derivation of the RfC for TMBs 2-6
Table 2-4. Isomer-specific DAFs using default dosimetric methods 2-13
Table 2-5. Summary of derivation of PODs for TMBs 2-14
Table 2-6. Summary of derivation of candidate inhalation values for TMBs 2-19
Table 2-7. Organ/system-specific RfCs and overall RfC for TMBs 2-22
Table 2-8. Summary of derivation of subchronic RfC values for TMBs 2-28
This document is a draft for review purposes only and does not constitute Agency policy.
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FIGURES
Figure LS-1. Literature search and study selection strategy for TMBs xxxiii
Figure 1-1. Exposure response array of neurological effects following inhalation exposure to
1,2,4-TMB, 1,2,3-TMB, or 1,3,5-TMB 1-24
Figure 1-2. Exposure response array of respiratory effects following inhalation exposure to
1.2.3-TMB, 1,2,4-TMB, or 1,3,5-TMB 1-36
Figure 1-3. Exposure response array of developmental effects following inhalation exposure to
1.2.4-TMB or 1,3,5-TMB 1-43
Figure 1-4. Exposure response array of hematological and clinical chemistry effects following
inhalation exposure to 1,2,4-TMB or 1,2,3-TMB 1-51
Figure 1-5. Exposure response array of hematological and clinical chemistry effects following
oral exposure to 1,3,5-TMB 1-51
Figure 2-1. Candidate values with corresponding POD and composite UF for TMBs 2-20
This document is a draft for review purposes only and does not constitute Agency policy.
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ABBREVIATIONS
AAQC
Ambient air quality criterion
Hb/g-A
animal blood:gas partition coefficient
ABR
amount of 1,2,4-TMB in the brain
Hb/g-H
human blood:gas partition coefficient
ADME
absorption, distribution, metabolism,
HEC
human equivalent concentration
and excretion
HED
human equivalent dose
AEGL
Acute Exposure Guideline Level
HERO
Health and Environmental Research
AIC
Akaike Information Criterion
Online
ALT
alanine aminotransferase
HFAN
High-Flash Aromatic Naphtha
ANCOVA
analysis of covariance
HLVOC
highly lipophilic volatile organic
ANOVA
analysis of variance
chemical
AP
alkaline phosphatase
HSDB
Hazardous Substances Data Bank
AST
aspartate aminotransferase
IL-8
interleukin-8
AUC
area under the curve
i.p.
intraperitoneal
BAL
bronchoalveolar lavage
IRIS
Integrated Risk Information System
BMCL
lower confidence limit on the
JP-8
jet propulsion fuel 8
benchmark concentration
KCCT
kaolin-cephalin coagulation time
BMD
benchmark dose
Km
Michaelis-Menten constant
BMDL
lower confidence limit on the
LLF
log-likelihood function
benchmark dose
LOAEL
lowest-observed-adverse-effect level
BMDS
benchmark dose software
MCH
mean corpuscular hemoglobin
BMR
benchmark response
MCHC
mean corpuscular hemoglobin
BrdU
5-bromo-2'-deoxyuridine
concentration
BUN
blood urea nitrogen
MCV
mean cell volume
BW
body weight
MMS
methyl methanesulfate
CAAC
Chemical Assessment and Advisory
MOE
Ministry of the Environment
Committee
NIOSH
National Institute for Occupational
CASRN
Chemical Abstracts Service Registry
Safety and Health
Number
NLE
neutral lipid equivalent
CE
cloning efficiency
NLM
National Library of Medicine
CHO
Chinese hamster ovary
N- methyl-D-aspartate
CI
confidence interval
NOAEL
no-observed-adverse-effect level
CMIX
average of arterial and venous blood
NOEL
no-observed-effect level
concentrations
NRC
National Research Council
CNS
central nervous system
NSC
normalized sensitivity coefficient
CV
concentration in venous blood
OSHA
Occupational Safety and Health
CVS
concentration in venous blood exiting
Administration
slowly perfused tissues
p-value
probability value
CXEQ
concentration in exhaled breath
PBPK
physiologically based pharmacokinetic
CYP450
cytochrome P450
(model)
DAF
dosimetric adjustment factor
PCV
packed cell volume
df
degree of freedom
Pg
picogram
DMBA
dimethylbenzoic acid
PMR
proportional mortality ratio
DMHA
dimethylhippuric acid
PND
postnatal day
DMSO
dimethylsulfoxide
POD
point of departure
DNA
deoxyribonucleic acid
PODadj
duration-adjusted POD
ECso
half maximal effective concentration
ppm
parts per million
EEG
electroencephalogram
QPC
alveolar ventilation rate
EPA
U.S. Environmental Protection Agency
OR
odds ratio
fMRI
functional magnetic resonance imaging
QRTOTC
sum of fractional flows to rapidly
GABA
gamma-aminobutyric acid
perfused tissues, liver, and brain
GD
gestational day
QSTOTC
sum of fractional flows to slowly
GGT
gamma-glutamyl transpeptidase
perfused tissues
This document is a draft for review purposes only and does not constitute Agency policy.
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RBC
red blood cell
TOXLINE
Toxicology Literature Online
RD
relative deviation
TWA
time-weighted average
RD50
50% respiratory rate decrease
UF
uncertainty factor
REL
recommended exposure limit
UFa
interspecies uncertainty factor
RfC
reference concentration
UFh
intraspecies uncertainty factor
RfD
reference dose
UFs
subchronic-to-chronic uncertainty
ROS
reactive oxygen species
factor
SAB
Science Advisory Board
UFl
LOAEL-to-NOAEL uncertainty factor
SCE
sister chromatid exchange
UFd
database deficiency uncertainty factor
SCI
Science Citation Index
VEP
visual evoked potential
SD
standard deviation
Vmax
% maximal enzyme rate
SDH
sorbitol dehydrogenase
voc
volatile organic compound
SE
standard error
w
watt
SMR
standardized mortality ratio
WBC
white blood cell
SOA
secondary organic aerosol
WOS
Web of Science
SVEP
short-latency visual evoked potential
I2
chi-squared
SWD
spike-wave discharge
TLV
threshold limit value
TMB
trimethylbenzene
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,
Currently CDC)
Paul Schlosser, Ph.D.
John Cowden, Ph.D.
Gary Foureman, Ph.D. (Currently ICF
Int.)
Andrew Kraft, Ph.D.
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
Assessment
Scientific Support Team
Reeder Sams, Ph.D. U.S. Environmental Protection Agency
John Stanek, Ph.D. Office of Research and Development
Rob Dewoskin, Ph.D. (Retired) National Center for Environmental Assessment
George Woodall, Ph.D.
Geniece Lehmann, Ph.D.
Connie Meacham, M.S.
Nina Wang, Ph.D
Martin Gehlhaus, MHS U.S. Environmental Protection Agency
Region 3
Philadelphia, PA
This document is a draft for review purposes only and does not constitute Agency policy.
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Production Team
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
Ryan Jones
Maureen Johnson
Vicki Soto
Ellen Lorang, M.S. (Retired)
Deborah Wales (Retired)
Gerald Gurevich (Currently NATO)
Connie Meacham
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
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 (Currently OSP)
(Associate Director for Health)
Vincent Cogliano, Ph.D.
(IRIS Program Director)
Samantha Jones, Ph.D.
(IRIS Associate Director for Science)
Jamie Strong, Ph.D. (Currently OW)
Ted Berner, MS
Reeder Sams, Ph.D.
Lyle Burgoon, Ph.D. (Currently Army Corp of
Engineers)
John Vandenberg, Ph.D.
Debra Walsh, M.S.
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
This document is a draft for review purposes only and does not constitute Agency policy.
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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 8, Denver, CO
Region 2, New York, NY
Office of the Administrator/Office of Children's Health Protection
Office of Air and Radiation
Office of Environmental Information
Office of Solid Waste and Emergency Response
Office of Pesticide Programs
Office of Water
This assessment was provided for review to other federal agencies and the Executive Office of the
President Comments were submitted by:
Department of Defense
DHHS/Agency for Toxic Substances and Disease Registry
DHHSNational Institute for Occupational Safety and Health
DHHS/National Institute of Environmental Health Sciences/National Toxicology Program
Executive Office of the President/Council on Environmental Quality
This assessment was released for public comment on June 26th, 2012 and comments were due on
August 28th, 2012. The public comments are available on Regulations.gov. A summary and EPA's
disposition of the comments from the public is available in the external peer review draft
assessment on the IRIS website. Comments were received from the following entities:
A public listening session was held by EPA on August 1st, 2012. Attendees external to the EPA are
listed below.
This assessment was peer reviewed by the Chemical Assessment Advisory Committee (of EPA's
Science Advisory Board). Reviewers included:
Leslie Berry
Allison Starmann
American Chemistry Council
Leslie Berry
Richard Becker
American Chemistry Council
Chair
Dr. Cynthia M. Harris, Florida A&M University
Members
Dr. James V. Bruckner, University of Georgia
Dr. Deborah Cory-Slechta, University of Rochester
Dr. Helen Goeden, Minnesota Department of Health
Dr. Sean Hays, Summit Toxicology
Dr. Lawrence Lash, Wayne State University
Dr. Lorenz Rhomberg, Gradient
Dr. Stephen M. Roberts, University of Florida
Consultants
Dr. Mitchell Cohen, New York University
Dr. Gary Ginsberg, Connecticut Department of Health
Dr. Robert A. Howd, ToxServices
This document is a draft for review purposes only and does not constitute Agency policy.
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Dr. Kannan Krishnan, University of Montreal
Dr. Frederick J. Miller, Independent ConsultanConsultant
Dr. EmanuelaTaioli, North Shore-LIJ School of Medicine
Dr. Raymond York, R.G. & Associates
Federal Experts
Dr. Frederick Beland, U.S. FDA
Designated Federal Officer
Mr. Thomas Carpenter, U.S. EPA
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzenes
PREFACE
This Toxicological Review critically reviews the publicly available studies on the three
isomers of trimethylbenzene (TMBs) (i.e., 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB) in order to identify
their adverse health effects and to characterize exposure-response relationships. Because more
types of studies are available for the 1,2,4-TMB isomer, it generally appears first when the
individual isomers are listed. This assessment was prepared under the auspices of the U.S.
Environmental Protection Agency (EPA) Integrated Risk Information System (IRIS) program.
This assessment was prepared because of the presence of TMBs at Superfund sites. Of sites
on EPA's National Priorities List that report TMB isomer contamination (38 sites), 93% report
1,3,5-TMB contamination, 85% report 1,2,4-TMB contamination, 12% report 1,2,3-TMB
contamination, and 17% report contamination by unspecified TMB isomers.
The Toxicological Review of Trimethylbenzenes is a new assessment; there is no previous
entry on the IRIS Database for 1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB. This assessment reviews
information on all health effects by all exposure routes.
This assessment was conducted in accordance with EPA guidance. The findings of this
assessment and related documents produced during its development are available on the IRIS
website (http://www.epa.gov/iris). Appendices for toxicokinetic information, summaries of
toxicity studies, and other supporting materials are provided as supplemental information to this
assessment (see Appendices C and D).
The IRIS Program released this assessment for public comment and peer review in June
2012, as it was beginning to implement systematic review. The approach to implementation is to
use procedures and tools available at the time, without holding assessments until new methods
become available. Accordingly, the IRIS Program edited this assessment to increase transparency
and clarity and to use more tables and figures. It conducted literature searches and evaluated
studies using tools and documentation standards then available. Problem formulation materials
and protocol development began with assessments started in 2015, after this assessment was well
into peer review. This assessment addresses peer-review comments and retains the structure of the
peer-review draft, to maintain fidelity with what the peer reviewers saw. Implementation of
systematic review is a process of continuous improvement subject to periodic review by the
Chemical Assessment Advisory Committee (of EPA's Science Advisory Board). This assessment
represents a step in the evolution of the IRIS Program.
For additional information about this assessment or for general questions regarding IRIS,
please contact EPA's IRIS Hotline at 202-566-1676 (phone), 202-566-1749 (fax), or
hotline.iris@epa.gov.
This document is a draft for review purposes only and does not constitute Agency policy.
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Assessments by Other National and International Health Agencies
Toxicity information on 1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB has been evaluated by the
National Institute for Occupational Safety and Health (NIOSH) and the National Advisory
Committee for Acute Exposure Guideline Levels (AEGLs) for Hazardous Substances. The results of
these assessments are summarized in Appendix B (Table B-l). It is important to recognize that
these assessments may have been prepared for different purposes and may utilize different
methods, and that newer studies may be included in the IRIS assessment
Chemical Properties and Uses
TMBs are aromatic hydrocarbons with three methyl groups attached to a benzene ring and
the chemical formula C9H12. The chemical and physical properties of the TMB isomers are similar to
one another. TMBs are colorless, flammable liquids with a strong aromatic odor; an odor threshold
of 0.4 parts per million (ppm) of air has been reported (U.S. EPA. 1994al. They are insoluble in
water but miscible with organic solvents such as ethyl alcohol, benzene, and ethyl ether (OSHA.
19961. Production and use of TMBs may result in their release to the environment through various
waste streams. If released to the atmosphere, 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB will exist solely
in the vapor phase in the atmosphere under ambient conditions, based on measured vapor
pressures of 1.69, 2.10, and 2.48 mm Hg at 25°C, respectively fHSDB. 2011a. b, c). All three isomers
are expected to have limited mobility through soil based on their log Koc values, but are expected to
volatilize from both moist and dry soil surfaces and surface waters based on their respective
Henry's law constants and vapor pressures. Degradation of trimethylbenzene isomers in the
atmosphere occurs by reaction with hydroxyl radicals; the half-life is 11-12 hours (HSDB. 2011a. b,
c). Non-volatilized TMBs may be subject to biodegradation under aerobic conditions fHSDB. 2011a.
b, c). The estimated bioconcentration factors (133-439) and high volatility of TMBs suggest that
bioaccumulation of these chemicals will not be significant fU.S. EPA. 19871. Additional information
on the chemical identities and physicochemical properties of TMBs is listed in Table P-l.
The commercially available substance known as trimethylbenzene, Chemical Abstracts
Service Registry Number (CASRN) 25551-13-7, is a mixture ofthree isomers in various
proportions, namely CASRN 526-73-8 (1,2,3-TMB or hemimellitene), CASRN 95-63-6 (1,2,4-TMB or
pseudocumene), and CASRN 108-67-8 (1,3,5-TMB or mesitylene). Production of TMB isomers
occurs during petroleum refining, and 1,2,4-TMB individually makes up approximately 40% of the
C9 aromatic fraction (i.e., aromatic hydrocarbons with nine carbons) fU.S. EPA. 1994al. The
domestic production of the C9 fraction in 1991 was estimated to be approximately 80 billion
pounds (40 million tons) (U.S. EPA. 1994a). Vehicle emissions are a major anthropogenic source of
TMBs, due to the widespread use of the C9 fraction as a component of gasoline (U.S. EPA. 1994a).
Other uses of TMBs include solvents in research and industry, dyestuff intermediate, paint thinner,
and as an ultraviolet oxidation stabilizer for plastics fHSDB. 20l ib, c).
This document is a draft for review purposes only and does not constitute Agency policy.
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Table P-l. Physical properties and chemical identities of TMB isomers
CAS Registry Number (CASRN)
95-63-6
108-67-8
526-73-8
Synonym(s)
1,2,4-T rimethylbenzene,
pseudocumene,
asymmetrical
trimethylbenzene
1,3,5-Trimethyl benzene,
mesitylene, symmetrical
trimethylbenzene
1,2,3-Trimethyl benzene,
hemimellitene,
hemellitol,
pseudocumol
Molecular formula
C9H12
Molecular weight
120.19
Chemical structure
cn3
= H3
/^t/CH3
ch3
CH3
Melting point, °C
-43.8
-44.8
-25.4
Boiling point, °C @ 760 mm Hg
168.9
164.7
176.1
Vapor pressure, mm Hg @ 25°C
2.10
2.48
1.69
Density, g/mL at 20 °C
0.8758
0.8637
0.8944
Flashpoint, °C
44
50
44
Water solubility, mg/L at 25 °C
57
48.2
75.2
Other solubilities
Ethanol, benzene, ethyl
ether, acetone,
petroleum ether
Alcohol, ether, benzene,
acetone, oxygenated
and aromatic solvents
Ethanol, acetone,
benzene, petroleum
ether
Henry's law constant,
atm m3/mol
6.16 x 10"3
8.77 x 10"3
4.36 x 10"3
Log Kow
3.78
3.42
3.66
Log Koc
2.73
2.70-3.13
2.80-3.04
Bioconcentration factor
439
234
133-259
Conversion factors
1 ppm = 4.92 mg/m3
1 mg/m3 = 0.2 ppm
1
2 Sources: HSDB (2011a); HSDB (2011b); HSDB (2011c); U.S. EPA (1987).
3
This document is a draft for review purposes only and does not constitute Agency policy.
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The Preamble summarizes the objectives and scope of the IRIS program, general principles and
systematic review procedures used in developing IRIS assessments, and the overall development
process and document structure.
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS
1. Objectives and Scope of the IRIS
Program "
Soon after the EPA was established in 1970, it36
was at the forefront of developing risk assessment
as a science and applying it in support of actions 38
to protect human health and the environment. ThS9
EPA's IRIS program1 contributes to this endeavor40
by identifying adverse health effects of chemicals41
in the environment and characterizing exposure-42
response relationships. IRIS assessments cover 43
the hazard identification and dose-response step$4
of risk assessment. Exposure assessment and risk^
characterization are outside the scope of IRIS 46
assessments, as are political, economic, and 47
technical aspects of risk management. 48
An IRIS assessment may cover one chemical, a49
group of structurally or toxicologically related 50
chemicals, or a chemical mixture. Exceptions 51
outside the scope of the IRIS program are ^
radionuclides, chemicals used only as pesticides, ^
and the "criteria air pollutants" (particulate
matter, ground-level ozone, carbon monoxide, 54
sulfur oxides, nitrogen oxides, and lead], 55
Enhancements to the IRIS program are 56
improving its science, transparency, and 57
productivity. To improve the science, the IRIS 58
program is adapting and implementing principles59
of systematic review (i.e., using explicit methods 60
to identify, evaluate, and synthesize study 61
findings]. To increase transparency, the IRIS 62
program releases a problem formulation and 63
other materials during draft development and
discusses key science questions with the scientific
community and the public. External peer review,
independently managed and in public, improves
both science and transparency. Increased
productivity requires that assessments be concise,
focused on EPA's needs, and completed without
undue delay.
IRIS assessments are conducted in accordance
with EPA guidance. This Preamble summarizes
and does not change IRIS operating procedures or
EPA guidance.
Periodically, the IRIS program asks for
nomination of agents for future assessment or
reassessment. Selection depends on EPA's
priorities, relevance to public health, and
availability of pertinent studies. The IRIS
multiyear agenda2 lists upcoming assessments.
The IRIS program may also assess other agents in
anticipation of public health needs.
2. Planning an Assessment: Scoping,
Problem Formulation, and Protocols
Early attention to planning ensures that IRIS
assessments meet EPA's needs and properly
frame science questions.
Scoping refers to the first step of planning
where the IRIS program consults with EPA's
program and regional offices to ascertain their
needs. Scoping specifies the agents an assessment
will address, routes and durations of exposure,
susceptible populations and lifestages, and other
questions of interest to the EPA.
1 IRIS program website: http://www.epa.gov/iris/
2 IRIS multiyear agenda: https://www.epa.gov/iris/iris-agenda
This document is a draft for review purposes only and does not constitute Agency policy.
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Problem formulation refers to the science 42
questions an assessment will address and included
input from the scientific community and the 44
public. A preliminary survey of secondary sources!l5
(e.g., assessments by national and international 46
health agencies and comprehensive review 47
articles] identifies potential health outcomes and48
science questions. It also identifies related 49
chemicals (e.g., toxicologically active metabolites 50
and compounds that metabolize to the chemical df 1
interest], 52
Each IRIS assessment comprises multiple 53
systematic reviews for multiple health outcomes.54
It also evaluates hypothesized mechanistic 55
pathways and characterizes exposure-response 56
relationships. An assessment may focus on 57
important health outcomes and analyses rather 58
than expand beyond what is necessary to support59
EPA's needs. 60
Protocols refer to the systematic review 61
procedures planned for use in an assessment. 62
They include strategies for literature searches, 63
criteria for study inclusion or exclusion, 64
considerations for evaluating study methods and 65
quality, and approaches to extracting data. As an 66
assessment progresses, additional science 67
questions may emerge and protocols may change68
3. Identifying and Selecting Pertinent^9
Studies
70
71
IRIS assessments conduct systematic literature
searches with criteria for inclusion and exclusion.
The objective is to retrieve the pertinent primary^
studies (i.e., studies with original data on health 74
outcomes or their mechanisms], PECO statementsj5
(Populations, Exposures, Comparisons, Outcomes^
govern the literature searches and screening yy
criteria. "Populations" and animal species yg
generally have no restrictions. "Exposures" refersyg
to the agent and related chemicals identified gQ
during scoping and problem formulation and majg-^
consider route, duration, or timing of exposure. g2
"Comparisons" means studies that allow
comparison of effects across different levels of
exposure. "Outcomes" may become more specific
(e.g., from "toxicity" to "developmental toxicity" to
"hypospadias"] as an assessment progresses.
For studies of absorption, distribution,
metabolism, and elimination, the first objective is
to create an inventory of pertinent studies.
Subsequent sorting and analysis facilitates
characterization and quantification of these
processes.
Studies on mechanistic events can be
numerous and diverse. Here, too, the objective is
to create an inventory of studies for later sorting
to support analyses of related data. The inventory
also facilitates generation and evaluation of
hypothesized mechanistic pathways.
IRIS assessments go beyond standard practices
of systematic review in including pertinent
studies. After posting search strategies on its
website and adding search results to the EPA's
HERO database,3 the IRIS program encourages the
scientific community and the public to provide
information on additional studies and ongoing
research. Assessments also consider data
submitted under the Toxic Substances Control Act
and the Federal Insecticide, Fungicide, and
Rodenticide Act. Even during the review process,
IRIS assessments consider late-breaking studies
that would impact the credibility of the
conclusions.4
4. Evaluating Study Methods and
Quality
IRIS assessments evaluate study methods and
quality, using uniform approaches for each group
of similar studies. The objective is that subsequent
syntheses can weigh study results on their merits.
Key concerns are bias (factors that affect the
magnitude or direction of an effect] and sensitivity
(factors that limit the ability of a study to detect a
true effect].
3 Health and Environmental Research Online: https://hero.epa.gov/hero/
4 IRIS "stopping rules": https://www.epa.gov/sites/production/files/2014-06/documents/
iris stoppingrules.pdf
This document is a draft for review purposes oniy and does not constitute Agency poiicy.
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For human and animal studies, the evaluation 50
of study methods and quality considers study 51
design, exposure characterization, outcome 52
assessment, data analysis, and selective reporting3
For human studies, this evaluation also considers54
selection of participant and referent groups and 55
potential confounding. Emphasis is on discerning56
bias that would substantively change an effect 57
estimate, considering also the expected direction 58
of the bias. Low sensitivity is a bias towards the 59
null. 60
Study-evaluation considerations are specific t(61
each study design, agent, and health effect. 62
Subject-matter experts evaluate each group of 63
studies to identify characteristics that would mal<64
results more or less informative. For 65
carcinogenicity, neurotoxicity, reproductive 66
toxicity, and developmental toxicity, there is EPA 67
guidance for study evaluation. As subject-matter 68
experts examine a group of studies, additional 69
methodologic concerns may emerge and a second70
pass become necessary. 71
Assessments use evidence tables to summarize
the design and results of pertinent studies. If 73
tables become too numerous or unwieldy, they 74
may focus on effects that are more important or 75
studies that are more informative. 76
The IRIS program posts on its website the 77
study-evaluation considerations and table entries78
for illustrative studies, then considers public inpuZ9
on these approaches as it completes study 80
evaluation and data extraction. 81
82
5. Integrating the Evidence of g3
Causation for Each Health Outcom^
Synthesis within lines of evidence. For each85
health outcome, IRIS assessments synthesize the 86
human evidence and the animal evidence, 87
augmenting each with informative subsets of 88
mechanistic data. Each synthesis considers 89
aspects of an association that may suggest 90
causation: consistency, exposure-response 91
relationship, strength of association, temporal 92
relationship, biological plausibility, coherence, 93
and "natural experiments" in humans. 94
Each synthesis seeks to reconcile ostensible 95
inconsistencies between studies, taking into 96
account differences in study methods and quality97
This leads to a distinction between conflicting 98
evidence (unexplained positive and negative
results in similarly exposed human populations or
in similar animal models] and differing results
(mixed results attributable to differences between
human populations, animal models, or exposure
conditions].
Each synthesis of human evidence explores
alternative explanations (e.g., chance, bias, or
confounding] and determines whether they
satisfactorily explain the results. Each synthesis of
animal evidence explores the potential for
analogous results in humans. Coherent results
across multiple species increase confidence that
the animal results are relevant to humans.
Mechanistic data are useful to augment the
human or animal evidence with information on
precursor events, to evaluate the human relevance
of animal results, or to identify susceptible
populations and lifestages. An agent may operate
through multiple mechanistic pathways, even if
one hypothesis dominates the literature.
Integration across lines of evidence. For
each health outcome, IRIS assessments integrate
the human, animal, and mechanistic evidence to
answer the question: What is the nature of the
association between exposure to the agent and the
health outcome?
For cancer, the EPA includes a standardized
hazard descriptor in characterizing the strength of
the evidence of causation. The objective is to
promote clarity and consistency of conclusions
across assessments.
Carcinogenic to humans: convincing epidemiologic
evidence of a causal association; or strong
human evidence of cancer or its key
precursors, extensive animal evidence,
identification of mode-of-action and its key
precursors in animals, and strong evidence
that they are anticipated in humans.
Likely to be carcinogenic to humans: evidence that
demonstrates a potential hazard to humans.
Examples include a plausible association in
humans with supporting experimental
evidence, multiple positive results in animals, a
rare animal response, or a positive study
strengthened by other lines of evidence.
Suggestive evidence of carcinogenic potential:
evidence that raises a concern for humans.
Examples include a positive result in the only
This document is a draft for review purposes only and does not constitute Agency policy.
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study, or a single positive result in an extensivSO
database. 51
Inadequate information to assess carcinogenic 52
potential: no other descriptors apply. ExampleS3
include little or no pertinent information, 54
conflicting evidence, or negative results not 55
sufficiently robust for not likely. 5 6
Not likely to be carcinogenic to humans: robust 57
evidence to conclude that there is no basis for 5 8
concern. Examples include no effects in well- 59
conducted studies in both sexes of multiple 60
animal species, extensive evidence showing 61
that effects in animals arise through modes-of62
action that do not operate in humans, or 63
convincing evidence that effects are not likely 64
by a particular exposure route or below a 65
defined dose. 66
67
If there is credible evidence of carcinogenicity^
an assessment determines whether the mode-of-
action involves mutagenicity, because this
influences the approach to dose-response 70
assessment and subsequent application of 71
adjustment factors for exposures early in life. 72
The EPA is discussing the potential use of 73
hazard descriptors for noncancer outcomes in 74
IRIS assessments. 75
76
6. Selecting Studies for Derivation of 77
Toxicity Values 78
The purpose of toxicity values (i.e., slope 79
factors, unit risks, reference doses, reference 80
concentrations; see section 7] is to estimate 81
exposure levels likely to be without appreciable 82
risk of adverse health effects. The EPA uses these 83
values to support its actions to protect human 84
health. 85
The health outcomes considered for derivatioi86
of toxicity values may depend on the hazard 87
descriptors. For example, IRIS assessments 88
generally derive cancer values for agents that are89
carcinogenic or likely to be carcinogenic, and 90
sometimes for agents with suggestive evidence. 91
Derivation of toxicity values begins with a new92
evaluation of studies, as some studies used 93
qualitatively for hazard identification may not be 94
useful quantitatively for exposure-response 95
assessment. Quantitative analyses require 96
quantitative measures of exposure and response.97
An assessment weighs the merits of the human
and animal studies, of various animal models, and
of different routes and durations of exposure.
Study selection is not reducible to a formula, and
each assessment explains its approach.
Other biological determinants of study quality
include appropriate measures of exposure and
response, investigation of early effects that
precede overt toxicity, and appropriate reporting
of related effects (e.g., combining effects that
comprise a syndrome, or benign and malignant
tumors in a specific tissue].
Statistical determinants of study quality
include multiple levels of exposure (to
characterize the shape of the exposure-response
curve] and adequate exposure range and sample
sizes (to minimize extrapolation and maximize
precision].
Studies of low sensitivity tend to
underestimate toxicity and may be less useful.
69 7. Deriving Toxicity Values
General approach. EPA guidance describes a
two-step approach to dose-response assessment:
analysis in the range of observation, then
extrapolation to lower levels. The analysis
considers studies by the exposure route of interest
and may include studies by other routes if dose
conversion is possible.
IRIS assessments derive a candidate value from
each suitable data set. Consideration of candidate
values yields a toxicity value for each organ or
system. Consideration of the organ/system-
specific values results in the selection of an overall
toxicity value to cover all health outcomes. The
organ/system-specific values are useful for
subsequent cumulative risk assessments that
consider the combined effect of multiple agents
acting at a common anatomical site.
Analysis in the range of observation. Within
the observed range, the preferred approach is
modeling to incorporate a wide range of data.
Toxicokinetic modeling has become increasingly
common for its ability to support target-dose
estimation, cross-species adjustment, or
exposure-route conversion. If data are too limited
to support toxicokinetic modeling, there are
standardized approaches to estimate daily
exposures and scale them from animals to
humans.
This document is a draft for review purposes only and does not constitute Agency policy.
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For human studies, an assessment may 44
develop exposure-response models that reflect 45
the structure of the available data. For animal 46
studies, the EPA has developed a set of empirical 47
("curve-fitting"] models5 that can fit typical data 48
sets. Such modeling yields a point of departure, 49
defined as a dose near the lower end of the 50
observed range, without significant extrapolation 1
to lower levels (e.g., the estimated dose associated2
with an extra risk of 10% for animal data or 1% 53
for human data, or their 95% lower bounds], 54
With complex data, an assessment may 55
develop specialized exposure-response models if56
compatible with the scope of the assessment. 57
Toxicodynamic ("biologically based"] modeling is58
possible if data are sufficient to ascertain the key 59
events of a mode-of-action and to estimate their 60
parameters. For a group of agents that act at a 61
common site or through common mechanisms, ai£2
assessment may derive relative potency factors 63
based on relative toxicity, rates of absorption or 64
metabolism, quantitative structure-activity 65
relationships, or receptor-binding characteristics.66
Extrapolation: slope factors and unit risks. 67
An oral slope factor or an inhalation unit risk 68
facilitates subsequent estimation of human cancel
risks at low levels of exposure. They presuppose 30
linear component to the dose-response curve 71
below the point of departure (e.g., if the mode-of-72
action involves mutagenicity], or there may be no73
established mode-of-action. Extrapolation 74
proceeds linearly (i.e., risk proportional to dose] 75
from the point of departure to the levels of 76
interest 77
Differences in susceptibility may warrant 78
derivation of multiple slope factors or unit risks. 79
For early-life exposure to known or likely 80
carcinogens whose mode-of-action involves 81
mutagenicity, the EPA has developed default age-82
dependent adjustment factors for agents without 83
chemical-specific susceptibility data. 84
If data are sufficient to ascertain the key event§5
of the mode-of-action and to conclude that they 86
87
are not linear at low levels, extrapolation may use
the reference-value approach.
Extrapolation: reference values. An oral
reference dose or an inhalation reference
concentration is an estimate of human exposure
(including in susceptible populations] likely to be
without appreciable risk of adverse health effects
over a lifetime. Reference values generally cover
effects other than cancer. They are also
appropriate for cancer if a well-characterized
mode-of-action indicates that a necessary key
event does not occur below a specific dose.
Calculation of reference values starts with a
point of departure, generally for an early effect
that precedes overt toxicity. To account for
different sources of uncertainty and variability, an
assessment applies uncertainty factors (each
typically 1, 3, or 10] to the point of departure.
Human variation: An uncertainty factor covers
susceptible populations and lifestages that may
respond at lower levels, unless the data
originate from a susceptible study population.
Animal-to-human extrapolation: For reference
values based on animal results, an uncertainty
factor reflects cross-species differences, which
may cause humans to respond at lower levels.
Subchronic-to-chronic exposure: For reference
values based on subchronic studies, an
uncertainty factor reflects the likelihood that a
lower level over a longer duration may induce
a similar response. This factor may not be
necessary for reference values of shorter
duration.
Adverse-effect level to no-observed-adverse-effect
level: For reference values based on a lowest-
observed-adverse-effect level, an uncertainty
factor reflects a level judged to have no
observable adverse effects.
Database deficiencies: If there is concern that
additional studies may identify a more
sensitive effect, target organ, population, or
lifestage, a database uncertainty factor reflects
the nature of the database deficiency.
5 Benchmark Dose Software: http://www.epa.gov/bmds/
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzenes
8. Process for Developing and Peer- 48
Reviewing IRIS Assessments 49
The IRIS process (revised in 2009 and 50
enhanced in 2013] involves extensive public 51
engagement and multiple levels of scientific 52
review. 53
Step 1: Draft development. As outlined in 54
section 2 of this Preamble, IRIS program 55
scientists specify the scope of an assessment 56
and formulate science questions for discussioi57
with the scientific community and the public. 58
Next, they release protocols for the systematic59
review procedures planned for use in the 60
assessment. IRIS program scientists then 61
develop a first draft, using structured 62
approaches to identify pertinent studies, 63
evaluate study methods and quality, integrate 64
the evidence of causation for each health 65
outcome, select studies for derivation of 66
toxicity values, and derive toxicity values, as 67
outlined in Preamble sections 3-7. 68
Step 2: Agency review. Health scientists across 69
the EPA review the draft assessment. 70
Step 3: Interagency science consultation. OtheFl
federal agencies and the Executive Office of th(F2
President review the draft assessment. 73
Step 4: Public comment, followed by external 74
peer review. The public reviews the draft 75
assessment. IRIS program scientists address 76
the public comments, then release a revised 77
draft for independent external peer review. 78
The peer reviewers consider whether the drafF9
assessment assembled and evaluated the 80
evidence according to EPA guidance and 81
whether the evidence justifies the conclusions82
Step 5: Revise assessment. IRIS program 83
scientists revise the assessment to address thd34
comments from the peer review. 85
Step 6: Final agency review and interagency 86
science discussion. The IRIS program 87
discusses the revised assessment with EPA's 88
program and regional offices and with other 89
federal agencies and the Executive Office of th©0
President. 91
Step 7: Post final assessment. The IRIS prograrr02
posts the completed assessment and a 93
summary on its website. 94
95
96
9. General Structure of IRIS
Assessments
Main text. IRIS assessments generally
comprise two major sections: (1] Hazard
Identification and (2] Dose-Response Assessment.
Section 1.1 briefly reviews chemical properties
and toxicokinetics to describe the disposition of
the agent in the body. This section identifies
related chemicals and summarizes their health
outcomes, citing authoritative reviews. If an
assessment covers a chemical mixture, this section
discusses environmental processes that alter the
mixtures humans encounter and compares them
to mixtures studied experimentally.
Section 1.2 includes a subsection for each
major health outcome. Each subsection discusses
the respective literature searches and study
considerations, as outlined in Preamble sections 3
and 4, unless covered in the front matter. Each
subsection concludes with evidence synthesis and
integration, as outlined in Preamble section 5.
Section 1.3 links health hazard information to
dose-response analyses for each health outcome.
One subsection identifies susceptible populations
and lifestages, as observed in human or animal
studies or inferred from mechanistic data. These
may warrant further analysis to quantify
differences in susceptibility. Another subsection
identifies biological considerations for selecting
health outcomes, studies, or data sets, as outlined
in Preamble section 6.
Section 2 includes a subsection for each
toxicity value. Each subsection discusses study
selection, methods of analysis, and derivation of a
toxicity value, as outlined in Preamble sections 6
and 7.
Front matter. The Executive Summary
provides information historically included in IRIS
summaries on the IRIS program website. Its
structure reflects the needs and expectations of
EPA's program and regional offices.
A section on systematic review methods
summarizes key elements of the protocols,
including methods to identify and evaluate
pertinent studies. The final protocols appear as an
appendix.
The Preface specifies the scope of an
assessment and its relation to prior assessments.
It discusses issues that arose during assessment
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzenes
1 development and emerging areas of concern. The 5
2 Preface also identifies assessment-specific 6 May 2016
3 approaches that may differ from the general
4 approaches outlined in this Preamble.
7
8
This document is a draft for review purposes only and does not constitute Agency policy.
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EXECUTIVE SUMMARY
Occurrence and Health Effects
Trimethylbenzenes (TMBs) are a commercially available mixture of three
individual isomers: 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB. TMB isomers are
produced during petroleum refining and production of aromatic hydrocarbons with
nine carbons (i.e., C9 aromatic fraction). As the vast majority of the C9 fraction is used
as a component of gasoline, vehicle emissions are expected to be the major
anthropogenic source of TMBs. TMBs are volatile hydrocarbons, and humans are
thus exposed to these isomers primarily through breathing air containing TMB
vapors, although ingestion through food or drinking water is also possible.
Effects on the nervous, respiratory, and hematological (i.e., blood) systems
have been reported in occupationally- and residentially-exposed humans, but these
effects were observed following exposure to complex mixtures containing TMB
isomers, thus making it difficult to determine the contribution of each TMB isomer to
the observed health effects. Health effects that are roughly analogous to those seen
in humans have been observed in animals exposed to the individual isomers. Effects
on the nervous system, including cognitive effects and decreased pain sensitivity, are
the most widely observed effects in animals. Effects on other systems, including the
respiratory and hematological systems, have also been observed in animals. Both
1,2,4-TMB and 1,3,5-TMB have been observed to elicit effects on pregnant animals
and developing fetuses, but at exposure levels greater than those that cause effects
on the nervous system. There is inadequate information to evaluate the
carcinogenicity of TMBs.
Effects Other Than Cancer Following Inhalation Exposure
The relationship between exposure to 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB and health
effects has been evaluated in studies of (1) exposed human adults, (2) animals exposed via
inhalation for acute, short-term, and subchronic durations, and (3) animals exposed gestationally
via inhalation.
Human studies included occupational exposure to various solvent mixtures containing
TMBs. Health effects noted in these studies were eye irritation, neurological effects (hand tremble,
abnormal fatigue, lack of coordination), and hematological effects. Residential exposure to mixtures
containing 1,2,4-TMB were observed to be associated with asthma. However, as these studies
involved exposures to mixtures containing multiple TMB isomers and other volatile organic
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Trimethylbenzenes
compounds (VOCs), it is difficult to ascertain the specific contribution of each TMB isomer to the
specific health effects reported. Studies involving controlled exposures of healthy adult volunteers
to individual isomers also exist, although these studies generally report little or no effect on
respiratory or sensory irritation. One controlled human exposure study reported some deficits in
attention following exposure to white spirit (WS), a complex mixture containing 1,2,4-TMB.
Animal inhalation studies included acute and short-term studies of TMBs that reported
respiratory irritation (decreased respiration rates) and neurological effects (decreased pain
sensitivity, altered cognitive function, and decreased anxiety and/or increased motor function) that
are consistent with effects seen in human studies. Four subchronic inhalation studies for 1,2,3-TMB
and 1,2,4-TMB observed exposure-response effects in multiple systems, including the nervous,
hematological, and respiratory systems. In these studies, disturbances in central nervous system
(CNS) function, including decreased pain sensitivity and decreased neuromuscular function and
coordination, appear to be the most sensitive endpoints following exposure to 1,2,3-TMB or
1,2,4-TMB. No subchronic studies were found that investigated exposure to 1,3,5-TMB. One
developmental toxicity study observed maternal and fetal toxicity (i.e., decreased maternal weight
gain and fetal weight) following exposure to either 1,2,4-TMB or 1,3,5-TMB; other indices of fetal
toxicity (i.e., fetal death and malformations) were not affected by exposure.
Inhalation Reference Concentration (RfC) for TMBs for Effects Other Than Cancer
The RfC for TMBs was derived using benchmark dose (BMD) modeling coupled with
physiologically-based pharmacokinetic (PBPK) modeling or default dosimetric methods. BMD
modeling was conducted using external exposure concentrations as the dose inputs and either a
benchmark response (BMR) level of 5% change (fetal weight) or 1 standard deviation (SD) of the
control mean (all other endpoints). Once a lower confidence limit on the benchmark dose (BMDL)
(or a no-observed-adverse-effect level [NOAEL] or lowest-observed-adverse-effect level [LOAEL] in
cases where no models fit the data) was identified as the point of departure (POD), a human
equivalent concentration (HEC) was calculated for each endpoint using either a PBPK model
(1,2,4-TMB) or default dosimetric adjustments (1,2,3-TMB and 1,3,5-TMB).
To each HEC, a composite uncertainty factor (UF) was applied to account for uncertainties
in the TMB database: 3 to account for uncertainty in extrapolating from laboratory animals to
humans (interspecies variability), 10 to account for variation in susceptibility among members of
the human population (interindividual variability), 3 to account for subchronic-to-chronic
extrapolation due to the use of a subchronic study, and 3 to account for deficiencies in the database
(no two-generation reproductive/developmental toxicity or developmental neurotoxicity studies
were available). Dividing the candidate HECs by this composite UF of 300 yielded the
organ/system-specific RfCs presented in Table ES-1.
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Table ES-1. Organ/system-specific RfCs for individual TMB isomers
Effect
Isomer
Basis
RfC
(mg/m3)
Composite
UF
Exposure
Description
Confidence
Neurological
1,2,4-TMB
Decreased pain
sensitivity
6 x 10"2
300
Subchronic
Low to medium
1,2,3-TMB
5 x 10"2
300
Subchronic
Low to medium
Hematological
1,2,4-TMB
Decreased
clotting time
8 x 10"2
300
Subchronic
Low to medium
1,2,3-TMB
Decreased
segmented
neutrophils
6 x 10"2
300
Subchronic
Low to medium
Respiratory
1,2,4-TMB
Inflammatory
lung lesions
2 x 10"1
300
Subchronic
Low to medium
1,2,3-TMB
2 x 10"1
300
Subchronic
Low to medium
Developmental
1,2,4-TMB
Fetal weight
4
100
Gestational
Low to medium
1,3,5-TMB
4
100
Gestational
Low to medium
Maternal
1,2,4-TMB
Decreased
maternal
weight
3
300
Subchronic
Low to medium
1,3,5-TMB
4 x 10"1
300
Subchronic
Low to medium
Overall RfC
(Neurological)
1,2,4-TMB
Decreased
pain
sensitivity
6 x 10"2
300
Subchronic
Low to medium
Neurotoxicity is the most consistently observed endpoint in the toxicological database for
TMBs, and decreased pain sensitivity was observed in multiple studies following exposures to
1,2,3- or 1,2,4-TMB for short-term or subchronic durations. Given the consistency of this effect and
the determination that decreased pain sensitivity is an appropriate adverse effect with which to
derive reference values (see Section 2.1.5), in accordance with the EPA's Guidelines for Neurotoxicity
Risk Assessment (U.S. EPA. 19981. decreased pain sensitivity was selected as the critical effect and
Korsak and Rvdzvriski f 199 61 was selected as the principal study for derivation of the RfC for TMBs.
No subchronic study was available that investigated neurotoxicity endpoints following exposure to
1,3,5-TMB, resulting in the lack of an isomer-specific neurotoxicity RfC for this isomer. However, as
discussed in Section 1.2.7, the available toxicological database for all three isomers, across all
exposure durations, indicates there are important similarities in the isomers' neurotoxicity that are
supportive of an RfC for 1,3,5-TMB that is not substantially different than the RfC derived for other
TMB isomers. Also supporting this conclusion is the observation that TMB isomers display
important similarities with regard to chemical properties and toxicokinetics, including similarities
in blood:air partition coefficients, respiratory uptake, and absorption into the bloodstream (see
Section 1.2.7 and Appendices C.l and C.2). These similarities support the conclusion that an RfC for
1,3,5-TMB would be similar to those calculated for 1,2,3- or 1,2,4-TMB. The RfC for 1,2,4-TMB was
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selected over the RfC for 1,2,3-TMB as the RfC for the entire TMB database due to increased
confidence in that it was calculated via the application of a validated PBPK model, whereas the
1,2,3-TMB value was estimated using default dosimetric methods. Therefore, the RfC for TMBs
was set at 6 x 10-2 mg/m3 based on decreased pain sensitivity following exposure to 1,2,4-
TMB.
In addition to providing an RfC for chronic exposures in multiple systems, this document
also provides an RfC for subchronic-duration exposures. In the case of TMBs, all of the studies used
to calculate the chronic RfCs were subchronic or gestational in duration. Therefore, the methods to
calculate subchronic RfCs are identical to those used for calculation of chronic RfCs, minus the
application of a subchronic-to-chronic UF (see Table ES-1). It should be noted that the subchronic
RfC values for the developing fetus are identical to the chronic RfC values as gestation represents a
critical window of susceptibility and no UFs was applied to account for less than chronic exposure
in either case. The subchronic inhalation RfC is intended for use with exposures for more than 30
days, up to approximately 10% of the lifespan in humans.
Table ES-2. Organ/system-specific subchronic RfCs for individual TMB
isomers
Effect
Isomer
Basis
RfC
(mg/m3)
Composite
UF
Exposure
Description
Confidence
Neurological
1,2,4-TMB
Decreased pain
sensitivity
2 x 10"1
100
Subchronic
Low to medium
1,2,3-TMB
2 x 10"1
100
Subchronic
Low to medium
Hematological
1,2,4-TMB
Decreased
clotting time
2 x 10"1
100
Subchronic
Low to medium
1,2,3-TMB
Decreased
segmented
neutrophils
2 x 10"1
100
Subchronic
Low to medium
Respiratory
1,2,4-TMB
Inflammatory
lung lesions
6 x 10"1
100
Subchronic
Low to medium
1,2,3-TMB
6 x 10"1
100
Subchronic
Low to medium
Developmental
1,2,4-TMB
Fetal weight
4
100
Gestational
Low to medium
1,3,5-TMB
4
100
Gestational
Low to medium
Maternal
1,2,4-TMB
Decreased
maternal
weight
8
100
Subchronic
Low to medium
1,3,5-TMB
1
100
Subchronic
Low to medium
Subchronic
Overall RfC
(Neurological)
1,2,4-TMB
Decreased
pain
sensitivity
2 x 10"1
100
Subchronic
Low to medium
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Confidence in the Chronic Inhalation RfC for 1,2,4-TMB
A confidence level of high, medium, or low is assigned to the study used to derive the RfC,
the overall database, and the RfC itself, as described in Section 4.3.9.2 of EPA's Methods for
Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA.
1994bJ.
Confidence in the study from which the critical effect was identified, is low to medium. The
study is a peer-reviewed study that utilized three dose groups plus untreated controls, employed an
appropriate number of animals per dose group, and performed appropriate statistical analyses.
However, sources of uncertainty exist that reduce confidence in this study.
One area of uncertainty regarding this study is the lack of reported actual concentrations.
However, as the methods by which the test atmosphere was generated and analyzed were reported
in sufficient detail, and given the fact that this laboratory has used this methodology in subsequent
studies and achieved appropriate actual concentrations (i.e., within 10% of target concentrations),
the concern regarding the lack of reported actual concentrations is reduced. Another source of
uncertainty is the fact that the principal study does not explicitly state that the reported measures
of variance in Table 1 of that reference are SDs. However, careful analysis of the reported levels of
variance and magnitude of statistical significance indicate that the measures of variance are SDs.
Supporting this conclusion is the observation that all other papers from this laboratory report
variance as SDs. The critical effect on which the RfC is based is well-supported as the weight of
evidence for TMB-induced neurotoxicity is coherent across species (i.e., human, mouse, and rat),
coherent across isomers, and consistent across multiple exposure durations (i.e., acute, short-term,
and subchronic).
The database for TMBs includes acute, short-term, subchronic, and developmental toxicity
studies in rats and mice. However, confidence in the overall database is low to medium because it
lacks chronic, multi-generation reproductive/developmental, and developmental neurotoxicity
studies, and the studies supporting the critical effect predominantly come from the same research
institute. The overall confidence in the RfC for TMBs is low to medium.
Effects Other Than Cancer Observed Following Oral Exposure
Only one subchronic study was identified that examined the effects of oral exposure to
1,3,5-TMB. Effects in the hematological system, including changes in clinical chemistry parameters
and differential white blood cell (WBC) numbers, were observed following exposure to 1,3,5-TMB
via gavage in rats. Altered organ weights were also observed in multiple systems. The alterations
to clinical chemistry parameters and organ weights were observed in the absence of
histopathological changes in relevant systems, and were thus considered to be compensatory in
nature. Discounting effects that could be non-adverse or compensatory in nature left an observed
increase in monocytes in male rats as the only statistically significant effect on which to base the
reference dose (RfD) derivation. While a slight increase in monocytes may be of questionable
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adversity if taken with no context of the larger TMB database, a number of endpoints involving the
alteration of WBC counts have been observed in the inhalation toxicity database. It was therefore
deemed that the observed increase in monocytes following oral exposures was possibly indicative
of an underlying toxicity to the hematological system also evident following inhalation exposure.
Oral Reference Dose (RfD) for TMBs for Effects Other Than Cancer
The RfD for TMBs was derived using BMD modeling coupled with default dosimetric
methods. BMD modeling was conducted using external exposure concentrations as the dose inputs
and a BMR level of 1 SD of the control mean. Once a BMDL was identified as the POD, a human
equivalent dose (HED) of 3.0 mg/kg-day was calculated for increased monocytes using default
dosimetric adjustments (i.e., body weight to the % power).
To the estimated HED, a composite UF was applied to account for uncertainties in the TMB
database: 3 to account for uncertainty in extrapolating from laboratory animals to humans
(interspecies variability), 10 to account for variation in susceptibility among members of the
human population (interindividual variability), 3 to account for subchronic-to-chronic
extrapolation due to the use of a subchronic study, and 3 to account for deficiencies in the database
(no two-generation reproductive/developmental toxicity or developmental neurotoxicity studies
were available). Dividing the HED by this composite UF of 300 yielded an RfD of
1 x 10_1 mg/kg-day.
In addition to the RfD calculated for TMBs from oral data, an RfD was calculated from
inhalation data using a route-to-route extrapolation to address the lack of suitable neurotoxicity
data in the oral TMB database. It is clear from the inhalation database for TMB that neurotoxicity is
an important endpoint for derivation of reference values, especially given the consistency with
which neurotoxicity is observed in the TMB database, across all isomers following acute oral and
acute, short-term, and subchronic inhalation exposures. Ultimately, the fact that oral and inhalation
neurotoxic endpoints are comparable, and that neurotoxic endpoints resulted in the most strongly
supported RfCs in the inhalation database, it is reasonable to expect that neurotoxicity-based PODs
would be critical for deriving RfDs. The available database for 1,2,4-TMB supports the use of route-
to-route extrapolation; sufficient evidence exists that demonstrates similar qualitative profiles of
metabolism (i.e., observation of dimethylbenzoic and hippuric acid metabolites) and patterns of
parent compound distribution across exposure routes (Section C.2, Appendix C).
Therefore, assuming that oral exposure would result in the same systemic effect as
inhalation exposure (i.e., altered CNS function, measured as decreased pain sensitivity), an oral
exposure component was added to the PBPK model by EPA (Section C.3.3.5, Appendix C), assuming
100% absorption of the ingested 1,2,4-TMB by constant infusion of the oral dose into the liver and
an idealized pattern of six ingestion events (see Section 2.2.3). Using the modified PBPK model
resulted in an HED of 3.5, which was divided by the composite UF of 300 to estimate an RfD
of 1 x 10"2 mg/kg-day. Although identical to the RfD calculated from the oral 1,3,5-TMB data for
increased monocytes, this value of 1 x 10~2 mg/kg-day was ultimately selected as the RfD for TMB
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isomers based on multiple lines of evidence in the oral and inhalation database, including
commonalities in the pattern of neurotoxic effects observed following oral and inhalation
exposures, similarities in blood:air and tissue:air partition coefficients and absorption into the
bloodstream between TMB isomers, and qualitative metabolic profiles that suggest that first-pass
metabolism through the liver is not expected to differ greatly between the three isomers.
In addition to providing RfDs for effects in the hematological and nervous systems, this
document also provides values for subchronic RfD values for exposures that may be of concern in a
less-than-lifetime context. In the case of TMBs, the oral 1,3,5-TMB study and 1,2,4-TMB inhalation
study used for the route-to-route extrapolation used to calculate the chronic RfDs were both of
subchronic duration. Therefore, the methods used to calculate subchronic RfDs is identical to that
used for calculation of chronic RfCs, minus the application of a subchronic-to-chronic UF. This
results in a composite UF of 100 (UFa = 3, UFh = 10, UFs = 1, and UFd = 3). Dividing the POD for
hematological effects (3.01 mg/kg-day) and neurotoxicity effects (3.5 mg/kg-day) by the composite
UF of 100 results in an RfD of 3 x 10~2 and 4 x 10"2 mg/kg-day for decreased monocytes and
decreased pain sensitivity, respectively. The subchronic oral RfD is intended for use with
exposures for more than 30 days, up to approximately 10% of the lifespan in humans.
Confidence in the Chronic Oral RfD for 1,2,4-TMB
The confidence in the oral database for TMB is low because it only contains acute oral
studies investigating neurotoxicity endpoints for multiple isomers, and one subchronic study
investigating general toxicity endpoints for one isomer (1,3,5-TMB). This database was used to
derive an RfD, but given the concern over the lack of a suitable neurotoxicity study, the confidence
in this RfD is low. A PBPK model was utilized to perform a route-to-route extrapolation to
determine a POD for the derivation of the RfD from inhalation data. The confidence in the study
from which the critical effect was identified, is low to medium (see Section 2.1.7). The inhalation
database for 1,2,4-TMB includes acute, short-term, subchronic, and developmental toxicity studies
in rats and mice. However, confidence in the database for TMB is low to medium because it lacks
chronic, multi-generation reproductive/developmental, and developmental neurotoxicity studies,
and the studies supporting the critical effect predominantly come from the same research institute.
Reflecting the confidence in the study and the database and the uncertainty surrounding the
application of the available PBPK model for the purposes of a route-to-route extrapolation, the
overall confidence in the RfD for TMB is low.
Evidence of Carcinogenicity
Under EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA. 20051. there is "inadequate
information to assess carcinogenic potential" of TMBs. No chronic inhalation studies that
investigated cancer outcomes were identified in the literature for 1,2,3-TMB, 1,2,4-TMB, or
1,3,5-TMB. One cancer study in which rats were exposed to 1,2,4-TMB via gavage at one
experimental dose of 800 mg/kg-day reported marginal increases in total malignant tumors and
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1 head tumors (e.g., neuroesthesioepitheliomas), but provided no statistical analyses of the results. A
2 number of methodological issues limit the utility of this study (e.g., only one dose group and no
3 discussion of histopathological analyses). Therefore, a quantitative cancer assessment for TMBs
4 was not conducted.
5 Susceptible Populations and Lifestages
6 No chemical-specific data that would allow for the identification of populations or lifestages
7 with increased susceptibility to TMB exposure exist.
8
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LITERATURE SEARCH STRATEGY | STUDY
SELECTION AND EVALUATION
1 A number of literature searches were conducted to identify references for inclusion in the
2 trimethylbenzene (TMB) assessment The initial literature search strategy used to identify primary,
3 peer-reviewed literature pertaining to TMBs was conducted using the databases and keywords
4 listed in Table LS-1. References from health assessments developed by other national and
5 international health agencies were also examined. Other peer-reviewed information, including
6 review articles, literature necessary for the interpretation of TMB-induced health effects, and
7 independent analyses of the health effects data were retrieved and included in the assessment
8 where appropriate. The U.S. Environmental Protection Agency (EPA) requested public submissions
9 of additional information in April 2008; no submissions in response to the data call-in were
10 received. The initial literature search was last conducted in December 2011.
Table LS-1. Details of the initial search strategy employed for TMBs
Databases
Termsa'b
EBSCO
DISCOVERY
SERVICE:
HERO
SCI
NLM
TOXLINE
WOS
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
11 Potentially relevant publications on TMBs were identified through a literature search conducted with the EBSCO
12 Discovery Service feature of Health and Environmental Research Online (HERO), a meta-search engine with access
13 to numerous databases including the Science Citation Index (SCI), Toxicology Literature Online (TOXLINE),
14 National Library of Medicine (NLM, PubMed/Medline), and Web of Science (WOS).
15 literature search was performed using related words (i.e., lemmatization) of included search terms. Search terms
16 were entered into the EBSCO Discovery Service portal with no qualifiers, and the results from individual search
17 engines were returned and exported to HERO.
18 CASRN = Chemical Abstracts Service Registry Number.
19
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Figure LS-1 depicts the literature search and study selection strategy and the number of
references obtained at each stage of the literature screening. Selection of studies for inclusion in
the Toxicological Review was based on consideration of the extent to which the study was
informative and relevant to the assessment and general study quality considerations. In general,
the relevance of health effect studies was evaluated as outlined in the Preamble and EPA guidance
(A Review of the Reference Dose and Reference Concentration Processes fU.S. EPA. 20021 and Methods
for Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S.
EPA. 1994b)). Approximately 2,600 references were obtained from the initial literature search for
1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB, including references retrieved from specific literature
searches necessary for the interpretation of TMB-induced health effects (e.g., literature on specific
modes of action, physiologically based pharmacokinetic [PBPK] analysis), or including references
identified from the primary literature. From this full list of references, there were 143 references
that were selected for inclusion in the Toxicological Review.
The initial literature search was augmented with an updated literature search to identify
references published since December 2011 that could provide information relevantto the
assessment This literature search identified approximately 960 references, 5 of which were
included in the assessment This literature search was conducted using similar search terms as the
initial literature search, but did not use the EBSCO Discovery Service and instead searched
TOXLINE, NLM, and Web of Science databases separately. This difference in search methodology
resulted in proportionally more references being identified over a shorter time span (2011-2016
vs. pre-2011), but fewer being deemed relevantto the assessment (5/962 vs. 143/2,608). During
the external peer review process, the Chemical Assessment Advisory Committee (CAAC)
recommended the inclusion of 15 studies, which were added to the assessment The CAAC further
recommended including studies investigating the health effects due to exposure of compounds
structurally similar to TMBs (e.g., toluene, xylene, etc.). In order to identify relevant studies, EPA
conducted a targeted literature search for review articles in PubMed using the following query:
(("styrene"[All Fields] or toluene[All Fields] or xylene[All Fields] or ethyl toluene [All Fields]) and
(neurotoxicity[All Fields] or "respiratory toxicity" [All Fields] or "hematological toxicity" [All Fields]
or (("developmental"[All Fields] or "respiratory"[All Fields]) and "toxicity"[All Fields]))) and
Review[Publication Type], A total of 70 review articles were identified. Of the identified review
articles, 14 were cited in the assessment, and 6 primary references identified from the review
articles were also included. In total, 183 references were cited in the assessment when considering
references identified in the initial, update, and related compound literature searches in addition to
the specific references recommended for inclusion by the CAAC. The references that are cited in the
document, as well as those that were considered but not included in the Toxicological Review of
TMBs, can be found within the Health and Environmental Research Online (HERO) website:
https://hero.epa.gov/hero/index.cfm/proiect/page/proiect id/1676. This site contains HERO
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1 links to lists of references, including bibliographic information and abstracts, which were
2 considered for inclusion in the Toxicological Review of Trimethylbenzenes.
3
Primary Literature Search - 2608
references identified based on initial
keyword search in listed Databases (see
Table LS-1)
Primary Literature Search — 2399
excluded references
Excluded by Journal (2059
references) - Atmospheric Chemistry
or Environmental (23), Chemistry or
Physical or Engineering (2036)
Excluded by Title/Abstract (340
references) - Atmospheric Chemistry
(50), Non-relevant Biological System
or Chemical (48), Case Studies and
Government Reports (6), Chemistry
or Physical or Engineering (152),
Exposure Analysis (39), Geological or
Ecological (39), Non-relevant Data or
Software (2), Non-scientific Literature
(4)
Literature Search Update - 962
references identified based on initial
keyword search in listed Databases (see
Table LS-1)
Literature Search Update - 949
excluded references
• Non-peer Reviewed - 125 references
• Excluded by Journal - 370 references
• Excluded by Title/Abstract (505
references) - Atmospheric Chemistry
(8), Non-relevant Biological System or
Chemical (308), Case Study or
Government Report (5), Chemistry or
Physical or Engineering (151),
Exposure Assessment (17), Geological
or Ecological (16)
~ *
Primary Literature Search + Literature Search Update -
223 considered references
SAB recommended references-15
references
References Cited in the Toxicological
Review or Supplemental Information
Document -183 references
Secondary Literature Search for
Related Compounds- 69 references
• Cited Review Articles-14
references
• Primary references Identified from
V Review Articles-6 references J
5
6
7 Note: Some references may provide information on more than one topic, and therefore, may be included in more
8 than one study type. Accordingly, the sum of the references for subcategories of studies is not expected to equal
9 the number of references for the larger category.
Figure LS-1. Literature search and study selection strategy for TMBs.
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1. HAZARD IDENTIFICATION
This Hazard Identification section critically reviews the publicly available studies on the
three isomers of trimethylbenzene (TMBs) (i.e., 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB) in order to
describe their toxicokinetics, identify their adverse health effects, and to characterize whether
these compounds pose organ/system-specific hazards to humans. In evaluating this isomer-
specific toxicity databases, the most consistently observed effect is neurotoxicity, and as such, most
of the hazard discussion is focused on effects in the nervous system. Neurotoxicity is observed in
human populations exposed to solvent mixtures containing TMBs, and in laboratory animals
exposed to single TMB isomers for acute, short-term, and subchronic durations. In addition to
neurotoxicity, TMB isomers are observed to elicit effects in the respiratory and hematological
systems, and are reported to result in adverse effects in pregnant animals and the developing fetus.
Each of these health effects, in addition to sections describing the general toxicity effects and
carcinogenicity of TMB isomers, are included in their own separate section, which includes
discussions of the various toxicities observed, as well as evidence tables and exposure-response
arrays that further summarize the data (Sections 1.2.1-1.2.6). Following each organ-specific
section, mode-of-action and summary sections are included to present evidence syntheses and
discuss possible modes of action and their potential relevance in hazard determinations. A general
paucity of chemical-specific information exists regarding possible modes of action, but some
insights can be gained by considering information on related compounds and/or mixtures
containing TMB isomers. Section 1.2.7 provides a summary of how the three TMB isomers are
similar to one another regarding observed toxicities. This determination that the three TMB
isomers are observed to result in largely similar toxicological profiles, especially neurotoxicity
effects, is of particular importance for subsequent dose-response analyses. The overall weight of
evidence for individual noncancer and cancer endpoints is synthesized in Section 1.3.1.
In addition to isomer-specific animal toxicity studies, four studies that investigated the
toxicity in rodent species following exposure to the C9 aromatic fraction are included. The C9
aromatic fraction is a mixture of volatile organic compounds (VOCs) containing an aromatic ring
and nine total carbons. Although the majority of the C9 fraction is composed of TMB isomers
(>50%), these studies generally report low levels of C9-induced toxicity. This discrepancy between
toxicity profiles of individual TMB isomers and the C9 fraction is also discussed.
This document is a draft for review purposes only and does not constitute Agency policy.
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1.1. OVERVIEW OF THE TOXICOKINETICS OF TMBs
1.1.1. Toxicokinetics of TMB isomers
In the existing toxicokinetic database for 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB, important
similarities have been observed in the chemical properties and absorption, distribution,
metabolism, and excretion (ADME) profiles for these isomers in animals and humans, although
some important differences also exist A summary of these comparisons across individual TMB
isomers is presented in Table 1-1; for comparison of metabolic schemes, see Figures C-l through
C-3.
All three isomers have very similar log Kow values (3.42-3.78), but a wider distribution of
Henry's law constants (4.36-8.77 x 10~3 atmxm3/mol). The isomers' blood:air partition coefficients
reported for humans and rats in the literature are similar: 43.0 and 55.7 for 1,3,5-TMB, 66.5 and
62.6 for 1,2,3-TMB, and 59.1 and 57.7 for 1,2,4-TMB (Meulenberg and Vijverberg. 20001. This gives
an indication that the three isomers would partition into the blood in a similar fashion. Supporting
this is the observation that 1,2,4-TMB and 1,3,5-TMB absorb equally into the bloodstream of
exposed humans (6.5 and 6.2 [J.M, respectively), although the absorption for 1,2,3-TMB was
observed to be higher (7.3 [iM) flarnbergetal.. 1998.1997a: larnberg et al.. 19961. Also, the net
respiratory uptake of 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB was similar among humans (48-60%),
and the respiratory uptake for 1,2,4-TMB was similar across humans and rats (50-60%) (larnberg
etal.. 1996: Da hi etal.. 19881. Although no data exist regarding the distribution of TMB isomers in
humans, experimentally-derived tissue-specific partition coefficients were similar for all three
isomers across a number of systems (Meulenberg and Viiverberg. 20001. strongly suggesting that
the individual isomers can be expected to distribute similarly to these various systems.
Distribution of 1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB throughout the body is qualitatively similar in
animals, although it appears that there are some quantitative differences. Liver concentrations for
1,2,4-TMB are greater than those for 1,3,5-TMB after both acute and short-term inhalation
exposures at all concentrations, whereas 1,2,4-TMB liver concentrations were only greater than
1,2,3-TMB concentrations at the mid- and high doses (Swiercz etal.. 2016: Swiercz etal.. 2006:
Swiercz etal.. 2003: Swiercz etal.. 20021. Although 1,2,4-TMB was observed to distribute to the
brain fSwiercz etal.. 2003: Eide and Zahlsen. 19961. distribution of 1,3,5-TMB or 1,2,3-TMB to the
brain was not experimentally measured in any study. However, the predicted brain:air partition
coefficient were similar between 1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB for both humans (206, 220,
and 199, respectively) and rats (552, 591, and 535, respectively) (Meulenberg and Viiverberg.
20001. This strongly suggests that 1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB can be expected to
distribute similarly to the brain in both humans and rats. The observation that organ
concentrations are lower following repeated exposures to TMB isomers compared to acute
exposures is mostly likely due to induction of metabolizing enzymes at higher exposure
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1 concentrations. This hypothesis is supported by observation of cytochrome P-450 (CYP450)
2 enzyme induction in the livers, kidneys, and lungs of rats exposed to 1,2,4-TMB fPvykko. 19801.
Table 1-1. Toxicokinetic similarities between TMB isomers
Toxicokinetic or metabolic parameter
Species
TMB isomer ranking
Absorption
Capillary blood concentration
Humans
1,2,3-TMB > 1,2,4-TMB * 1,3,5-TMB
Respiratory uptake
Humans
1,2,4-TMB > 1,3,5-TMB > 1,2,3-TMB
Blood:air partition coefficient
Humans
1,2,4-TMB * 1,2,3-TMB > 1,3,5-TMB
Blood:air partition coefficient
Rats
1,2,4-TMB * 1,2,3-TMB * 1,3,5-TMB
Distribution
Liver concentration (4-wk exposure)
123 mg/m3
492 mg/m3
1,230 mg/m3
Rats
1.2.3-TMB > 1,2,4-TMB * 1,3,5-TMB
1.2.4-TMB * 1,2,3-TMB * 1,3,5-TMB
1,2,4-TMB > 1,2,3-TMB * 1,3,5-TMB
Kidney concentration (4-wk exposure)
123 mg/m3
492 mg/m3
1,230 mg/m3
Rats
1,2,3-TMB > 1,3,5-TMB
1,3,5-TMB > 1,2,3-TMB
1,2,3-TMB * 1,3,5-TMB
Lung concentration (4-wk exposure)
123 mg/m3
492 mg/m3
1,230 mg/m3
Rats
1.2.3-TMB > 1,2,4-TMB * 1,3,5-TMB
1.2.4-TMB > 1,2,3-TMB * 1,3,5-TMB
1,2,4-TMB * 1,2,3-TMB > 1,3,5-TMB
Brain:air partition coefficient
Humans
1,2,4-TMB * 1,2,3-TMB * 1,3,5-TMB
Brain:air partition coefficient
Rats
1,2,4-TMB * 1,2,3-TMB * 1,3,5-TMB
Metabolism
Urinary hippuric acids (% inhaled TMB dose, 25 ppm)
Humans
1,2,4-TMB > 1,2,3-TMB » 1,3,5-TMB
Urinary hippuric acids (% oral TMB dose, 1.2 g/kg)
Rats
1,3,5-TMB » 1,2,4-TMB > 1,2,3-TMB
Excretion
Total blood clearance
Humans
1,3,5-TMB > 1,2,4-TMB * 1,2,3-TMB
Terminal half-life of elimination
Humans
1,3,5-TMB > 1,2,4-TMB * 1,2,3-TMB
Terminal half-life of elimination (123 mg/m3)
Rats
1,2,4-TMB * 1,2,3-TMB * 1,3,5-TMB
Terminal half-life of elimination (1,230 mg/m3)
Rats
1,2,4-TMB > 1,2,3-TMB * 1,3,5-TMB
3
4 All three TMB isomers were observed to primarily metabolize to benzoic and hippuric acids
5 in humans and rats flarnbergetal.. 1996: Huo etal.. 1989: Mikulski and Wiglusz. 19751. although
6 the amounts of inhaled TMB recovered as hippuric acid metabolites following exposure to
7 1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB were dissimilar in humans (11, 22, and 3%, respectively) and
8 rats flO. 24-38. and 59%. respectively! flarnberg etal.. 1996: Mikulski and Wiglusz. 19751. Greater
9 amounts of urinary benzoic acid and hippuric acid metabolites (73%) were observed after
10 exposure to higher amounts of 1,3,5-TMB (up to 30.5 ppm) for 8 hours (Kostrzewski etal.. 1997:
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Kostrewski and Wiaderna-Brvcht. 19951. Other terminal metabolites included mercapturic acids
(~14-19% total dose), phenols (~12% total dose), and glucuronides and sulphuric acid conjugates
(4-9% total dose) for 1,2,4-TMB; mercapturic acids (~5% total dose), phenols (<1-8% total dose),
and glucuronides and sulphuric acid conjugates (8-15% total dose) for 1,2,3-TMB; and phenols
(—4—8% total dose) and glucuronides and sulphuric acid conjugates (~5-9% total dose) for
1,3,5-TMB fTsuiimoto etal.. 2005: Tsuiimoto etal.. 2000. 1999: Huo etal.. 1989: Wiglusz. 1979:
Mikulski and Wiglusz. 19751.
In humans, the half-lives of elimination from blood were observed to be similar for all
isomers in the first three phases of elimination: 1,2,4-TMB (1.3 ± 0.8 minutes, 21 ± 5 minutes,
3.6 ± 1.1 hours), 1,2,3-TMB (1.5 ± 0.9 minutes, 24 ± 9 minutes, 4.7 ± 1.6 hours), and 1,3,5-TMB
(1.7 ± 0.8 minutes, 27 ± 5 minutes, 4.9 ± 1.4 hours) (larnberg etal.. 19961. The half-life of
elimination for 1,3,5-TMB in the last and longest phase is much greater than those for 1,2,4-TMB or
1,2,3-TMB (120 ± 41 versus 87 ± 27 and 78 ± 22 hours, respectively). Urinary excretion of
unchanged parent compound was extremely low (<0.002%) for all three isomers flanasik etal..
2008: larnberg etal.. 1997b). The difference observed in half-lives between the three isomers in
the last elimination phase may be due to small sample sizes and difficulties in measuring slow
elimination phases rather than a true difference in half-lives. All three isomers were eliminated via
exhalation: 20-37% of the absorbed dose of 1,2,4-TMB, 1,2,3-TMB, or 1,3,5-TMB was eliminated via
exhalation during exposure to 123 mg/m3 (25 ppm) for 2 hours flarnberg etal.. 19961.
Following exposure of rats to 25 ppm (123 mg/m3) 1,2,4-TMB, 1,2,3-TMB, or 1,3,5-TMB for
6 hours, the terminal half-life of elimination of 1,3,5-TMB from the blood (2.7 hours) was shorter
than that for 1,2,4-TMB (3.6 hours) or 1,2,3-TMB (3.1 hours) (Swiercz etal.. 2016: Swiercz etal..
2006: Swiercz etal.. 20021. As dose increased, the half-lives for elimination from blood following
single exposures to 1,2,4-TMB (17.3 hours) became much longer than those for 1,3,5-TMB (4.1
hours) or 1,2,3-TMB (5.3 hours). In repeated-dose experiments (4 weeks), the terminal half-lives of
elimination ofTMB isomers in venous blood were similar for 1,2,4-TMB and 1,2,3-TMB (9.9 and 8.0
hours, respectively), but larger than that of 1,3,5-TMB (4.6 hours) (Swiercz etal.. 2016: Swiercz et
al.. 2006: Swiercz etal.. 2003: Swiercz etal.. 20021.
For a full discussion of the toxicokinetics of 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB, see
Appendix C, Sections C.l.
1.1.2. Description of Toxicokinetic Models
There were three deterministic physiologically-based pharmacokinetic (PBPK) models
identified in the literature for TMB isomers. larnberg and lohanson (1999) developed a PBPK
model for inhalation of 1,2,4-TMB in humans in order to investigate how various factors (work load,
exposure level, fluctuating exposure) influence potential biomarkers of exposure (end-of-shift and
prior-to-shift concentrations of parent compound in blood and metabolites in urine). There was no
animal component to this PBPK model and it was not parameterized for other TMB isomers.
Emond and Krishnan f2 0061 developed a PBPK model not for any TMB isomer specifically, but to
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test whether a model could developed for highly lipophilic VOCs (HLVOCs) using the neutral lipid
equivalent (NLE) content of tissues and blood as the basis. Lastly, Hissink etal. (2007) developed a
PBPK model to characterize internal exposure following white spirit inhalation. This model
contained both a rat and a human component and was constructed to be able to predict levels of
1,2,4-TMB and n-decane in the blood and brain following exposure to white spirit.
All three of the available 1,2,4-TMB PBPK models were evaluated for potential use in this
assessment. Of the three deterministic PBPK models available for 1,2,4-TMB (Hissink etal.. 2007:
Emond and Krishnan. 2006: larnberg and lohanson. 19991. the Hissink etal. (2007) model was
chosen to utilize in this assessment because it was the only published 1,2,4-TMB model that
included parameterization for both rats and humans, the model code was available, and the model
adequately predicted experimental data in the dose range of concern. The Hissink etal. (2007)
model was thoroughly evaluated, including a detailed computer code analysis. Full details on
model verification, parameterization, optimization, and validation (including all analyses to confirm
published model outputs), discussions of the uncertainties in model structure and choice of dose
metric, and sensitivity analyses are provided in Appendix C, Section C.3.3.
1.2. SYNTHESIS OF EVIDENCE
1.2.1. Neurological Effects
There is evidence in humans and animals that inhalation exposure to TMBs induces
neurotoxic effects. The human evidence comes from occupational studies involving complex VOC
mixtures that include TMBs; thus, effects cannot be attributed to any TMB isomer specifically.
Prevalence rates of neuropsychological symptoms increased with exposure duration in dockyard
painters, with symptoms related to motor coordination exhibiting the strongest association (Chen
etal.. 19991. Similarly, significant associations between exposure and impaired performance in
short-term memory (symbol digit substitution), motor speed/coordination (finger tapping), and
peripheral nerve function tests were observed in other studies of shipyard painters exposed to
mixtures possibly containing TMBs (isomers were not specified) and other solvents (Lee etal..
2005: Ruiiten et al.. 1994). Other neuropsychological symptoms (mood changes, equilibrium
complaints, and sleep disturbances) were also reported by Ruiiten et al. f 19941. Detrimental
neuropsychological effects (memory problems, dizziness, hand tremble) have also been reported in
paint factory workers exposed to multiple unspecified solvents; working in jobs with a ostensible
higher exposure to solvents (production versus packaging) was observed to be among the strongest
predictors of symptoms (El Ha mid Hassan et al.. 20131. A significant, positive association between
exposure symptoms (e.g., abnormal fatigue) and 1,2,4-TMB exposure, but not exposure to lower
levels of 1,2,3-TMB or 1,3,5-TMB, was reported in asphalt workers (Norseth et al.. 19911.
Nervousness, tension, headaches, vertigo, and anxiety were reported in paint shop workers
exposed to 49-295 mg/m3 of a solvent mixture containing 50% 1,2,4-TMB, 30% 1,3,5-TMB, and
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unspecified amounts of 1,2,3-TMB (listed as possibly present) (Battig etal. (1956). as reviewed by
MOE (2006) and Battig etal. (1958)).
Additional evidence suggests damage or dysfunction of the inner ear and increased
occurrence of vertigo following exposure to TMBs and other organic solvents in paint and varnish
factories and histology laboratories fluarez-Perez etal.. 2014: Fuente etal.. 2013: Sulkowski etal..
20021. However, an analysis using naphthalene as a marker for jet propulsion fuel 8 (JP-8) (which
contains TMB isomers among multiple other aliphatic and aromatic solvents) did not indicate that
exposure to complex solvent mixtures resulted in increased postural sway (Maule etal.. 2013).
Similar to suggestive evidence of visual impairment following exposure to complex solvent
mixtures (i.e., altered color vision and contrast in exposed furniture factory workers f Gong etal..
2003) and increased latencies for visual evoked potentials [VEPs] in gasoline-exposed exposed
workers f Pratt etal.. 200011. increased reaction time was significantly and consistently associated
with exposure in controlled, acute volunteer studies in which adults were exposed to mixtures
containing 1,2,4-TMB (Lammers etal.. 2007). although it is unclear whether 1,2,4-TMB or other
constituents within the mixtures were responsible for the observed effects. However, in another
volunteer study in which participants were exposed to aromatic or dearomatized white spirit for
4 hours, neurobehavioral impairments were either weakly or inconsistently associated with
exposure flu ran etal.. 2014). Uptake of TMBs was reported in volunteers exposed for 2 hours to
either: 300 mg/m3 white spirit (corresponding to 11 mg/m31,2,4-TMB); 11 or 123 mg/m3
1,2,4-TMB; 123 mg/m31,2,3-TMB; or 123 mg/m31,3,5-TMB. However, effects on the central
nervous system (CNS) were based on measures of overt CNS depression (heart rate and pulmonary
ventilation) and a subjective rating of CNS symptoms (i.e., headache, fatigue, nausea, dizziness, and
intoxication) (larnberg etal.. 1997a: larnberg et al.. 1996). For full details of the epidemiologic and
controlled human exposures studies (including human subjects research ethics procedures), see
individual study summary tables in Appendix C.
In two studies examining the toxicokinetics of TMBs following controlled human exposures
to 5-150 mg/m31,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB, no neurological abnormalities in routine
clinical examinations were reported following exposure, although neither results data nor details
regarding the specific tests performed were provided (Kostrzewski etal.. 1997: Kostrewski and
Wiaderna-Brvcht. 1995). Studies identifying an association between occupational exposure to TMB
isomers and neurological effects are limited due to an inability to attribute effects due to 1,2,3-TMB,
1,2,4-TMB, or 1,3,5-TMB individually versus those due to the other isomers or additional
constituents within the mixture. The studies detailing controlled exposures to volunteers are also
limited for evaluating neurotoxicity to TMBs due to a lack of methods to adequately assess CNS
function, a lack of no-exposure controls, short exposure duration, and exposure of individual
subjects to different concentrations of TMB isomers.
In animals, there is consistent evidence of neurotoxicity following inhalation exposure, and
to a lesser extent following oral exposure, to either 1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB; a summary
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of the evidence pertaining to neurotoxic effects for TMBs is shown in Tables 1-2 and 1-3 for
inhalation and oral exposures, respectively. This information is presented graphically in Figure 1-1.
Pain sensitivity
No data on pain-related behaviors were identified from studies of humans exposed to
individual TMB isomers or mixtures containing TMBs. Decreased pain sensitivity has been
observed following inhalation exposure to TMBs in multiple studies conducted in male Wistar rats
(Table 1-2; Figure 1-1). To test pain responses following TMB exposure, animal studies have
employed the hot plate test In this test, animals are exposed to a thermal stimulus by placing them
on a copper plate heated to 54.5°C in order to determine pain sensitivity fAnkier. 19741. The
latency for the animals is recorded as the time it takes for the tested animals to remove their paws
from the heated plate and lick their paws, with longer latencies reflecting decreased pain
sensitivity. In order to investigate potential TMB-induced sensitizing and/or latent effects, the
short-term exposure studies introduced an additional challenge to the testing paradigm in the form
of a footshock. By incorporating the footshock, which itself decreases pain sensitivity, before
testing hotplate responses, the short-term exposure studies were able to investigate potentially
more subtle TMB-induced neurotoxic effects long after the termination of exposure (testing began
>50 days post-exposure). Specifically, the footshock challenge introduces a stressor on nervous
system processes related to pain perception (essentially straining the capabilities of this system
either through desensitization of sensory pain receptors or damage to the peripheral nerves),
which appears to be capable of unmasking latent nervous system effects that may persist after TMB
isomer exposure weeks earlier.
Decreases in pain sensitivity have been observed at concentrations >492 or >123 mg/m3
following subchronic exposure to 1,2,4-TMB or 1,2,3-TMB, respectively fKorsak and Rydzvhski.
19961. Decreased pain sensitivity after a footshock challenge was observed at concentrations
>492 mg/m3 following short-term exposure to 1,2,4-TMB fGralewicz and Wiaderna. 2001:
Gralewicz etal.. 1997b). 1,3,5-TMB (Wiaderna etal.. 2002: Gralewicz and Wiaderna. 2001). or
1,2,3-TMB (Wiaderna et al.. 1998). although changes were not observed at 492 mg/m31,2,3-TMB
(latencies 75% longer than controls were not statistically significant) in another short-term
exposure study (Gralewicz and Wiaderna. 2001). No statistically significant decreases in pain
sensitivity were observed 24 hours after exposure in rats exposed to concentrations of the C9
fraction up to 1,500 ppm for up to 13 weeks (approximately 4,059 mg/m3 TMB isomers) (Douglas
etal.. 19931. There was a statistically significant increase in thermal response time in C9-exposed
groups when the effect was measured immediately prior to the exposure period; however, this was
most likely due to an unusually low control group response at this time point Specifically
regarding pain sensitivity measured immediately after exposure, effects were more pronounced
following subchronic exposure as compared to acute exposure (e.g., for 1,2,4-TMB, a 50% response,
or 30-second latency, required levels nearly 5-fold higher: 5,682 versus <1,230 mg/m3, with
exposure for 4 hours as compared to for 13 weeks) fKorsak and Rvdzvriski. 19961.
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In the subchronic exposure study (Korsak and Rvdzyhski. 19961. inhalation of 1,2,4-TMB or
1,2,3-TMB resulted in reduced pain sensitivity, which occurred in a concentration-dependent
manner. In short-term exposure studies that examined a range of concentrations fWiaderna etal..
2002.1998: Gralewicz etal.. 1997bl. decreases in pain sensitivity after footshock challenge
following exposure to TMB isomers were non-monotonic. Additionally, no effect on pain sensitivity
was observed in rats exposed to the C9 fraction. Differences in experimental design (discussed
below) or mechanism of action may account for the lack of monotonicity in TMB short-term
exposure studies and no effect in the C9 study, in contrast to the observations in Korsak and
Rvdzyhski (1996). Similar to the subchronic study, acute exposures to 1,2,3-TMB, 1,2,4-TMB,
1,3,5-TMB, or Farbasol (a solvent mixture containing 44% TMB isomers) induced concentration-
dependent decreases in pain sensitivity, with EC50 values of 4,172, 5,682, 5,963, or 6,589 mg/m3,
respectively, for increased latency to paw-lick compared to controls fKorsaketal.. 1999: Korsak
and Rvdzyhski. 1996: Korsak etal.. 19951.
The decreases in pain sensitivity measured in the subchronic and acute TMB-only studies
were observed immediately after exposure (Korsak and Rvdzyhski. 1996: Korsak etal.. 1995). with
no statistically significant effects persisting 2 weeks after subchronic exposures were terminated
(i.e., increases in latency of 95 or 78% greater than controls were reduced to 12 or 13% greater
than controls at 1,230 mg/m3 for 1,2,4- or 1,2,3-TMB, respectively) fKorsak and Rvdzyhski. 1996:
Korsak etal.. 19951. Similarly, short-term TMB exposure without the footshock challenge did not
result in statistically significant effects on pain sensitivity in the hot plate test several weeks after
exposures had ended, although latencies were increased up to 200%. In contrast, performance in
the hot plate test after footshock challenge was significantly impaired following short-term
exposure to the TMB isomers when tested 51 days after exposure fWiaderna etal.. 1998)
fWiaderna etal.. 2002: Gralewicz and Wiaderna. 2001: Gralewicz etal.. 1997bl. indicating a
persistence of these pain sensitivity-related effects. The footshock data suggest that, following
short-term exposure, TMB isomers caused a long-term (potentially permanent) reduction in the
ability of the nervous system to respond to and/or recover from the stress-induced analgesia
caused by footshock.
The addition of a footshock challenge to the hotplate tests following short-term (i.e.,
4-week), inhalation exposure to TMB isomers makes these experiments somewhat distinct from
those performed following subchronic exposure, as the footshock challenge can elicit a cognitive
response from the animals in later hot plate test trials (see below) fWiaderna etal.. 2002: Gralewicz
and Wiaderna. 2001: Wiaderna et al.. 1998: Gralewicz etal.. 1997bl. In the short-term studies
fWiaderna etal.. 2002: Gralewicz and Wiaderna. 2001: Wiaderna et al.. 1998: Gralewicz etal..
1997b). treatment-related, statistically significant changes at >492 mg/m31,2,3-TMB, 1,2,4-TMB, or
1,3,5-TMB were observed 24 hours after rats were given a footshock; no consistent, significant
effects at any concentration were observed immediately following footshock, when pain sensitivity
is maximally suppressed by footshock. Additionally, no statistically significant effects were
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observed prior to footshock at 50 days post-exposure; studies did tend to observe increases in
latency in non-shocked rats that were not statistically significant at >492 mg/m31,2,4-TMB (up to
206% longer than controls), 1,3,5-TMB (up to 215% longer than controls), or 1,2,3-TMB (up to 95%
longer than controls), but these responses were highly variable and not consistently observed
across studies. As footshock alone is known to cause transient reductions in pain sensitivity, these
findings suggest that inhalation exposure to TMBs prolongs footshock-induced reductions in pain
sensitivity. However, although a lengthening of the footshock-induced decrease in pain sensitivity
by TMB exposure is the most likely reason for the observed effects, and, accordingly, these
responses are discussed in this context herein, this is not the only possible explanation. It is also
possible that cognitive effects resulting from TMB exposure might contribute to the responses
observed 24 hours after footshock. Specifically, control groups may better associate the hotplate
environment with the previously-applied aversive stimulus and more quickly withdraw their paws
than their TMB-exposed counterparts that may exhibit a decreased fear response or shorter
retention of that fear-associated memory. Alternatively, since this test paradigm can cause the hot
plate test apparatus to become associated with the effects of footshock, inducing stress-related
responses in the shocked animal such that subsequent exposure to the hot plate test apparatus
alone can reduce sensitivity to pain (possibly via the release of endogenous opioids), prior TMB
exposure could amplify this effect From the data available, the relative contribution(s) of these
behaviors to the observed effects cannot be easily distinguished. Despite the possible overlap
between contributing neurological processes in this test paradigm, these observations are still
regarded as significant and adverse, and indicate a persistence of neurological effects long after
TMB exposures have ceased.
Differences in study design may also partially explain the difference in results observed in
the subchronic studies, in which statistically significant decreases in pain sensitivity were observed
when measured immediately after termination of exposure fKorsak and Rydzvhski. 1996). but not
when measured 24 hours after exposure fDouglas etal.. 19931: (information provided in written
comments submitted during public comment period). As stated above, decreased pain sensitivity
was mostly reversible when measured 2 weeks post-exposure in a subchronic exposure study
fKorsak and Rydzvhski. 1996) and 24 hours after the termination of exposure (Douglas etal..
1993). Additionally, when the endpoint was measured up to 50 days after the termination of short-
term exposures (i.e., pre-footshock), there was no observable effect fWiaderna etal.. 2002:
Gralewicz and Wiaderna. 2001: Wiadernaetal.. 1998: Gralewicz etal.. 1997b). However, when
animals were subjected to an environmental stimulus (i.e., footshock), decreased pain sensitivity
was observed in exposed animals, demonstrating a persistence of effect. While the U.S.
Environmental Protection Agency (EPA) Guidelines for Neurotoxicity Risk Assessment (U.S. EPA.
1998) note that effects that are reversible in minutes, hours, or days after the end of exposure and
appear to be associated with the pharmacokinetics of the agent and its presence in the body may be
of less concern than effects that persist for longer periods of time, they also note that it is important
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to consider that effects that may seem reversible may re-appear later or be predictive of later
adverse outcomes. While the Douglas etal. (1993) study appears to present differing results
compared to the subchronic and short-term exposure studies fWiaderna etal.. 2002: Gralewicz and
Wiaderna. 2001: Wiaderna etal.. 1998: Gralewicz etal.. 1997b: Korsak and Rydzvhski. 19961. it
actually comports with the subchronic and short-term studies when considering the potential time-
course of the decreased pain sensitivity endpoint Decreased pain sensitivity is clearly observed
immediately after termination of subchronic exposure to either 1,2,4-TMB or 1,2,3-TMB (Korsak
and Rvdzvnski. 19961. and to a lesser extent following acute exposure; this effect then becomes
undetectable when measured at time points further from the end of the exposure period (24 hours:
Douglas etal. Q9931: 2 weeks: Korsak and Rvdzvnski Q9961: and 50 days: Gralewicz etal. f!997bl
and Wiaderna etal. (199811. and then becomes evident again as a latent effect following challenge
with an environmental stimulus fWiaderna etal.. 2002: Gralewicz and Wiaderna. 2001: Wiaderna
etal.. 1998: Gralewicz etal.. 1997b). Additional differences in study design that may explain some
discrepancies in observed effects include the fact that Douglas etal. T19931 used the C9 aromatic
fraction as the test agent and not a single TMB isomer or mix thereof, and how the hot plate test
was administered Douglas etal. (19931 did not provide exact details on the hot plate temperature.
It is not currently known how constituents of the C9 fraction, including TMBs, would be absorbed
and distributed throughout the body after exposure, as no toxicokinetic studies are available that
have investigated this topic. Therefore, it is not known whether TMB isomers in the C9 fraction
partition to the target organ (i.e., the brain) in a similar pattern as TMB isomers when used as the
sole exposure agent
Substantial differences in study design between short-term and subchronic exposure
studies, and a lack of knowledge regarding the specific mode(s) of action, make it impossible to
distinguish the particular aspects of the pain sensitivity phenotype that appear to be latent and only
manifest with an environmental challenge from those that appear to be at least partially reversible.
Regardless, the ability of male Wistar rats to respond to a thermal stimulus in the hot plate test was
consistently impaired following inhalation exposure to individual TMB isomers. The overall
database indicates that individual TMB isomers are similar in their capacity to decrease pain
sensitivity following inhalation exposure (Table 1-2; Figure 1-1). Pain sensitivity was not examined
following oral exposure.
Neuromuscular function and coordination
Human exposures to solvent mixtures containing 1,2,4-TMB fLammers etal.. 20071.
multiple TMB isomers (Battigetal. (1956). as reviewed by MOE (2006) and (Lee etal. (2005):
Sulkowski etal. (2002): Battig etal. (1958)1. or complex solvent mixtures (El Ha mid Hassan et al..
2013: Ruiiten et al.. 19941 result in effects that suggest alterations to neuromuscular function and
balance, including increased reaction time, increased hand tremble, decreased hand eye
coordination, and vertigo. Animal studies using rotarod performance, which tests motor
coordination, balance, and overall neuromuscular function, indicate that inhalation of TMB isomers
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can affect neuromuscular system function (Table 1-2; Figure 1-1). Significant decreases in rotarod
performance were observed at 1,230 mg/m31,2,4-TMB (40% response) and >492 mg/m3
1,2,3-TMB (50-70% response) when tested immediately after exposure for 13 weeks fKorsak and
Rvdzyhski. 1996): an exposure duration-dependency for this effect was observed, with less robust,
but statistically significant, decreases in performance also reported at 1,230 mg/m3 after 4 (40 and
30% response) or 8 (60 and 40% response) weeks of exposure to 1,2,3-TMB or 1,2,4-TMB,
respectively. This impaired function was still evident at 2 weeks post-exposure, indicating a
persistence of this effect Specifically, failures in 70 and 40% of animals after 13 weeks of exposure
to 1,230 mg/m31,2,3-TMB and 1,2,4-TMB, respectively (compared to 0% of animals in control
groups at any time), were 50 and 30% at 2 weeks post-exposure, although 30% failures at 15 weeks
for 1,2,4-TMB was no longer significantly different from controls (note: statistical comparisons did
not appear to include a repeated measures component and comparisons to the 13-week time-point
were not performed). The observations of substantial decrements in rotarod performance are
interpreted as a biologically relevant responses in light of the lack of failures in controls and the
similarities in response magnitude across isomers. Inhalation studies of acute TMB exposure
support this observation. Effects such as loss of reflexes and righting responses have been
observed following acute inhalation exposure to 1,250-45,000 mg/m31,2,4-TMB fMOE. 2007.
2006: Henderson. 2001: Cameron etal.. 1938). Similarly, acute exposure to 1,2,3-TMB, 1,2,4-TMB,
1,3,5-TMB, or Farbasol resulted in decreased performance in rotarod tests immediately following
exposure, with ECr.o values of 3,779, 4,693, 4,738, or 5,497 mg/m3, fKorsak etal.. 1999: Korsak and
Rvdzyhski. 1996: Korsak et al.. 1995). The potential for reversibility of effects following acute
exposure has not been tested. These results indicate that 1,2,4-TMB and 1,3,5-TMB are similar in
their ability to impair neuromuscular function, balance, and coordination, while 1,2,3-TMB
exposure may elicit effects at lower concentrations compared to the other two isomers. Other tests
of neuromuscular function and/or coordination (grip strength, hindfoot splay) were not affected at
any exposure concentration (up to 1,500 ppm C9; approximately 4,059 mg/m3 TMB isomers) in
exposed rats (Douglas etal.. 1993). No studies evaluating oral exposure to TMB isomers address
this endpoint
The neurobehavioral tests administered (i.e., hot plate and rotarod) in the subchronic and
acute exposure studies by Korsak and Rvdzyhski (1996) and Korsak etal. (1995) appear to have
been conducted on the same days; however, it is unclear whether the tests were performed
sequentially in the same cohorts of animals. Performing the hot plate test immediately following
the rotarod test could introduce a potential confounder, as shock alone (such as that used as
negative reinforcement following rotarod failure, see Table C-29, Appendix C) can cause reductions
in pain sensitivity. Thus, if the tests were performed sequentially in the same animals, TMB-
exposed animals failing more often in the rotarod test may exhibit increases in paw-lick latency
unrelated to treatment, as compared to controls receiving less shock reinforcement However, the
observations by Korsak and Rvdzvhski f 19961 and Korsak etal. f 19951 are supported by 2-3-fold
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increases in latency to paw-lick that, although not statistically significant but possibly biologically
relevant given the magnitude and consistency of response across isomers, were observed 50 days
after termination of short-term exposures to 492 mg/m31,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB
fGralewicz and Wiaderna. 20011: increases of this magnitude were not present in the studies
evaluating multiple concentrations of the isomers fWiaderna etal.. 2002.1998: Gralewicz etal..
1997b).
Motor function and/or anxiety
Effects in open field testing have been consistently reported in oral and inhalation studies of
exposure to 1,2,4-TMB and 1,3,5-TMB, but not 1,2,3-TMB or the C9 fraction, in male rats fWiaderna
etal.. 2002: Gralewicz and Wiaderna. 2001: Wiaderna et al.. 1998: Gralewicz etal.. 1997bl
(Table 1-2; Figure 1-1); however, open field locomotion following injections with the stimulant,
amphetamine, were amplified by prior 1,2,3-TMB exposure, but not by prior 1,2,4-TMB exposure
(Lutz etal.. 2010). suggesting a more complicated pattern to these effects. Altered behaviors and
locomotion in open field tests can be attributed to anxiety responses due to open spaces and bright
light, as well as changes to motor system function. Factors other than anxiety and motor function
(e.g., interpretation of olfactory or visual cues) may also contribute to alterations in open field
behavior, but these are less likely to be drivers of the responses. As a majority of studies observed
animals for only 5 or 10 minutes, these experimental tests are insufficient to identify the relative
contribution(s) of the various neurological responses to the observed increases in open field
behaviors. Thus, EPA has concluded that decreased anxiety and/or increased motor function are
the most likely explanations for the TMB-induced effects.
Decreased anxiety and/or increased motor function at >492 mg/m31,2,4-TMB or 1,3,5-TMB
has been reported in short-term exposure studies several weeks after exposures ceased, as
evidenced by increases in horizontal locomotion or grooming activities fLutz etal.. 2010: Gralewicz
and Wiaderna. 2001: Gralewicz etal.. 1997bl. Statistically significant increases in horizontal
locomotion were observed in short-term studies assessing open field behavior following inhalation
exposure to 1,2,4-TMB or 1,3,5-TMB (Lutz etal.. 2010: Gralewicz and Wiaderna. 2001). Non-
monotonic increases in grooming were reported following short-term exposure to 1,2,4-TMB,
although changes in horizontal locomotion were not statistically significant (increases of 3-35%
were also non-monotonic) fGralewicz etal.. 1997b). No statistically significant effects on open field
activity have been observed following short-term exposure of male rats to 1,2,3-TMB fLutz et al..
2010: Gralewicz and Wiaderna. 2001: Wiaderna etal.. 19981. No short-term inhalation exposure
studies evaluated open field behaviors immediately after exposure. Open field locomotion
following injections with the stimulant amphetamine was amplified by previous short-term
exposure to 1,2,3-TMB, but not 1,2,4-TMB (which actually tended to inhibit amphetamine-induced
increases in activity at 492 mg/m3), suggesting possible effects of 1,2,3-TMB on sensitization-type
responses. As open field testing was conducted 14 or 25 days after termination of repeated
exposures for 4 weeks in these studies and TMB isomers are cleared rapidly from the body
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following the end of inhalation exposures (Section C.2, Appendix C), the results suggest persistence
of the effects of 1,2,4-TMB and 1,3,5-TMB on anxiety and/or motor function following clearance of
the toxic moiety from the nervous system.
Slight, transient increases in locomotor activity were also observed in open field tests
immediately following acute, oral exposure to the TMB isomers (Table 1-3). Significant increases in
locomotor activity—measured as number of squares crossed after exposure compared with prior to
exposure—were observed at 3,850 mg/kg for 1,2,4-TMB and 1,2,3-TMB, and at >1,920 mg/kg for
1,3,5-TMB, with minimal concentration-effect or time-effect relationships and negligible differences
in the magnitude of the change in activity between isomers (Tomas et al.. 1999b). Increases in
locomotor activity were biphasic in nature. At early timepoints immediately following acute
exposure, increased locomotor activity was associated with perturbed motor coordination and
tremor, whereas after 90 minutes, this apparent motor ataxia progressed to hindlimb paralysis, full
immobility, and respiratory distress (e.g., tachypnea), leading to several deaths by 24 hours (Tomas
etal.. 1999b).
As mentioned previously, open field tests cannot easily distinguish between anxiety-related
responses and changes in motor activity. However, mixed effects on motor activity were observed
following inhalation exposure to elevated concentrations of TMBs in several studies, and some
findings appear inconsistent with observations in open field tests. No consistent treatment-related
effects on motor activity were reported in male rats exposed to up to 1,500 ppm C9 fraction
(approximately 4,059 mg/m3 TMB isomers) for 13 weeks (Douglas etal.. 1993). Transient
increases in motor activity (horizontal movement and total activity) were observed in rats exposed
to 1,500 ppm C9 at minutes 10-20 of the test during week 9 of the exposure period. However,
motor activity in this exposure group returned to control levels during minutes 20-30 of the test,
and no effects were observed at the termination of exposure (i.e., week 13). When results were
summarized across the entire 30-minute test period, no effects on motor activity were reported at
any time during the 13-week exposure period. Decreased motor activity was observed in male rats
immediately after exposure to 5,000 mg/m31,2,4-TMB or similar levels of the C9 aromatic fraction
(Mckee etal.. 2010). Decreased motor activity was also reported in rats acutely exposed via
inhalation to a mixture containing TMB isomers (Lammers etal.. 20071. but the use of a mixture
precludes a determination of the toxicity specifically associated with individual isomers. As
biphasic changes in activity (i.e., initial increases followed by decreases in movement) are
frequently observed immediately following acute exposures to solvents, it is likely that the timing of
the evaluations across studies, as well as the differing isomer concentrations, may influence the
consistency of these results.
Overall, exposure to 1,2,4-TMB and 1,3,5-TMB affects anxiety and/or motor function at
concentrations above 492 mg/m3, although the exact, sometimes non-monotonic, concentration-
response relationship remains unclear, and the potential contribution of motor activity changes
based on activity tests of longer duration were mixed and difficult to interpret The results for
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1.2.3-TMB are difficult to interpret, as no effects were observed following short-term inhalation
exposure, while acute oral exposure elicited responses consistent with 1,2,4-TMB and 1,3,5-TMB.
Although an explanation for this disparity is lacking, these data highlight a potential difference
between 1,2,3-TMB and the other isomers, regarding altered motor function and/or anxiety.
Cognitive function
Cognitive function following exposure to TMB isomers alone has not been extensively
evaluated in humans or following oral exposure in animals. Controlled exposure of volunteers to
mixtures containing TMBs did not indicate any effects on short-term learning or memory tests
(Lammers etal.. 20071. However, in one study, solvent-exposed construction workers were
observed to have decreased performance in memory tasks fTang etal.. 20111. In animals, short-
term spatial memory (radial maze performance) was unaffected by exposure to either 1,2,4-TMB or
1,3,5-TMB via inhalation fWiaderna etal.. 2002: Gralewicz and Wiaderna. 2001: Gralewicz etal..
1997b). Similarly, although one study indicates a significant decrement in radial maze performance
following exposure to 123 mg/m31,2,3-TMB fWiaderna etal.. 19981. higher concentrations had no
effect (Wiaderna etal.. 19981. preventing interpretations regarding the significance of this finding.
In contrast, effects on cognitive function in passive and active avoidance tests of conditioning
behaviors were consistently observed across multiple studies in male rats 6-8 weeks following
short-term inhalation exposure to the TMB isomers, although clear concentration-effect
relationships were not observed (Table 1-2; Figure 1-1). Comparing the results of the behavioral
tests reveals that there are differences in cognitive effects reported for each TMB isomer, as well as
differences in the concentrations at which the cognitive effects were observed.
In the passive avoidance tests, rats were conditioned to avoid stepping down from a small,
elevated platform (the impulse of rats is to step down in order to escape the bright light and
constrained, elevated space of the platform) through the use of a brief series of footshocks applied
on the lower level. It is important to clarify that these tests are distinct from tests of pain sensitivity
and that observations of decreased step-down latency in these tests do not contrast with the
increases in paw-lick latency observed in hot plate tests; in fact, they may be complementary (see
below; note: the footshocks used are of a much shorter duration than those used to induce
decreased pain sensitivity in the hot plate tests). Decreases in step-down latency in passive
avoidance tests, particularly at 7 days following footshock conditioning, were observed 6-7 weeks
after short-term inhalation exposure to >123 mg/m31,2,3-TMB and 1,3,5-TMB or >492 mg/m3
1.2.4-TMB fWiaderna etal.. 2002: Gralewicz and Wiaderna. 2001: Wiaderna etal.. 1998: Gralewicz
etal.. 1997b). Differences in latency prior to footshock were not observed. Decreases in latency
were consistently observed and were similar in magnitude across all studies at 7 days post-
footshock, although the decreases were not statistically significant for 1,2,4-TMB or 1,2,3-TMB in
the study by Gralewicz and Wiaderna (2001). At 3 days post-footshock, decreases in latency were
less consistent (i.e., statistically significant decreases were observed at 123 mg/m31,2,3-TMB and
at 492 mg/m31,2,4-TMB, but not at other concentrations, and were not observed following
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exposure to 1,3,5-TMB), and only 123 mg/m31,2,3-TMB was shown to have an effect at 1 day after
footshock. In these tests, the effects occurring several days following conditioning with footshock
are most likely attributable to a reduced ability to inhibit motor reactions (or a lowered motor
threshold) in response to the fear-inducing environment. Alternative explanations involve possible
contributions of the following in the TMB-exposed rats: a diminished fear response to the
footshock; decreased pain sensitivity leading to a less effective negative reinforcement by the (less
painful) footshock; or diminished retention of the fear-associated memory (i.e., from the footshock).
However, as statistically significant changes were observed <24 hours following footshock only
after exposure to 123 mg/m31,2,3-TMB, neither diminished fear responses to the footshock nor
decreases in pain sensitivity are likely to be the sole driver(s) of these effects. This suggests that, in
this particular test paradigm, TMB exposure causes latent effects on neurological functions
associated with the persistence of adaptive behaviors to a fear-inducing stimulus. Despite the
consistency of the results at 7 days post-footshock, these tests are insufficient to pinpoint whether
the effects of TMB exposure are specific to diminished memory retention, increased impulsivity,
and/or decreased motor control.
Reduced performance in two-way active avoidance tests was observed in male rats
following short-term inhalation exposure to >492 mg/m31,2,4-TMB fGralewicz and Wiaderna.
2001: Gralewicz etal.. 1997b). >123 mg/m31,3,5-TMB (Wiaderna etal.. 2002: Gralewicz and
Wiaderna. 20011. and 492 mg/m31,2,3-TMB fWiaderna et al.. 19981. The effects of TMBs were
particular to the learning component of the test (acquisition/training session), rather than the
memory component (retention session 7 days later) (Wiaderna etal.. 2002: Gralewicz and
Wiaderna. 2001: Wiaderna etal.. 1998). The conditioning or training of active avoidance behaviors
was based on avoiding a painful footshock (the unconditioned stimulus) upon presentation of a
tone (conditioned stimulus). Similar to the interpretation of results from passive avoidance tests, it
is unclear whether and to what extent potential alterations in locomotor activity (rats had to shuttle
between compartments) and/or pain sensitivity following exposure to TMB isomers could
contribute to learning deficits in these tests.
Acute inhalation exposure studies provide some support for the observed effects of TMB
isomers on learned behaviors. Significant increases in response latency in psychomotor tasks,
observed immediately after exposure (effects did not persist to 24 hours later), were reported in
male rats following acute exposure to 5,000 mg/m31,2,4-TMB fMckee etal.. 20101 or to
4,800 mg/m3 of a mixture containing TMBs (Lammers etal.. 2007). The effects on active and
passive avoidance behaviors indicate that learning and/or long-term memory processes are
affected by exposure to the TMB isomers. The data suggest that 1,3,5-TMB may be a more potent
inducer of toxic effects on cognitive function than 1,2,4-TMB and 1,2,3-TMB, as the effects following
exposure to 1,3,5-TMB were more consistent and sometimes occurred at lower concentrations than
those reported following exposure to the other two isomers. Overall, however, these differences
were slight.
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Controlled human exposure studies suggest that exposures of <123 mg/m3 ofthe TMB
isomers do not cause overt CNS depression (measured as heart rate and respiration) (larnberg et
al.. 19961. although symptoms related to this effect (e.g., lightheadedness, fatigue) have been
reported in workers occupationally exposed to mixtures containing TMBs. In mice, CNS depression
has been observed following acute inhalation exposure to >25,000 mg/m31,3,5-TMB, with similar
effect levels for 1,2,4-TMB fACGIH. 20021
Other Sensory-related behaviors
Very little information exists for TMB isomers or mixtures containing TMBs regarding the
association between exposure and decrements in sensory-related behaviors (typically visual
and/or auditory function). Evidence from a number of occupational studies indicate that exposure
to complex solvent mixtures possibly containing TMB isomers can result in visual and auditory
dysfunction as measured by the latencies of evoked potentials (luarez-Perez etal.. 2014: Fuente et
al.. 2013: Ouevedo etal.. 2012: Gong etal.. 2003: Pratt et al.. 20001. Although acute inhalation
studies of rats exposed to TMBs or TMB mixtures observe some effects on visual discrimination
tasks (Mckee etal.. 2010: Lammers et al.. 20071. these studies incorporate components of cognition
(e.g., learning or habituation; memory) in their design, complicating interpretations of possible
effects on sensory function alone. The C9 fraction, a complex mixture of VOCs, including TMB
isomers, did not affect the auditory startle response in rats exposed up to 1,500 ppm
(approximately 4,059 mg/m3 TMBs) (Douglas etal.. 19931. Overall, the data are inadequate to
interpret the potential for TMB exposure to affect sensory-related behaviors.
Electrocortical activity
The only electrophysiological data available from TMB isomer or mixture studies were
measures most likely to be associated with attention, alertness, or memory-related behaviors.
Neurophysiological evidence from short-term inhalation studies in animals, as well as supportive
evidence from acute oral and injection studies, suggests that exposures to TMB isomers at lower
concentrations (at least for 1,2,4-TMB) may affect parameters associated with brain excitability.
Decreases in a particular component of electrocortical arousal (i.e., spike-wave discharge, SWD,
bursts in recordings from cortical-hippocampal electroencephalograms, EEGs) were observed in
male rats 120 days after short-term exposure to >492 mg/m31,2,4-TMB (statistically significant at
1,230 mg/m3), suggesting persistent functional changes in the rat CNS fGralewicz etal.. 1997al.
Altered EEG patterns can be induced by anesthetics as well as stimuli that produce arousal, and
may precede other measures of neurotoxicity (U.S. EPA. 19981. In recordings from rats that were
awake, but immobile (not exhibiting pronounced exploratory activity, as determined by EEG
morphology), statistically significant decreases in the frequency of SWD episodes were observed at
24 hours following short-term exposure to 492 mg/m31,2,4-TMB (decreases that were not
statistically significant were also observed at >492 mg/m31,2,4-TMB at 30 and 120 days after
exposure) fGralewicz etal.. 1997al.
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Complementing these findings, dose-related decreases in the duration and number of SWD
bursts (termed high-voltage spindles) were observed in rats at >240 mg/kg of the TMB isomers
subsequent to acute oral exposure fTomas et al.. 1999a) (Table 1-3). The stronger and more
persistent effects on electrocortical activity followed a pattern of 1,2,3-TMB > 1,3,5-TMB >
1,2,4-TMB fTomas etal.. 1999a). Similarly, electrophysiological alterations in cortical and
hippocampal EEGs were more pronounced following intraperitoneal (i.p.) injection of 1,2,3-TMB,
with 1,2,4-TMB and 1,3,5-TMB exerting lesser effects (Tomas etal.. 1999c). Although it is unclear
whether these changes affect related processes such as memory and seizure initiation/propagation,
the observed EEG abnormalities following inhalation (Gralewicz etal.. 1997a). oral fTomas etal..
1999a), and i.p. fTomas etal.. 1999c) exposure to TMB isomers provide supportive evidence of
possible acute CNS depression by TMB isomers fTomas etal.. 1999a: Tomas etal.. 1999c) and
indicate persistent (up to 120 days post-exposure) fGralewicz etal.. 1997a) alterations in CNS
activity that may reflect an adaptive response to TMB exposure.
Table 1-2. Evidence pertaining to neurological effects of TMBs in animals—
inhalation exposures
Study design3'15 and reference
Assav 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 d; Rat, Wistar, male, N = 10
Korsak and Rvdzvnski (1996),
Table C-29c
Hot plate (exposure-dependent increase in paw-lick latencv, which
recovers by 2 wks post-exposure)
Response immediately post-exposure: 0,18, 79*, 95*%
Response at 2 wks post-exposure: 0, ND, ND, 12%
0, 492 mg/m3
4 wks; Rat, Wistar, male, N = 11
Gralewicz and Wiaderna (2001),
Table C-24
Hot plate (increased paw-lick latencv 24 hrs after footshock)
Response at 50 d post-exposure: 0, 206%
Response at 50 d post-exposure seconds after footshock: 0, 25%
Response at 51 d post-exposure 24 hrs after footshock: 0,191*%
0, 123, 492, 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 15
Gralewicz et al. (1997b), Table C-22
Hot plate (increased paw-lick latencv 24 hrs after footshockd)
Response at 50 d post-exposure: 0, -6, 7, -9%
Response at 50 d post-exposure seconds after footshock: 0, -8,17,
-11%
Response at 51 d post-exposure 24 hrs after footshock: 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 d; Rat, Wistar, male, N = 10
Korsak and Rvdzvnski (1996), Table C-29
Rotarod (exposure-dependent increase in percent failures at 13 wks,
which does not recover by 2 wks post-exposure)
Response after 13 wks of exposure: 0,10, 20, 40*%
Response at 2 wks post-exposure: 0, ND, ND, 30%
This document is a draft for review purposes only and does not constitute Agency policy.
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Study design3'15 and reference
Assav and results (as response relative to control)
Motor function and/or anxiety
0, 123, 492, 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 15
Lutz et al. (2010), Table C-34
Open field (increased horizontal locomotion (distance traveled); no
overall effects with amphetamine challenge6)
Response at 2 wks 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%
0, 492 mg/m3
4 wks; Rat, Wistar, male, N = 11
Gralewicz and Wiaderna (2001),
Table C-24
Open field (increased horizontal locomotion; number of crossings)
Response at 25 d post-exposure: 0, 61*%
No change in exploration (rearings) or grooming episodes
0, 123, 492, 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 15
Gralewicz et al. (1997b), Table C-22
Open field (increased grooming at middle concentration):
Response at 25 d post-exposure: 0, 82,147*, 76%
No 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 C-24
Passive avoidance (decreased step-down latencv 7 d post-footshockf)
Response at 39 d post-exposure prior tofootshock: 0, 34%
Response at 42 d post-exposure 1 d after footshock: 0, -23%
Response at 44 d post-exposure 3 d after footshock: 0, -51 %
Response at 48 d post-exposure 7 d after footshock: 0, -43%
Note: statistical significance 7 d after footshock 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 d post-exposure: 0, 58*%
No differences were noted during retraining (retention)
Radial maze
No notable change in performance 14-18 d post-exposure
0,123, 492, or 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 15
Gralewicz et al. (1997b), Table C-22
Passive avoidance (decreased step-down latencv 3-7 d post-footshock)
Response at 39 d post-exposure prior tofootshock: 0, 26, 41, -31%
Response at 42 d post-exposure 1 d after footshock: 0, 95, -28, -87%
Response at 44 d post-exposure 3 d after footshock: 0, 7, -67*, -36%
Response at 48 d post-exposure 7 d after footshock: 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 d post-exposure
This document is a draft for review purposes only and does not constitute Agency policy.
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Study design3'15 and reference
Assav and results (as response relative to control)
Electrocortical activity
0, 123, 492, 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 9
Gralewicz et al. (1997a), Table C-23
EEG recordingsh (decreased SWD bursts/hr)
Response at 120 d post-exposure: 0,13, -35, -55*%
No change in global arousal level or in SWD/hr at 1 or 30 d post-
exposure
1,2,3-TMB
Pain sensitivity
0,123, 492,1,230 mg/m3 (recovery:
1,230 mg/m3 at 2 wks post-exposure)
90 d; Rat, Wistar, male, N = 10
Korsak and Rvdzvnski (1996), Table C-29
Hot plate (exposure-dependent increase in paw-lick latencv, which
recovers by 2 wks post-exposure)
Response immediately post-exposure: 0, 22*, 68, 78*%
Response at 2 wks post-exposure: 0, ND, ND, 13%
0, 492 mg/m3
4 wks; Rat, Wistar, male, N = 11
Gralewicz and Wiaderna (2001),
Table C -24
Hot plate (no statistically significant change in paw-lick latencv)
Response at 50 d post-exposure: 0, 95%
Response at 50 d post-exposure seconds after footshock: 0, -1%
Response at 51 d post-exposure 24 hrs after footshock: 0, 75%
0, 123, 492, 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 15
Wiaderna et al. (1998), Table C-43
Hot plate (increased paw-lick latencv 24 hrs after footshock at middle
concentration)
Response at 50 d post-exposure: 0, -28, -13, -12%
Response at 50 d post-exposure seconds after footshock: 0, -9, -16,
-5%
Response at 51 d post-exposure 24 hrs after footshock: 0, -19, 45*, 8%
Neuromuscular function and coordination
0,123, 492,1,230 mg/m3 (recovery:
1,230 mg/m3 at 2 wks post-exposure)
90 d; Rat, Wistar, male, N = 10
Korsak and Rvdzvnski (1996), Table C-29
Rotarod (exposure-dependent increase in percent failures at 13 wks,
which does not recover by 2 wks post-exposure)
Response after 13 wks of exposure: 0, 20, 40*, 70*%
Response at 2 wks post-exposure: 0, ND, ND, 50*%
Motor function and/or anxiety
0, 123, 492, 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 15
Lutz et al. (2010), Table C-34
Open field (statistically significant increase in horizontal locomotion
[distance traveled] only after amphetamine challenge6)
Response at 2 wks 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%
0, 492 mg/m3
4 wks; Rat, Wistar, male, N = 11
Gralewicz and Wiaderna (2001),
Table C-24
Open field (no change in horizontal locomotion; crossings)
Response at 25 d post-exposure: 0, -9%
No change in exploration (rearings), or grooming
0, 123, 492, 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 15
Wiaderna et al. (1998), Table C-43
Open field (no significant change in horizontal locomotion; crossings)
Response at 25 d post-exposure: 0,19, 51, 37%
No statistically significant change' in exploration (rearings) or grooming
This document is a draft for review purposes only and does not constitute Agency policy.
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Study design3'15 and reference
Assav and results (as response relative to control)
Cognitive function
0, 492 mg/m3
4 wks; Rat, Wistar, male, N = 11
Gralewicz and Wiaderna (2001),
Table C-24
Active avoidance (decreased performance during training; learning)
Trials to reach avoidance criteria at 54-60 d post-exposure: 0, 53*%
No differences were noted during retraining (retention)
Passive avoidance (no significant change in step-down latency')
Response at 39 d post-exposure prior tofootshock: 0, -39%
Response at 42 d post-exposure 1 d after footshock: 0, -40%
Response at 44 d post-exposure 3 d after footshock: 0, -23 %
Response at 48 d post-exposure 7 d after footshock: 0, -28%
Radial maze
No notable change in performance 14-18 d post-exposure
0, 123, 492, 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 15
Wiaderna et al. (1998), Table C-43
Passive avoidance (decreased step-down latencv after footshock)
Response at 39 d post-exposure prior tofootshock: 0, -41, -37,19%
Response at 42 d post-exposure 1 d after footshock: 0, -74*, -52, -43%
Response at 44 d post-exposure 3 d after footshock: 0, -54*, -49, -14%
Response at 48 d post-exposure 7 d after footshock: 0, -50*, -62*,
-37%
Active avoidance (decreased performance during training; learning)
Trials to reach avoidance criteria at 54-60 d post-exposure: 0, 3, 41*,
14%
No 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%
No notable change in trial duration at any day (14-18 d post-exposure)
1,3,5-TMB
Pain sensitivity
0, 492 mg/m3
4 wks; Rat, Wistar, male, N = 11
Gralewicz and Wiaderna (2001),
Table C-24
Hot plate (increased paw-lick latencv 24 hrs after footshock)
Response at 50 d post-exposure: 0, 215%
Response at 50 d post-exposure seconds after footshock: 0, 26%
Response at 51 d post-exposure 24 hrs after footshock: 0, 246*%
0, 123, 492, 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 12
Wiaderna et al. (2002), Table C-44
Hot plate (increased paw-lick latencv 24 hrs after footshock at middle
concentration)
Response at 50 d post-exposure: 0, -6, 36, 24%
Response at 50 d post-exposure seconds after footshock: 0, -14, 8, -4%
Response at 51 d post-exposure 24 hrs after footshock: 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 C-24
Open field (increased horizontal locomotion; number of crossings)
Response at 25 d post-exposure: 0, 65*%
No change in exploration (rearings) or grooming
This document is a draft for review purposes only and does not constitute Agency policy.
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Study design3'15 and reference
Assav and results (as response relative to control)
Cognitive function
0, 123, 492, 1,230 mg/m3
4 wks; Rat, Wistar, male, N = 12
Wiaderna et al. (2002), Table C-44
Passive avoidance (decreased step-down latencv 7 d post-footshock)
Response at 39 d post-exposure prior tofootshock: 0, -5,146, 40%
Response at 42 d post-exposure 1 d after footshock: 0, 99,108,113%
Response at 44 d post-exposure 3 d after footshock: 0, -32, -41, -40%
Response at 48 d post-exposure 7 d after footshock: 0, -47*, -53*,
-3*%
Active avoidance (decreased performance during training; learning)
Trials to reach avoidance criteria at 54-60 d post-exposure: 0, 40*, 35*,
50*%
Radial maze
No notable change in performance 14-18 d post-exposure
0, 492 mg/m3
4 wks; Rat, Wistar, male, N = 11
Gralewicz and Wiaderna (2001),
Table C-24
Passive avoidance (decreased step-down latencv 7 d post-footshockg)
Response at 39 d post-exposure prior tofootshock: 0, -3%
Response at 42 d post-exposure 1 d after footshock: 0, -61%
Response at 44 d post-exposure 3 d after footshock: 0, -65%
Response at 48 d post-exposure 7 d after footshock: 0, -57*%
Note: statistical significance 3 d after footshock 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 d post-exposure: 0, 65*%
Radial maze
No notable change in performance 14-18 d post-exposure
C9 Fraction
Pain sensitivity
0,100, 500,1,500 ppm C9 fraction
(approximately 0, 270,1,353,
4,059 mg/m3 TMB isomers)
13 wks; Rat, Wistar, male, N = 18-20
Douglas et al. (1993), Table C-21
Decreased pain sensitivity (increase in paw-lick latency)
Response relative to control
Exposure period:
Owks: 0, +52**, +34**, +19
5 wks: 0, +31, -5, +47
9 wks: 0, 0, -4, +9
13 wks: 0, +4, -1, +17
^Significantly different from controls (p < 0.05).
aRotarod and hot plate tests were administered immediately after termination of exposure or following a 2 week
recovery period by Korsak and Rvdzvriski (1996). EEG recordings were acquired prior to exposure and 1, 30, or
120 days after exposure by Gralewicz et al. (1997a). Motor behavior in an open field (tested for 30 minutes) was
assessed 14 days after exposure and re-tested following single and multiple (to induce sensitization) injections
with amphetamine for 120 minutes by Lutz et al. (2010). For the remaining studies (Wiaderna et al., 2002;
Gralewicz and Wiaderna, 2001; Wiaderna et al., 1998; Gralewicz et al., 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.
This document is a draft for review purposes only and does not constitute Agency policy.
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bln instances where authors reported exposures in ppm, EPA converted these values to mg/m3. See Table P.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 C.
Observations of hot plate latency were made prior to (LI); immediately following (L2); or 24 hours after footshock
(L3). Values for L3 in Gralewicz 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 minutes, 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 five daily injections and evaluated for
120 minutes.
'Results of passive avoidance tests in Gralewicz and Wiaderna (2001) may reflect adjusted data where, due to large
individual differences, two rats (the highest and lowest responders to footshock) 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.
gAt 54 days post-exposure, TMB-exposed rats were slower to increase the percentage of avoidance responses
across blocks (one block = five 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.
hEEGs 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.
JData represent percent change relative to control in same trial day, but statistical significance determined by the
authors is based on comparison to trial day 1 responses within the same group.
ND = not determined
Note: For studies other than Korsak and Rvdzvriski (1996), percent change from control was calculated from
digitized data using Grab It! XP software.
Table 1-3. Evidence pertaining to neurological effects of TMBs in animals—
oral exposures
Study design3'15 and reference
Assay and results
1,2,4-TMB
Motor function and/or anxiety
0, 960,1,920, 3,850 mg/kg single gavage
Rat, Wag/Rij, male, N = 10
Tomas et al. (1999b), Table C-41c
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*%
No significant changes were reported at 10, 30, 40, 50, 60, or 70 min
Electrocortical activity
0, 240, 960, 3,850 mg/kg, single gavage
Rat, Wag/Rij, male, N = 6
Tomas et al. (1999a), Table C-40
EEG recordingsd (reduction of the duration and number of high
voltage spindle episodes) (response relative to vehicle control):
20 min
40 min
60 min
Duration
0, -72, -58,
-83%
0, -80*, -97*,
-45%
0,11, -67, -45%
Number
0, -26, -44,
-62*%
0, -53*, -88*,
-73*%
0, 7, -53*, -22%
This document is a draft for review purposes only and does not constitute Agency policy.
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Study design3'15 and reference
Assay and results
1,2,3-TMB
Motor function and/or anxiety
0, 960,1,920, 3,850 mg/kg single gavage
Rat, Wag/Rij, male, N = 10
Tomas et al. (1999b), Table C-41
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
No significant changes were noted at 10, 40, 50, 60, or 70 min
Electrocortical activity
0, 960, 3,850 mg/kg, single gavage
Rat, Wag/Rij, male, N = 6
Tomas et al. (1999a), Table C-40
EEG recordingsd (reduction of the duration and number of high
voltage spindle episodes) (response relative to vehicle control):
20 min
40 min
60 min
Duration
0, -86, -97*,
-76*%
0, -95, -98*,
-97*%
0, -81, -94*,
-99*%
Number
0, -71*, -86*,
-48%
0, -84*, -93*,
-86*%
0, -70*, -99*,
-96*%
1,3,5-TMB
Motor function and/or anxiety
0, 960,1,920, 3,850 mg/kg single gavage
Rat, Wag/Rij, male, N = 10
Tomas et al. (1999b), Table C-41
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
No significant changes were noted at 10, 30, or 70 min
Electrocortical activity
0, 240, 960, 3,850 mg/kg, single gavage
Rat, Wag/Rij, male, N = 6
Tomas et al. (1999a), Table C-40
EEG recordingsd (reduction of the duration and number of high
voltage spindle episodes) (response relative to vehicle control):
20 min
40 min
60 min
Duration
0, -76*, -79,
-86%
0, -85*, -97*,
-95*%
0, -66*, -94*,
-88*%
Number
0, -57, -67,
-77%
0, -52*, -93*,
-91*%
0, -49*, -91*,
-89*%
1 ^Significantly different from controls (p < 0.05).
2 locomotor activity in open field tests and electrocortical arousal were assessed prior to exposure and
3 immediately after exposure every 10 minutes for up to 70 minutes.
4 bln instances where authors reported exposures in ppm, EPA converted these values to mg/m3. See Table P.l for
5 conversion factor, and individual study summary tables for ppm values.
6 Tables referenced in Study Design and Reference column correspond to study summary tables in Appendix C.
7 dEEGs were recorded prior to exposure and at 20, 40, and 60 minutes after exposure via electrodes implanted in
8 the fronto-parietal cortex.
9 Note: Percent change from control was calculated from digitized data using Grab It! XP software.
This document is a draft for review purposes only and does not constitute Agency policy.
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Effect Summary
Pain Sensitivity
Duration
Sub-chronic
Pain Sensitivity (Footshock) Short-term
Neuromuscular function
Motor Activity/Anxiety
Sub-chronic
Short-term
Cognitive Function
Short-term
Electrocortical Activity Short-term
indpolnt
? Paw Lick Latency (a)
! Paw Lick Latency (a)
1 Paw Lick Latency (b)
J Paw lick Latency fe)
? Paw Lick Latency (e)
nlc Paw tick latency (e)
t Paw Li* Latency
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Mode-of-Action Analysis—Neurological Effects
The observation of neurotoxicity following acute-, short-term-, and subchronic-duration
exposure to TMB fLutz etal.. 2010: Lammers etal.. 2007: Wiaderna etal.. 2002: Gralewicz and
Wiaderna. 2001: Wiaderna etal.. 1998: Gralewicz etal.. 1997b: Gralewicz etal.. 1997a: Korsak and
Rvdzvriski. 1996: Korsak etal.. 19951 may indicate that TMB perturbs normal neurotransmission in
exposed animals, although the specific key events necessary for TMB-induced neurotoxicity are not
established. Although mechanistic and mode-of-action data are lacking for TMBs, structurally
similar compounds like toluene and xylene have been more thoroughly characterized and it is
reasonably anticipated that TMBs may interact with similar biological receptors. However, while
TMB isomers may operate through similar mechanisms as other alkylbenzenes, the specific
neurotoxicological effects mediated via those interactions may be highly disparate. Gralewicz and
Wiaderna (20011. citing Kvrklund f!9921. note that alkylbenzenes are known to target
catecholaminergic systems. Inhalation exposures to toluene and xylene have been shown to
significantly change concentration and turnover rate of both dopamine and norepinephrine in
various regions of the rat brain (Rea etal.. 1984: Anderssonetal.. 1983: Andersson etal.. 1981:
Andersson etal.. 19801. Although these changes have been hypothesized to be due to the action of
potential metabolites on catecholamine receptors f Andersson etal.. 1983: Anderssonetal.. 1981:
Anderssonetal.. 19801. the role of the parent compound has not been fully characterized. T oluene
exposure also results in persistent dopamine dysfunction in basal ganglia in chronically exposed
rats (Tormoehlen etal.. 20141 and styrene can cause decreased dopamine levels and corresponding
increases in dopamine receptor expression (Costa. 19961.
The observation of catecholaminergic changes following toluene exposure, and the
observation of similar neurological effects following exposure to toluene and TMB isomers,
increases the plausibility that the mechanisms of neurotoxicity are similar between the two
compounds. For example, subchronic inhalation exposures of rats to low concentrations of toluene
(as low as 80 ppm [300 mg/m3]) have been shown to decrease spatial learning and memory,
increase dopamine-mediated locomotor activity, increase the number of dopamine D2 receptors,
and increase dopamine D2 agonist receptor binding (Hillefors-Berglund etal.. 1995: von Euler etal..
1994: von Euler et al.. 19931. Dopamine-dependent locomotor hyperactivity has been shown to be
biphasic in nature fRiegel and French. 19991. and toluene has also been shown to exert biphasic
effects on locomotor activity fWood and Colotla. 1990: H in man. 19871. These effects were
observed to persist up to 4 weeks after the termination of the toluene exposure. Both of these
observations are consistent with the open field behavioral effects observed to persist weeks after
short term exposure to 1,2,4- or 1,3,5-TMB, and they are not easily attributed to brain
concentrations of the solvents.
Activation of the dopaminergic system may also result in an inability to inhibit locomotor
responses (lackson and Westlind-Danielsson. 19941. Direct application of dopamine to the nucleus
accumbens of rats has been observed to result in retardation of the acquisition of passive avoidance
This document is a draft for review purposes only and does not constitute Agency policy.
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learning at concentrations that also stimulated locomotor activity (Braes etal.. 19841. Increases in
catecholaminergic neurotransmission (through exposure to norepinephrine or dopamine agonists)
result in dose-dependent reductions in the duration of SWDs in rats fSnead. 1995: Waiter etal..
19881. These observations and findings are in concordance with those resulting from exposure to
TMBs, including increased locomotion in open field tests and altered SWDs after TMB isomer
exposure fWiaderna etal.. 2002: Gralewicz and Wiaderna. 2001: Tomas etal.. 1999a: Tomas etal..
1999c: Gralewicz etal.. 1997b: Gralewicz etal.. 1997al. Effects on the dopamine system, with its
well-established role in reinforcement, would also be consistent with the observations of sustained
perturbations in active and passive avoidance tasks following short-term TMB exposure.
Additionally, with regard to toluene and related aromatic hydrocarbons, it is known that there is
direct interaction with these compounds on various ion channels (ligand and voltage gated) that
are present in the CNS fBowen etal.. 2006: Balster. 19981.
Exposure to alkylbenzene compounds has also been shown to affect other neurotransmitter
pathways. Exposure to benzene, xylene, toluene, and ethylbenzene inhibited N-methyl-D-aspartate
(NMDA) receptor function inXenopus oocytes (Tormoehlen et al.. 20141. Toluene was additionally
observed to exert dose-dependent effects on rat hippocampal neuron NDMA receptor activity:
acute exposures decreased NMDA receptor activity, whereas chronic exposures increased activity,
indicating increased expression of NMDA receptors fTormoehlen etal.. 20141. Toluene has
additionally been shown to increase the function of gamma-aminobutyric acid (GABA) and glycine
receptors and decrease the function of nicotinic acetylcholine receptors (Tormoehlen etal.. 2014:
Manto. 2012: Win-Shwe and Fuiimaki. 20101. Win-Shwe and Fuiimaki (20101 conclude that the
evidence that toluene inhibits excitatory receptors (NMDA and nicotinic acetylcholine) and
enhances inhibitory receptors (GABA and glycine) indicates that toluene exerts a range of both
inhibitory and stimulatoiy effects on exposed animals. By extension, TMB isomers can reasonably
be assumed to exert the same neuropharmacological effects in exposed animals, possibly explaining
the nonlinearities observed in some endpoints (i.e., effects at lower doses but not higher ones).
Further, observations that toluene exposure alters NMDA receptor activities in rat hippocampal
neurons may explain the observed effects on the visual system in rodents as well as decreased
spatial memory and learning (Win-Shwe and Fuiimaki. 20101.
There is not enough information to ascertain the specific molecular sites of action, and how
the resultant changes correlate to the observed neurotoxicological effects. However, it is widely
believed that the interactions with the neuronal receptors in the brain (e.g., ion channels,
catecholaminergic systems) may influence these changes. There is suggestive evidence from
functional magnetic resonance imagining (fMRI) (Tang etal.. 20111 that exposure to complex
solvent mixtures results in deficits in the brain neuronal circuitry responsible for attention and
working memory tasks. In exposed subjects, performance in memory tasks was significantly
decreased relative to controls, and activity in brain regions responsible for attention and working
memory (anterior cingulate, prefrontal, and parietal cortices) was also significantly lowered.
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Aromatic hydrocarbons may also perturb neurotransmitter activity via direct action on the
phospholipids that comprise nerve cell membranes (Anderssonetal.. 19811. Perturbation of the
phospholipids on the cell membrane could indirectly affect the binding of neurotransmitters to the
catecholamine or other receptors and potentially lead to alterations in receptor activity or uptake-
release mechanisms. Uneven distribution of the solvent or its metabolites within differing regions
of the brain, or spatial variations in phospholipid composition of nerve cell membranes, may
explain the differential effects seen in regard to catecholamine levels and turnover (Andersson et
al.. 1981). Based on effect levels with other related solvents (e.g., toluene) (see Balster (1998)). it is
hypothesized that with TMBs, there may be an initial interaction with the neuronal receptors
(e.g., catecholaminergic systems, ion channels) followed by, at much higher exposures, interaction
with the lipid membrane when the available sites on the neuronal receptors are completely
occupied.
Additional mechanisms that may play a role in TMB neurotoxicity include production of
reactive oxygen species (ROS). Mvhre etal. (2000) observed increased respiratory burst in
neutrophils after 1,2,4-TMB exposure demonstrated by fluorescence spectroscopy, hydroxylation of
4-hydroxybenzoic acid, and electron paramagnetic resonance spectroscopy. The authors suggested
that the observation of solvent-induced ROS production may be relevant to brain injury, as
microglia cells and neutrophils have similar respiratory burst characteristics. Stronger evidence of
potential ROS-related mechanisms of neurotoxicity was observed in a related study by Mvhre and
Fonnum (2001) in which rat neural synaptosomes exposed to 1,2,4-TMB produced a dose-
dependent increase in reactive oxygen and nitrogen species demonstrated by the formation of the
fluorescence of 2'7'-dichlorofluorescein. This observation of ROS production in rat synaptosomes
may potentially explain the observed TMB-induced neurotoxicity in acute, short-term, and
subchronic inhalation studies. In experiments investigating the neurotoxic effect of toluene
exposures, toluene has been shown to increase the concentration of oxygen radicals, with the
hippocampus identified as a particularly vulnerable region fTormoehlen et al.. 20141. Increased
production of oxygen radicals in the hippocampus of exposed animals possibly explains the
observation of decreased performance in memory and learning tasks. Additional evidence for a
possible oxidative stress mode of action is the observation that styrene decreases the levels of
glutathione in brains of exposed rats (Costa. 1996).
Summary of Neurological Effects
Neurotoxicity is strongly and consistently associated with exposure to TMBs in multiple
studies, and these associations are coherent in human populations exposed to mixtures containing
TMBs and in laboratory animals exposed to individual TMB isomers. All three TMB isomers are
absorbed readily in humans (larnbergetal.. 1998.1997a: larnbergetal.. 19961. and occupational
studies involving exposure to TMBs and other VOCs demonstrate neuropsychological effects (Chen
etal.. 1999). deficits in short term memory and reduced motor speed/coordination (Lee etal..
20051. abnormal fatigue fNorseth etal.. 19911. and nervousness, anxiety, and/or vertigo fBattig et
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al. (1956). as reviewed by MOE (2006) and Battigetal. (1958)1. These effects, however, cannot be
attributed to any specific compound. None of the available human studies have addressed the
potential for latent neurological effects, and no studies examined the potential for neurological
effects in sensitive populations.
There is strong, consistent evidence of neurotoxicity in male Wistar rats exposed to any
TMB isomer via inhalation across multiple concentrations and multiple exposure durations;
however, the studies were all conducted at the same institute (Wiaderna etal.. 2002: Gralewicz and
Wiaderna. 2001: Wiaderna etal.. 1998: Gralewicz etal.. 1997b: Gralewicz etal.. 1997a: Korsak and
Rvdzvnski. 1996: Korsak et al.. 1995). These studies were well-conducted and used common
neurobehavioral assays relevant to neurotoxicity in humans. Across studies and TMB isomers, an
array of behavioral effects were consistently induced by exposure which, when taken together,
provide internally consistent evidence of neurotoxicity amongst the disparate adverse effects.
Many of these changes exhibited a large response magnitude (e.g., 150-200% change compared to
controls) and, although infrequently tested, increased in magnitude with increasing exposure
duration (i.e., tests of pain sensitivity and neuromuscular function). Some endpoints exhibited clear
exposure-response relationships, including measures of pain sensitivity and neuromuscular
function, when tested immediately after exposure. Most other endpoints did not show a clear
concentration-effect relationship, although the direction and magnitude of responses was relatively
consistent across studies. In most cases, effects at 1,230 mg/m3 were less robust than those
observed at lower TMB concentrations (i.e., responses were nonlinear). However, nonlinear
relationships are not uncommon for solvents and, as they were observed across multiple studies
using each of the three isomers, they are considered to be biologically-relevant observations rather
than experimental artifacts, and this does not necessarily detract from the evidence. By gavage,
similar effects were observed (e.g., altered EEG recordings; increased locomotor activity in open
field tests) f To mas et al.. 1999a: Tomas etal.. 1999b). although testing by this route was not as
extensive as by inhalation. Altogether, these data support a high level of confidence in the
conclusion that inhalation exposure to TMB isomers causes neurotoxicity.
Considering all of the available data, there is a strong indication for a lack of reversibility of
TMB-induced neurotoxicity. Although findings of reduced pain sensitivity in naive animals
appeared to be mostly reversible 2 weeks after subchronic exposure to 1,2,4-TMB or 1,2,3-TMB
f Korsak and Rvdzvnski. 19961 and no decrease in pain sensitivity was observed in rats exposed to
the C9 fraction when tested 24 hours following the termination of exposure (Douglas etal.. 1993). a
variety of other neurotoxic findings contradict this suggestive evidence of reversibility. Specifically,
statistically significant effects on measures of pain sensitivity (i.e., following footshock challenge),
neuromuscular function, open field activity, and cognitive function (learning and memory) were all
observable several weeks to months after short-term or subchronic exposure to the various TMB
isomers. For pain sensitivity in particular, the possibility of reversibility following subchronic
exposure was only tested at the highest concentration of TMB used in the Korsak and Rvdzvhski
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(1996) study (i.e., 1,230 mg/m3). In multiple other tests of neurological function (including pain
sensitivity following a footshock challenge), it was shown that exposure to any of the TMB isomers
can show a non-monotonic dose-response relationship when tested some period of time after
exposure, with 1,230 mg/m3 TMB usually resulting in no response or a substantially reduced
response as compared to lower TMB concentrations (e.g., 492 mg/m3). Thus, from the data
available, a determination regarding the reversibility of TMB-induced decreases in pain sensitivity
at other concentrations (i.e., 492 mg/m3) at 2 weeks post-exposure cannot be made with
confidence. Additionally, while decreased pain sensitivity is observed to possibly resolve following
termination of exposure, the determination of reversibility is based solely on statistical significance.
In both the Korsak and Rvdzvriski f 19961 and Douglas etal. Q9931 studies, latency to paw-lick was
still increased relative to control after termination of exposure in the high-dose animals: 12-13% 2
weeks after exposure to 1,230 mg/m31,2,4-TMB or 1,2,3-TMB, and 37% 24 hours after exposure to
1,500 ppm C9 fraction (approximately 4,059 mg/m3 TMB isomers). While these effects are not
statistically significant, they may still represent biologically significant impairments in pain
sensitivity at time points following the termination of exposure.
Other evidence from TMB isomer studies demonstrates that decreased pain sensitivity in
particular, and neurotoxicity in general, is not a transient effect of exposure, and that these effects
are long-lasting (potentially permanent) in exposed rats. In several short-term studies
investigating the neurotoxicity of all of the TMBs individually, a footshock challenge was
incorporated into the testing paradigm in order to test whether TMB exposure resulted in long-
lasting, latent effects. Footshock itself reduces pain sensitivity and when applied to all groups
(exposed animals and controls), it should elicit a similar response. By measuring pain sensitivity
24 hours following the footshock challenge, the short-term studies observed that treated animals
retained decreased pain sensitivity, whereas control animals' responses had returned to
background levels. This demonstrates that exposure to TMB results in long-lasting latent changes
in how the nervous system is able to respond to negative environmental stimuli. It is important to
consider that an environmental challenge was not included in the design of Korsak and Rydzvhski
(1996). If it had been, it can be reasonably assumed that the animals exhibiting reduced pain
sensitivity immediately after exposure in Korsak and Rydzvhski (1996) would also exhibit the
persistent, latent neurotoxicity observed in the short-term studies. Of particular note is that this
evidence indicates that effects on nervous system processes associated with pain sensitivity are not
rapidly reversible or associated with clearance of the chemical from the body. TMB isomers have
been observed to clear rapidly from blood (Section C.2, Appendix C), and decreased pain sensitivity
following footshock persisted 51 days after termination of short-term exposures (Wiaderna etal..
2002: Gralewicz and Wiaderna. 2001: Gralewicz etal.. 1997b! The observed effects on pain
sensitivity 51 days after exposure cannot be related to the presence of TMB isomers in the blood
given TMB isomer's half-lives of elimination from venous blood (4.6-10 hours! f Swiercz etal..
2016: Swiercz etal.. 2006: Swiercz etal.. 2003: Swiercz etal.. 20021. Even when considering the
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longest half-life of elimination (10 hours for 1,2,4-TMB), more than 99% of the chemical would be
eliminated from blood 70 hours after termination of exposure (i.e., approximately 3 days). As
pointed out in A Review of the Reference Dose and Reference Concentration Processes fU.S. EPA.
20021. "[i]t is also important to keep in mind that effects that may initially appear to be reversible
may re-appear later or be predictive of later adverse outcomes." (pg. 4-16). Additionally, the
Neurotoxicity Guidelines fU.S. EPA. 19981 state that "latent effects (those that become evident only
after an environmental challenge [e.g., in this case, footshock]) have a high level of concern." These
consistent latent effects on pain sensitivity after footshock are considered adverse. Thus, it is
concluded that the neurotoxic changes in rats exposed to TMB isomers are persistent
The hotplate test is a relatively simple assessment that may not be sensitive enough to
detect subtle changes (U.S. EPA. 19981. suggesting that the large changes observed immediately
after TMB exposure may represent gross effects. It is possible that, at longer durations after
exposure, an environmental challenge is necessary for the more subtle perturbations that persist to
become manifest at a detectable level. Decrements in pain sensitivity following footshock appear to
reflect a lengthening of the numbing effects of footshock following exposure to TMBs weeks earlier;
the immediate effects of footshock, which essentially temporarily strains the nociceptive
capabilities of the nervous system, were unchanged by prior TMB exposure. Although these
measures may be complicated by less likely, but possible, effects on cognition, the results suggest
that some aspect(s) of the altered pain sensitivity phenotype fail to resolve following termination of
exposure. No environmental challenge was applied in the subchronic study by Korsak and
Rvdzvnski (1996): such an experiment may have uncovered similar latent responses. Conversely,
the short-term TMB exposure studies testing pain sensitivity failed to analyze effects shortly after
exposure, as these evaluations only occurred at >50 days post-exposure.
Some differing results are observed in neurotoxicity tests involving exposure to the C9
fraction (Douglas etal.. 1993). It is notable, however, that not all of the C9 studies' neurological
endpoints failed to show an effect of exposure: exposure of male rats to 5,000 mg/m3 C9 fraction or
1,2,4-TMB resulted in similar decrements in motor activity immediately after exposure (Mckee et
al.. 20101. As the C9 fraction is comprised of multiple solvents, the effects on motor activity due to
1,2,4-TMB in the mixture are more pronounced than the effects due to exposure to the isomer
alone. However, the observed differences in other neurotoxicological tests may be due to
differences in the neurotoxicity of single isomers of TMB as compared to a mixture of multiple
aromatic compounds including TMB isomers. As the specific modes of action associating any of the
various neurotoxic endpoints with exposure to TMB isomers or to the other components of the
mixture are not known, it is plausible that there are mechanistic that modify the observed
phenotypes with mixtures exposure. Alternatively, it is also possible that differences in
experimental designs rather than real differences in toxicity are contributing to any observed
differences. The experimental details varied substantially between the C9 fraction and TMB isomer
studies. Additionally, the possibility remains that differences in study findings may also reflect
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differences in study design, as the C9 fraction studies were not conducted in a manner completely
similar to the TMB isomer-specific short-term and subchronic studies. Specifically regarding pain
sensitivity, when all studies (TMB isomer and C9 studies) are considered in the proposed time-
course of effects, the C9 study by Douglas etal. f 19931 is consistent with the observed reversibility
(to levels not significantly different from controls) of the robust increase in paw-lick after some
time has passed since termination of exposure, a finding also observed in Korsak and Rvdzvriski
(1996) and the numerous short-term studies (Wiaderna etal.. 2002: Gralewicz and Wiaderna.
2001: Wiaderna etal.. 1998: Gralewicz etal.. 1997b). However, whereas these same short-term
studies demonstrate that some latent neurotoxic effects of TMB exposure persist long after
exposures have ended, including the observation of decreased pain sensitivity following
environmental challenge, the C9 studies did not measure these potentially more subtle
manifestations of neurotoxicity. Overall, the available data from C9 fraction studies do not reduce
the confidence in the conclusion that exposure to TMB isomers results in neurotoxicity.
The spectrum of observed effects suggests that TMBs affect multiple, possibly overlapping,
CNS systems rather than a single brain region or neuronal nuclei (suggested by the solvent activity
of the compounds). Almost all tests (including pain sensitivity) involve a contributing component
of motor system function. It is notable that none of the identified short-term or subchronic studies
on individual TMB isomers employed protocols capable of distinguishing effects on motor activity
alone (e.g., the majority of studies used open field tests 5-10 minutes in duration); thus, it remains
to be determined whether TMB exposure specifically affects motor system function. Similarly,
while latent neurological effects following TMB exposure were consistently observed across studies
and isomers, these effects were difficult to characterize as deficits in a single neurological function.
For example, latent measures of pain sensitivity following TMB exposure, although consistent, were
only statistically significant when the rats were challenged with a footshock on the prior day. The
most likely explanation for this observation is that TMB exposure extends the duration of
footshock-induced decreases in pain sensitivity, since the immediate response to footshock was
similar across groups; yet, it cannot be ruled out that TMB exposure could alter cognitive function,
resulting in the observed responses.
Evidence from related compounds (i.e., alkylbenzene derivatives such as toluene, styrene,
and xylene) supports the neurotoxicological evidence available for TMB isomers. Multiple review
articles identified in the literature describe human populations exposed to toluene, styrene, or
hydrocarbon solvent mixtures that suffer neurotoxic effects (e.g., color discrimination,
neuropsychological symptoms, decreased reaction times, impaired postural equilibrium, etc.) that
are functionally similar to those observed in adults exposed to mixtures containing TMB isomers
(Ritchie etal.. 2001: Costa. 1996). Unfortunately, sensory-related endpoints have not been well-
studied in animals exposed to TMB isomers, preventing conclusions regarding similarities in these
types of responses across related compounds. Somewhat less relevant to chronic, low-level
exposures but still useful in qualitative hazard identification, evidence from the reviewed inhalant
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abuse literature (largely focused on toluene) also includes the observation of numerous
neurological effects (some of which are similar to those in the TMB database): cerebellar
dysfunction (e.g., ataxia), cranial nerve abnormalities, and impairments in memory, attention, and
learning fTormoehlen etal.. 2014: Manto. 2012: Arora etal.. 2008: Grandiean and Landrigan. 2006).
Evidence from the integrated reviews of the animal literature also identifies effects such as
ototoxicity, altered operant conditioning, and increased locomotor activity in adults resulting from
exposure to toluene or xylene (Ritchie etal.. 2001).
Evidence from related compounds also helps identify possible gaps in the current TMB
database, the largest of which is uncertainty surrounding the degree to which TMB isomers can be
expected to result in developmental neurotoxicity. Although no studies on the possible
developmental neurotoxicity of individual TMB isomers exist, the potential for developmental
neurotoxicity has been studied in related compound and/or mixture studies. In a study
investigating developmental neurotoxicity due to exposure to a mixture containing TMB isomers
(i.e., Aromatol, a mixture of ethyltoluenes and TMBs indicated by the study authors to be a mild CNS
depressant), no signs of developmental neurotoxicity (startle reaction time, righting response, open
field behavior, or altered avoidance tasks) were observed in rats exposed gestationally to
concentrations of up to 2,000 mg/m3 Aromatol fLehotzkv etal.. 19851. although methodological
details and data were not reported. However, another gestational exposure study of a less
informative mixture containing TMBs (i.e., white spirit, which has a lower TMB content and greater
proportion of non-TMB compounds) did report long-lasting learning and memory deficits, but not
neuromuscular or motor activity changes, at 800 ppm (Hass etal.. 1999: Hass etal.. 1997: Hasset
al.. 1995). In addition, suggestive evidence for developmental neurotoxicity does exist when
considering other related alkylbenzene compounds. In reviews of the human literature, a range of
cognitive, behavioral, and visual dysfunction effects have been observed in children whose mothers
were exposed occupationally or via inhalant abuse (Grandiean and Landrigan. 2014: Hannigan and
Bowen. 2010: Win-Shwe and Fuiimaki. 2010: Grandiean and Landrigan. 20061. Integrated reviews
of the animal literature (Hannigan and Bowen. 2010: Bowen and Hannigan. 2006: Ritchie et al..
2001) also found that some developmental neurotoxicity studies (Bowen and Hannigan. 2006: Hass
etal.. 1999: Hougaardetal.. 1999: Hass etal.. 1997: tones andBalster. 1997: Hass etal.. 1995)
report that gestational exposures to toluene or xylene result in delayed development of the air
righting reflex, impaired performance in behavioral tests, sex-specific decrements in spatial
learning, or lower absolute brain weights. Notably, the studies that do report effects on
developmental neurotoxicity tended to either utilize fairly high exposure concentrations
(500-1,800 ppm) or unconventional dosing paradigms meant to approximate the non-continuous,
episodic exposures experienced by inhalant abusers.
In summary, the TMB neurotoxicity database supports a determination that TMBs are
neurotoxic following inhalation or oral exposure, based on strong and consistent effects in
experimental animals that are coherent with observations in exposed humans; biological
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plausibility based primarily on similarities to findings from related chemicals; evidence of effects
that worsen with increasing duration of exposure; delayed-onset and/or latent neurological effects
in animals several weeks following exposure; and observed exposure-response relationships in
animals tested immediately after exposure. Further supporting this determination is evidence
drawn from the larger alkylbenzene database reporting strong evidence for neurotoxicity in both
human and animal studies.
1.2.2. Respiratory Effects
There is evidence in humans and animals that inhalation exposure to TMBs induces
respiratory toxicity. The human evidence comes from occupational and residential studies
involving complex VOC mixtures that include TMBs; thus, effects cannot be attributed to any TMB
isomer specifically. TMB isomers are associated with increased measures of respiratory irritation,
such as laryngeal and/or pharyngeal irritation (Norseth etal.. 19911 and asthmatic bronchitis
(Battigetal. (1956). as reviewed in MOE (2006) and Battigetal. (1958)) following occupational
exposures. Residential exposures have demonstrated significant associations between 1,2,4-TMB
and asthma (Billionnetetal.. 2011). Controlled human exposures (lones etal.. 2006: larnbergetal..
1997a: larnbergetal.. 1996) have failed to observe substantial irritative symptoms following acute
(<4 hours) inhalation exposures to TMB isomers of up to 25 ppm (123 mg/m3). For full details of
the epidemiologic and controlled human exposures studies (including human subjects research
ethics procedures), see individual study summary tables in Appendix C.
In animals, there is consistent evidence of respiratory toxicity following inhalation exposure
of rodents to the TMB isomers (Table 1-4; Figure 1-2). Markers of inflammation and irritation in
the lungs of rats have been observed following subchronic inhalation exposures of Wistar rats to
1,2,4-TMB or 1,2,3-TMB. Increases in immune and inflammatory cells in bronchoalveolar lavage
(BAL) fluid have been observed following subchronic exposures of male Wistar rats to 1,2,4-TMB at
concentrations >123 mg/m3 fKorsak etal.. 19971. Specifically, the number of cells in the BAL fluid
of exposed rats was increased for both total cells (>123 mg/m3) and macrophages (>492 mg/m3).
However, some attenuation of these effects was observed at high concentrations (i.e., at
1,230 mg/m3) compared to lower concentrations. For example, the number of macrophages was
increased 2.7-fold relative to control at 492 mg/m3, but only 2.2-fold at 1,230 mg/m3. This may
indicate either adaptation to the respiratory irritation effects of 1,2,4-TMB, saturation of metabolic
pathways, or immune suppression at higher doses. Subchronic exposure of male Wistar rats also
significantly increased the BAL fluid content of polymorphonuclear leukocytes and lymphocytes;
however, the specific concentrations eliciting these significant increases were not reported by
study authors. A small, but not significant, decrease in cell viability (all cells) was observed
following subchronic exposure to 1,2,4-TMB at >123 mg/m3 fKorsak etal.. 19971.
In addition to increases in immune and inflammatory cells in BAL fluid following exposure
to 1,2,4-TMB, histopathological alterations characterized by increases in lymphatic tissue in the
lower respiratory tract have also been observed following subchronic exposures of male and female
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Wistar rats to 1,2,4-TMB or 1,2,3-TMB (Korsak etal.. 2000a. b). Significant proliferation of
peribronchial lymphatic tissue was observed in male rats exposed to 492 mg/m31,2,4-TMB,
although trend analysis demonstrated that this increase was not concentration-dependent due to
lack of an effect at the 1,230 mg/m3. Statistically significant increases in interstitial lymphocytic
infiltrations were also observed in male rats exposed to 492 mg/m31,2,4-TMB and in male and
female rats exposed to 1,230 mg/m31,2,3-TMB or 1,2,4-TMB, respectively. The later increases
were concentration-dependent based on trend analysis. Slight increases in pulmonary macrophage
infiltration and alveolar wall thickening were also observed in male rats exposed to 450, 900, or
1,800 mg/m3 C9 fraction for 12 months fClark etal.. 19891.
In some 1,2,4-TMB- or 1,2,3-TMB-exposed rats exhibiting peribronchial lymphatic
proliferation, the bronchial epithelium lost its cuboidal shape and formed lymphoepithelium
f Korsak et al.. 2000a. b). However, this formation of lymphoepithelium was apparently non-
monotonic and not dependent on concentration. Alveolar macrophages were increased in both
sexes exposed to 1,230 mg/m31,2,4-TMB (significant only for males), with trend analysis
demonstrating concentration-dependence across the entire concentration range. Goblet cells were
statistically significantly increased in a concentration-dependent manner in female rats exposed to
>492 mg/m31,2,3-TMB. When the incidences of all pulmonary lesions were analyzed in aggregate,
lesions were significantly increased in males at 492 mg/m31,2,4-TMB, but not at any concentration
in females. However, trend-analysis demonstrated significant increases in aggregate pulmonary
lesions in both sexes across the entire concentration range. In rats exposed to 1,2,3-TMB, the
aggregate incidences of pulmonary lesions were not statistically significantly increased at any
single concentration in males or females. Male rats, however, did exhibit a concentration-
dependent increase in aggregate lesions according to trend analysis. Studies on the respiratory
effects of subchronic exposures to 1,3,5-TMB were not available.
Additional effects on clinical chemistry including increased total protein (37% increase at
exposures of both 123 and 492 mg/m3), decreased mucoprotein (13% decrease at 123 mg/m3
exposure), increased lactate dehydrogenase (170 and 79% increase at 123 and 492 mg/m3,
respectively), and increased acid phosphatase activity (47-75% increase at >123 mg/m3) were
observed in animals exposed to 1,2,4-TMB, suggesting pulmonary irritation or inflammation
(Korsak etal.. 1997). All of these effects also exhibited either some attenuation of effect at high
concentrations compared to lower concentrations. Therefore, some adaptation to the respiratory
irritation effects of 1,2,4-TMB may be occurring.
Decreased respiration, a symptom of sensory irritation, has been observed in male BALB/C
mice during acute inhalation exposures to individual TMB isomers or Farbasol (a solvent mixture
containing TMB isomers) for 6 minutes. These acute exposures were observed to result in dose-
dependent depression of respiratory rates, with the maximum decrease in respiration occurring in
the first 1 or 2 minutes of exposure f Korsak et al.. 1997: Korsak etal.. 19951. The concentration of
1,2,3-TMB, 1,2,4-TMB, 1,3,5-TMB, or Farbasol that was observed to result in a 50% depression in
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1 the respiratory rate (RD50) was similar between the three isomers and the mixture: 578, 541, 519,
2 or 638 ppm (2,844, 2,662, 2,553, or 3,139 mg/m3), respectively.
Table 1-4. 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 d (6 hrs/d,
5 d/wk)
Rat, Wistar, male, N = 6-7
Korsak et al. (1997), Table C-30b
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**%
0,123, 492,1,230 mg/m3, 90 d (6 hrs/d,
5 d/wk)
Rat, Wistar, male and female, N = 10
Korsak et al. (2000a), Table C-31
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 d (6 hrs/d,
5 d/wk)
Rat, Wistar, male, N = 10
Korsak et al. (1997), Table C-30
Increased acid phosphatase activity with evidence of attenuation at
high exposure.
Response relative to control: 0, 47*, 74*, 45*%
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 C-30 and C-28
Decreased respiratory rate as measured during first minute of
exposure.
Response relative to control: RD50 = 2,844
1,2,3-TMB
Pulmonary inflammation/irritation
0,123, 492,1,230 mg/m3, 90 d (6 hrs/d,
5 d/wk)
Rat, Wistar, male and female, N = 10
Korsak et al. (2000b), Table C-32
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), Table C-30
Decreased respiratory rate as measured during first minute of
exposure.
Response relative to control: RD50 = 2,662
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Study design3 and reference
Results
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 C-30
Decreased respiratory rate as measured during first minute of
exposure.
Response relative to control: RDso = 2,553
C9 Fraction
Pulmonary inflammation/irritation
0, 450, 900,1,800 C9 fraction
(approximately 0, 203, 405, 810 mg/m3
TMB isomers), 12 months (5 days/week, 6
hours/day)
Rats, Wistar, male, N = 50
Clark et al. (1989), Table C-20
Increased pulmonary macrophage infiltration
Response relative to control: 0, +260, +260, +260 (mean severity
grade)
Increased alveolar wall thickening
Response relative to control: 0, +23, +15, +23 (mean severity grade)
^Statistically different from controls at p < 0.05.
**Statistically different from controls at p < 0.01.
***Statistically different from controls at p < 0.001.
aln instances where authors reported exposures in ppm, EPA converted these values to mg/m3. See Table P.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 C.
Effect Summary Duration End point
Pulmonary Inflammation/Irritation Sub-chronic t Total BAL celis (at
f Macrophage count (a;
f Lung Lesions {SI
f Lung Lesions ic,J
Clinical Chemistry Effects Sub-chronic 1 Acid Phosphatase Activity (a>
_0_
LOAEL: • 1 2.4 TMB ¦ 1,2.3-TMB ~ 1 3.5 TM6
NOAEL: O 1,2,4-TMB ~ 1,2.3-TMB ® 1.3.5-TMB
250
500
I I I
750 1000 1250
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. (2000b); (d) Korsak et al. (1995). All effects are in male Wistar rats,
except for increased pulmonary lesions, which occur in both male and female Wistar rats. Effects
in italics are from studies that reported actual exposure concentrations.
Figure 1-2. 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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Mode-of-Action Analysis—Respiratory Effects
Data regarding the potential mode of action for the respiratory effects resulting from TMB
inhalation exposures are limited and the key events for TMB-induced respiratory toxicity are not
established. However, the available toxicity data suggest that TMB isomers act as potent acute
respiratory irritants and induce inflammatory responses following longer exposures
(i.e., subchronic) in animals. Korsak etal. (1995) and Korsak etal. (1997) have suggested that
decreased respiratory rate following TMB inhalation exposure is indicative of irritation, and
proposed that respiratory irritants such as TMB may activate a "sensory irritant receptor" on the
trigeminal nerve ending in the nasal mucosa leading to an inflammatory response. Korsak et al.
f 19971 and Korsak et al. Q9951 further suggested that activation of this irritant receptor follows
either adsorption of the agonist, or adsorption and chemical reaction with the receptor. The
authors referenced a proposed model for the receptor protein that includes two main binding sites
for benzene moieties and a thiol group. Further, they suggested that in the case of organic solvents
(i.e., toluene, xylene, and TMB), a correlation between the potency of the irritating effect and the
number of methyl groups is likely, given the observation that RD50 values for depressed respiratory
rates following exposure to TMB isomers are approximately 8-fold lower than toluene and 4-fold
lower than xylene.
Following subchronic inhalation exposure of rats to 1,2,4-TMB, inflammatory cell
(i.e., macrophages, polymorphonuclear leukocytes, and lymphocytes) numbers were increased
along with markers of their activation (i.e., total lactate dehydrogenase and acid phosphatase
activity in BAL) (Korsak etal.. 1997). further indicating the inflammatory nature of responses in the
respiratory tract of TMB-exposed animals. Inflammatory pulmonary lesions were also observed
following subchronic inhalation exposures in rats. However, many of these effects were not
observed to be concentration-dependent in repeat exposure studies (i.e., no progression of effect
over an order of magnitude of concentrations), suggesting that there may be adaptation to
respiratory irritation that occurs following extended inhalation exposure to TMB. The processes
responsible for the respiratory inflammatory responses observed in subchronically exposed
animals are unknown. However, a major inflammatory mediator, interleukin-8 (IL-8), was
increased following exposure of porcine and human macrophages to secondary organic aerosol
(SOA) particles derived from 1,3,5-TMB fGaschen et al.. 20101. The observation that IL-8 levels
increase following exposure to 1,3,5-TMB-derived SOA is noteworthy, as a major function of IL-8 is
to recruit immune cells to sites of inflammation. Therefore, the observation of inflammatory
lesions involving immune cells (i.e., macrophages and leukocytes) may be partially explained by
increases in inflammatory cytokines following TMB exposures. Additionally, ROS-generation has
been observed in cultured neutrophil granulocytes and rat neural synaptosomes exposed to TMB
(Mvhre and Fonnum. 2001: Mvhre etal.. 2000). and the related compounds benzene and toluene
have been shown to induce oxidative stress in cultured lung cells fMogel etal.. 20111. Although
pulmonary ROS-generation has not been observed following in vivo or in vitro TMB exposures,
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there is suggestive evidence that it could play a role in the irritative and inflammatory responses
seen in exposed animals.
In a study investigating jet fuel-induced cytotoxicity in human epidermal keratinocytes,
aromatic hydrocarbons were more potent inducers of cell death than aliphatic constituents, even
though the aromatic compounds only accounted for less than one-fourth of aliphatic constituents
fChou etal.. 20031. Of the single aromatic ring hydrocarbons, 1,2,4-TMB and xylene were the most
lethal to human epidermal keratinocytes. Increased cytotoxicity may explain the small, but
insignificant, decrease in BAL cell viability observed in Korsak etal. (1997).
Summary of Respiratory Effects
Respiratory toxicity is associated with inhalation exposure to TMBs based on coherent and
consistent evidence in humans and animals. All three TMB isomers are absorbed by humans
flarnbergetal.. 1998.1997a: larnbergetal.. 19961. and occupational and residential studies
involving exposure to TMBs and other VOCs suggest an association between TMB exposure and
asthmatic symptoms (Billionnet etal.. 2011: Battig etal.. 19561 and sensory irritation (Norsethet
al.. 19911. These effects, however, cannot be attributed to any specific compound, and a causal
association cannot be determined. Evidence from related compounds also demonstrates an
association between benzene and alkylbenzenes (toluene, xylene, and ethylbenzene) and asthma,
increased risk of bronchitis, and decreases in lung function fBolden etal.. 20151.
There is strong, consistent evidence of respiratory toxicity in male and female Wistar rats
exposed to any TMB isomer via inhalation across multiple concentrations and multiple durations,
although the studies were conducted at the same institute (Korsak etal.. 2000a. b; Korsak etal..
1997: Korsak etal.. 19951. Some endpoints (i.e., BAL macrophages and alkaline phosphatase [AP])
showed concentration-dependence at low- and mid-exposures, all effects were observed to exhibit
some attenuation of effect at high doses, potentially indicating either adaptation to the respiratory
irritation effects, saturation of metabolic and/or toxicity pathways, or immune suppression at
higher doses. As stated above, the observed respiratory effects following TMB exposure are most
likely irritative and/or inflammatory in nature. This conclusion is supported by the nature of the
observed lung lesions (increased macrophages, interstitial lymphocytic infiltrations, etc.), but also
by the observation of similar hematological effects (increases in white blood cells [WBCs],
increased monocytes, etc.). Alterations in red blood cell [RBC] counts may also be attributable to
the respiratory irritative and inflammatory effects of TMB isomers. An irritative/inflammatory
mode of action for respiratory effects observed at high doses in animal models may increase
concern that these effects are relevant to human populations. Although not attributable to
individual TMB isomers, the respiratory effects observed at lower concentrations in exposed
humans (respiratory irritation, asthma) may be analogous to the effects seen in rats (inflammatory
lung lesions, increases in inflammatory markers in BAL fluid, etc.).
In summary, the evidence supports a determination that TMBs are respiratory toxicants
following inhalation exposure, based on consistency and coherency of effects observed in humans
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and animals, biological plausibility, observed exposure-response relationships, and evidence drawn
from related compounds such as benzene, toluene, and xylene.
1.2.3. Reproductive and Developmental Effects
There are no studies in humans that investigated the reproductive or maternal toxicity of
the TMB isomers by any route of exposure. Maternal toxicity in the form of decreased corrected
body weight (i.e., maternal body weight minus the weight of the gravid uterus) was observed in
Sprague-Dawley rat dams following inhalation exposure during gestation to 1,2,4-TMB or
1,3,5-TMB (Saillenfaitetal.. 20051 (Table 1-5; Figure 1-3). Dams exposed to 2,952 mg/m3
1,2,4-TMB gained only 86% of the weight gained by control animals, whereas dams exposed to
2,952 mg/m31,3,5-TMB gained only 70% of the weight gained by controls. Decreased maternal
food consumption (across gestational days [GDs] 6-21) was also observed at >2,952 mg/m3
1,2,4-TMB (88-83%) and >1,476 mg/m31,3,5-TMB (92-75%). Maternal toxicity was also observed
following exposure of CD-I mice to the C9 fraction. Forty-four percent of dams died after exposure
to 1,500 ppm C9 (4,059 mg/m3 TMB isomers). Maternal body weights were significantly decreased
at all exposure concentrations, and body weight gains were significantly decreased 13% at
500 ppm and 38% at 1,500 ppm C9 (1,353 and 4,059 mg/m3 TMB isomers, respectively). Clinical
observations of dams revealed some evidence of gross neurobehavioral toxicity including abnormal
gait (18 animals), labored breather (9 animals), weakness (7 animals), circling (8 animals), and
ataxia (8 animals).
There are no studies in humans that investigated the developmental toxicity of either
1,2,4-TMB or 1,3,5-TMB by any route of exposure. Developmental toxicity (reported as decreased
fetal body weight) has been observed in male and female rats following gestational exposure to
1,2,4-TMB and 1,3,5-TMB on GDs 6 through 20 via inhalation for 6 hours/day fSaillenfait etal..
20051 (Table 1-6). Fetal body weights were decreased (statistically significantly) by 5-13% at
concentrations >2,952 mg/m3 of 1,2,4-TMB and 1,3,5-TMB. No adverse effects were noted on
embryo/fetal viability, and no increase in skeletal, visceral, or external morphology (i.e.,
teratogenesis) was observed up to the highest concentrations for either isomer. Studies on the
developmental or reproductive effects of 1,2,3-TMB by any route of exposure were not available.
Developmental toxicity was also observed in mice exposed gestationally to the C9 aromatic fraction
(>55% TMB isomers) on GDs 6-15 via inhalation for 6 hours/day (Mckee etal.. 19901. Exposure to
1,500 ppm C9 (approximately 4,059 mg/m3 TMBs) resulted in statistically significant decreases in
live fetuses per litter, increases in post-implantation loss, and increases in the incidences of cleft
palate and unossified sternebrae (#5 and/or 6). No other evidence of teratogenicity was observed.
Fetal body weights (sex not reported) were statistically significantly decreased 7 and 34%
following exposure to 500 ppm C9 (1,353 mg/m3 TMB isomers) and 1,500 ppm C9 (4,059 mg/m3
TMBs), respectively. Developmental toxicity was also observed in a study investigating the effects
of multiple alkylbenzenes (benzene, toluene, xylene, ethylbenzene, Aromatol) derivatives in several
species (rats, mice, rabbits) fUngvarv and Tatrai. 19851. In rats, ethylbenzene was observed to
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increase dead/resorbed fetuses (>600 mg/m3), decrease fetal weights (2,400 mg/m3), and increase
"skeletal retarded fetuses" (>600 mg/m3). Xylene and Aromatol elicited similar results, but xylene
was seemingly more potent at inducing "skeletal retarded fetuses" (>250 mg/m3) compared to
either ethylbenzene or Aromatol (>600 mg/m3). In mice, benzene and xylene induced weight- and
skeletal-retarded fetuses at >500 mg/m3, toluene induced these effects at 1,000 mg/m3, and
Aromatol did not induce these effects at any dose. Ethylbenzene, xylene, and Aromatol all increased
the rate of malformations in rats and mice. In rabbits, benzene, toluene, and xylene reduced fetal
weights at 500 mg/m3, and all solvents resulted in spontaneous abortion at 1,000 mg/m3.
No studies were available that investigated the reproductive toxicity of any TMB isomer in
humans or animals. Mckee etal. Q9901 investigated the reproductive toxicity of the C9 fraction in a
multi-generational study of CD rats exposed to 100, 500, or 1,500 ppm C9 fraction (271,1,353, or
4,059 mg/m3 TMB isomers). No pathological lesions in the reproductive organs were noted in Fo
generation animals (or in any Fi, F2, or F3 animals). In the Fo generation, there were no observed
alterations in female or male fertility, number of females delivering a litter, or litter size at birth.
However, there was a small, but not statistically significant, increase in time necessary for
successful mating. No differences in Fi postnatal survival were observed. Male fertility (number of
fertile males/number of mated males) was significantly decreased at 1,500 ppm (4,059 mg/m3
TMBs) in the Fi generation. There were statistically significant reductions in the number of live F2
offspring delivered per litter and the percentage of live F2 births. F2 generation birth weights were
also decreased, but not significantly. The authors reported that among mated Fi females, mating of
24 females (6 in the control group, 8 at 100 ppm [271 mg/m3TMB isomers], 1 at 500 ppm [1,353
mg/m3TMB isomers], and 9 at 1,500 ppm [4,059 mg/m3TMB isomers]) was not confirmed, and
exposure was carried out until delivery, rather than being terminated on GD 20. When the dams
were analyzed as separate groups, the F2 litter size was only statistically significantly decreased in
litters delivered from the dams that were exposed until delivery. In dams with exposure
termination on GD 20, F2 litter size was slightly, but not significantly, decreased. The percentage of
F3 live births was decreased in both groups of dams; among the dams that were exposed until
delivery, pup survival was still decreased at postnatal day (PND) 4. There were no observed effects
on the mean number of live F3 births or postnatal survival. Birth weights of the F3 generation were
statistically significantly decreased (5%) in the 1,500 ppm group (4,059 mg/m3 TMB isomers).
Throughout the multi-generational study, increased mortality was observed in the
1,500 ppm C9 (4,059 mg/m3 TMB isomers) exposure group: 7 Fo females, 6 Fi females, and 36 and
34 F2 males and females, respectively. Also, body weights were consistently decreased in exposed
animals throughout the study, with decreases in postnatal weights occurring at lower doses in later
generations compared to earlier generations. In the Fo generation, male and female body weight
gains were decreased 5-7% in the 500 ppm C9 exposure group (1,353 mg/m3 TMB isomers), and
decreased 5-7% (females) or 14-16% (males) in the 1,500 ppm group (4,059 mg/m3 TMB
isomers). Although birth weights were not decreased in the Fi generations, mean body weights
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were decreased 12 and 21% in the 1,500 ppm (4,059 mg/m3) group on PNDs 7 and 14
(respectively), and remained decreased in both males (24%) and females (23%) at PND 21. These
decrements in body weight continued throughout the Fi exposure period (which began on
approximately PND 31 and continued for 10 weeks) with male and female body weights decreased
21-25 and 9-14% in the 1,500 ppm group (4,059 mg/m3 TMB isomers). Additionally, male body
weights were decreased 5-7% in males in the 500 ppm group (1,353 mg/m3 TMB isomers). A
similar pattern was observed in the F2 generation, with decreases in body weights in the 1,500 ppm
group (4,059 mg/m3 TMB isomers) observed starting on PND 7 and continuing throughout the
adult exposure and mating phase. Decreases in the adult F2 generation body weights were more
substantial compared to the Fi generation: 31-38% in males and 21-30% in females. Body
weights were also decreased 6-10% in both sexes in the 100 ppm group (271 mg/m3 TMB
isomers), and 16% in both sexes in the 500 ppm group (1,353 mg/m3 TMB isomers). In the F3
generation, no decreases in body were observed on PND 4, but body weights were decreased 14%
in the 1,500 ppm group (4,059 mg/m3) on PND 7, and 11 and 21% in the 500 and 1,500 ppm
groups (1,353 and 4,059 mg/m3 TMB isomers, respectively) on PND 14. These decrements in body
weight were also observed in males (500 ppm: 10%; 1,500 ppm: 24%) and females (500 ppm:
10%; 1,500 ppm: 23%) on PND 21. Lastly, Fi males and females in the 1,500 ppm group exhibited
some gross signs of neurotoxicity in the form of ataxia (18 males, 23 females) and/or decreased
motor activity (11 males, 8 females).
Table 1-5. Evidence pertaining to reproductive and developmental effects of
TMBs in animals—inhalation exposures
Study design3 and reference
Results
1,2,4-TMB
Developmental toxicity
0, 492, 1,476, 2,952, 4,428 mg/m3, GDs
6-20 (6 hrs/d)
Rat, Sprague-Dawley, female and male, N
= 275-342
Saillenfait et al. (2005), Table C-38b
Decreased fetal body weight of male and female fetuses.
Response relative to control:
Male: 0, -1, -2, -5*, -11**%
Female: 0, -1, -3, -5*, -12**%
Maternal toxicity
0, 492, 1,476, 2,952, 4,428 mg/m3, GDs
6-20 (6 hrs/d)
Rat, Sprague-Dawley, female,
N = 24-25 dams
Saillenfait et al. (2005), Table C-38
Decreased maternal weight gain, GDs 6-15.
Response relative to control: 0, -5, -4,-11**, -27**%
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Study design3 and reference
Results
1,3,5-TMB
Developmental toxicity
0, 492, 1,476, 2,952, 5,904 mg/m3, GDs
6-20 (6 hrs/d)
Rat, Sprague-Dawley, female and male,
N = 217-314
Saillenfait et al. (2005), Table C-38
Decreased fetal body weight of male and female.
Response relative to control:
Male: 0, -1, -5, -7*, -12**%
Female: 0, -1, -4, -6, -13**%
Maternal Toxicity
0, 492, 1,476, 2,952, 5,904 mg/m3, GDs
6-20 (6 hrs/d)
Rat, Sprague-Dawley, female,
N = 24-25 dams
Saillenfait et al. (2005), Table C-38
Decreased maternal weight gain, GDs 6-15.
Response relative to control: 0, +2, -13*, -30**, -46**%
C9 fraction
Developmental toxicity
0,100, 500,1,500 ppm C9 fraction
(approximately 0, 270,1,353, 4,059
mg/m3 TMB isomers), GDs 6-15 (6 hrs/d)
Mice, CD-I, female and male,
N = 22-27 litters
Mckee et al. (1990), Table C-36
Live fetuses/litter
Response relative to control: 0, -19*, -13, -26*
Post-implantation loss/dam
Response relative to control: 0, +255, +222, +478**
Fetal body weight (g)
Response relative to control: 0, -1, -7*, -34**
Maternal toxicity
0,100, 500,1,500 ppm C9 fraction
(approximately 0, 270,1,353, 4,059
mg/m3 TMB isomers), GDs 6-15 (6 hrs/d)
Mice, CD-I, female and male,
N = 28-30 litters
Mckee et al. (1990), Table C-36
Decreased maternal body weight gain (GDs 6-15)
Response relative to control: 0, -13, -13*, -38*
Reproductive toxicity
0,100, 500,1,500 ppm C9 fraction
(approximately 0, 270,1,353, 4,059
mg/m3 TMB isomers), GDs 6-15 (6 hrs/d)
Rats, CD, female and male,
N = 28-30 litters
Mckee et al. (1990), Table C-36
Male fertility index (Fi generation)
Response relative to control: 0, -3, +4, -28*
Litter size at birth
Response relative to control: 0, -10, -6, -30**
Gestational Survival Index
Response relative to controls: 0, -2, -6, -13*
This document is a draft for review purposes only and does not constitute Agency policy.
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Study design3 and reference
Results
Intergenerational toxicity
0,100, 500,1,500 ppm C9 fraction
(approximately 0, 270,1,353, 4,059
mg/m3 TMB isomers), GDs 6-15 (6 hrs/d)
Rats, CD, female and male,
N = 28-30 litters
Mckee et al. (1990), Table C-36
Decreased birth/pup/adult body weights, response relative to controls
Birth weight
Fi! 0, +2, +6, 0
F2: 0, +2, 0, -5
Fs:0, 0, +2,-5**
Day 4 body weights
Fi! 0, +1, +4, —5
F2: 0, +5, +4, -2
F3: 0, +3, +1, -5
Day 7 body weights
Fi: 0, -3, +2,-12**
F2:0, 0,+1,-12**
Fs:0,+1,-4,-14**
Day 14 body weights
Fi: 0, -7,-4,-21**
F2:0, -3, -2, -21**
F3:0, -2,-11**,-21**
Day 21 male body weights
Fi: 0, -6,+1,-24**
F2: 0, -4, -3, -26
Fs:0, 0,-10*,-24**
Day 21 female body weights
Fi: 0, -6, 0, -23**
F2:0, -4, -3, -27**
Fs:0, 0,-10**,-23**
1 ^Statistically significantly different from controls at p < 0.05.
2 **Statistically significantly different from controls at p < 0.01.
3 aln instances where authors reported exposures in ppm, EPA converted these values to mg/m3. See Table P.l for
4 conversion factor, and individual study summary tables for ppm values.
5 bTables referenced in Study Design and Reference column correspond to study summary tables in Appendix C.
6
Doses: -O-
LOAEl: • 1,2,4-TMi • 1,2,3-TMB ~ 1,3,5-TMB
NOAH,; O 1,2.4-TMB 8 1.2,3-TMB © 1.3,5-TMB
o———q, ^ o
o 0 '~ ——————o
o 0 • o
0 o 0 ~
a o © •
0——•——o— «
0—0———0
<>J^==.J0g==== 0
! 1 1—¦—I 1 1
1000 2000 3000 4000 5000 6000
Concentration, mg/m3
8 Note: Solid lines represent range of exposure concentrations. All effects from Saillenfait et al. (2005).
9 Effects in italics are from studies that reported actual exposure concentrations.
Figure 1-3. Exposure response array of developmental effects following inhalation
exposure to 1,2,4-TMB or 1,3,5-TMB.
10
Effect Summitry
Developmental Toxicity
Maternal Toxicity
Duration
Gestational
Gestational
Endpoint
J Fetal imight (U)
j Fmi weight (Uj
I F§tal weight (Fj
J Fetal might {F}
1 Maternal might change
i Maternal weigh! change
i Maternal weight gain (cormcted)
i Maternal weight gain (cmeclmi)
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Mode-of-Action Analysis—Reproductive and Developmental Effects
Data regarding the potential mode of action for the reproductive and developmental effects
resulting from TMB inhalation exposures are not available, and the key events for TMB-induced
reproductive and developmental toxicity are not established. However, evidence drawn from the
literature regarding related alkylbenzene compounds provides suggestive evidence that
alkylbenzene exposures modulate endocrine function and signaling (Bolden et al.. 20151.
Alkylbenzenes (especially benzene and toluene) are associated with endpoints such as altered
menstrual cycles and sperm production, and have been shown to alter the concentrations of
luteinizing hormone, follicle stimulating hormone, and testosterone in occupationally exposed
populations fBolden etal.. 20151. Further, alkylbenzene-induced alterations in the concentrations
of other hormones (insulin-like growth factor, thyroid hormone) and perturbation of endocrine
signaling (glucocorticoid, estrogen, and progesterone) may explain observed alterations in fetal
growth and development of inflammatory responses (respectively) similar to those observed in
TMB studies (Boldenetal.. 2015: Billionnetetal.. 2011: Saillenfaitetal.. 2005: Korsak etal.. 2000a.
b).
Summary of Reproductive and Developmental Effects
The database for reproductive and developmental toxicity following inhalation exposure to
1,2,4-TMB and 1,3,5-TMB is limited to one animal developmental study; no studies in humans are
available. Thus, these isomers may cause developmental toxicity, although this is based on only one
study that demonstrated clear, exposure-related effects on fetal and maternal body weights. There
is possibly confirmatory evidence of TMB-induced developmental and/or reproductive effects
given the results of studies of the C9 fraction that observed increased fetal loss and decreased fetal
weights in mice, and decreased measures of fertility in rats. Information from integrated reviews of
the alkylbenzene literature provides evidence that compounds are associated with a number of
reproductive and/or developmental endpoints such as decreased semen quality/sperm count
(benzene, toluene), altered menstrual cycling and fecundity (benzene, toluene), miscarriage and
stillbirth (benzene, toluene), malformations (benzene), and low birth weight (toluene,
ethylbenzene, xylene) (Bolden etal.. 2015: Webb etal.. 20141. Additionally, there was suggestive
evidence of intergenerational TMB-toxicity as decreases in fetal/pup/adultbody weights were
observed to occur at lower doses in later generations compared to earlier ones in the exposed rats.
However, comprehensive reviews of the styrene (a related alkylbenzene) database indicate that
styrene is likely not a reproductive or developmental toxicant (Luderer et al.. 2006: Brown etal..
20001.
In summary, the evidence supports a determination thatTMBs are developmental toxicants
following inhalation exposure, based on biological plausibility and observed exposure-response
relationships. Supportive evidence from related compounds and mixtures containing TMB isomers
support this determination. Although no direct evidence exists for TMB isomers, evidence from
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related compounds and mixtures containing TMB isomers indicate thatTMB isomers may be
reproductive toxicants as well.
1.2.4. Hematological and Clinical Chemistry Effects
There is limited evidence in humans, and stronger evidence in animals, that exposure to
TMB isomers via inhalation induces hematological toxicity and alterations in clinical chemistry
parameters. Alterations in blood clotting and anemia in workers exposed to a paint solvent
containing 50% 1,2,4-TMB, 30% 1,3,5-TMB, and unspecified amounts of 1,2,3-TMB (listed as
possibly present) was reported by Battigetal. (1956). as reviewed by MOE (2006): effects were
observed at 295 mg/m3. Studies identifying an association between occupational exposure to TMB
isomers and hematological and clinical chemistry effects are limited due to an inability to attribute
effects due to 1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB individually versus those due to the other
isomers or additional constituents within the solvent mixtures. For example, Gerarde (1960)
suggested that the hematological effects observed in Battigetal. fl9561 may have been due to trace
amounts of benzene present in the solvent mixture.
In animals, there is evidence of hematological toxicity following subchronic inhalation
exposure to 1,2,4-TMB or 1,2,3-TMB and short-term inhalation exposure to 1,3,5-TMB (Table 1-6;
Figure 1-4). Subchronic exposures to 1,2,4-TMB or 1,2,3-TMB have been shown to result in
hematological effects and changes in serum chemistry in rats fKorsak etal.. 2000a. b). In male rats
exposed to 1,230 mg/m31,2,4-TMB or 1,2,3-TMB, RBC counts were significantly decreased 23 and
15%, respectively. The observed alterations in RBCs were concentration-dependent as determined
by trend analysis. Exposure to 1,2,4-TMB or 1,2,3-TMB did not significantly decrease RBCs in
female rats, but trend analysis demonstrated that decreases in RBC counts in female rats exposed to
1,2,3-TMB were concentration dependent, with a maximum decrease of 9% at 1,230 mg/m3. RBCs
in both sexes were observed to still be depressed relative to controls 2 weeks following termination
of exposure to both isomers, but these decreases were not statistically significant
WBC counts were significantly increased 80% in male rats and increased 30% (not
statistically significant) in female rats exposed to 1,230 mg/m31,2,4-TMB. After a 2-week follow-
up after termination of exposure, WBC counts had returned to normal in female rats and were
slightly depressed (18%) in male rats. WBC numbers were unchanged in male rats exposed to
1.2.3-TMB, but were increased (not statistically significant) 22% in female rats exposed to
1,230 mg/m3. After 2 weeks following termination of exposure, WBC counts in male and female
rats had fallen to roughly 60% of controls.
Significant decreases in reticulocytes (71% decrease relative to controls) and clotting time
(37% decrease relative to controls) were observed in female rats exposed to 1,230 and 492 mg/m3
1.2.4-TMB, respectively. Both of these effects were concentration-dependent across the entire
range of concentrations as determined by trend-analysis; animals fully recovered within 2 weeks
after termination of exposure. Reticulocyte numbers were statistically significantly increased 60%
in male rats exposed to 1,230 mg/m31,2,3-TMB, with reticulocyte numbers even further increased
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(150%) 2 weeks following the termination of exposure. Reticulocyte numbers in females exposed
to 1,2,3-TMB were significantly increased 77 and 100% at 123 and 492 mg/m3, and increased 69%
(not statistically significant) at 1,230 mg/m3. Reticulocyte numbers were still increased in males
and females 2 weeks after the termination of exposure to 1,2,3-TMB. Segmented neutrophils were
statistically significantly decreased 29% in male rats exposed to 1,230 mg/m31,2,3-TMB;
statistically significant decreases of 29 and 48% were observed in female rats exposed to 492 and
1,230 mg/m31,2,3-TMB. Lymphocytes were statistically increased 11 and 15% in male and female
rats exposed to 1,230 mg/m3, respectively. Numbers of segmented neutrophils and lymphocytes
returned to control values 2 weeks after termination of exposure. In a study investigating the
hematological toxicity of the C9 aromatic fraction (~45% TMB isomers), few consistent trends
were reported for any of the hematological parameters investigated (Clark etal.. 1989). A decrease
(0.42 versus 0.40) in osmotic fragility (50%) was noted in rats exposed to 450, 900, and
1,800 mg/m3 C9 fraction (corresponding to 203, 405, and 810 mg/m3 TMB isomers, respectively)
for 12 months. Additional effects included increased WBCs and absolute lymphocytes in males
exposed to 1,800 mg/m3 C9. Another study investigating a complex solvent mixture (white spirit)
containing TMB isomers (albeit at a much lower proportion of the mixture, total C9 fraction 8-11%,
TMB content not reported) reported alterations in some hematological and clinical parameters
(decreased packed cell volume [PCV], decreased RBCs, increased mean cell volume [MCV], and
increased AP and aspartate aminotransferase [AST]) fCarrillo etal.. 20141.
Exposure to TMB isomers was also observed to have an effect on clinical chemistry markers
that possibly indicate hepatic injury. Sorbitol dehydrogenase (SDH) was increased at >123 mg/m3
in male rats exposed to 1,2,4-TMB (18-23% relative to controls) and at 1,230 mg/m3 in male rats
exposed to 1,2,3-TMB (69% relative to controls) (Korsak etal.. 2000a. b). However, the increases
following exposure to 1,2,4-TMB were not concentration-dependent SDH activity was also higher
in female rats exposed to 1,2,4-TMB (19-23% relative to controls), but the increases in activity
were not significantly higher when compared to controls. SDH activity was not affected in female
rats exposed to 1,2,3-TMB. Alanine aminotransferase (ALP) was decreased (23% relative to
controls) and AP was increased (42-45% relative to controls) at 1,230 and >492 mg/m3
(respectively) in female rats exposed to 1,2,3-TMB. Absolute liver weights were only observed to
increase (9%) in male rats exposed to 1,230 mg/m31,2,3-TMB, and no histopathological changes
were observed in either sex exposed to 1,2,3-TMB or 1,2,4-TMB. Therefore, the adversity of the
observed changes in clinical chemistry parameters is unclear. In rats exposed to the C9 fraction for
12 months, alterations in clinical chemistry parameters were generally mild, with females exposed
to 1,800 mg/m3 C9 fraction exhibiting increased sodium and decreased albumin (Clark etal.. 1989).
The only clinical chemistry effect in exposed males was increased creatinine at 1,800 mg/m3.
An increase (30% relative to controls) in AST, but no other substantial hematological
effects, was observed in rats 14 days following short-term exposure (6 hours/day, 6 days/week for
5 weeks) fWiglusz etal.. 1975b: Wiglusz etal.. 1975al. The adversity of AST is uncertain given the
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lack of a clear pattern in temporality (effects at some days post-exposure, but not others) and the
lack of accompanying liver histopathology.
Acute inhalation exposures of male Wistar rats to 1,500-6,000 mg/m31,3,5-TMB for
6 hours did not result in substantial effects on hemoglobin or RBC or WBC count fWiglusz etal..
1975b). However, the number of segmented neutrophilic granulocytes was increased in 1,3,5-TMB-
exposed rats up to 28 days following exposure (statistics not reported). The greatest increase in
granulocyte numbers (100%) was observed the day of exposure and 1 day following in rats
exposed to 6,000 mg/m3, although attenuation was seen 7-28 days following exposure, possibly
indicating induction of metabolizing enzymes or saturation of toxicity pathways. Investigation of
clinical chemistry parameters in rats acutely exposed to 300-3,000 mg/m3 for 6 hours did not
reveal any consistent pattern in the levels of AST or ALT, although AP was statistically increased
84% in rats 7 days following exposure to 3,000 mg/m3 fWiglusz etal.. 1975al.
Slight alterations in clinical chemistry parameters and differential WBC counts were also
observed in rats following subchronic, oral exposure to 1,3,5-TMB (Table 1-7; Figure 1-5) (Adenuga
etal.. 2014: Koch Industries. 1995b). While no hematological parameters (i.e., RBC counts,
hematocrit) were observed to differ between exposed rats and controls, the number of monocytes
were observed to increase (100-200% increase) in male rats exposed to >200 mg/kg-day
1,3,5-TMB. Additionally, a number of clinical chemistry parameters were altered in exposed rats.
In female rats exposed to 600 mg/kg-day, sodium and chloride levels were statistically significantly
decreased (2.3 and 2.7%, respectively) relative to controls, and cholesterol and phosphorus were
statistically significantly increased (41 and 23%, respectively). In male rats, exposure to
600 mg/kg-day resulted in a significant decrease (19%) in glucose levels, and significant increases
in phosphorus levels and AP activity (17 and 46%, respectively). In a related, preliminary study
fKoch Industries. 1995a). hematological and clinical chemistry effects were also observed following
14 days of oral exposure. Female Sprague-Dawley rats exposed to either 150 or 600 mg/kg-day
1,3,5-TMB had increased cholesterol levels, and high-dose males exhibited increased WBC counts
with corresponding increased neutrophil and lymphocyte numbers.
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Table 1-6. Evidence pertaining to hematological and clinical chemistry effects
of TMBs in animals—inhalation exposures
Study design3 and reference
Results
1,2,4-TMB
Hematological toxicity
0,123, 492,1,230 mg/m3, 90 d (6 hrs/d, 5 d/wk)
Rat, Wistar, female and male, N = 10
Korsak et al. (2000a), Table C-31b
Decreased RBCs in males only.
Response relative to control: 0, -1, -15, -23**%
(recovery = -24%)
Increased WBCs in males only.
Response relative to control: 0, 2, 4, 80**%
(recovery = -18%)
Decreased reticulocytes in females only.
Response relative to control: 0, -51, -49, -71*%
(recovery = 65%)
Decreases in clotting time in females only.
Response relative to control: 0, -23, -37**, -27*%
(recovery = 60%)
Clinical chemistry effects
0,123, 492,1,230 mg/m3, 90 d (6 hrs/d, 5 d/wk)
Rat, Wistar, female and male, N = 10
Korsak et al. (2000a), Table C-31
Non-monotonic increases in SDH in males only.
Response relative to control: 0, 73**, 74*,73**%
1,2,3-TMB
Hematological toxicity
0,123, 492,1,230 mg/m3, 90 d (6 hrs/d, 5 d/wk)
Rat, Wistar, female and male, N = 10
Korsak et al. (2000b), Table C-32
Decreased RBCs in males only.
Response relative to control: 0, 8, 6, -15*% (recovery = -9%)
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)
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)
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/m3, 90 d (6 hrs/d, 5 d/wk)
Rat, Wistar, female and male, N = 10
Korsak et al. (2000b), Table C-32
Decreased ALT in females only.
Response relative to control: 0, -1, -6, -23*%
Increased alkaline AP in females only.
Response relative to control: 0, 20, 45*, 42*%
Increased SDH in males only.
Response relative to control: 0, 44, 56, 69*%
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Study design3 and reference
Results
1,3,5-TMB
Hematological toxicity
1,500, 3,000, 6,000 mg/m3, 6 hrs
Samples collected 0,1, 7,14, and 28 d post-
exposure
Rat, Wistar, male, N = 5-8
Wielusz et al. (1975b), Table C-45
Increased segmented neutrophilic granulocytes (1-28 d 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 hrs/day, 6 d/wk)
Samples collected 1, 3, 7,14, and 28 d during
exposure
Rat, Wistar, male, N = 6
Wielusz et al. (1975a), Table C-46
Increased AST on d 14.
Response relative to control (d 14): 12*%
Increased AP on d 7 post-exposure.
Response relative to control (on d 7.0, -0.1, 0.03, 84*%
C9 fraction
Hematological toxicity
0, 450, 900,1,800 C9 fraction (approximately 0,
203, 405, 810 mg/m3 TMB isomers), 12 mo
(5 d/wk, 6 hrs/d)
Rats, Wistar, male, N = 50
Clark et al. (1989), Table C-20
Decreased osmotic fragility
Response relative to controls: 0, -5, -5, -5
Increased WBCs
Response relative to controls: 0, -3, +9, +27
Increased absolute lymphocytes
Response relative to controls: 0, +5, -5, +29*
Clinical chemistry effects
0, 450, 900,1,800 C9 fraction (approximately 0,
203, 405, 810 mg/m3 TMB isomers), 12 mo
(5 d/wk, 6 hrs/d)
Rats, Wistar, male and female, N = 50
Clark et al. (1989), Table C-20
Increased creatinine (males)
Response relative to controls: 0, 0, +7, +9*
Increased sodium (females)
Response relative to controls: 0, 0, +1, +1*
Decreased albumin (females)
Response relative to controls: 0, +1, -5, -9*
1 ^Statistically different from controls at p< 0.05.
2 **Statistically different from controls at p < 0.01.
3 aln instances where authors reported exposures in ppm, EPA converted these values to mg/m3. See TableP.l for
4 conversion factor, and individual study summary tables for ppm values.
5 bTables referenced in Study Design and Reference column correspond to study summary tables in Appendix C.
6
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Table 1-7. Evidence pertaining to hematological and clinical chemistry effects
of TMBs in animals—oral exposures
Study design and reference
Results
1,3,5-TMB
Hematological toxicity
0, 50, 200, 600 mg/kg-day, 90 d (once daily,
5 d/wk)
Rat, Sprague-Dawley, female and male, N = 10
Koch Industries (1995b), Adenuga et al. (2014),
Tables C-27a and C-17
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 d (once daily,
5 d/wk)
Rat, Sprague-Dawley, female and male, N = 10
Koch Industries (1995b), Adenuga et al. (2014),
Tables C-27a and C-17
Increased phosphorus levels in males and females
Response relative to control:
Male: 0, 3, 8,17*% (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 AP activity in males only
Response relative to control: 0, 5,13, 46*%
(recovery = 28% decrease)
1 ^Statistically different from controls at p< 0.05.
2 **Statistically different from controls at p < 0.01.
3 aTables referenced in Study Design and Reference column correspond to study summary tables in Appendix C.
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Effect Summary
Htmalologicai Toxicity
Clinical Chemisliy
Duration Endpoint
Sub-chronic | Pm Blood Cells M M
I Rett Blood Celts
! te (MJ (b)
! H«eeulmftm {F) (b)
1 Segeiuenferf Numtmptiiis iui (b)
i SegamenltKl Neutrophils (F) (b)
! Lymphocytes m M
? Lymphocytes (Fj #)
I Ctotttogtime(F)(al
Sui»-d*orsic| Alanine Amimtmmftimse IP} fit
T Alkaline Phosphaiase (F) f6|
I Sorbitol Dehydrogenase 'Mi (»}
Costs;
LOAEL.
NOAEL:
• 1,2,4-TMB • 1,2,3-TMB
O 1,2,4-TMB e 1,2,3-THi
0 280 500
Concentration, mgliuS
750 1000 1250
2
3
4
Note: Solid lines represent range of exposure concentrations, (a) Korsak et al. (2000a); (b) Korsak
et al. (2000b). Effects in italics are from studies that provided actual exposure concentrations as
measured by gas chromatography.
Figure 1-4. Exposure response array of hematological and clinical chemistry effects
following inhalation exposure to 1,2,4-TMB or 1,2,3-TMB.
Doses; -O
LOAEL; ~
NOAEL; ~
Effect Summary
Hematological Toxicity
Clinical Chemistry
Duration Endpoint
Sub-chronic f Monocytes (M)
Sub-chronic f Phosphorus {MS
f Phosphorus (F)
j, Chloride (F)
f Cholesterol (Fj
i Glucose (M).
f Alkaline Phosphatase (M)
100 200 300 400 500 600
Dose, mg/kg-day
6
7
8
Note: Solid lines represent range of exposure concentrations. All effects from Adenuga et al.
(2014). Effects in italics are from studies that provided actual exposure concentrations as
measured by gas chromatography.
Figure 1-5. Exposure response array of hematological and clinical chemistry effects
following oral exposure to 1,3,5-TMB.
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Mode-of-Action Analysis—Hematological and Clinical Chemistry Effects
The mode of action for TMB-induced hematological and clinical chemistry effects has not
been established. Increased SDH and AP activity are both markers for hepatic injury f Adenuga et
al.. 2014: Ramaiah. 20071 and therefore, underlying hepatotoxicity could explain their increase in
rats exposed to 1,2,4-TMB or 1,2,3-TMB via inhalation, or 1,3,5-TMB via oral ingestion. However,
absolute and relative liver weights were not observed to increase with inhalation exposure to
1,2,4-TMB (Korsak et al.. 2000a). and relative liver weights were only observed to increase 9% over
controls in male rats exposed to 1,230 mg/m31,2,3-TMB. Increases in relative liver weights were
also observed males and females exposed orally to 1,3,5-TMB. However, in studies that observed
liver weight increases, microscopic histopathological analyses of the liver did not demonstrate any
observable changes in either sex following exposure to TMB isomers. Therefore, the adversity of
the observed changes in clinical chemistry parameters is unclear. Similarly, although increased
cholesterol levels could also indicate hepatic dysfunction, the lack of gross or histopathological
lesions in animals orally exposed to 1,3,5-TMB calls into question the adversity of this particular
finding. The increases in WBC counts in exposed animals could be secondary to the observed
respiratory irritative and inflammatory effects of 1,2,4-TMB exposure in Korsak etal. f2000al and
Korsak etal. Q9971.
Summary of Hematological and Clinical Chemistry Effects
Hematological and clinical chemistry toxicity was consistently observed following
inhalation and oral exposure to TMBs based on coherent evidence in humans and animals. The
information regarding hematological toxicity in humans is limited to one study involving exposure
to a complex VOC mixture containing both 1,2,4-TMB and 1,3,5-TMB fBattigetal. Q9561. as
reviewed in MOE (2006) and Battig etal. (1958)1. Although this study reported hematological
effects coherent with those observed in animal studies (alterations in clotting and anemia),
exposure was to a mixture of TMB isomers and other VOCs. Therefore, it is impossible to attribute
the effects to any TMB isomer. There is strong and consistent evidence of hematological effects in
male and female Wistar rats following inhalation exposure (Korsak et al.. 2000a. b) that are roughly
analogous to those observed in humans. Additionally, there is some evidence of hematological and
clinical chemistry effects in male and female Sprague-Dawley rats following oral exposure
f Adenuga et al.. 2014: Koch Industries. 1995b). Given the observation of increased WBC counts,
decreased RBC counts, and alteration of clinical chemistry parameters following exposure to all
TMB isomers, it is possible that these effects are markers of underlying TMB-induced
hepatotoxicity. However, no study that investigated histopathological changes in the liver observed
any changes that might indicate toxicity or injury to that organ. Similarly, the observed alterations
in WBC counts might be a biomarker of effect for the consistently reported respiratory effects, most
of which are observed to be irritative or inflammatory in nature. An integrated review of the health
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effects literature for related compounds indicates that exposure to benzene is associated with
decreased hemoglobin, hematocrit, and RBC counts (Bolden et al.. 20151.
In summary, the evidence supports a determination that 1,2,4-TMB and 1,2,3-TMB result in
hematological toxicity following inhalation exposure, based on consistency and coherency of effects
across species (human and rats) and the observation of similar effects following exposure to the
related compound benzene.
1.2.5. General Toxicity
Decreased body weight as a marker of general systemic toxicity has not been observed
consistently in TMB studies. For instance, no study investigating the toxicity of individual TMB
isomers in adult animals has observed decreased body weight following inhalation or oral
administration to TMB fAdenuga etal.. 2014: Wiaderna etal.. 2002: Gralewicz and Wiaderna. 2001:
Korsak etal.. 2000a: Wiaderna etal.. 1998: Gralewicz etal.. 1997b: Korsak and Rydzvhski. 19961.
However, decreased body weight has been observed in pregnant dams as a marker of maternal
toxicity following gestational exposure to 1,2,4-TMB or 1,3,5-TMB via inhalation (Saillenfaitetal..
20051. Similarly, decreased fetal weight has been observed as a marker of developmental toxicity
in animals exposed to either 1,2,4-TMB, 1,3,5-TMB, or the C9 fraction f Saillenfaitetal.. 2005: Mckee
etal.. 19901. A possible cumulative intergenerational effect on postnatal body weight was observed
in a multi-gene rational reproductive toxicity study fMckee etal.. 19901.
Decreased body weight gains in Fo adults (exposure beginning prior to mating) were
observed to be decreased at 500 ppm and 1500 ppm C9 fraction (approximately 1,353 and 4,059
mg/m3 TMB isomers) (Mckee etal.. 19901. Although birth weights were not decreased in the Fi
generation, decreases in body weights were observed in the 1500 ppm C9 (4,059 mg/m3 TMB
isomers) group beginning at PND7 and continuing through adulthood; a similar pattern was
observed in F2 animals. In the F3 generation, birth weight and PND7 body weight weredecreased at
1500 ppm (4,059 mg/m3 TMB isomers). Beginning on PND14, body weight was decreased in both
the 500 ppm and 1500 ppm exposure groups (1,353 and 4,059 mg/m3 TMB isomers). Decreases in
adult body weights have been observed in other C9 fraction toxicity studies. Body weights were
slightly decreased relative to control during the first 4 weeks of a 12 month study in male rats
exposed to 1830 mg/m3C9 and females exposed to 970 mg/m3 and during the first 12 weeks of
exposure in females exposed to 1830 mg/m3 (Clark etal.. 19891. In a 13 week neurotoxicity study
fDouglas etal.. 19931. animals in the high exposure group (i.e., 1500 ppm C9 [4,059 mg/m3 TMB
isomers]) exhibited statistically decreased body weights at every time point during exposure;
animals in the 500 ppm group (1,353 mg/m3 TMB isomers) had decreased body weights early
during exposure, with a statistically significant decrease at week 4. However, by the end of the
exposure period, these animals weighted more than controls. Decreases in body weight were also
observed in male and female rats exposed to white spirit containing 8-11% C9 aromatics
(proportion of individual TMB isomers not specified) fCarrillo etal.. 20141.
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Other markers of possible systemic toxicity included alternations in organ weight in
multiple organs, most commonly the liver. Relative liver weights were increased in male and
female rats following oral exposure to 600 mg/kg-day 1,3,5-TMB fAdenuga etal.. 20141. male rats
exposed to 1,230 mg/m31,2,3-TMB fKorsak et al.. 2000b). and male and female rats exposed to
>4,000 mg/m3 white spirit fCarrillo etal.. 20141. Absolute liver weight was also increased in male
rats exposed to 1,800 mg/m3 C9 fraction f Clark etal.. 19891. Possible evidence of hepatic injury
was further demonstrated by the evidence of alterations in clinical chemistry parameters in some
studies. For example, increased AP, SDH, and cholesterol were all observed in treated animals
(Adenuga et al.. 2014: Carrillo etal.. 2014: Korsak et al.. 2000a. b). However, no indications of
hepatic toxicity was apparently in microscopic, histopathological examinations. Given this
observation, it is mostly likely that the changes in liver weight are not markers of adversity, but
rather markers of adaptive and compensatory changes. Alterations in spleen, kidney, and heart
weights were also sporadically noted (Carrillo et al.. 2014: Korsak etal.. 2000a. b; Clark etal..
19891. but as with the liver, gross and histopathological examinations revealed no consistent
treatment-related lesions.
1.2.6. Carcinogenicity
There are no studies in humans that investigated the carcinogenic potential of the TMB
isomers by any route of exposure. One animal study was identified that investigated the association
of chronic oral exposure (via gavage) to 1,2,4-TMB and cancer endpoints (Maltoni etal.. 19971.
Male and female Sprague-Dawley rats were exposed to a single dose of 800 mg/kg-day of
1,2,4-TMB in olive oil by stomach tube for 4 days/week starting at 7 weeks of age. Exposures were
terminated at the end of 104 weeks (i.e., at 111 weeks of age) and the animals were kept under
observation until natural death. The authors reported that chronic oral exposure to 1,2,4-TMB
resulted in an "intermediate" reduction of survival in male rats and a "slight" reduction in females
(no quantitative information on survival was reported). A slight increase in total malignant tumors
in both sexes of rats was observed, with the incidence of head cancers being specifically increased
in male rats. The predominant type of head cancer identified was neuroesthesioepithelioma, which
arises from the olfactory neuroepithelium and is normally rare in Sprague-Dawley rats. Other head
cancers observed included those in the Zymbal gland, ear duct, and nasal and oral cavities. No tests
of statistical significance were reported for these data. The carcinogenicity of the C9 fraction was
investigated in rats exposed to C9 for 12 months f Clark etal.. 19891. Although there were no
treatment-related increases in tumors at the end of the exposure period, some sporadic tumors
were noted: one leiomyoma of the left uterine horn (female, 1,800 mg/m3), one lymphoma of the
spleen (male, 1,800 mg/m3), and one glioblastoma of the cerebellum (male, 450 mg/m3). A number
of pituitary adenomas were observed in both sexes in all exposure groups, including the control
group.
Some evidence exists that 1,2,4-TMB can be used as a biomarker for cancer incidence.
1,2,4-TMB was detected as frequently, and in some cases more frequently, in the urine of cancer
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patients (breast, colorectal, lymphoma, and leukemia) compared to noncancer controls (Silva etal..
2012: Silva etal.. 20111. However, the total amount of 1,2,4-TMB recovered in the patients' urine
was consistently greater by 38-99% compared to controls. Neither 1,2,3-TMB nor 1,3,5-TMB, nor
any metabolites of any of the TMB isomers, were observed to be increased in cancer patients
relative to controls. While this evidence is suggestive of an association between 1,2,4-TMB and the
presence of various tumor type, the study's cross-sectional nature cannot support a firm conclusion
that 1,2,4-TMB was a potential cause of the particular cancers under investigation.
lanik-Spiechowicz etal. (1998) investigated the genotoxicity of TMB isomers by measuring
three genotoxic endpoints: mutation frequency in bacteria, micronucleus formation in mice, and
sister chromatid exchanges (SCEs) in mice. Neither 1,2,4-TMB nor 1,3,5-TMB induced gene
mutations in any Salmonella typhimurium strain tested (TA102, TA100, TA98, and TA97a).
However, 1,2,3-TMB induced gene mutations in all four strains in the absence of rat S9 fraction.
When cells were incubated in the presence of S9,1,2,3-TMB did not induce gene mutation, possibly
indicating that 1,2,3-TMB itself is the primary mutagen. No isomer induced the formation of
micronuclei in Imp:BALB/c mice following i.p. injection. Males in the high-dose groups for
1,2,4-TMB and 1,3,5-TMB, but not 1,2,3-TMB, exhibited a statistically significant reduction in the
ratio of polychromatic erythrocytes to normochromatic erythrocytes, indicating bone marrow
cytotoxicity. All three isomers significantly increased the frequency of SCEs in Imp:BALB/c mice
following i.p. injection, with 1,2,4-TMB eliciting the more significant response. These results appear
to have occurred at doses that did not induce significant bone marrow cytotoxicity.
Schreiner etal. (1989) assessed the mutagenic potential of the C9 fraction (total TMB
content = 55.05%) via multiple in vitro and in vivo assays. In a bacterial mutagenicity assay, five
S. typhimurium test strains (TA98, TA100, TA1535, TA1537, andTA1538) were exposed to either
negative controls (DMSO), positive controls, or to 0.0025-0.50 [iL/plate C9 fraction in the presence
or absence of the S9 microsomal mixture. There was no evidence that the C9 fraction induced gene
mutations with or without S9 activation in any S. typhimurium strain up to the highest test
concentration, at which signs of cellular toxicity became apparent. Multiple in vitro assays in
Chinese hamster ovary (CHO) cells were similarly negative in the absence and presence of the S9
microsomal mixture. Mutation frequencies were not increased in CHO cells exposed to the C9
fraction for 4 hours after a 7-day incubation period. Neither SCEs nor chromosomal aberrations
were increased in CHO cells at any concentration of the C9 fraction (up to concentrations of
90.2 (ig/mL). In order to investigate the potential in vivo mutagenicity of the C9 fraction, Sprague-
Dawley rats (30 per exposure group, 15 male and 15 female) were exposed via inhalation to 0,150,
500, or 1,500 ppm C9 fraction for 6 hours on 5 consecutive days. Following the termination of
exposure, 10 rats from each treatment group were sacrificed at 6, 24, and 48 hours, and their bone
marrow was examined for chromosome/chromatid aberrations. No induction of chromosomal/
chromatid aberrations was observed at any exposure concentration in animals sacrificed any time
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point In general, the results of Schreiner et al. (1989) indicate that, as tested, the C9 fraction did
not induce in vitro or in vivo mutagenicity in multiple assays.
In summary, very little genotoxicity data are available on individual isomers of TMBs. lanik-
Spiechowicz etal. (1998) observed varying results in the Ames mutation assay in Salmonella, with
1,2,3-TMB, but not 1,2,4-TMB or 1,3,5-TMB, inducing gene mutations. Results for the in vivo assays
for micronucleus and SCE formation were consistent across isomers: TMB isomers were observed
to induce SCEs, but not micronuclei, in mouse bone marrow cells. Increased frequency of SCEs
indicates that DNA damage occurred as a result of exposure to these isomers, but it does not
provide a specific indication of mutagenic potential, as there is no known mechanistic association
between SCE induction and a transmissible genotoxic effect. A similar lack of evidence of
genotoxicity is provided by Schreiner et al. (1989) in that the C9 fraction was observed to not
induce increased mutation frequencies, SCEs, or chromosomal aberrations. With only one isomer
(1,2,3-TMB) demonstrating a positive result for gene mutation and positive SCE results for all three
isomers, there is currently inadequate evidence to conclude that any isomer is directly genotoxic.
1.2.7. Similarities among TMB Isomers Regarding Observed Inhalation and Oral Toxicity
In the existing toxicological database for 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB, important
similarities have been observed in the potency and magnitude of effect resulting from exposure to
these three isomers in male and female Wistar rats, although some important differences also exist
(Table 1-8).
Measures of acute inhalation neurotoxicity, namely EC50 values for decreases in rotarod
performance (4,694 and 4,738 mg/m3) and pain sensitivity (5,683 5,963 mg/m3), were also similar
for 1,2,4-TMB and 1,3,5-TMB, respectively (Korsak and Rydzyriski. 19961. However, the EC.™ values
for both measures were lower following exposure to 1,2,3-TMB (3,779 and 4,172 mg/m3,
respectively). The observation that 1,2,3-TMB may be slightly more neurotoxic than 1,2,4-TMB or
1,3,5-TMB was also observed following acute oral and injection exposures. Although all three
isomers were observed to result in altered EEG readings, stronger and more persistent effects
followed a pattern of 1,2,3-TMB > 1,3,5-TMB > 1,2,4-TMB after oral exposures (Tomas etal.. 1999a)
and 1,2,3-TMB > 1,2,4-TMB > 1,3,5-TMB after i.p. injections (Tomas etal.. 1999c! Acute exposure
to both 1,2,4-TMB and 1,2,3-TMB affected motor function and/or anxiety at similar exposure levels,
whereas 1,3,5-TMB appeared to be slightly more potent, although the magnitude of the response
across isomers suggests that this difference is negligible fTomas etal.. 1999bl.
In short-term neurotoxicity studies, a qualitatively similar pattern of effects (inability to
learn passive and/or active avoidance and decreased pain sensitivity following footshock
challenge) indicating altered neurobehavioral function was observed for TMBs, although some
quantitative differences were noted (Wiaderna etal.. 2002: Gralewicz and Wiaderna. 2001:
Wiaderna etal.. 1998: Gralewicz etal.. 1997b). Exposure to any isomer resulted in statistically
significant decreases in pain sensitivity following footshock challenge at the same concentration,
although the magnitude of effect and consistency across studies was greater for 1,3,5-TMB and
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1,2,4-TMB compared to 1,2,3-TMB (Wiaderna etal.. 2002: Gralewicz and Wiaderna. 2001:
Wiaderna etal.. 1998: Gralewicz etal.. 1997b). 1,2,4-TMB and 1,3,5-TMB were also observed to
increase motor function and/or decrease anxiety in open field tests, whereas 1,2,3-TMB was
observed to have no statistically significant effects (Lutz etal.. 2010: Wiaderna etal.. 2002.1998:
Gralewicz etal.. 1997bl. In contrast, increased locomotor activity elicited by amphetamine was
amplified following exposure to 1,2,3-TMB, but not 1,2,4-TMB fLutz etal.. 20101. All three isomers
elicited effects on cognitive function, as measured by learning decrements in two-way active
avoidance or by decreased fear responses in a passive avoidance test paradigm (Wiaderna etal..
2002: Gralewicz and Wiaderna. 2001: Wiaderna etal.. 1998: Gralewicz etal.. 1997b). 1,3,5-TMB
was observed to be the most potent isomer in this regard, eliciting effects on both passive and
active avoidance at >123 mg/m3. 1,2,3-TMB and 1,2,4-TMB affected passive avoidance
performance at >123 and >492 mg/m3, respectively, and both 1,2,3-TMB and 1,2,4-TMB affected
the ability to learn active avoidance at 492 mg/m3. For all isomers, short-term exposure to
1,230 mg/m3 TMB was nearly always less effective (or ineffective), as compared to lower TMB
concentrations, at eliciting responses (i.e., responses were nonlinear).
Following subchronic exposure to either 1,2,4-TMB or 1,2,3-TMB, both isomers decreased
pain sensitivity and decreased rotarod performance were observed. With regard to decreased pain
sensitivity, although 1,2,3-TMB was observed to decrease pain sensitivity at a lower concentration
than 1,2,4-TMB, the magnitude of effect was similar between isomers at every concentration
(Korsak and Rvdzvnski. 1996). For either isomer, effects on pain sensitivity appeared to be
reversible at 1,230 mg/m3 TMB; lower concentrations were nottested. 1,2,3-TMB was more potent
than 1,2,4-TMB in reducing rotarod performance. Specifically, 1,2,3-TMB elicited effects at a lower
concentration and caused a greater magnitude of effect at each concentration, as well as following a
period of recovery f Korsak and Rvdzvnski. 19961.
In acute studies investigating respiratory irritative effects (i.e., decreased respiratory rate),
the RD so for the three isomers were very similar, ranging from 2,553 to 2,844 mg/m3 f Korsak et al..
1997). Similarities were also observed in 1,2,4-TMB- and 1,3,5-TMB-induced developmental and
maternal effects (Saillenfaitetal.. 2005). Male fetal weights were significantly reduced in animals
exposed gestationally to 2,952 mg/m31,2,4-TMB (5% decrease) or 1,3,5-TMB (7% decrease).
1,2,4-TMB also significantly decreased female fetal weights by approximately 5% in animals
exposed to the same concentration. Although, 1,3,5-TMB significantly reduced female fetal weights
by 13% in animals exposed to 5,904 mg/m3, female fetal weights were decreased at 2,952 mg/m3 to
a similar degree (6%) as animals exposed to the same concentration of 1,2,4-TMB. Maternal
toxicity, measured as decreased corrected maternal weight gain, was significantly decreased in
animals exposed to 2,952 mg/m31,2,4-TMB or 1,3,5-TMB. However, 1,3,5-TMB exposure resulted
in a 75% reduction of maternal weight gain compared to controls, whereas 1,2,4-TMB exposure
reduced maternal weight gain by 50%.
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Lastly, 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB were observed to elicit hematological toxicity
in exposed animals. Although all three isomers were observed to qualitatively affect similar
hematological parameters, the direction and magnitude of effect often differed between isomers.
RBCs were significantly decreased in male rats exposed to 1,230 mg/m31,2,3-TMB (23% decrease)
or 1,2,4-TMB (15% decrease) fKorsak etal.. 2000a. b). Reticulocyte numbers were also altered in
rats following exposure to these isomers, although 1,2,4-TMB was observed to significantly
decrease reticulocytes in male rats at 1,230 mg/m3 (71% decrease), while exposure to 1,2,3-TMB
increased reticulocytes in male rats at 1,230 mg/m3 (61% increase) and female rats at 123 and
492 mg/m3 (77 and 100% increases, respectively). 1,2,3-TMB and 1,2,4-TMB also altered the
numbers of WBCs in exposed animals following subchronic exposures. In male rats exposed to
1,230 mg/m31,2,4-TMB, WBC numbers were significantly increased by 80%. Exposure to
1,230 mg/m31,2,3-TMB also increased lymphocyte numbers by 11 and 15% in male and female
rats, respectively. Exposure to 1,230 mg/m31,2,3-TMB decreased segmented neutrophils by 29%
in male rats, whereas exposure to 492 mg/m3 and 1,230 mg/m3 decreased neutrophil numbers in
female rats by 29 and 48%, respectively. Acute exposure (6 hours) to 1,500-6,000 mg/m3
1,3,5-TMB was also reported to result in increased numbers of segmented neutrophils that
persisted for up to 28 days post-exposure fWiglusz etal.. 1975bl.
In addition to similarities in observed toxicities among the individual TMB isomers, there
are some similarities observed when considering the C9 aromatic fraction studies as well. For
example, Clark et al. (1989) observed slight alterations to some hematological and clinical
chemistry parameters that are similar to effects observed after exposure to 1,2,4-TMB and
1.2.3-TMB via inhalation. For example, in all studies, effects on the number of total WBCs, or
specific types of lymphocytes, are observed. These alterations are possibly related to the
respiratory irritative and/or inflammatory effects observed in the respiratory system following
exposure. The effects observed in Clark etal. (1989) occur at similar concentrations of TMB
isomers (approximately 800 mg/m3) as in the individual TMB studies (492-1,230 mg/m3).
Similarly, Mckee etal. (1990) also observed developmental toxicity following exposure to the C9
fraction in the form of decreased fetal weight, similar to effects observed following exposure to
1.2.4-TMB and 1,3,5-TMB (Saillenfait etal.. 2005). although Clark etal. (1989) additionally
observed increases in fetal death and increased incidences of some malformations (e.g., cleft
palate). However, some of the C9 fraction studies are also discordant with the larger TMB database.
For example, Schreiner etal. (1989) did not observe any increase in SCE following in vitro assays,
whereas lanik-Spiechowicz etal. T19981 observed that all three TMB isomers induced SCE when
mice were exposed via i.p. injection. The starkest difference in toxicity between TMB isomers and
the C9 fraction regards neurotoxicity. There is strong and consistent evidence that exposure to
individual isomers of TMB causes neurotoxicity following short-term and subchronic exposures
(Wiaderna etal.. 2002: Gralewicz and Wiaderna. 2001: Wiaderna et al.. 1998: Gralewicz etal..
1997b: Korsak and Rvdzvriski. 19961. whereas subchronic exposure to the C9 fraction does not
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appear to result in similar effects (Douglas etal.. 19931. There are a number of possibilities for the
observed differences in toxicity between TMB isomers and the C9 fraction. The specific test
compound used in the C9 fraction was a complex aromatic hydrocarbon mixture reported to
contain between 45 and 55% TMB isomers, with the remaining mixture primarily consisting of
ethyltoluene isomers. However, the precise test agent to which animals were exposed was likely
variable depending on the conditions under which they were generated across the different C9
fraction studies. Tertiary constituents (xylene, n-propyl- and isopropylbenzene, and unspecified
CIO aromatic hydrocarbons) comprised as much as 16% of the test compound. Although a
conclusion of sufficient toxicokinetic and toxicological similarity is used in the Toxicological Review
to support the adoption of consistent, cross-isomer reference values, such a conclusion has not
been tested for the other constituents of the C9 mixture. For some constituents (i.e., the CIO
compounds), such a comparison is not possible as they were not specifically identified in the
compositional analysis. Complex toxicokinetic interactions between different constituents of the C9
fraction are also possible that would result in altered distribution or metabolism of the individual
constituents. For example, some components of the C9 fraction may have induced metabolic
enzymes that cleared TMB isomers more rapidly, or may have competed for distributional
pathways into target organs (i.e., preferentially distributed to the brain over TMB isomers). Some
evidence does exist that co-exposures of rats to 1,3,5-TMB with the solvent ethyl acetate decreases
the absorption of 1,3,5-TMB into the blood compared to rats only exposed 1,3,5-TMB fFreundt et
al.. 1989). It is possible that similar effects on TMB isomer uptake and/or absorption are mediated
by the other constituents or impurities contained in the C9 fraction mixture.
Specifically considering decreased pain sensitivity, the difference in observed effects
between TMB isomers and the C9 fraction may be due to study design differences rather than a
fundamental difference in effects (see Section 1.2.1). Briefly, the failure of Douglas etal. Q9931 to
observe decreases in pain sensitivity may be due to when the endpoint was examined (24 hours
post-exposure). This would be consistent with the findings of Korsak and Rvdzvriski T1996I where
no effect was observed 2 weeks post-exposure, and short-term studies (Wiaderna etal.. 2002:
Gralewicz and Wiaderna. 2001: Wiaderna etal.. 1998: Gralewicz etal.. 1997b). where no
statistically significant effect was observed 50 days post-exposure. However, the observation of a
latent effect at 51 days post-exposure (1 day post-environmental challenge) demonstrates that a
persistent alteration of the nervous system of rats exposed to TMB isomers exists. Therefore, the
apparent difference between the Douglas etal. (1993) C9 study and the TMB studies is potentially
not a difference per se, but one piece of possibly confirmatory evidence when describing the entire
time-course of TMB-induced decreases in pain sensitivity.
A summary of these comparisons across individual TMB isomers is presented in Table 1-8.
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Table 1-8. Similarities between 1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB
regarding observed inhalation and oral toxicity
Health outcome measure
Exposure
duration
TMB isomer potency
Pain sensitivity
Acute
1,2,3-TMB > 1,2,4-TMB * 1,3,5-TMB
Subchronic
1,2,4-TMB * 1,2,3-TMB
Pain sensitivity following footshock
challenge
Short-term
1,2,4-TMB * 1,3,5-TMB > 1,2,3-TMB
Neuromuscular function
Acute
1,2,3-TMB > 1,2,4-TMB * 1,3,5-TMB
Subchronic
1,2,3-TMB > 1,2,4-TMB
Motor function/anxiety
Short-term
1,2,4-TMB * 1,3,5-TMB » 1,2,3-TMB
Sensitization
Short-term
1,2,3-TMB > 1,2,4-TMB
Cognitive function
Short-term
1,3,5-TMB > 1,2,4-TMB * 1,2,3-TMB
Electrocortical activity
Acute
1,2,3-TMB » 1,3,5-TMB > 1,2,4-TMB
Respiratory effects
Acute
1,2,4-TMB * 1,3,5-TMB * 1,2,3-TMB
Developmental effects
Gestational
1,2,4-TMB = 1,3,5-TMB
Hematological effects
Subchronic
1,2,4-TMB * 1,2,3-TMB
1.3. SUMMARY AND EVALUATION
1.3.1. Weight of Evidence for Effects Other than Cancer
TMB isomers have been observed to exhibit many similarities in toxicokinetics in humans
and animals across isomers. All three isomers have similar physiochemical properties and readily
absorb into the bloodstream following inhalation exposures. Net respiratory uptake was similar
across all three isomers in humans, and was similar in rats and humans for 1,2,4-TMB. The
distributional pattern was similar across isomers in exposed rats, with the liver, lung, and kidneys
identified as targets. 1,2,4-TMB was observed to distribute heavily to the brain, and while there
was no distributional information for 1,2,3-TMB or 1,3,5-TMB, similarities in brain:air partition
coefficients strongly suggest that distributional patterns would be similar across isomers. Brain:air
partition coefficients are also similar in humans, suggesting that no appreciable difference in
nervous system distributions patterns should be expected. All TMB isomers primarily metabolize
to benzoic and hippuric acid metabolites, although the proportion of total metabolites that these
individual metabolites comprised differed slightly across enzymes. Other metabolites included
phenols, mercapturic acids, and glucuronides or sulphuric acid conjugates. The half-lives of
elimination are multi-phasic for all isomers, with similar half-lives for the first three phases for all
three isomers. However, the half-life for the last phase of elimination was much longer for
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1.2.4-TMB compared to 1,3,5-TMB. Overall, these data support the conclusion that the TMB
isomers are very similar to one another regarding their toxicokinetic characteristics.
In both humans and animals, inhalation exposure to TMBs has been shown to result in
toxicity in multiple systems, including the nervous, respiratory, and hematological systems. In
addition, developmental toxicity has been observed in animals exposed to either 1,2,4-TMB or
1.3.5-TMB. Generally, the information regarding inhalation toxicity in humans is limited for a
number of reasons, including that the majority of human studies involved exposure to complex VOC
mixtures containing several TMB isomers and other VOCs, and not the individual isomers
themselves. Therefore, the observed health effects cannot be attributed to specific TMB isomers.
However, these studies observe effects in exposed human populations that are generally analogous
to effects observed in animal toxicity studies, and provide qualitative, supportive evidence for
hazard identification. Currently, no human studies exist that investigate the oral toxicity of any
TMB isomer.
The most strongly and widely supported manifestation of toxicity in humans and animals
following inhalation exposure to 1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB is neurotoxicity (see
Summary in Section 1.2.1). In humans exposed to TMB-containing VOC mixtures, a multitude of
effects, including neuropsychological effects fEl Hamid Hassan et al.. 2013: Chen etal.. 19991.
deficits in short-term memory and reduced motor speed/coordination (Lee etal.. 2005: Ruiiten et
al.. 19941. abnormal fatigue fNorseth et al.. 19911. dysfunction of the inner ear/vertigo fluarez-
Perez etal.. 2014: Fuente etal.. 2013: Sulkowski etal.. 2002). visual dysfunction (Gongetal.. 2003:
Pratt etal.. 2000). and nervousness, anxiety, and/or vertigo (Battig etal. (1956). as reviewed by
MOE (2006) and Battig etal. (1958)1 have been observed. None of the available human studies
have addressed the potential for latent neurological effects, and no studies examined the potential
for neurological effects in sensitive populations. Although the reported human symptoms do not
directly parallel the animal data, exposure of male Wistar rats to the TMB isomers has been shown
to consistently result in a multitude of neurotoxic effects, including decreased pain sensitivity,
impaired neuromuscular function and coordination, altered cognitive function, decreased anxiety
and/or increased motor function, and neurophysiological effects (e.g., decreased electrocortical
activity) across multiple concentrations and durations (Wiaderna etal.. 2002: Gralewicz and
Wiaderna. 2001: Wiaderna etal.. 1998: Gralewicz etal.. 1997b: Gralewicz etal.. 1997a: Korsak and
Rvdzvriski. 1996: Korsak et al.. 19951. Some differing evidence does exist regarding the potential
for neurotoxicity following exposure to C9 mixtures: exposure to the C9 fraction (resulting in
concentrations of total TMB isomers much higher than those used in individual TMB isomer
studies) failed to elicit a similar pattern of neurotoxic responses to those observed in isomer-
specific studies. However, this may be due more to the particular study designs used rather than
true differences in toxicity between the total C9 fraction and its individual constituents (see
Sections 1.2.1 and 1.2.7).
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The effects observed in animals in the TMB isomer neurotoxicity studies are recognized in
EPA's Guidelines for Neurotoxicity Risk Assessment (U.S. EPA. 19981 as possible indicators of
neurotoxicity. The effects include concentration-dependent decrements in pain sensitivity in hot
plate tests and neuromuscular function in rotarod tests following subchronic exposure. Although
effects on pain sensitivity appeared to be reversible at the highest concentration (i.e., 1,230
mg/m3), a multitude of other adverse endpoints indicates that neurotoxicity is not reversible,
including latent effects of short-term TMB exposure on potentially more sensitive indicators of pain
sensitivity (i.e., hot plate tests following an environmental [footshock] challenge), as well as
persistent neuromuscular dysfunction in rotarod tests following subchronic exposure, and
reproducible learning decrements in passive and active avoidance experiments, altered EEG
patterns, and increased locomotor activity in open field tests weeks to months after short-term
exposure. The data from short-term exposure studies indicated a consistent nonlinearity in many
of the TMB-elicited responses observed sometime after exposures had ended (e.g., 1,230 mg/m3
was nearly always substantially less effective than 123 or 492 mg/m3), which may be related to the
specific mode(s) of action for these latent effects, which remains unknown. The neurotoxic effects
are biologically plausible and analogous to effects that could occur in humans. Thus, the evidence
for TMBs identifies neurotoxicity as a toxicity hazard based on consistency and coherency of effect
across multiple studies and durations of exposure.
Three acute oral studies fTomas etal.. 1999a: Tomas etal.. 1999b: Tomas etal.. 1999cl exist
that reported similar effects as observed in the available inhalation neurotoxicity studies
(i.e., increased locomotor activity and altered brain wave activity). However, these studies are
limited with regard to their duration (i.e., acute) and nature of endpoints investigated, and as such,
no weight-of-evidence determination can be made regarding the oral toxicity of the TMB isomers.
In addition to neurotoxicity, both respiratory and hematological toxicity have been
observed in human populations and animals exposed to TMBs, or to mixtures containing the three
isomers. In humans, occupational and residential exposures to VOC mixtures containing TMB
isomers have resulted in number of effects characterized as respiratory toxicity, including
asthmatic bronchitis (Battigetal. (1956). as reviewed in MOE (2006) and Battig etal. (1958)).
asthma (Billionnetetal.. 2011). or laryngeal/pharyngeal irritation fNorseth etal.. 1991).
Additionally, workers exposed to a VOC mixture containing 1,2,4-TMB and 1,3,5-TMB, and possibly
1,2,3-TMB, were reported to exhibit hematological effects including alterations in clotting time and
anemia (Battigetal. (1956). as reviewed in MOE (2006) and Battigetal. (1958)). Again, as workers
were exposed to complex VOC mixtures containing TMB isomers, the observed health effects cannot
be attributed to any single TMB isomer.
The observation of respiratory irritation and inflammation in Wistar rats and BALB/C mice
following exposure to 1,2,4-TMB was consistent across multiple concentrations, and subchronic
and acute exposure durations fKorsak etal.. 2000a: Korsak etal.. 1997: Korsak et al.. 19951.
Respiratory toxicity was also observed in multiple studies involving exposure to 1,2,3-TMB fKorsak
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etal.. 2000b: Korsak et al.. 19951. Some inflammatory lesions similar to those observed in
individual isomer studies (pulmonary macrophage infiltration and alveolar wall thickening) were
also observed following exposure to the C9 fraction fClarketal.. 19891. Although the reported
symptoms in humans (laryngeal and/or pharyngeal irritation, asthmatic bronchitis, and asthma) do
not directly parallel the effects observed in animal studies, the observation of irritative and/or
inflammatory responses in multiple species (including humans) demonstrates a consistency in
TMB-induced respiratory toxicity. Additionally, multiple measures of hematological toxicity have
been observed in rats subchronically exposed to 1,2,4-TMB or 1,2,3-TMB, including decreased
RBCs, increased WBCs, decreased clotting time, and decreased reticulocytes (1,2,4-TMB) and
decreased RBCs, decreased segmented neutrophils, increased lymphocytes, and increased
reticulocytes (1,2,3-TMB) fKorsak etal.. 2000a. b). At least two of these effects, decreased RBCs
and decreased clotting time, are roughly analogous to the hematological effects (alterations in
clotting and anemia) observed in occupationally exposed humans, thereby demonstrating a
consistency and coherency of effect across species. Some hematological and clinical chemistry
effects (decreased osmotic fragility, decreased PCV, decreased RBCs, increased AP and AST) were
also observed following exposure to the C9 fraction (Carrillo etal.. 2014: Clark etal.. 19891.
Therefore, the respiratory and hematological effects observed in animals are biologically plausible
and analogous to effects that could occur in exposed human populations. The available weight of
evidence for 1,2,4-TMB and 1,2,3-TMB identified respiratory and hematological toxicity as a hazard.
Currently, no human studies exist that investigate the reproductive or developmental
toxicity of 1,2,3-TMB, 1,2,4-TMB, or 1,3,5-TMB. However, one animal study (Saillenfait etal.. 20051
observed effects on fetal body weights and maternal body weight gains due to exposure during
gestation to 1,2,4-TMB or 1,3,5-TMB. Exposure to the C9 fraction also induced developmental
toxicity: mice exposed to the C9 fraction experienced decreased fetal survival and increased
malformations (Mckee etal.. 19901. In rats exposed to the C9 fraction, decreased male fertility was
observed and some suggestions of intergenerational effects on growth (exacerbated body weight
decrements in later generations) were observed in a multi-generational reproductive toxicity study
(Mckee etal.. 19901: no individual isomer reproductive toxicity studies currently exist. Although
the weight of evidence regarding developmental toxicity is not as strong compared to other
measures of toxicity in the TMB database, these effects observed in animals are considered
biologically plausible and potentially analogous to effects that could occur in humans. The available
evidence for 1,2,4-TMB and 1,3,5-TMB identifies maternal and developmental toxicity as a hazard.
Although no compelling TMB isomer-specific data exist by which to draw firm conclusions
on possible modes of action, data from other lines of evidence (related compounds, mixtures) can
be used to tentatively identify possible modes of action. One possible overarching mode of action
for effects in multiple systems is oxidative stress. 1,2,4-TMB has been observed to result in
increased reproductive burst in neutrophils, which may possibly be relevant to brain injury, as
microglia cells have a respiratory burst similar to neutrophils fMvhre etal.. 20001. Additional
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evidence of an oxidative stress mode of action is the observation that rat neural synaptosomes
exposed to 1,2,4-TMB produced a dose-dependent increase in reactive oxygen and nitrogen species
demonstrated by the formation of the fluorescence of 2'7'-dichlorofluorescein fMvhre and Fonnum.
20011 and the observation that toluene exposure increases the concentration of oxygen radicals in
the brain fTormoehlen et al.. 20141. These observations of ROS production in rat synaptosomes
may potentially explain the observed TMB-induced neurotoxicity in acute, short-term, and
subchronic inhalation studies. Although pulmonary ROS-generation has not been observed
following in vivo or in vitro TMB exposures, related compound such as benzene and toluene have
been shown to induce oxidative stress in cultured lung cells (Mogel etal.. 20111. This suggests that
TMB-induced irritative and inflammatory responses may also be due to an oxidative stress mode of
action.
Although the TMB toxicity database is relatively complete in some areas (e.g., neurotoxicity
testing in adult animals), there do exist some important limitations and data gaps in the database.
In general, the available human data are not specific to TMBs, and provide only limited qualitative
support Most notably, there are no chronic studies available that investigate health effects in
human populations exposed to TMB isomers individually via inhalation. While there are a number
of occupational epidemiologic studies in the database, the workers in these studies were exposed to
complex solvent mixtures that contained not only TMB isomers, but also other aliphatic and
aromatic compounds. Therefore, as stated above, these studies can provide qualitative support for
hazard identification as they report effects that are generally analogous to effects observed in
animal toxicity studies. Currently, no human studies exist that investigate the oral toxicity of any
TMB isomer.
There are also a number of potential limitations in the animal inhalation and oral toxicity
database for TMBs, including the lack of a chronic study for any individual TMB isomer, the lack of a
subchronic neurotoxicity test for 1,3,5-TMB, and the fact that all of the available short-term or
subchronic inhalation animal studies were conducted by the same research group: The Nofer
Institute of Occupational Medicine, Lodz Poland. Although no chronic studies exist for individual
TMB isomers, a chronic study investigating the effects of exposure to the C9 fraction is available
that reports effects similar to those observed in the subchronic inhalation studies of individual TMB
isomers (i.e., effects on respiratory, hematological, and clinical chemistry effects), but failed to
assess the potential for the most prominent health effect caused by the individual isomers (i.e.,
neurotoxicity). Other data gaps in the TMB toxicity database include the lack of a developmental
neurotoxicity study or a multi-generational reproductive toxicity study for any individual TMB
isomer. There is a multi-generational reproductive/developmental toxicity study for the C9
fraction (Mckee etal.. 19901 that reported possible reproductive toxicity in rats (increased time to
mating, decreased male fertility), and a suggestive intergenerational effect on pup weight in rats.
While a study on the developmental neurotoxicity of Aromatol reported no observed effects
fLehotzkv etal.. 19851. a number of review articles on related compounds report that gestational
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exposure to toluene (humans) or toluene or xylene (animals) results in developmental
neurotoxicity (delayed righting reflexes, decrements in spatial learning, and altered behavioral
responses) fGrandiean and Landrigan. 2014: Hannigan and Bowen. 2010: Win-Shwe and Fuiimaki.
2010: Bowen and Hannigan. 2006: Grandiean and Landrigan. 2006: Ritchie etal.. 2001).
While evidence from related compounds can identify possible data gaps in the TMB
database, this additional evidence stream can also provide confirmatory support for health effects
that are observed in the TMB database. Multiple reviews of the alkylbenzene literature report that
occupationally exposed humans suffer neurotoxic effects (e.g., color discrimination,
neuropsychological symptoms, decreased reaction times, impaired postural equilibrium, etc.) that
are functionally similar to those observed in adults exposed to mixtures containing TMB isomers
(Gobbaand Cavalleri. 2003: Ritchie etal.. 2001: Costa. 1996). while alkylbenzene exposure is
observed to result in ototoxicity, altered operant conditioning, and increased locomotor activity in
adult animals (Ritchie etal.. 2001). Information from the C9 fraction or related compounds and/or
mixtures support the observation of developmental toxicity following TMB exposure. Exposure to
the C9 fraction resulted in substantial developmental toxicity in mice, including increased fetal
death, decreased fetal weights, and increases in particular malformations and developmental
variations (i.e., cleft palate and unossified sternebrae) fMckee etal.. 19901. Developmental toxicity
(fetal death, skeletal malformations, spontaneous abortions, and decreased fetal weights) was
observed in rats, mice, and rabbits exposed to benzene, toluene, xylene, ethylbenzene, or Aromatol
(Ungvarv and Tatrai. 1985). Limited evidence also exists that demonstrates associations between
alkylbenzenes and increased risk of asthma and decreased lung function and between benzene and
decreased hemoglobin, hematocrit, and RBC counts (Bolden etal.. 2015).
In summary, the overall evidence for TMB isomers strongly supports a determination that
exposure to TMBs results in adverse health effects in numerous systems, including the nervous,
respiratory, and hematological systems. This determination is based on consistency and coherency
of effects in humans and animals, biological plausibility, and observed exposure-response
relationships in animals. The evidence for reproductive and developmental toxicity is weaker than
the aforementioned effects, but evidence in animals indicates that exposure to TMBs does elicit
some measures of reproductive and developmental toxicity. Given the observation of similar types
of toxicity endpoints in animals exposed to related alkylbenzenes, there is increased confidence
that observed TMB isomer hazards have been adequately identified, even when considering that
the TMB database is lacking in some areas (e.g., no chronic studies, limited to no information on
mode of action). The health effects identified as hazards for TMBs will be considered further for
their utility in quantitatively deriving reference values for the individual TMB isomers; these
considerations and derivations are included in Section 2: Dose-Response Analysis.
1.3.2. Weight of Evidence for Carcinogenicity
Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005). there is "inadequate
information to assess carcinogenic potential" of TMBs. This characterization is based on the fact
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that there is no information regarding the carcinogenicity of TMBs in humans and that the only
animal study available on the carcinogenicity of 1,2,4-TMB observed no statistically significant
carcinogenic effects. No studies regarding the carcinogenicity of 1,2,3-TMB or 1,3,5-TMB were
identified in the available scientific literature.
In the animal carcinogenicity study fMaltoni et al.. 19971. involving exposure to 1,2,4-TMB
by gavage, an increased incidence of total malignant tumors in both sexes and head cancers
(including the rare neuroethesioepithelioma in one male and two females) was observed in
exposed rats, no statistical analyses were reported.
In the only study investigating the genotoxicity of TMB isomers, lanik-Spiechowicz etal.
f 19981 observed negative results in in vitro genotoxicity assays (i.e., Ames mutation assay in
Salmonella) involving 1,2,4-TMB and 1,3,5-TMB. However, 1,2,3-TMB was observed to induce gene
mutations in all S. typhimurium strains tested. All three isomers failed to induce micronuclei in
mouse bone marrow cells. lanik-Spiechowicz etal. (19981 observed an increased incidence of SCE
in mice exposed to all three TMB isomers (individually); however, this observation does not
provide a specific indication of mutagenic potential. Given the findings regarding the in vitro
genotoxicity of the TMB isomers, and the fact that increased frequency of SCEs does not provide
specific indication of mutagenic potential, the evidence is inadequate to conclude that any TMB
isomer is genotoxic.
1.3.3. Susceptible Populations and Lifestages
Although there are no TMB-specific data that would allow for the identification of
susceptible populations and lifestages, the reduced metabolic and elimination capacities in children
relative to adults may be a source of susceptibility (Ginsberg etal.. 20041. TMB isomers are
metabolized following inhalation and oral exposure via side-chain oxidation to form alcohols and
aromatic carboxylic/mercapturic acids or by hydroxylation to form phenols, which are then
conjugated with glucuronic acid, glycine, or sulfates for urinary excretion. The activities of multiple
CYP450 mono-oxygenase isozymes have been shown to be reduced in children up to 1 year of age
compared to adult activities (Ginsberg etal.. 20041. Currently, it is not known which CYP450
isozyme is responsible for TMB metabolism, although CYP2E1 is the major CYPP450 isozyme
responsible for the metabolism of low molecular weight VOCs (Nong et al.. 20061. If that is also the
case for TMB isomers, the lower activity of the CYP2E1 isozyme in children up to 1 year of age
f27-47% compared to Ginsberg et al.. 20041 could result in newborns and young infants
experiencing higher and more persistent blood concentrations of the un-metabolized TMB isomers.
This is important as it is currently assumed that the parent TMB isomers are the toxicity moieties.
When modeling inter-child differences in pharmacokinetics of toluene, a clear difference was
observed in the intrinsic clearance of toluene based on CYP2E1 content and liver weight in young
children <11 years old (0.03-1.05 L/minute) versus adolescents and adults (3.49-3.52 L/minute)
(Nong etal.. 20061. Incorporating these differences into a PBPK model and estimating venous
blood concentrations of toluene, neonates (<1 month old) had maximum (Cmax) toluene blood levels
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ranging from 0.21 to 0.70 |J.g/mL compared to 0.110.29 |J.g/mL in adults and other children. The
difference was even larger when comparing the toluene blood levels of low-metabolizing neonates,
who had blood toluene levels of 0.51-0.71 [ig/mL. Similar effects were obtained when
investigating cumulative doses (area under the curve, AUC). In order to estimate a chemical-
specific intra-human pharmacokinetic uncertainty factor (UFh-pk) for toluene, the mean adult AUC
was compared to the 95th percentile AUC for low metabolizing neonates resulting in an estimated
UFh-pk of 3.9, slightly higher than the EPA value of 3.16 (i.e., VlO); a value of 2.5 was estimated for
high metabolizing infants, and no other age-group returned values >1.6.
Ginsberg etal. f20041 also demonstrates that the rate of glucuronidation and sulfation is
decreased in neonates and children up to 2 months of age (34-47% compared to adults), resulting
in possible prolonged exposure to the metabolites of TMB isomers. If TMB metabolites also confer
some toxicity to exposed children, decreased glucuronidation in young children may increase their
susceptibility to the various hazards identified for TMB isomers. Reduced renal clearance in
children may be another important source of potential susceptibility. TMB isomers and their
metabolites are excreted in the urine of exposed laboratory animals and occupationally exposed
humans. Data indicating reduced renal clearance for infants up to 2 months of age f Ginsberg etal..
20041 may suggest a potential to affect TMB excretion, thus possibly prolonging its toxic effects.
Additionally, those with pre-existing respiratory diseases (e.g., asthma) may be more sensitive to
the respiratory irritative and inflammatory effects of TMB isomers. Genetic polymorphisms that
alter the expression or activity of enzymes that mediate the normal, homeostatic metabolism of
neurotransmitters may also increase the susceptibility of carriers of those polymorphisms to
environmental neurotoxicants fCosta. 19961.
Some research into toluene provides evidence that the postnatal period may be a sensitive
time point for VOC-induced neurotoxicity. During the early neonatal period, a number of
neurodevelopmental processes, including synaptogenesis, gliogenesis, and myelination, are
ongoing (Win-Shwe and Fuiimaki. 20101. Exposure of mouse pups to 5 ppm toluene via inhalation
on PNDs 8-12 resulted in the upregulation of multiple neuroimmune markers in the hippocampus
fWin-Shwe etal.. 20121. Additionally, inhalation exposure to the same level of toluene (5 ppm) on
PNDs 8-12 was shown to upregulate the expression of signal transduction receptors in the
hippocampus that are essential to spatial learning and memory (Win-Shwe etal.. 20101. which
correlated with the results of neurobehavioral testing of pups on PND 49, including treatment-
related decrements in spatial learning using a water maze. As these effects were not observed in
animals exposed on PNDs 14-18, these data identify PNDs 8-12 as a potential sensitive window of
susceptibility for neurodevelopmental effects. Consistent with this, decreases in spatial learning
and memory were observed in postnatally exposed animals at 5 ppm, whereas similar effects were
only observed in animals exposed as adults at 80 ppm fvon Euler et al.. 19931. Exposure to toluene
during early life has also been observed to perturb pathways related to pain sensitivity: neonatal
rats exposed to 500 mg/kg toluene i.p. on PNDs 4-9 had reduced sensitivity to nicotine-induced
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antinociception (meaning that toluene exposure prevented nicotine-induced decreases in pain
sensitivity) (Chan etal.. 20081. Together, these data indicate that infants may represent a
particularly susceptible lifestage for the effects of exposure to compounds related to TMB isomers.
Other reviews of the VOC literature further indicate that toluene exposure during gestation or early
life can result in developmental neurotoxicity fGrandiean and Landrigan. 2014: Hannigan and
Bowen. 2010: Win-Shwe and Fuiimaki. 2010: Bowen and Hannigan. 2006: Grandiean and
Landrigan. 2006: Ritchie etal.. 20011. although often due to maternal inhalant abuse in human
populations or resulting from animals studies utilizing study designs meant to approximate
inhalant abuse patterns (i.e., high doses, intermittent exposures). It is unclear how these paradigms
would relate to constant, low-level environmental exposures.
Therefore, although there exist no TMB-specific data with which to estimate early-life
vulnerability, some data gleaned from the related compound, toluene, does provide some
suggestive evidence that periods in early life do represent periods of susceptibility to solvent
exposure. Therefore, it can be reasonably assumed that exposures in early life to individual TMB
isomers are of particular concern.
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2. DOSE-RESPONSE ANALYSIS
This Dose-Response Analysis section details the derivation of chronic reference
concentration (RfC) and reference dose (RfD) values for all three trimethylbenzene (TMB) isomers
for specific organ-systems (nervous, respiratory and hematological systems as well as pregnant
animals and the developing fetus), as well as an overall chronic RfC and RfD value for TMB isomers
as a group. This last derivation stems from the conclusions of the Hazard Identification section in
which the similarity in toxicological profiles between TMB isomers was extensively reported. This
conclusion supports decisions in this section to adopt RfC or RfD values for one isomer as the RfC or
RfD value for another isomer when data are lacking for the second isomer. Regarding the RfD, only
one isomer-specific oral study was available that could support the derivation of a chronic RfD.
This study reported low levels of toxicity in the hematological system, and did not investigate
neurotoxicity endpoints. As neurotoxicity was identified as the critical effect domain in the Hazard
Identification section, a route-to-route extrapolation was performed to extrapolate inhalation data
to oral exposures to support the derivation of a neurotoxicity-based RfD. This neurotoxicity RfD
was then compared to the hematological RfD for final selection of the overall TMB RfD. Lastly,
subchronic RfC and RfD values were derived for less than lifetime exposures.
2.1. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER
THAN CANCER FOR TMBs
The RfC (expressed in units of mg/m3) is defined as an estimate (with uncertainty spanning
perhaps an order of magnitude) of a continuous inhalation exposure to human population
(including sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects
during a lifetime. It can be derived from a no- or lowest-observed-adverse-effect level (NOAEL or
LOAEL), or the 95% lower bound on the benchmark concentration (BMCL), with uncertainty factors
(UFs) generally applied to reflect limitations of the data used.
2.1.1. Identification of Studies and Effects for Dose-Response Analysis and Derivation of
Reference Concentrations for TMBs
Multiple systems have been identified as targets of inhaled TMB isomers in humans and
experimental animals, and effects in these systems have been identified as hazards following
inhalation exposure to these isomers. In humans and experimental animal models, the nervous,
respiratory, and hematological systems have been identified as targets following exposure to
1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB. Additionally, pregnant animals and the developing fetus
have been identified as targets of inhaled 1,2,4-TMB and 1,3,5-TMB in experimental animal models.
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No human studies exist to characterize the potential toxicity for pregnant women and fetuses
following inhalation exposure to TMB isomers, and no toxicological study exists to characterize this
hazard in animals exposed to 1,2,3-TMB.
The selection of studies and general procedures for dose-response analysis are discussed in
Sections 7 and 8 of the Preamble. Human data are preferred over animal data for deriving
reference values when possible because the use of human data is more relevant in the assessment
of human health and avoids the uncertainty associated with interspecies extrapolation introduced
when animal data serve as the basis for the reference value. In this case, while literature exists on
the effects of 1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB exposure in humans, including neurological,
respiratory, and hematological toxicities, no human studies are available that would allow for dose-
response analysis. The human studies evaluated TMB exposures occurring as complex solvents or
volatile organic carbon (VOC) mixtures, and this confounding along with other uncertainties
including high imprecision in effect measures due to low statistical power, lack of quantitative
exposure assessment, and lack of control for co-exposures, limit their utility in derivation of
quantitative human health toxicity values. However, these studies provide supportive evidence for
the neurological, respiratory, and hematological toxicity of TMB isomers in humans and indicate a
coherency of effects in both humans and laboratory animals.
The available animal toxicity studies evaluating health effects following exposure to the C9
fraction fDouglas etal.. 1993: Mckee etal.. 1990: Clark etal.. 19891 were similarly not considered
for the derivation of RfC values given that approximately half of the mixture was comprised of
chemicals other than the three TMB isomers. Significant uncertainty exists in these studies
regarding whether or not co-exposure with other alkylbenzenes altered the distribution or
metabolism of TMB isomers, and whether antagonistic interactions between constituents
influenced the toxicodynamics of individual components of the mixture. As such, given this lack of
knowledge, an assumption that the C9 fraction would be an adequate surrogate for individual TMB
isomers is not justified. Therefore, given this uncertainty, and the existence of multiple suitable
individual TMB isomer studies, the C9 fraction studies were excluded from consideration for the
purpose of identifying a principal study.
Several studies investigating 1,2,4-TMB-, 1,2,3-TMB-, and 1,3,5-TMB-induced effects in
experimental animal models were identified in the literature. No chronic studies were available,
although acute, short-term, subchronic, and developmental toxicity studies were identified.
1,2,4-TMB- and 1,2,3-TMB-induced toxicity was observed across several systems in four subchronic
studies by Korsak and colleagues fKorsak et al.. 2000a. b; Korsak etal.. 1997: Korsak and
Rvdzvnski. 19961. One developmental toxicity study investigating maternal and fetal toxicity
following exposure to either 1,2,4-TMB or 1,3,5-TMB was identified in the literature (Saillenfait et
al.. 20051. These studies were the only subchronic or developmental studies identified in the peer-
reviewed literature. Data from these studies pertaining to the hazards observed in humans and
animals identified in Chapter 1 (neurological, respiratory, and hematological toxicity) or in animals
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only (maternal and developmental toxicity) were considered as critical effects for the purpose of
determining the point of departure (POD) for derivation of the inhalation RfC for TMB isomers. In
addition to effects following subchronic exposure, neurotoxicity was observed in both acute and
short-term inhalation studies and respiratory toxicity was also observed in acute studies. However,
the high concentrations used in acute studies and the short exposure durations employed in both
acute and short-term studies limit their utility for the quantitation of chronic human health effects
(e.g., several studies investigating relevant neurotoxicity endpoints (Wiaderna etal.. 2002:
Gralewicz and Wiaderna. 2001: Wiaderna etal.. 1998: Gralewicz etal.. 1997b) only exposed animals
for 28 days, and are therefore less suitable for derivation of chronic RfCs). Thus, the studies of
subchronic exposure duration were preferred and the short-term and acute studies on these health
endpoints were not selected. Nevertheless, as with the human mixture studies, these studies of
shorter exposure durations do provide qualitative information regarding hazard identification,
especially the observation of the consistency and coherency of these effects across the TMB
database.
The four subchronic studies by Korsak and colleagues fKorsak etal.. 2000a. b; Korsak etal..
1997: Korsak and Rvdzyhski. 1996). and the developmental toxicity study by Saillenfait et al.
f20051. are adequate for dose-response analysis. All of these studies used rats as an appropriate
laboratory animal species, and utilized appropriate sham-exposed controls. Animals were exposed
to 1,2,4-TMB, 1,2,3-TMB, or 1,3,5-TMB reported as >97-99% pure (impurities not reported). These
studies utilized an appropriate route (inhaled air) and duration (subchronic or gestational) of
exposure. The subchronic and gestational studies used a reasonable range of appropriately-spaced
exposure levels to facilitate dose-response analysis. An appropriate latency between exposure and
development of toxicological outcomes was used, and the persistence of some outcomes
(i.e., neurotoxicity and hematological effects) after termination of exposure was investigated.
Adequate numbers of animals per exposure group were used, and appropriate statistical tests
including pair-wise and trend analyses were performed. With regard to reporting of exposure
methodologies, actual concentrations, as measured by gas chromatography, were reported to be
within 10% of target concentrations (Saillenfait etal.. 2005: Korsak etal.. 2000a. b). This increases
the confidence in the overall evaluation and adequacy of these studies. Although Korsak and
Rvdzyhski (1996) and Korsak et al. (1997) do not report actual, measured concentrations, these
studies use the same exposure methodology as Korsak etal. f2000al. suggesting that it is likely that
the actual concentrations in these studies were also within 10% of target concentrations. Target
and actual concentrations are presented in Table 2-1.
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Table 2-1. Target and actual exposure concentrations used in BMD modeling
of 1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB endpoints considered for the
derivation of the RfC
Reference
Species/sex
Isomer
Target exposure
concentration (mg/m3)
Actual exposure
concentration (mg/m3)a
Korsak and
Rvdzvriski
(1996)
Rat, male
1,2,4-TMB;
1,2,3-TMB
123
N/A
492
N/A
1,230
N/A
Korsak et al.
(1997)
Rat, male
1,2,4-TMB
123
N/A
492
N/A
1,230
N/A
Korsak et al.
(2000a)
Rat, male and
female
1,2,4-TMB
123
129
492
492
1,230
1,207
Korsak et al.
(2000b)
Rat, male and
female
1,2,3-TMB
123
128
492
523
1,230
1,269
Saillenfait et al.
(2005)
Rat, female
(pregnant dam);
male and
female (fetuses)
1,2,4-TMB
492
492
1,476
1,471
2,952
2,913
4,428
4,408
1,3,5-TMB
492
497
1,476
1,471
2,952
2,974
5,904
5,874
1
2 aActual exposure concentrations, when available, were used for dose inputs for benchmark dose (BMD) modeling.
3
4 These subchronic and developmental toxicity studies examined TMB-induced toxicity in
5 multiple systems and neurological, respiratory, hematological, maternal, or developmental toxicity
6 endpoints that demonstrated statistically significant pair-wise increases or decreases relative to
7 control were considered for the derivation of the RfC for TMBs (Tables 2-2 and 2-3).
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Table 2-2. Endpoints observed in rats in the Korsak studies (Korsak etal..
2000a. b; Korsak et al.. 1997: Korsak and Rvdzvnski. 1996) considered for the
derivation of the RfC for TMBs
Exposure concentration (mg/m3)a
Endpoint
Isomer
Sex
0
123
492
1,230
Neurological
Decreased pain sensitivity
(sec)b
1,2,4-TMB
M
15.4 ±5.8
(n = 9)
18.2 ±5.7
(n = 10)
27.6 ±3.2**
(n = 9)
30.1 ±7.9**
(n = 10)
1,2,3-TMB
M
9.7 ±2.1
11.8 ±3.8*
16.3 ± 6.3c
17.3 ±3.4**
(n = 30)
(n = 20)
o
1
II
c
o
1
II
c
Decreased neuromuscular
1,2,4-TMB
M
0
30
40**
40**
function (% failure)d
o
1
II
c
o
1
II
c
o
i
ii
c
o
i
ii
c
1,2,3-TMB
M
0
40**
60**
70**
o
1
II
c
o
i
ii
c
o
1
II
c
o
1
II
c
Hematological
Decreased RBCs (106/mm3)e
1,2,4-TMB
M
9.98 ± 1.68
9.84 ± 1.82
8.50 ± 1.11
7.70+ 1.38**
o
1
II
c
o
1
II
c
o
1
II
c
(n = 10)
1,2,3-TMB
M
9.49 ± 2.03
10.2 ± 1.29
10.11 ± 1.27
8.05 ± 1.38*
o
1
II
c
o
1
II
c
(n = 10)
o
1
II
c
Increased WBCs (103/mm3)e
1,2,4-TMB
M
8.68 ±2.89
8.92 ± 3.44
8.30 ± 1.84
15.89 ± 5.74**
o
1
II
c
o
1
II
c
o
1
II
c
o
1
II
c_
Decreased segmented
1,2,3-TMB
M
24.8 ±4.5
25.4 ±5.8
20.7 ±5.8
17.7 ±8.3*
neutrophils (%)f
(n = 10)
(n = 10)
(n = 10)
o
1
II
c
F
23.1 ±6.1
19.7 ±3.4
16.4 ±4.2*
11.9 ±7.1**
(n = 10)
(n = 10)
o
1
II
c
o
1
II
c
Decreased reticulocytes
1,2,4-TMB
F
3.5 ±2.6
1.7 ±2.0
1.8 ±0.9
1.0 ±0.6*
(%)e
o
1
II
c
o
1
II
c
o
1
II
c
(n = 10)
Increased reticulocytes (%)f
1,2,3-TMB
M
2.8 ± 1.3
(n = 10)
2.1 ± 1.7
(n = 10)
3.8 ±2.1
(n = 10)
4.5 ± 1.8*
(n = 10)
Decreased clotting time
1,2,4-TMB
F
30 ± 10
23 ±4
19 ± 5"
22 ±7*
(sec)d
o
1
II
c
(n = 10)
o
1
II
c
(n =10)
Respiratory
Inflammatory lung lesionse f
1,2,4-TMB
M
h
(n = 10)
h
(n = 10)
h
(n = 10)
h
(n = 10)
1,2,3-TMB
F
h
h
h
h
O
1
II
c
O
1
II
c
O
1
II
c
O
1
II
c
Increased bronchoalveolar
1,2,4-TMB
M
1.93 ±0.79
5.82 ±1.32***
5.96 ±2.80**
4.45 ± 1.58*
total cells (106/cm3)g
(n = 6)
(n = 6)
(n = 7)
(n = 7)
*p < 0.05; **p < 0.01; ***p < 0.001.
aValues are expressed as mean ± 1 standard deviation. Korsak and Rvdzvnski (1996) does not explicitly state that
the reported measures of variance in Table 1 of that reference are SDs. However, independent analysis
conducted by EPA confirms that the reported measures of variance are SDs.
bAdapted from Korsak and Rvdzvnski (1996), measured as latency to paw-lick.
cLevel of significance not reported in Table 1 from Korsak and Rvdzvnski (1996); however, the results of an ad-hoc
t-test (performed by EPA) indicated significance at p< 0.01.
dAdapted from Korsak and Rvdzvnski (1996), measured as percent failure on the rotarod apparatus.
e f gAdapted from Korsak et al. (2000a), Korsak et al. (2000b), and Korsak et al. (1997), respectively,
incidences for individual exposure groups not reported; however, based on qualitative information reported in
the study (i.e., that male or female rats exhibited a statistically significant increase in inflammatory lung lesions at
492 mg/m3), a NOAEL of 123 mg/m3 was identified for both isomers.
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Table 2-3. Endpoints observed in rats in the Saillenfait et al. (2005) study
considered for the derivation of the RfC for TMBs
Endpoint
Isomer
Sex
Exposure concentration (mg/m3)a
0
492
1,476
2,952 4,428
5,904
Maternal
Decreased
maternal weight
gain (g)
1,2,4-TMB
Female
131 ± 33
(n = 24)
124 ± 18
(n = 22)
126 ± 24
(n = 22)
116 ± 23
(n = 22)
95 ±19**
(n = 24)
N/A
1,3,5-TMB
Female
135 ± 15
(n = 21)
138 ± 11
(n = 22)
118 ± 24*
(n = 21)
95 ± 24**
(n = 17)
N/A
73 ±28**
(n = 18)
Developmental
Decreased fetal
weightb
1,2,4-TMB
N
23
22
22
22
24
N/A
Male
5.86 ±
0.34
5.79 ±
0.30
5.72 ±
0.49
5.55 ±
0.48*
5.20 ±
0.42**
N/A
Female
5.57 ±
0.33
5.51 ±
0.31
5.40 ±
0.45
5.28 ±
0.40*
4.92 ±
0.40**
N/A
1,3,5-TMB
N
21
22
21
17
N/A
18
Male
5.80 ±
0.41b,c
5.76 ±
0.27
5.50 ±
0.31
5.39 ±
0.55*
N/A
5.10 ±
0.57**
Female
5.50 ±
0.32
5. 47 ±
0.21
5.27 ±
0.47
5.18 ±
0.68
N/A
4.81
0.45**
*p < 0.05; **p < 0.01.
aValues are expressed as mean ± 1SD.
bTotal number of fetuses examined for 1,2,4-TMB and 1,3,5-TMB (respectively): 319, 297 (controls); 275,
314 (492 mg/m3), 293, 282 (1,476 mg/m3), 310, 217 (2,952 mg/m3), 342 (1,2,4-TMB only, 4,428 mg/m3), and
236 (1,3,5-TMB only, 5,904 mg/m3). Number of fetuses per sex was not reported.
Endpoints considered for derivation of the RfC included decreased pain sensitivity
(1,2,4-TMB, 1,2,3-TMB), decreased neuromuscular function (1,2,4-TMB, 1,2,3-TMB), decreased red
blood cells (RBCs) in male rats (1,2,4-TMB and 1,2,3-TMB), increased white blood cells (WBCs) in
male rats (1,2,4-TMB), decreased reticulocytes in female rats (1,2,4-TMB), increased reticulocytes
in male rats (1,2,3-TMB), decreased clotting time in female rats (1,2,4-TMB), decreased segmented
neutrophils in male and female rats (1,2,3-TMB), increased bronchoalveolar lavage (BAL) total cells
in male rats (1,2,4-TMB), increased inflammatory lung lesions in male and female rats (1,2,4-TMB
or 1,2,3-TMB), decreased fetal weights in males and female rats (1,2,4-TMB and 1,3,5-TMB), and
decreased maternal weight gain (1,2,4-TMB and 1,3,5-TMB) in rats (Saillenfait etal.. 2005: Korsak
etal.. 2000a. b; Korsak etal.. 1997: Korsak and Rvdzynski. 1996).
Increases in BAL polymorphonuclear leukocytes and lymphocytes observed in the Korsak et
al. (1997) study following exposure to 1,2,4-TMB were not selected for possible RfC derivation due
to a lack of reporting of exposures at which statistically significant increases occurred. Additionally,
Korsak etal. T19971 reported that 123 mg/m3 was the LOAEL for increased BAL total cells, but the
NOAEL for increased BAL macrophages. Therefore, the increase in BAL macrophages was not
selected for RfC derivation as this effect was not observed at concentrations that elicited an
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increase in total BAL cells. Changes in BAL protein and enzyme activity level following 1,2,4-TMB
exposure were not selected for RfC derivation due to non-monotonically increasing dose-responses.
A number of endpoints possibly indicating compensatory changes rather than adverse effects were
also not selected for RfC derivation: increases in sorbitol dehydrogenase (SDH) following
1,2,4-TMB exposure fKorsak etal.. 19971. changes in liver and splenic organ weights and altered
clinical chemistry parameters following exposure to 1,2,3-TMB fKorsak et al.. 2000bl. and changes
in serum chemistry parameters in rats exposed to 1,3,5-TMB in a short-term (5 weeks) inhalation
study (Wiglusz etal.. 1975a). These changes were considered to be possibly compensatory in
nature given the lack of accompanying histopathological changes in the relevant organs.
Inconsistent temporal patterns of effect were also considered in the decision to not use altered
clinical chemistry parameters fWiglusz etal.. 1975a) for the RfC derivation. Increases in
reticulocytes in female rats exposed to 1,2,3-TMB fKorsak etal.. 2000bl were not selected due to
non-monotonicity in response (increases in high concentration animals that were not statistically
significant). Increased lymphocytes observed in the same study were excluded from further
consideration due to the unusually high standard deviations (SDs) reported in the high-
concentration group.
Impaired neuromuscular function and coordination, measured as performance deficits on
the rotarod apparatus, was also observed in rats exposed to 1,2,4-TMB or 1,2,3-TMB. The use of
rotarod data from Korsak and Rvdzvhski f19961 was initially considered as a candidate critical
effect for these isomers. However, upon critical evaluation of the exposure-response information in
the study, it was determined that the endpoint was reported in a manner that reduced the
confidence in the observed effect levels. The primary limitation noted for these data relates to the
presentation of rotarod performance, which is best represented as a continuous variable, as
opposed to a quantal variable such as that presented by Korsak and Rvdzvhski f 19961. In contrast
to the percent failures reported by the study authors, the most widely used and accepted measure
of rotarod performance in rodents is latency to fall from the rotating rod f Brooks and Dunnett.
2009: Kaspar et al.. 2003: Bogo etal.. 19811. typically with an arbitrary upper limit on the maximum
latency allowed to prevent confounding by fatigue. Although the quantal percent failures data can
provide useful hazard information, these measures require an arbitrary selection of the length of
time required for successful performance; there is no scientific consensus on an optimal time for
this parameter. In addition, when identifying effect levels based on the data presented by Korsak
and Rvdzvhski f 19961. latencies on the rod of 1 second versus 119 seconds would be treated
identically as failures when, in fact, they indicate very different levels of neurological dysfunction
(Bogo etal.. 19811. This adds uncertainty when trying to extrapolate to a concentration associated
with a minimally adverse effect Finally, this quantal presentation of data does not allow for
interpretations related to intra-rat and intra-group variability in performance. Due to these
reporting limitations, impaired neuromuscular function and coordination, measured as
performance deficits on the rotarod apparatus, was considered to be less informative in
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representing the identified neurotoxicity hazard than the data supporting decreases in pain
sensitivity, and thus, was not further evaluated for derivation of the RfC for 1,2,4-TMB.
2.1.2. Methods of Analysis for Derivation of Reference Concentrations for TMBs
This assessment uses physiologically based pharmacokinetic (PBPK) model estimates of
internal blood dose metrics (for 1,2,4-TMB) and default dosimetric methods (for 1,2,3-TMB and
1,3,5-TMB) coupled with the benchmark dose (BMD) approach to estimate a POD for the derivation
of an RfC for TMBs (see Section C.2 of Appendix C and Section D.l of Appendix D for details
regarding PBPK model estimates and BMD modeling, respectively).
The BMD approach involves fitting a suite of mathematical models to the observed dose-
response data using the U.S. Environmental Protection Agency (EPA) Benchmark Dose Software
(BMDS, version 2.6.0.1). Each fitted model estimates a BMD and its associated 95% lower
confidence limit (BMDL) corresponding to a selected benchmark response (BMR). For continuous
data (i.e., decreased pain sensitivity, increased BAL total cells, decreased RBCs, decreased
reticulocytes, and decreased clotting time) from the Korsak and colleagues studies (Korsak etal..
2000a: Korsak etal.. 1997: Korsak and Rvdzvnski. 1996). and maternal weight gain from Saillenfait
etal. (2005). no information is available regarding the change in these responses that would be
considered biologically significant In cases such as this, EPA's Benchmark Dose Technical Guidance
fU.S. EPA. 20121 recommends against using BMRs based on percent change in the control mean.
Therefore, a BMR equal to a 1 SD change in the control mean was used in modeling these endpoints,
consistent with EPA's Benchmark Dose Technical Guidance (U.S. EPA. 20121. When lacking a
biological rationale for setting a BMR, the BMR ultimately chosen should reflect a minimally
biologically significant effect level. Although there is some uncertainty surrounding the selection of
1 SD for the BMR (see Section 2.1.6), there is no indication that selecting a lower BMR (i.e., 0.5 SD)
would be a more appropriate choice. As such, a BMR equal to 1 SD was used for the endpoints
listed above. For the decreased male and female fetal body weight endpoints identified from the
Saillenfait etal. (2005) study, a BMR of 5% relative deviation (RD) from the control mean was
selected. A 5% decrease in fetal body weight relative to control was determined to be a minimal,
biologically significant response. This determination is based on the fact that decreased body
weight gain in fetuses and/or pups is considered indicative of altered growth, which has been
identified by EPA as one of the four major manifestations of developmental toxicity (U.S. EPA.
19911. In addition, a 10% decrease in adult body weight in animals is generally recognized as a
biologically significant response associated with identifying a maximum tolerated dose, but since
fetuses and/or pups are generally recognized as a susceptible lifestage, and thus are assumed to be
more greatly affected by decreases in body weight than adult animals, a 5% decrease in fetal body
weight is considered a biologically significant response. Finally, in humans, reduced birth weight is
associated with a series of adverse effects including neonatal and postnatal mortality, coronary
heart disease, arterial hypertension, chronic renal insufficiency, and diabetes mellitus (Barker.
2007: Reyes and Manalich. 20051. For these reasons, the selection of a BMR of 5% for decreased
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fetal body weight was considered appropriate. Following BMD modeling, the estimated BMDL is
used as the POD for deriving the RfC (see Table 2-6).
The suitability of the above methods to determine a POD is dependent on the nature of the
toxicity database for a specific chemical. Some endpoints in the TMB database were not modeled
for a variety of reasons, including equivalent responses in the mid and high doses (e.g., increased
BAL total cells and decreased reticulocytes [1,2,4-TMB]), responses only in the high exposure group
with no changes in responses in lower exposure groups (e.g., increased WBCs [1,2,4-TMB] and
decreased RBCs [1,2,3-TMB]), and absence of incidence data (e.g., increased inflammatory lung
lesions [1,2,4-TMB and 1,2,3-TMB]). Additionally, some datasets were modeled, but appropriate
model fit (to either the mean response or the reported variance) was not achievable for a variety of
reasons. Correctly characterizing the variance in a dataset is critical for estimating accurate BMDL
values. When a constant (i.e., homogenous) variance model was not able to fit the reported
variances, a non-homogenous variance model was used. For example, the reported variances for
decreased pain sensitivity (1,2,4-TMB and 1,2,3-TMB) were not homogenous, with variances at 492
mg/m3 being lower or higher (1,2,4-TMB and 1,2,3-TMB, respectively) than reported variances in
other dose groups. Fitting a homogenous variance model to these data resulted in poor model fit,
and model fit was not improved by running the non-homogenous variance model (see Tables D-2
and D-3). In cases such as this, the high dose was dropped and the models were re-run on the
truncated datasets. Dropping the high dose resulted in adequate model fit in some cases (e.g.,
decreased pain sensitivity; 1,2,4-TMB or 1,2,3-TMB, constant or non-constant variance
[respectively], Table D-4), but not others (e.g., decreased clotting time; 1,2,4-TMB). In cases where
BMD modeling was not feasible or modeling failed to appropriately describe the dose-response
characteristics, the NOAEL/LOAEL approach was used to identify a POD. Detailed modeling
methodology and results are provided in Appendix D.
Because an RfC is a toxicity value that assumes continuous human inhalation exposure over
a lifetime, data derived from inhalation studies in animals need to be adjusted to account for the
non-continuous exposures used in these studies. For 1,2,4-TMB-induced systemic effects
(e.g., neurotoxicological effects), the available deterministic PBPK model (Hissink et al.. 2007) was
used to convert non-continuous external inhalation concentrations (in mg/m3) of 1,2,4-TMB to the
internal blood dose metric of average weekly venous blood concentration (in mg/L) of 1,2,4-TMB
for Korsak etal. T19971: Korsak etal. f2000al: and Korsak and Rvdzvriski f 19961 only. Weekly
average venous blood 1,2,4-TMB concentration was chosen as the internal dose metric on which to
base the POD as it is assumed that the parent compound is the toxic moiety of interest and that
average venous blood concentration of 1,2,4-TMB adequately represents the target tissue dose
across the multiple tissues of interest The use of concentration of parent compound in venous
blood as the relevant dose metric in non-metabolizing, non-first-pass organs is recommended by
Avlward etal. f2011I Furthermore, toluene-induced neurological effects in the brain are provided
by Avlward etal. (20111 as an example of a chemically induced toxic endpoint for which this dose
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metric is relevant. As discussed in Section 1.2.1 (Mode-of-Action Analysis—Neurotoxic Effects),
1,2,4-TMB is reasonably expected to have a mode of action for neurotoxic effects similar to toluene,
further supporting the selection of venous blood concentration as the relevant internal dose metric.
For pulmonary/tracheobronchial effects (e.g., inflammatory lung lesions), the internal dose metric
used was the average lung deposition rate per unit surface area (mg/m2/hour, pulmonary +
tracheobronchial area).
Although dosimetry can often be nonlinear due to metabolic saturation, and internal dose
metrics are expected to correlate more closely to toxic response than external concentrations
(McLanahan etal.. 2012). the order of analysis employed in this assessment for 1,2,4-TMB is the use
of BMD modeling methods with external concentrations used as the dose inputs, followed by
conversion of the resulting PODs into human equivalent concentrations (HECs) using the available
PBPK model. The order of analysis was conducted in this manner primarily to account for
inaccuracies in the animal PBPK model at high exposure concentrations. During the validation and
optimization of the animal PBPK model (Hissink et al.. 2007) against available animal toxicokinetic
datasets, the model accurately reproduced venous blood concentrations of 1,2,4-TMB following
repeated (6 hours/day, 5 days/week, 4 weeks) exposures to 123 or 492 mg/m3 (see Section C.2.3.2,
Appendix C). However, the PBPK model consistently over-predicted venous blood concentrations
following exposure to 1,230 mg/m3. It was concluded that the optimized animal PBPK model
produces acceptable simulations of venous blood 1,2,4-TMB concentrations for chronic exposures
of up to 100 ppm [492 mg/m3] in rats following inhalation exposure to 1,2,4-TMB (Section C.2.3.2,
Appendix C). Therefore, as the model-estimated internal blood dose metrics at the high
concentration are not representative of empirically observed blood concentrations, using the high-
dose model estimates as dose inputs for BMD modeling is not appropriate. To account for this,
BMD modeling was performed on external exposure concentrations in order to identify a POD that
was within the validated region of the PBPK model (i.e., <492 mg/m3) and then use the PBPK model
to convert that POD into a HEC.
The Hissink et al. (2007) PBPK model was not parameterized for pregnant animals, did not
include a fetal compartment, and was not parameterized for either 1,2,3-TMB or 1,3,5-TMB. For
these reasons, the model could not be used to account for non-continuous exposures utilized in
studies that investigated effects following subchronic exposures to 1,2,3-TMB or 1,3,5-TMB or
gestational exposures to 1,2,4-TMB. In order to calculate PODs adjusted for continuous exposures,
default dosimetric adjustments were used for these endpoints. For example, in the Saillenfait et al.
f20051 study, rats were exposed to 1,2,4-TMB or 1,3,5-TMB for 6 hours/day for 15 consecutive
days (gestational days [GDs] 6-20). Therefore, the duration-adjusted PODs for
developmental/maternal effects were calculated as follows:
PODadj (mg/m3) = POD (mg/m3) x hours exposed per day/24 hours
For example, for decreased fetal weight in males following exposure to 1,2,4-TMB, the
PODadj would be calculated as follows:
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PODadj (mg/m3) = 1,640.07 mg/m3 x 6 hours/24 hours
PODadj (mg/m3) = 410.82 mg/m3
For subchronic 1,2,3-TMB-induced effects observed in the Korsak etal. f2000b) and Korsak
and Rvdzvnski f19961 studies, rats were exposed to 1,2,3-TMB for 6 hours/day, 5 days/week for
3 months. For these endpoints, the duration-adjusted PODs for effects in rats were calculated as
follows:
PODadj (mg/m3) = POD (mg/m3) x hours exposed per day/24 hours x days exposed
per week/7 days
Therefore, for decreased pain sensitivity from Korsak and Rydzvhski (1996). the PODadj
would be calculated as follows:
PODadj (mg/m3) = 97.19 mg/m3x 6 hours/24 hours x 5 days/7 days
PODadj (mg/m3) = 17.36 mg/m3
For the derivation of an RfC based upon animal data, the calculated PODadj values for
1.2.4-TMB-induced effects were converted to HECs using the available human PBPK model (Hissink
etal.. 2007) for the selected endpoints from the Korsak and colleagues studies (Korsak etal.. 2000a:
Korsak etal.. 1997: Korsak and Rydzvhski. 1996). The human PBPK model was run (as described in
Appendix C), assuming a continuous (24 hours/day, 7 days/week) exposure, to estimate a human
PODhec that would result from the same weekly average venous blood concentration or rate of
pulmonary deposition reflected in the PODadj in animals.
The majority of 1,2,3-TMB-induced subchronic endpoints and all of the 1,2,4-TMB- and
1.3.5-TMB-induced gestational endpoints under consideration for the critical effect result primarily
from systemic distribution of the TMB isomers. As the Hissink etal. (2007) PBPK model is not
parameterized for 1,2,3-TMB or 1,3,5-TMB, the HECs for subchronic hematological and
neurotoxicological 1,2,3-TMB endpoints and maternal/developmental 1,2,4-TMB and 1,3,5-TMB
endpoints were calculated by the application of the appropriate dosimetric adjustment factor (DAF)
for systemically acting gases with effects distal to the portal of entry, in accordance with the EPA's
RfC Methodology (U.S. EPA. 1994b). This determination is supported by other factors, including the
isomer's relatively low water solubility and non-reactivity. Gases with these properties are
expected to preferentially distribute to the lower regions of the respiratory tract where larger
surface areas and thin alveolar-capillary boundaries facilitate uptake. Respiratory absorption of
1,2,3-TMB into the bloodstream has been observed to be relatively high (~60%) following
inhalation exposures to humans flarnbergetal.. 19961. TMB isomers are also observed to elicit
inflammatory lung lesions. Currently, it is not known whether these effects are due to localized
deposition or to systemic redelivery of the active agent to the pulmonary region. For the purpose of
deriving reference values, 1,2,3-TMB-induced respiratory endpoints were assumed to be portal-of-
entry effects; treating 1,2,3-TMB in such a manner is consistent with HEC calculations for 1,2,4-TMB,
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in which the dose metric used for respiratory effects was the average lung deposition rate per unit
surface area (mg/m2/hour, pulmonary + tracheobronchial area), rather than a dose metric based
on venous blood concentrations.
DAFs are ratios of animal and human physiologic parameters, and are dependent on the
nature of the contaminant (i.e., particle or gas) and the target site (i.e., respiratory tract or remote to
the portal-of-entry [i.e., systemic]) fU.S. EPA. 1994bl. For gases with systemic effects, the DAF is
expressed as the ratio between the animal and human blood:air partition coefficients:
DAF = (Hb/g)A/(Hb/g)H
where:
(Hb/g)A = the animal bloochair partition coefficient and (Hb/g)H = the human blood:air
partition coefficient
For gases that elicit portal-of-entry effects, the DAF is expressed as the ratio between the
quotient of ventilation rate divided by respiratory tract surface area for animals and humans,
respectively:
DAF = (Va/SAa) / (V h/SAh)
where:
VA and VH are the ventilation rates for rats and humans, respectively; and SAa and SAH
are the surface areas of the tracheobronchial and pulmonary regions in rats and
humans, respectively
Calculation of the isomer-specific DAF values are summarized in Table 2-4.
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Table 2-4. Isomer-specific DAFs using default dosimetric methods
1,2,4-TMB
1,2,3-TMB
1,3,5-TMB
(Hb/g)Aa
57.7
62.6
55.7
(Hb/g)Hb
59.1
66.5
43
Systemic DAF
0.98
0.94
1.3
\/A(L/min)
-
0.178 (M); 0.125(F)0
-
\/h (L/min)
-
8.28d
-
SAa (m2)
-
0.422 (M); 0.280(F)6
-
SAh (m2)
-
54 (default)
-
Respiratory DAF
-
2.75 (M); 2.91 (F)
-
1
2 aDerived from Jarnberg and Johanson (1995).
3 bDerived from Meulenberg and Viiverberg (2000).
4 Calculated using the equation ln(V)=bo + biln(BW). Body weight (BW) was the average of all body weights in
5 Korsak et al. (2000b) for males (0.404 kg) and females (0.263). Values for bo (-0.578) and bi (0.821) for rats were
6 drawn from the 1994 RfC Guidance (U.S. EPA. 1994b). Values for total ventilation (M = 0.267, F = 0.187) were
7 multiplied by (2/3) to calculate alveolar ventilation.
8 Calculated in L/minute from the value of 0.497 m3/hour used in 1,2,4-TMB PBPK modeling
9 Calculated by scaling BW095.
10
11 In cases where the animal blood:air partition coefficient is lower than the human value,
12 resulting in a DAF <1, the calculated value is used for dosimetric adjustments fU.S. EPA. 1994bl.
13 When the resulting DAF >1, a default value of 1 is substituted. The calculated PODhec (mg/m3)
14 values for all endpoints considered for candidate value derivation are presented in Table 2-5.
15
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Table 2-5. Summary of derivation of PODs for TMBs
Endpoint
Isomer
Sex
Model; BMR
PODa
PODadj
PODhec
Neurological
Decreased pain sensitivity
1,2,4-TMB
Male
Linear, 1SD
140.54
0.099b
18.156
1,2,3-TMB
Male
Linear, 1 SD
97.19
17.36°
16.31f
Hematological
Decreased RBCs
1,2,4-TMB
Male
Exponential 2,1 SD
451.51
0.737b
116.24e
1,2,3-TMB
Male
NOAEL
523.00
93.39°
cn
00
Increased WBCs
1,2,4-TMB
Male
NOAEL
492.00
0.868b
131.60e
Decreased segmented
neutrophils
1,2,3-TMB
Male
Exponential 2,1 SD
534.81
95.50°
00
ID
Female
Hill, 1SD
99.21
17.72°
to
ID
UD
1
Decreased reticulocytes
1,2,4-TMB
Female
NOAEL
492.00
0.889b
133.85e
Increased reticulocytes
1,2,3-TMB
Male
Linear, 1 SD
652.80
116.57°
109.58f
Decreased clotting time
1,2,4-TMB
Female
NOAEL
129.00
0.1335b
24.306
Respiratory
Inflammatory lung lesions
1,2,4-TMB
Male
NOAEL
129.00
0.564d
61.656
1,2,3-TMB
Female
NOAEL
128.00
22.86°
66.56f
Increased BAL total cells
1,2,4-TMB
Male
LOAEL
123.00
0.533d
58.21s
Developmental
Decreased fetal weight
1,2,4-TMB
Male
Linear, 5% RD
1640.07
410.02°
401.82f
Female
Linear, 5% RD
1612.89
403.22°
395.16f
1,3,5-TMB
Male
NOAEL
1471.00
367.75°
367.75f
Female
NOAEL
2974.00
743.50°
743.50f
Maternal
Decreased maternal weight
1,2,4-TMB
Female
Polynomial 3,1 SD
3094.13
773.53°
758.06f
1,3,5-TMB
Female
NOAEL
497.00
124.25°
124.25f
aExternal air concentration (mg/m3).
bAverage venous blood concentration (mg/L) predicted for the rat using the PBPK model, given a 6-hour/day,
5-day/week exposure.
cDuration-adjusted external concentrations (mg/m3).
dAverage lung deposition rate per unit surface area (mg/m2/hour, pulmonary + tracheobronchial region), predicted
for the rat using the available PBPK model, given a 6-hour/day, 5-day/week exposure.
Continuous exposure level (mg/m3) for a 70-kg human predicted to result in a blood concentration equal to
PODadj, calculated with PBPK model.
'Continuous exposure level (mg/m3) for a human as calculated using default dosimetric methods.
gHEC (mg/m3) = PODadj x SAhum / Venthum, where SAhum = 54.32 m2 (pulmonary + tracheobronchial in a 70-kg adult)
and Venthum = 0.497 m3/hour (total ventilation for a 70-kg adult).
2.1.3. Derivation of Candidate Inhalation Values for TMBs
Under EPA's A Review of the Reference Dose and Reference Concentration Processes (U.S. EPA.
20021. also described in the Preamble, five possible areas of uncertainty and variability were
considered in deriving the candidate values for TMBs. An explanation of these five possible areas of
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uncertainty and variability and the values assigned to each as a designated UF to be applied to the
candidate PODhec are as follows.
An interspecies uncertainty factor, UFa, of 3 (101/2 = 3.16, rounded to 3) was applied to
account for uncertainty in characterizing the toxicokinetic and toxicodynamic differences between
rats and humans following inhalation exposure to TMB isomers. In this assessment, the use of a
1,2,4-TMB PBPK model to convert internal doses in rats to administered doses in humans reduces
toxicokinetic uncertainty in extrapolating from the rat to humans for that isomer. For 1,2,3-TMB
and 1,3,5-TMB, the use of default dosimetric methods to extrapolate external concentrations from
rats to humans reduces the toxicokinetic uncertainty for those isomers to the same degree.
However, neither of these methods accounts for the possibility that humans may be more sensitive
to TMB due to interspecies differences in toxicodynamics. Therefore, a UFa of 3 was applied to
account for this remaining toxicodynamic and any residual toxicokinetic uncertainty not accounted
for by application of the PBPK model or default dosimetric methods.
An intraspecies uncertainty factor, UFh, of 10 was applied to account for potentially
susceptible individuals in the absence of data evaluating variability of response in the human
population following inhalation of TMB isomers. No information is currently available to predict
potential variability in human susceptibility, including variability in the expression of enzymes
involved in TMB metabolism. However, PBPK modeling on other methylated benzene derivatives
(i.e., toluene and xylene) in order to derive chemical-specific UFh-pk values (the portion of the UFh
accounting for toxicokinetic uncertainty) resulted in UFh-pk values ranging from 0.96-0.52 for
sensitive adults to 0.5-3.9 for extrapolating to children. Both values indicate that the UFh-pk of 3.16
is sufficient to account for uncertainty due to toxicokinetic differences between possible sensitive
subpopulations and the general population (Nongetal.. 2006: Pelekis etal.. 20011. However, this
information does not inform how toxicodynamics may differ between possible sensitivity
subpopulations and the general population. As such, a UFh-td of 3.16 is used, resulting in a total UFh
of 10 to account for potentially susceptible individuals.
A LOAEL to NOAEL uncertainty factor, UFl, of 1 was applied because the current approach is
to address this factor as one of the considerations in selecting a BMR for BMD modeling. In other
words, when selecting a BMR value, care should be taken to select a response level that constitutes
a minimal, biologically significant change so that the estimated BMDLs can be assumed to
conceptually correspond to a NOAEL. In the case of TMBs, BMRs were preferentially selected based
on biological information on what constitutes a biologically significant change for these effects,
when such information was available. For example, a 5% reduction in fetal body weight was
selected as the BMR for that endpoint based on the fact that a 10% reduction in adult body weight
is considered adverse, the assumption that fetuses are a susceptible population and thus more
vulnerable to body weight changes, and that decreases in fetal weight in humans are associated
with a number of chronic diseases such as hypertension and diabetes. For endpoints for which
there was no information available to make assumptions about what constitutes a minimal,
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biologically significant response, a BMR equal to a 1 SD change in the control mean was selected.
For endpoints that could not be modeled, a LOAEL to NOAEL UF of 1 was applied as a NOAEL was
used, except for increased BAL cells following 1,2,4-TMB exposure, to which a UF of 10 was applied
due to the use of a LOAEL for this endpoint.
A subchronic to chronic uncertainty factor, UFs, of 3 was applied to account for
extrapolation from a subchronic exposure duration study to derive a chronic RfC for all endpoints
except decreases in fetal weight, to which a UFS of 1 was applied. The 3-fold UF is applied to the
POD identified from the subchronic studies on the assumption that effects observed in a similar
study utilizing a chronic exposure duration would be observed at lower concentrations for a
number of possible reasons, including potential cumulative damage occurring over the duration of
the chronic study or an increase in the magnitude or severity of effect with increasing duration of
exposure. Given that the adaptive responses of the nervous system appear to be impaired several
weeks after short-term exposure, including prolongation of decreased pain sensitivity phenotypes
following environmental challenge using a footshock, there is concern that chronic exposures may
more thoroughly overwhelm adaptive responses in the nervous system, and thus lead to more
severe responses, compared to shorter duration exposures. In addition, there is some evidence that
neurotoxicity worsens with continued exposure, and thus, effects are expected to be more severe
following chronic exposure. For example, decrements in rotarod function were shown to increase
in magnitude as a function of exposure duration, worsening from 4 to 8 weeks of exposure (1,2,3-
TMB and 1,2,4-TMB), and worsening further from 8 to 13 weeks of exposure (1,2,3-TMB only)
(Korsak and Rvdzvnski. 1996). Although a similar time-course is not available for reduced pain
sensitivity, reduced pain sensitivity is observed at approximately 5-fold lower concentrations
following subchronic exposure, as compared to acute exposure (see discussion in Section 1.2.1).
However, there does not seem to be an exacerbation of other neurotoxic effects at lower doses
when comparing subchronic exposures to short-term exposures. Further, evidence from
toxicokinetic studies indicates that blood and organ concentrations ofTMBs are similar following
repeated vs. acute exposures (approximately 600 hours vs. 6 hours, respectively; see Table C-9) and
the PBPK model predicts less than a 5% increase between the first day and subsequent days of
repeated exposures. By extension, it can be reasonably assumed that TMB isomers would not
accumulate to an appreciably greater degree following a longer chronic exposure and thus may not
lead to effects at lower doses compared to shorter duration studies. Taken together, the
toxicokinetic and toxicological data support the application of a UFs of 3 for neurotoxic,
hematological, and respiratory endpoints. Additionally, for maternal weight gain following
exposure to 1,2,4-TMB or 1,3,5-TMB, a subchronic to chronic UF of 3 was applied given there was
no observed decrease in adult body weights in rats exposed to either 1,2,4-TMB or 1,2,3-TMB for
longer durations (i.e., 90 days). For decreases in fetal weight, a UFs of 1 was applied as the
gestational period is presumed to be a critical window of susceptibility and no adjustment for
duration of exposure is necessary.
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A database uncertainty factor, UFd, of 3 (101/2 = 3.16, rounded to 3) was applied to account
for database deficiencies. Strengths of the database include the three well-designed subchronic
studies that observe exposure-response effects in multiple systems (nervous, respiratory, and
hematological systems) in Wistar rats exposed to 1,2,4-TMB or 1,2,3-TMB via inhalation. A major
deficiency of the database is the lack of a chronic study for any isomer; however, this particular data
gap is accounted for in the UFs. An additional strength of the database is the well-designed
developmental toxicity study that investigated standard measures of maternal and fetal toxicity in a
different strain of rat (Sprague-Dawley) following exposure to 1,2,4-TMB or 1,3,5-TMB. Supporting
the observation of developmental toxicity following exposure to TMB isomers, is the observation
that exposure to mixtures containing TMB isomers or related compounds (the C9 fraction,
Aromatol, toluene, xylene, or ethylbenzene) also elicits developmental toxicity (increased fetal
death, decreased fetal weight, cleft palate) in rats, mice, and rabbits fMckee etal.. 1990: Ungvarv
and Tatrai. 1985). albeit at doses >500 mg/m3, which is higher than the lowest LOAEL for
neurotoxicity effects in rats (i.e., 123 mg/m3 for decreased pain sensitivity following exposure to
1,2,3-TMB). However, the lack of a multi-generation reproductive toxicity study for TMB isomers is
a possible deficiency in the TMB database. There is suggestive evidence that exposure to the C9
fraction may produce reproductive toxicity: exposure of rats to 4,059 mg/m3 TMB isomers
(1,500 ppm C9 fraction, containing ~55% TMB isomers) resulted in decreased male fertility (Mckee
etal.. 19901. Additionally, there was a possible intergenerational effect on body weight in which
decreases in fetal/pup/adult weight occurred at lower doses in later generations compared to
earlier ones. However, the lowest concentration of TMB isomers (as part of the total mixture) that
resulted in decreased body weights was 1,353 mg/m3. Therefore, while reproductive toxicity and
progressive, intergenerational health effects appear to be a concern following exposure to mixtures
containing TMB isomers, the effects observed occur at concentrations much greater than TMB
concentrations that elicit neurotoxicity and hematological effects in adult animals (1,353 mg/m3
versus 123 mg/m3 [1,2,3-TMB] and 492 mg/m3 [1,2,4-TMB]), somewhat reducing the level of
concern for observing reproductive effects at much lower concentrations in a multi-generational
study.
The lack of a developmental toxicity study for any individual isomer is also a potential
weakness of the database. EPA's A Review of the Reference Dose and Reference Concentration
Processes fU.S. EPA. 20021 recommends that the database UF take into consideration whether there
is concern from the available toxicology database that the developing organism may be particularly
susceptible to effects in specific organs/systems. TMBs (unspecified isomer) are able to cross the
placenta (Cooper etal.. 2001: Dowtv etal.. 1976): therefore, as neurotoxicity is observed in adult
animals following exposure to any TMB isomer, there is the concern that exposure to TMB isomers
may result in neurotoxicity in the developing organism. EPA's Guidelines for Neurotoxicity Risk
Assessment fU.S. EPA. 19981 identifies specific effects observed in adult animals (e.g., cognitive and
motor function) that can also affect the developing organism exposed in utero. The Neurotoxicity
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Guidelines (U.S. EPA. 19981 also indicate that neurotoxicants may have greater access to the
nervous system in developing organisms due to an incomplete blood-brain barrier and immature
metabolic detoxifying pathways. Lastly, EPA's A Review of the Reference Dose and Reference
Concentration Processes (U.S. EPA. 20021 also states that effects that may be mild or reversible in
adults may produce more robust or permanent effects in offspring following developmental
exposures. Therefore, there is some concern that the lack of a developmental neurotoxicity study is
a major deficiency in the database and that inclusion of such a study would potentially result in a
lower POD than the POD for neurotoxicity identified from the available adult TMB toxicity database.
Although TMB-specific studies are lacking, inferences can be drawn from developmental studies of
related chemicals. Unfortunately, much of the human and animal literature demonstrating the
developmental neurotoxicity of related alkylbenzenes comes from epidemiological studies of
inhalant abuse or animal studies using exposure paradigms intended to approximate inhalant
abuse patterns (i.e., high exposure concentrations and intermittent and non-continuous exposures)
(Bowen and Hannigan. 2006: Hass etal.. 1999: Hougaard etal.. 1999: Hass etal.. 1997: lones and
Balster. 1997: Hass etal.. 19951. which are difficult to interpret in the context of an RfC. However,
information from the related compound, toluene, indicates that, while toluene is able to cross the
placenta, and that toluene levels in the placenta, amniotic fluid, and fetal brains increased with
increasing exposure concentrations, concentrations of toluene in the amniotic fluid were less than
those in maternal blood f Hannigan and Bowen. 20101. Although this fails to account for potential
differences in sensitivity of the developing organism to induced effects, or for differences in
metabolism, it does suggest that gestational exposure to TMBs might result in lower exposure
concentrations to the fetus, which raises uncertainty in the TMB and related compound database
regarding whether sufficient amounts of the toxic agent crosses the placenta to elicit effects, and
whether the concentrations necessary to elicit effects are lower than those that result in
neurotoxicity in the adult organism. In contrast to this, there is evidence from perinatal toxicity
studies in rats that low-level exposures to toluene (as low as 5 ppm) early in life (postnatal days
[PNDs] 4-12) may disrupt developmental processes that began early in embryogenesis
(synaptogenesis, myelination, etc.) and result in neurotoxicity (decrements in spatial learning)
(Win-Shwe etal.. 2010: Win-Shwe and Fuiimaki. 20101. Additionally, evidence of measures of
neurotoxicity in children born to occupationally exposed women exists (Grandiean and Landrigan.
2014: Hannigan and Bowen. 2010: Grandiean and Landrigan. 20061 and analogies drawn between
related alkylbenzenes and TMBs illustrate reasonable modes of action for developmental
neurotoxicity (see Section 1.2.1). Therefore, concern still exists regarding the possibility that
exposure to TMB may result in developmental neurotoxicity, potentially at lower TMB
concentrations and, as such, a 3-fold database UF was applied to account for the lack of a
developmental neurotoxicity study in the available database for TMB isomers.
Table 2-6 is a continuation of Table 2-5, and summarizes the application of UFs to each POD
to derive a candidate inhalation value for each data set The candidate values presented in Table 2-
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1 6 are preliminary to the derivation of the organ/system-specific RfCs for TMBs. These candidate
2 values are considered individually in the selection of a representative RfC for a specific hazard and
3 subsequent overall RfC for all three of the TMB isomers. Figure 2-1 presents graphically these
4 candidate values, UFs, and PODs, with each bar corresponding to one data set described in
5 Tables 2-5 and 2-6.
Table 2-6. Summary of derivation of candidate inhalation values for TMBs
Endpoint
Isomer
Sex
HEC
(mg/m3)a
UFa
UFh
UFl
UFs
UFd
Composite
UF
Candidate
Value
(mg/m3)a
Neurological
Decreased pain
sensitivity
1,2,4-TMB
Male
18.15
3
10
1
3
3
300
6.05 x 10"2
1,2,3-TMB
Male
16.31
3
10
1
3
3
300
5.44 x 10"2
Hematological
Decreased RBCs
1,2,4-TMB
Male
116.24
3
10
1
3
3
300
3.87 x 10"1
1,2,3-TMB
Male
87.79
3
10
1
3
3
300
2.93 x 10"1
Increased WBCs
1,2,4-TMB
Male
131.60
3
10
1
3
3
300
4.39 x 10"1
Increased segmented
neutrophils
1,2,3-TMB
Male
89.77
3
10
1
3
3
300
2.99 x 10"1
Female
16.65
3
10
1
3
3
300
5.55 x 10"2
Decreased reticulocytes
1,2,4-TMB
Female
133.85
3
10
1
3
3
300
4.46 x 10"1
Increased reticulocytes
1,2,3-TMB
Male
109.58
3
10
1
3
3
300
3.65 x 10"1
Decreased clotting time
1,2,4-TMB
Female
24.30
3
10
1
3
3
300
8.10 x 10"2
Respiratory
Inflammatory lung
lesions
1,2,4-TMB
Male
61.65
3
10
1
3
3
300
2.06 x 10"1
1,2,3-TMB
Female
66.56
3
10
1
3
3
300
2.22 x 10"1
Increased BAL total cells
1,2,4-TMB
Male
58.21
3
10
10
3
3
3,000b
N/A
Developmental
Decreased fetal weight
1,2,4-TMB
Male
401.82
3
10
1
1
3
100
4.02
Female
395.16
3
10
1
1
3
100
3.95
1,3,5-TMB
Male
367.75
3
10
1
1
3
100
3.68
Female
743.50
3
10
1
1
3
100
7.44
Maternal
Decreased maternal
weight
1,2,4-TMB
Female
758.06
3
10
1
3
3
300
2.53
1,3,5-TMB
Female
124.25
3
10
1
3
3
300
4.14 x 10"1
6 aAs calculated by application of UFs, not rounded to 1 significant digit.
7 b Endpoint excluded from further consideration due to increased uncertainty relative to other endpoints (as illustrated
8 by a UFcomposite 10-fold higher than any other endpoint.
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2.1.4. Derivation of Organ/System-Specific Reference Concentrations for TMBs
Table 2-7 distills the candidate values from Table 2-6 into a single value for each isomer,
where possible, for each organ or system. The single isomer-specific RfC selected for a particular
organ/system was preferably chosen using biological and toxicological information regarding that
endpoint. If no compelling biological information exists on which to base the selection, the lowest
RfC for that organ/system was selected. These organ- or system-specific RfCs may be useful for
subsequent cumulative risk assessments that consider the combined effect of multiple agents acting
at a common site. In the TMBs Toxicological review, a UFd =3 is applied to derive each candidate
value to account for the lack of a developmental neurotoxicity study in the TMB database.
However, for the purposes of cumulative risk assessments, it may not be necessary to employ a UFd
to calculate RfCs for endpoints that only account for toxicity in that respective organ/system (i.e.,
hematological and respiratory endpoints). In the case of the present assessment, calculation of RfCs
that do not take into account database deficiencies would incorporate either a composite UF = 100
(UFa = 3, UFh = 10, UFs = 3, UFl = 1, UFd = 1 [hemtatological, respiratory, and maternal endpoints])
or a composite UF = 30 (UFa = 3, UFH = 10, UFS = 1, UFL = 1, UFD = 1 [developmental endpoints]),
instead of 300.
The individual organs and systems for which specific RfC values were derived were the
neurological, hematological, and respiratory systems, along with specific RfCs derived for the
pregnant animal (maternal) and developing fetus (developmental). The RfC values for the
neurological system, based on decreased pain sensitivity following exposure to 1,2,4-TMB or 1,2,3-
TMB, were similar (6 x 10~2 mg/m3vs. 5 x 10"2 mg/m3, respectively). The RfC for 1,2,4-TMB was
selected for the overall RfC for all TMBs (see Section 2.1.5 for details). The RfC values for the
hematological system (based on decreased clotting time for 1,2,4-TMB and decreased segmented
neutrophils for 1,2,3-TMB) were similar to those calculated for neurological effects. The
respiratory RfCs were somewhat higher compared to the neurological and hematological RfCs: 2 x
10_1 mg/m3. This generally indicates that effects in all of these systems may be of concern at
similar levels of environmental exposure. However, effects to pregnant animals and the developing
fetus may be of less concern, as the RfCs for these types of effects (based on decreased maternal
weight gain and decreased male and female fetal weight, respectively) are much higher (6-70-fold
higher) than those derived for the neurological or hematological systems.
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Table 2-7. Organ/system-specific RfCs and overall RfC for TMBs
Effect
Isomer
Basis
RfC
(mg/m3)
Composite
UF
Exposure
Description
Confidence
Neurological
1,2,4-TMB
Decreased pain
sensitivity
6 x 10"2
300
Subchronic
Low to medium
1,2,3-TMB
5 x 10"2
300
Subchronic
Low to medium
Hematological
1,2,4-TMB
Decreased
clotting time
8 x 10"2
300
Subchronic
Low to medium
1,2,3-TMB
Decreased
segmented
neutrophils
6 x 10"2
300
Subchronic
Low to medium
Respiratory
1,2,4-TMB
Inflammatory
lung lesions
2 x 10"1
300
Subchronic
Low to medium
1,2,3-TMB
2 x 10"1
300
Subchronic
Low to medium
Developmental
1,2,4-TMB
Fetal weight
4
100
Gestational
Low to medium
1,3,5-TMB
4
100
Gestational
Low to medium
Maternal
1,2,4-TMB
Decreased
maternal
weight
3
300
Subchronic
Low to medium
1,3,5-TMB
4 x 10"1
300
Subchronic
Low to medium
Overall RfC
(Neurological)
1,2,4-TMB
Decreased
pain
sensitivity
6 x 10"2
300
Subchronic
Low to medium
2.1.5. Selection of the Overall Reference Concentration for TMBs
Neurotoxicity is the most consistently observed endpoint in the toxicological database for
TMBs. According to EPA's Guidelines for Neurotoxicity Risk Assessment (U.S. EPA. 19981. many
neurobehavioral changes reported in the available TMB studies are regarded as adverse, and, in
particular, decreased pain sensitivity, measured as an increased latency to paw-lick in hot plate
tests, represents an alteration in neurobehavioral function fU.S. EPA. 19981. It is important to
consider that the choice of the critical effect is intended as a representation of the overall
neurotoxic hazard of TMB, which, as discussed in Section 1.2.1, is not limited to observations of
reduced pain sensitivity in only one study. Rather, the TMB database includes the observations of
multiple correlated and replicated measures of neurotoxicity, which in turn, strengthen the overall
evidence for a neurotoxic hazard. Decreased pain sensitivity (Korsak and Rydzvhski. 19961 or
decreased pain sensitivity following a footshock challenge was observed in multiple studies across
multiple exposure durations (Wiaderna etal.. 2002: Gralewicz and Wiaderna. 2001: Wiaderna etal..
1998: Gralewicz etal.. 1997bl. and in the presence of other measures of altered neurobehavior,
including impaired neuromuscular function and altered cognitive function. Additionally,
neurological symptoms (e.g., hand tremble, weakness) were observed in worker populations
exposed to complex VOC mixtures containing TMB isomers. Notably, pain sensitivity has not been
evaluated in any occupational epidemiologic or controlled human exposure studies. However,
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other measures of neurotoxicity were noted in human populations, including altered
neuromuscular function (hand tremble, altered motor control), altered cognition (memory
problems), and increased nervousness and/or anxiety, suggesting a consistency and coherency of
neurotoxic effects in humans and animals following exposure to TMBs. This supports the
assumption that neurotoxic effects observed in animals, but not yet evaluated in human
populations, are relevant to human populations.
Although a superficial consideration of the available evidence may lead to a determination
that decreased pain sensitivity may be a reversible endpoint related to intoxication and the
presence of TMB isomers in the body, consideration of the neurotoxicological database as a whole
supports the conclusion that decreased pain sensitivity is a persistent effect lasting up to 2 months
post exposure (see Section 1.2.1: Summary of Neurological Effects for more details). Additionally,
although it is important to consider the potential for reversibility of neurological effects, "for
chronic lifetime exposures, designation of an effect as irreversible or reversible is academic, as
exposure is presumed to be lifetime (i.e., there is no post-exposure period)" (U.S. EPA. 2002) (pg. 3-
27). In other words, the nature of an RfC precludes the possibility of recovery of the critical effect
This supports the choice of the principal study even when all aspects of the pain sensitivity
phenotype were identified as transient, which, notably, does not appear to be the case. Ultimately, a
consideration of the entire body of evidence for decreased pain sensitivity supports the conclusion
that exposure to TMB isomers elicits a long-lasting, non-reversible insult to the CNS.
Taken as a whole, the database supports the characterization of decreased pain sensitivity
associated with exposure to TMBs as relevant to human health. Given the consistency of
observations from hot plate tests with or without footshock challenge across several studies from
the same research group using multiple durations of exposure in male Wistar rats, the abundant
supportive evidence of neurotoxic effects at similar concentrations across a range of behavioral
measures and exposure durations, as well as the evidence and biological plausibility of similarities
in neurological effects between rats and humans, there is strong evidence that neurotoxicity is the
primary hazard associated with exposure to TMB isomers. Based on the above considerations,
Korsak and Rydzyriski (1996) was selected as the principal study for derivation of the RfC for TMBs,
and decreased pain sensitivity measured immediately after subchronic exposure is identified as an
adverse neurotoxic effect and is thus an appropriate critical effect on which to base the RfC.
Unfortunately, there are no subchronic data pertaining to the neurotoxicity of 1,3,5-TMB. However,
the available evidence regarding toxicokinetic and toxicological similarities between the isomers
supports the assumption that decreased pain sensitivity is also a concern of exposure to 1,3,5-TMB.
Additionally, although decreased pain sensitivity was observed in multiple short-term studies
(Wiaderna etal.. 2002: Gralewicz and Wiaderna. 2001: Wiaderna et al.. 1998: Gralewicz etal..
1997b). those studies used a shorter exposure duration of 28 days and were determined to be less
suitable for the derivation of chronic RfCs (as stated above). However, these studies qualitatively
contribute to the determination that neurotoxicity is the most consistently observed effect in the
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TMB toxicity database, and support the determination that Korsak and Rvdzvnski (1996) is the
most appropriate principal study for derivation of the RfC, as it is the only subchronic study with
quantitative data appropriate for dose-response evaluation investigating the neurotoxic effects of
individual TMB isomers. Therefore, the RfC for neurotoxicity, based on decreased pain sensitivity
from Korsak and Rvdzvnski T1996I was selected as the RfC for TMBs.
A PODhec of 18.15 mg/m3 for decreased pain sensitivity following exposure to 1,2,4-TMB
(Korsak and Rvdzvnski. 19961 was used as the POD from which to derive the chronic RfC for TMBs
(see Table 2-6). The PODhec for 1,2,4-TMB was selected over that of 1,2,3-TMB due to increased
confidence in that value given that it was calculated via the application of a validated PBPK model,
whereas the 1,2,3-TMB value was estimated using default dosimetric methods. However, this
distinction is largely academic, as the candidate RfCs for the two isomers are almost identical
(6 x 10"2 mg/m3 vs. 5 x 10~2 mg/m3, respectively) after application of UFs. The UFs, selected and
applied in accordance with the procedures described in EPA's A Review of the Reference Dose and
Reference Concentration Processes (U.S. EPA. 2002) (Section 4.4.5 of the report), were discussed
previously in Section 2.1.3. Application of the composite UF of 300 to the PODhec yields the
following chronic RfC for TMBs:
RfC = PODhec tUF = 18.15 mg/m3 -r 300 = 0.0605 mg/m3 = 6 x 10"2 mg/m3 (rounded
to one significant digit)
2.1.6. Uncertainties in the Derivation of the Reference Concentration for TMBs
As presented above, the UF approach, following EPA practices and RfC guidance (U.S. EPA.
2002.1994b). was applied to the PODhec for 1,2,4-TMB in order to derive the chronic RfC for all
TMB isomers. Factors accounting for uncertainties associated with a number of steps in the
analyses were adopted to account for extrapolation from animals to humans, a diverse human
population of varying susceptibilities, duration of exposure, POD determination methodologies
(NOAEL, LOAEL, or BMDL), and database deficiencies.
The critical effect selected, decreased pain sensitivity, does not introduce substantial
uncertainty into the RfC calculation, as selection of alternative hematological effects would result in
RfCs that would be equivalent when rounding to one significant digit (i.e., 6 x 10~2 mg/m3, see
Figure 2-1). The two isomer-specific RfC values for 1,2,4-TMB and 1,2,3-TMB are identical to one
another. While it may seem inconsequential as to which value to ultimately select as the RfC for all
TMB isomers, there is increased confidence in the 1,2,4-TMB value regarding its calculation via a
PBPK model rather than the use of default dosimetric methods. Additionally, there is some utility in
selecting the RfC for 1,2,4-TMB as the final RfC given its use in ultimately calculating the RfD for
TMBs (see Section 2.2.3). There is some uncertainty in adopting this RfC for 1,3,5-TMB in light of
the lack of subchronic neurotoxicity data with which to calculate an isomer-specific RfC for 1,3,5-
TMB. However, as stated above in Section 2.1.5,1,3,5-TMB shares many commonalities and
similarities with 1,2,4-TMB and 1,2,3-TMB regarding their chemical, toxicokinetic, and toxicological
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properties that support the adoption of the value of one isomer for the other. The majority of
uncertainty regarding 1,3,5-TMB's database involves the lack of a chronic, subchronic, or multi-
generational reproductive study for this isomer. Given the similarities in toxicity from the
developmental toxicity study, and neurotoxicity and respiratory toxicity observed in the available
acute and short-term studies, there is strong evidence that the toxicity resulting from subchronic
exposure to any isomer can be expected to be similar. Regarding the selection of the DAF and dose
metric for respiratory effects due to 1,2,3-TMB or 1,2,4-TMB exposure (respectively), there is some
uncertainty regarding whether these endpoints are due to localized effects or to systemic
redelivery of the toxic moiety to the pulmonary region. However, there is evidence that TMBs act
as acute respiratory irritants and induce inflammatory responses following longer exposures.
These observations support the conclusion that TMB-induced respiratory effects most likely act as
portal-of-entry effects within the pulmonary region.
Some uncertainty exists regarding the selection of the BMRs for use in BMD modeling due to
the absence of information to determine the biologically significant level of response associated
with the endpoints. In cases such as this, the selection of a BMR of 1 SD for continuous endpoints is
supported by EPA guidance (U.S. EPA. 20121: using a BMR of 1 SD assumes that a change of that
magnitude constitutes a minimally biologically significant response. Using decreased pain
sensitivity as an example, there is a lack of information as to what constitutes a biologically
significant response and statistical significance testing was not definitive in identifying exposure
levels that may be considered biologically significant Given the uncertainty in whether a change of
1 SD is biologically significant, one option would be to use a BMR equal to 0.5 SD. For decreased
pain sensitivity, using 0.5 SD as the BMR would lower the POD by 50%. However, there is no
compelling evidence that reducing the BMR to 0.5 SD is necessary and doing so may be overly
conservative. Therefore, for the purposes of this assessment, a BMR of 1 SD change is used for
endpoints lacking information on what constitutes a biologically significant response.
Uncertainty regarding the selection of particular models for individual endpoints exists, as
selection of alternative models could decrease or increase the estimated POD and consequently, the
RfC. The selection criteria for model selection was based on a practical approach as described in
EPA's Benchmark Dose Technical Guidance (U.S. EPA. 20121. Uncertainty may exist in the PBPK
model estimates of internal blood dose metrics for the rat, and subsequent HEC calculations for
humans, including parameter uncertainty, but such uncertainties would apply equally to all
endpoints. Equivalently, any uncertainty regarding default dosimetric adjustments for 1,2,3-TMB
and 1,3,5-TMB endpoints would apply equally to all endpoints for each individual endpoint There
is some uncertainty in comparing the isomer-specific RfC values calculated via PBPK methods and
default dosimetric methods. However, this uncertainty does not seem to be overly concerning as
the two RfC values for 1,2,4-TMB and 1,2,3-TMB are identical to one another. The RfC value for
1,2,4-TMB was ultimately selected for use as the overarching RfC value for TMBs due to slightly
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more confidence in the validated PBPK model versus default dosimetric adjustments, and its utility
in route-to-route conversions for calculating RfD values.
2.1.7. Confidence Statement for the Reference Concentration for TMBs
A confidence level of high, medium, or low is assigned to the study used to derive the RfC,
the overall database, and the RfC itself, as described in Section 4.3.9.2 of EPA's Methods for
Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA.
1994b).
Confidence in the study from which the critical effect was identified, Korsak and Rydzvhski
(1996). is low to medium. The study is a peer-reviewed study that utilized three dose groups plus
untreated controls, employed an appropriate number of animals per dose group, and performed
appropriate statistical analyses. However, sources of uncertainty exist that reduce confidence in
this study.
One area of uncertainty regarding this study is the lack of reported actual concentrations.
However, as the methods by which the test atmosphere was generated and analyzed were reported
in sufficient detail, and given the fact that this laboratory has used this methodology in subsequent
studies (Korsak et al.. 2000a. b) and achieved appropriate actual concentrations (i.e., within 10% of
target concentrations), the concern regarding the lack of reported actual concentrations is minimal.
Another source of uncertainty is the fact that the Korsak and Rvdzvriski f!9961 study does not
explicitly state that the reported measures of variance in Table 1 of that reference are SDs.
However, careful analysis of the reported levels of variance and magnitude of statistical significance
reported indicate that the measures of variance are SDs. Supporting this conclusions is the
observation that all other papers by Korsak and colleagues (Korsak et al.. 2000a. b; Korsak etal..
1997: Korsak etal.. 1995) report variance as SDs. The critical effect on which the RfC is based is
well-supported, as the weight of evidence for TMB-induced neurotoxicity is coherent across species
(i.e., human and rat), coherent across isomers, and consistent across multiple exposure durations
(i.e., acute, short-term, and subchronic) and outcome measures (Gralewicz and Wiaderna. 2001:
Chen etal.. 1999: Wiaderna etal.. 1998: Gralewicz etal.. 1997b: Gralewicz etal.. 1997a: Korsak and
Rydzvhski. 1996: Norseth etal.. 19911.
The database for TMBs includes acute, short-term, subchronic, and developmental toxicity
studies in rats and mice. However, confidence in the overall database is low to medium because it
lacks chronic, multi-generation reproductive/developmental, and developmental neurotoxicity
studies, and the studies supporting the critical effect predominantly come from the same research
institute. The overall confidence in the RfC for TMBs is low to medium.
2.1.8. Calculation of Subchronic Reference Concentrations for TMBs
In addition to providing RfCs for chronic exposures in multiple systems, this document also
provides an RfC for subchronic- duration exposures. In the case of TMBs, all of the studies used to
calculate the chronic RfCs were subchronic or gestational in duration. Therefore, the methods to
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calculate subchronic RfCs is identical to that used for calculation of chronic RfCs, minus the
application of a subchronic-to-chronic UF (see Table 2-8). The individual organs and systems for
which specific subchronic RfC values were derived were the neurological, hematological, and
respiratory systems, along with specific RfCs derived for the pregnant animal (maternal) and
developing fetus (developmental). The subchronic RfC values for the neurological system, based on
decreased pain sensitivity following exposure to 1,2,4-TMB or 1,2,3-TMB, were identical (2 x 101
mg/m3). The RfC for 1,2,4-TMB was selected for the overall subchronic RfC for TMBs for the
reasons outlined in Section 2.1.5. The subchronic RfC values for the hematological system (based on
decreased clotting time for 1,2,4-TMB and decreased segmented neutrophils for 1,2,3-TMB) were
identical to those calculated for neurological effects. The respiratory subchronic RfCs were
somewhat higher compared to the neurological and respiratory RfCs: 6 x 101 mg/m3. This
generally indicates that effects in all of these systems may be of concern at similar levels of
environmental exposure. However, effects to pregnant animals and the developing fetus may be of
less concern as the subchronic RfCs for these types of effects (based on decreased maternal weight
gain and decreased male and female fetal weight, respectively) are much higher (5- to 40-fold
higher) than those derived for the neurological or hematological endpoints. It should be noted that
the subchronic RfC values for the developing fetus are identical to the RfCs derived in Sections 2.1.3
and 2.1.4 as gestation represents a critical window of susceptibility and no UFs was applied to
account for less than chronic exposure in either case. The subchronic inhalation RfC is intended for
use with exposures for more than 30 days, up to approximately 10% of the lifespan in humans.
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Table 2-8. Summary of derivation of subchronic RfC values for TMBs
Effect
Isomer
Basis
RfC
(mg/m3)
Composite
UF
Exposure
Description
Confidence
Neurological
1,2,4-TMB
Decreased pain
sensitivity
2 x 10"1
100
Subchronic
Low to medium
1,2,3-TMB
2 x 10"1
100
Subchronic
Low to medium
Hematological
1,2,4-TMB
Decreased
clotting time
2 x 10"1
100
Subchronic
Low to medium
1,2,3-TMB
Decreased
segmented
neutrophils
2 x 10"1
100
Subchronic
Low to medium
Respiratory
1,2,4-TMB
Inflammatory
lung lesions
6 x 10"1
100
Subchronic
Low to medium
1,2,3-TMB
6 x 10"1
100
Subchronic
Low to medium
Developmental
1,2,4-TMB
Fetal weight
4
100
Gestational
Low to medium
1,3,5-TMB
4
100
Gestational
Low to medium
Maternal
1,2,4-TMB
Decreased
maternal
weight
8
100
Subchronic
Low to medium
1,3,5-TMB
1
100
Subchronic
Low to medium
Subchronic
overall RfC
(Neurological)
1,2,4-TMB
Decreased
pain
sensitivity
2 x 10"1
100
Subchronic
Low to medium
2.2. ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER FOR
TMBs
The RfD (expressed in units of mg/kg-day) is defined as an estimate (with uncertainty
spanning perhaps an order of magnitude) of a daily exposure to the human population (including
sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a
lifetime. It can be derived from a NOAEL, a LOAEL, or a 95% lower bound on the benchmark dose
(BMDL), with UFs generally applied to reflect limitations of the data used.
2.2.1. Identification of Studies and Effects for Dose-Response Analysis and Derivation of
Reference Doses for TMBs
No chronic or subchronic studies were identified for any isomer of TMB that utilized the
oral route of exposure. However, one subchronic study (Adenuga etal.. 2014) investigated the oral
toxicity of 1,3,5-TMB (this study is a peer-reviewed summary report of the more detailed Koch
Industries Q995bl report). This study investigated general toxicity endpoints in rats exposed to
1,3,5-TMB via gavage, including organ weight changes, clinical chemistry parameters, and
hematological endpoints. Data from this study pertaining to the hazards observed in animals
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(hematological toxicity) were considered as a critical effect for the purpose of determining the POD
for derivation of the oral RfD for TMB isomers. The Adenugaetal. (2014) study is adequate for
dose-response analysis. This study used an appropriate laboratory animal species, and utilized
appropriate sham-exposed controls. Animals were exposed to 1,3,5-TMB, reported as 99.2% pure
(1,2,4-TMB reported as impurity at 0.7% fKoch Industries. 1995bll. This study utilized an
appropriate route (gavage) and duration (subchronic) of expose. This study also used a reasonable
range of appropriately-spaced exposure levels to facilitate dose-response analysis. An appropriate
latency between exposure and development of toxicological outcomes was used, and the
persistency of some outcomes after termination of exposure was investigated. Adequate numbers
of animals per exposure group were used, and appropriate statistical tests (pair-wise comparisons)
were performed. Regarding the reporting of exposure methodology, the study reported actual
concentrations in mg/mL for the low and high exposure groups (10 and 120 mg/mL) at the
beginning, middle, and end of the exposure period (weekly values given in Koch Industries
(1995b)). Actual concentrations were given for weeks 1 and 2 for the middle exposure group (40
mg/mL). No actual dosages in mg/kg were given for any of the exposure groups (expressed as 50,
200, and 600 mg/kg). Although no actual dosage information was given, all of the reported test
article concentrations in mg/mL were within 10% of the target concentrations, which increases the
confidence in the overall evaluation and adequacy of this study.
This study examined 1,3,5-TMB-induced toxicity in multiple target organs/systems and any
endpointthat demonstrated statistically significant increases or decreases relative to control were
considered for the derivation of the RfD for TMBs. A number of endpoints possibly indicating
compensatory changes rather than adverse effects were not considered for the RfD derivation.
These endpoints include changes in various clinical chemistry parameters and kidney and liver
weights. These changes were considered to be possibly compensatory in nature given the lack of
accompanying histopathological changes in the relevant organs. Discounting these endpoints left
an observed increase in monocytes in male rats as the only statistically significant effect (0.1 ± 0.09
[0 mg/kg-day], 0.2 ± 0.09 [50 mg/kg-day], 0.3 ± 0.17 [200 mg/kg-day], 0.2 ± 0.18 [600 mg/kg-day],
units = x 106/mm3). Although a slight increase in monocytes may be of questionable adversity if
taken out of context of the TMB database, a number of endpoints involving the alteration of WBC
counts have been observed in the inhalation toxicity database. Given that, it was deemed that the
observed increase in monocytes following oral exposures was possibly indicative of an underlying
toxicity to the hematological system also evident following inhalation exposure.
2.2.2. Methods of Analysis for Derivation of Reference Doses for TMBs
This assessment uses the BMD approach to estimate a POD for the derivation of an RfD for
TMBs. The BMD approach involves fitting a suite of mathematical models to the observed dose-
response data using EPA's BMDS (version 2.6.0.1). Each fitted model estimates a BMD and its
associated BMDL corresponding to a selected BMR For continuous data (i.e., increased monocytes)
from the Adenuga etal. f20141 study, no information is available regarding the change in these
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responses that would be considered biologically significant; thus, a BMR equal to a 1 SD change in
the control mean was used in modeling these endpoints, consistent with EPA's Benchmark Dose
Technical Guidance fU.S. EPA. 20121. A1 SD shift in the control mean roughly corresponds to a 10%
increase in the number of animals responding adversely (i.e., number animals either falling below
the 2nd percentile or above the 98th percentile for decreasing and increasing responses,
respectively) fU.S. EPA. 20121. Thus, using a 1 SD change in response approximates a BMR based
on 10% extra risk, partially harmonizing BMR selection for continuous and dichotomous endpoints.
The estimated BMDL is then used as the POD for deriving the RfD. The suitability of the above
methods to determine a POD is dependent on the nature of the toxicity database for a specific
chemical. However, no issues were encountered when modeling the dose-response data for
increased monocytes. Detailed modeling methodology and results are provided in Appendix D. For
increased monocytes, the best-fitting model was the Exponential 4 model (using non-constant
variance), which returned BMD and BMDL values of 51.99 and 13.92 mg/kg-day, respectively. The
BMDL value is subsequently used as the POD.
Because an RfD is a toxicity value that assumes continuous human inhalation exposure over
a lifetime, data derived from inhalation studies in animals need to be adjusted to account for the
non-continuous exposures used in these studies. In the Adenuga et al. f20141 study, rats were
exposed to 1,3,5-TMB via gavage 5 days/week for 90/91 days. Because no PBPK model exists for
1,3,5-TMB, the duration-adjusted POD for increased monocytes in male rats was calculated as
follows:
PODadj (mg/kg-day) = POD (mg/kg-day) x days exposed per week/7 days
Therefore, for increased monocytes from Adenuga etal. f 20141. the PODadj would be
calculated as follows:
PODadj = 13.92 mg/kg-day x 5 days/7 days = 9.94 mg/kg-day
Because increased monocytes results from systemic distribution of 1,3,5-TMB, and no PBPK
model exists for this isomer, the human equivalent dose (HED) for 1,3,5-TMB was calculated by
allometrically scaling the PODadj by a DAF equal to the ratio of animal and human body weights,
scaled to the % power (U.S. EPA. 20111. In other words, the DAF is:
DAF = (BWa/BWh)1-P/fl
Where BWa and BWh are the animal and human body weights, respectively. At the end of
the exposure period, male rats in the Adenuga etal. (20141 study weighed an average of 0.605 kg
across all exposure groups (detailed body weights provided in Koch Industries (1995bll: a default
human body weight of 70 kg was used. Therefore, the DAF for increased monocytes was:
DAF = (0.605/70)025 = 0.305
Calculation of the PODhec would then follow as:
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PODhed = PODadj * DAF = 9.94 mg/kg-day x 0.305 = 3.03 mg/kg-day
2.2.3. Derivation of the Reference Dose for TMBs
A PODhed of 3.03 mg/kg-day was derived for the oral database using default dosimetric
adjustment (allometric scaling by body weight) based on hematological effects (i.e., increased
monocytes) observed by Adenuga etal. f 20141 following gavage administration. The UFs, selected
and applied in accordance with the procedures described in EPA's A Review of the Reference Dose
and Reference Concentration Processes (U.S. EPA. 2002) (Section 4.4.5 of the report), also described
in the Preamble, address five possible areas of uncertainty and variability were considered in
deriving the candidate oral values for TMBs. An explanation of these five possible areas of
uncertainty and variability and the values assigned to each as a designated UF to be applied to the
PODhed are as follows.
AUFAof 3 (101/2 = 3.16, rounded to 3) was applied to account for uncertainty in
characterizing the toxicokinetic and toxicodynamic differences between rats and humans following
oral exposure to TMB isomers. In this assessment, the use of default dosimetric methods to
extrapolate oral doses from rats to humans reduces the toxicokinetic uncertainty for 1,3,5-TMB to
the same degree. However, neither of these methods accounts for the possibility that humans may
be more sensitive to TMB due to interspecies differences in toxicodynamics. Therefore, a UFa of 3
was thus applied to account for this remaining toxicodynamic and any residual toxicokinetic
uncertainty not accounted for by application of the default dosimetric methods.
A UFh of 10 was applied to account for potentially susceptible individuals in the absence of
data evaluating variability of response in the human population following inhalation of TMB
isomers. No information is currently available to predict potential variability in human
susceptibility, including variability in the expression of enzymes involved in TMB metabolism.
A UFl of 1 was applied because the current approach is to address this factor as one of the
considerations in selecting a BMR for BMD modeling. In this case, BMRs were preferentially
selected based on biological information on what constitutes a biologically significant change for
these effects. When no information was available to make assumptions about what constitutes a
minimal, biologically significant response, a BMR equal to a 1 SD change in the control mean was
selected.
A UFs of 3 (101/2 = 3.16, rounded to 3) was applied to account for extrapolation from a
subchronic exposure duration study to derive a chronic RfD. The 3-fold UF is applied to the POD
identified from the subchronic study on the assumption that effects observed in a similar chronic
study would be observed at lower RfD concentrations for a number of possible reasons, including
potential cumulative damage occurring over the duration of the chronic study or an increase in the
magnitude or severity of effect with increasing duration of exposure. However, evidence from
inhalation toxicokinetic studies and PBPK modeling of inhalation data indicate that blood and organ
concentrations of TMBs are similar following repeated vs. acute exposures (approximately 600
hours vs. 6 hours, respectively; see Table C-9). It is reasonable to assume similar behavior following
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oral exposures. This reduces, but does not completely remove, the concern that longer oral
exposures may result in adverse effects at lower doses. As such, a UFs = 3 was applied to account
for the possibility of a lower POD following chronic exposures.
A UFd of 3 (101/2 = 3.16, rounded to 3) was applied to account for database deficiencies.
Strengths of the database include a single well-designed subchronic study that observed exposure-
response effects in Wistar rats exposed to 1,3,5-TMB via gavage. Beyond this study, however, there
is a lack of any other studies in the oral TMB database, including no neurotoxicity, developmental
neurotoxicity, or reproductive/developmental toxicity studies. The lack of these types of studies is
a potentially serious weakness in the TMB oral database. A chemical-specific deficiency in the oral
TMB database is the lack of neurotoxicity study. Given the ample evidence of neurotoxicity
following inhalation exposure to TMB isomers in rats, it is reasonable to expect that neurotoxicity
would also be a concern following oral exposures. However, the PODs for hematological effects in
the inhalation database (16.65 mg/m3 [decreased segmented neutrophils, 1,2,3-TMB] and
24.30 mg/m3 [decreased clotting time, 1,2,4-TMB]) were similar to the POD for decreased pain
sensitivity (18.15 and 16.32 mg/m3) for 1,2,4-TMB and 1,2,3-TMB, respectively. After application
of UFs, the RfCs for all of the above inhalation effects were similar: 6 x 10 2 to 8 x 10 2 mg/m3. This
somewhat alleviates the concern that the availability of a neurotoxicity study in the TMB oral
database would result in a substantially lower POD currently available for decreased monocytes.
EPA's A Review of the Reference Dose and Reference Concentration Processes fU.S. EPA. 20021
recommends that the database UF take into consideration whether there is concern from the
available toxicology database that the developing organism may be particularly susceptible to
effects in specific organs/systems. TMBs (unspecified isomer) are able to cross the placenta
(Cooper etal.. 2001: Dowtv etal.. 1976): therefore, some degree of developmental toxicity is
expected as it was observed following inhalation exposure. While general developmental toxicity is
not expected to be a concern, as those types of endpoints (e.g., decreased fetal weight) were
observed at concentrations much higher than effects observed in other systems, including the
nervous system, there is also the possibility that exposure to TMB isomers may result in
neurotoxicity in the developing organism. EPA's Guidelines for Neurotoxicity Risk Assessment (U.S.
EPA. 1998) identifies specific effects observed in adult animals (e.g., cognitive and motor function)
that can also affect the developing organism exposed in utero. The Neurotoxicity Guidelines (U.S.
EPA. 19981 also indicate that neurotoxicants may have greater access to the nervous system in
developing organisms due to an incomplete blood-brain barrier and immature metabolic
detoxifying pathways. Lastly, EPA's A Review of the Reference Dose and Reference Concentration
Processes (U.S. EPA. 2002) also states that effects that may be mild or reversible in adults may
produce more robust or permanent effects in offspring following developmental exposures.
However, as discussed in Section 2.1.3, while the developing brain is potentially at greater risk of
permanent injury due to exposure to neurotoxicants, there is uncertainty in the TMB and related
compound database whether sufficient amounts of the toxic agent cross the placenta to elicit effects
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and whether the concentrations necessary to elicit effects are lower than those that result in
neurotoxicity in the adult organism. There is evidence from perinatal inhalation toxicity studies in
rats that low-level exposures to toluene (as low as 5 ppm) early in life (PNDs 4-12) may disrupt
developmental processes that began early in embryogenesis (neuronal proliferation,
synaptogenesis, myelination, etc.) and result in neurotoxicity (decrements in spatial learning)
fWin-Shwe etal.. 2010: Win-Shwe and Fuiimaki. 20101. Therefore, concern still exists regarding
the possibility that exposure to TMB may result in developmental neurotoxicity, possibly at levels
lower than neurotoxicity in adult animals. In summary, a 3-fold database UF was applied to
account primarily for the lack of both an adult and developmental neurotoxicity studies in the oral
database for TMB isomers.
While the derivation of organ- or system-specific RfDs may be useful for subsequent
cumulative risk assessments that consider the combined effect of multiple agents acting at a
common site, only one adverse effect was identified as a critical effect in the available oral TMB
database. Thus, only one chronic RfD was derived for TMBs via the application of the 300-fold
composite UF (see above), based on increased monocytes following exposure to 1,3,5-TMB, as
follows:
RfD = PODhed tUF = 3.01 mg/kg-day -r 300 = 0.0101 mg/kg-day = lx 10-2 mg/kg-day
(rounded to one significant digit)
While Adenuga et al. (2014) is a well-conducted study that evaluates a wide range of
general toxicity endpoints in multiple organs/systems, the oral TMB database suffers from the lack
of a neurotoxicity study. It is clear from the inhalation database for TMB that neurotoxicity is a
critical endpoint for derivation of reference values. While Adenuga et al. f20141 (and the earlier
Koch Industries (1995b) industry report) do report some clinical signs possibly indicative of
neurotoxicity, they are too general to be used in the quantitative derivation of an RfD. However, the
consistency with which neurotoxicity is observed in the TMB database, across all isomers following
acute oral and acute, short-term, and subchronic inhalation exposures, is quite strong. Ultimately,
the fact that oral and inhalation neurotoxic endpoints are qualitatively comparable, and that
neurotoxic endpoints resulted in the most strongly supported RfCs in the inhalation database, it is
reasonable to expect that neurotoxicity-based PODs would be important when calculating oral
RfDs. Therefore, the available PBPK model for 1,2,4-TMB was used to perform a route-to-route
extrapolation.
A route-to-route extrapolation from inhalation to oral for the purposes of deriving an RfD is
possible using the existing inhalation data and the available 1,2,4-TMB PBPK model (Hissink etal..
2007). and was conducted to address the lack of suitable neurotoxicity data in the oral TMB
database. The Hissink etal. f20071 model was chosen as an appropriate model because it was the
only published 1,2,4-TMB model that included parameterization for both rats and humans, the
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model code was available, and the model adequately predicted experimental data in the dose range
of interest. Using route-to-route extrapolation via application of PBPK models is supported by EPA
guidance fU.S. EPA. 2002.1994b) given enough data and the ability to interpret that data with
regard to differential metabolism and toxicity between different routes of exposure. The available
database for 1,2,4-TMB supports the use of route-to-route extrapolation; sufficient evidence exists
that demonstrates similar qualitative profiles of metabolism (i.e., observation of dimethylbenzoic
and hippuric acid metabolites) and patterns of parent compound distribution across exposure
routes (Section C.2, Appendix C). Further, no evidence exists that would suggest that toxicity
profiles would differ to a substantial degree between oral and inhalation exposures. In fact, in acute
oral studies in rats fTomas etal.. 1999a: Tomas etal.. 1999bl. the observed neurotoxic effects of
exposure to 1,2,4-TMB (i.e., alterations in motor function and electrocortical activity) are similar to
effects observed following short-term exposures to 1,2,4-TMB via inhalation. This consistency of
effect is also true for 1,3,5-TMB. Additional evidence of concordance of effects within the inhalation
database is the similarity in types and magnitude of effects (i.e., decreased fetal weight) observed in
rats exposed gestationally to either 1,2,4-TMB or 1,3,5-TMB (Saillenfaitetal.. 20051 (see Section
1.2.7 for a full description of the toxicological and toxicokinetic similarities among the TMB
isomers). Lastly, it is reasonable to assume that had Adenuga etal. f 20141 investigated more
subtle markers of neurotoxicity, they would have been observed as the PBPK model-predicted
blood TMB levels were approximately 10-fold higher when estimated using the exposure paradigm
in Adenuga et al. (20141 compared to blood levels estimated from the inhalation exposures used in
Korsak and Rydzvhski Q9961.
Therefore, assuming that oral exposure would result in the same systemic effect as
inhalation exposure (i.e., altered central nervous system [CNS] function, measured as decreased
pain sensitivity fKorsak and Rvdzvriski. 199611. an oral exposure component was added to the
Hissink etal. (20071 PBPK model by EPA (Section C.2.3.5, Appendix C), assuming 100% absorption
of the ingested 1,2,4-TMB by constant infusion of the oral dose into the liver. This is a common
assumption when information about the oral absorption of the compound is unknown. The
contribution of the first-pass metabolism in the liver for oral dosing was evaluated by simulating
steady-state venous blood levels (at the end of 50 days of continuous exposure) for an average
human at rest (70 kg) for a range of concentrations and doses; at low daily doses
(0.1-10 mg/kg-day), equivalent inhalation concentrations result in steady-state blood
concentrations 4-fold higher than those resulting from oral doses, indicating the presence of first-
pass metabolism following oral exposure (see Figure C-18, Appendix C). This difference became
insignificant for daily doses exceeding 50 mg/kg-day. To more accurately approximate patterns of
human oral ingestion, ingestion was simulated as an idealized pattern of six events, each lasting
30 minutes. Twenty-five percent of the total daily dose was assumed to be ingested at each of three
events beginning at 7 am, 12 pm (noon), and 6 pm (total of 75%). Ten percent of the daily dose was
assumed to be ingested at events beginning at 10 am and 3 pm (total of 20%). The final 5% was
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assumed to be ingested in an event beginning at 10 pm. After the daily blood concentration profile
achieved a repeating pattern, or periodicity, the weekly average blood concentration was then used
to determine the HED.
The human PBPK model inhalation dose metric (weekly average blood concentration,
mg/L) for the PODadj (0.099 mg/L) for decreased pain sensitivity was used as the target for the oral
dose metric. The human PBPK model was run to determine what oral exposure would yield an
equivalent weekly average blood concentration, and the resulting value of 3.5 mg/kg-day was then
used as the PODhed for the RfD derivation, as follows:
RfD = PODhed tUF = 3.5 mg/kg-day -r 300 = 0.0117 mg/kg-day = lx 10-2 mg/kg-day
(rounded to one significant digit)
This calculated value of 1 x 10~2 mg/kg-day is equal to the RfD calculated from the oral
1,3,5-TMB data for increased monocytes. This is consistent with what was observed in the
inhalation database. The RfCs for hematotoxicity endpoints (decreased segmented neutrophils for
1.2.3-TMB [6 x 10"2 mg/m3] and decreased clotting time for 1,2,4-TMB [8 x 10~2 mg/m3]) were
identical or similar to the value estimated for decreased pain sensitivity following exposure to
1.2.4-TMB or 1,2,3-TMB (5 x 10~2 to 6 x 10~2 mg/m3). This indicates that some endpoints in the
hematological system are equally as sensitive to exposure to TMB isomers as endpoints in the
nervous system. Further supporting this conclusion is the fact that a route-to-route extrapolation
of the most sensitive hematological effect following inhalation exposure to 1,2,4-TMB (decreased
clotting time, PODadj = 0.1335 mg/L) results in a PODhed of 4.3 mg/kg-day and a final RfD of 1 x 10~2
mg/kg-day (after application of the composite 300-fold UF). Confidence in the route-to-route
derived RfD is additionally strengthened given that the PBPK model used is a well-characterized
model determined to be appropriate for this assessment Ultimately choosing the route-to-route
neurotoxicity-based RfD (based on 1,2,4-TMB) as the RfD for all TMB isomers is supported by
multiple lines of evidence in the oral and inhalation database. First, all three isomers are observed
to elicit similar neurotoxic effects in rats in acute oral and inhalation studies and short-term and
subchronic inhalation studies. For example, following inhalation exposure, while 1,2,3-TMB is
observed to decrease pain sensitivity at lower concentrations than 1,2,4-TMB (LOAEL values of
123 versus 492 mg/m3, respectively), the magnitude of decreased pain sensitivity is similar for
1,2,4-TMB and 1,2,3-TMB, especially at the low- and mid-concentrations. This similarity of effect in
the low-dose region of the dose-response curve is exhibited by identical RfC values derived from
isomer-specific data: 6 x 10~2 mg/m3. Similarities in blood:air and tissue:air partition coefficients
and absorption into the bloodstream between TMB isomers support the conclusion that internal
blood dose metrics for 1,3,5-TMB and 1,2,3-TMB would be similar to those calculated for 1,2,4-TMB
using the available PBPK model. Also, the qualitative metabolic profiles for the two isomers are
similar, with dimethylhippuric acids being the major terminal metabolite for both isomers, so that
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first-pass metabolism through the liver is not expected to differ greatly between the three isomers.
Based on these considerations, and the confidence in the route-to-route extrapolation, the
RfD for TMBs is 1 x 10"2 mg/kg-day, based on decreased pain sensitivity observed in rats
exposed to 1,2,4-TMB.
2.2.4. Uncertainties in the Derivation of the Reference Dose for TMBs
As the oral RfD for TMBs was based on a route-to-route extrapolation in order to determine
the oral dose that would result in the same effect (i.e., decreased pain sensitivity) as inhalation
exposure in Korsak and Rvdzyhski (1996). many of the uncertainties regarding this derivation are
the same as those for the RfC for TMBs (see Section 2.1.6), with the exception of the uncertainty
surrounding the route-to-route extrapolation. The model used to perform this route-to-route
extrapolation is a well-characterized model considered appropriate for the purposes of this
assessment. One source of uncertainty regarding the route-to-route extrapolation is the
assumption of 100% bioavailability (i.e., 100% of the ingested 1,2,4-TMB would be absorbed and
passed through the liver). If not all of the compound is bioavailable, a lower blood concentration
would be expected compared to the current estimate, and thus, a higher RfD would be calculated.
Although there is uncertainty surrounding the assumption of 100% bioavailability, this is a
common assumption when data on oral absorption are unknown. Further, the use of this
assumption results in an RfD value that is lower than one that would be derived using a
bioavailability less than 100%. Therefore, even in the consideration of the uncertainty surrounding
this decision, the assumption of 100% bioavailability results in a health- protective RfD. There is
additionally some uncertainty about adopting the RfD based on the route-to-route extrapolation of
decreased pain sensitivity in rats exposed to 1,2,4-TMB for the RfD for both 1,3,5-TMB and
1,2,3-TMB. However, as discussed above (Section 2.1.6), all three TMB isomers share multiple
commonalities and similarities regarding their chemical, toxicokinetic, and toxicological properties
that support adopting one isomer's value for the other.
2.2.5. Confidence Statement for the Reference Dose for TMB
The confidence in the oral database for TMB is low as it only contains acute oral studies
investigating neurotoxicity endpoints for multiple isomers, and one subchronic study investigating
general toxicity endpoints for one isomer (1,3,5-TMB). This database was used to derive an RfD,
but given the concern over the lack of a suitable neurotoxicity study, the confidence in this RfD is
low. A PBPK model was utilized to perform a route-to-route extrapolation to determine a POD for
the derivation of the RfD from the Korsak and Rvdzyhski (1996) inhalation study and
corresponding critical effect. The confidence in the study from which the critical effect was
identified, Korsak and Rvdzyhski (1996). is low to medium (see Section 2.1.7). The inhalation
database for 1,2,4-TMB includes acute, short-term, subchronic, and developmental toxicity studies
in rats and mice. However, confidence in the database for TMB is low to medium because it lacks
chronic, multi-generation reproductive/developmental, and developmental neurotoxicity studies,
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and the studies supporting the critical effect predominantly come from the same research institute.
Reflecting the confidence in the study and the database and the uncertainty surrounding the
application of the available PBPK model for the purposes of a route-to-route extrapolation, the
overall confidence in the RfD for TMB is low.
2.2.6. Calculation of Subchronic Reference Doses for TMBs
In addition to providing RfDs for effects in the hematological and nervous systems, this
document also provides subchronic RfDs for less-than-lifetime exposures. In the case of TMBs, the
oral 1,3,5-TMB study and 1,2,4-TMB inhalation study used for the route-to-route extrapolation to
calculate the chronic RfDs were both subchronic duration. Therefore, the methods used to calculate
subchronic RfDs is identical to that used for calculation of chronic RfCs, minus the application of a
subchronic-to-chronic UF. This results in a composite UF of 100 (UFa = 3, UFh = 10, UFs = 1, and UFd
= 3). Dividing the POD for hematological effects (3.03 mg/kg-day) and neurotoxicity effects
(3.5 mg/kg-day) by the composite UF of 100 results in a subchronic RfD of 3 x 10~2 or 4 x 10"2
mg/kg-day for decreased monocytes and decreased pain sensitivity, respectively. The subchronic
oral RfD is intended for use with exposures for more than 30 days, up to approximately 10% of the
lifespan in humans.
2.3. CANCER RISK ESTIMATES FOR TMBs
Under the U.S. EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA. 20051. the database
for 1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB provides "inadequate information to assess carcinogenic
potential." Information available on which to base a quantitative cancer assessment is lacking, and
thus, no cancer risk estimates for either oral or inhalation exposure are derived.
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This document is a draft for review purposes only and does not constitute Agency policy.
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This document is a draft for review purposes only and does not constitute Agency policy.
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This document is a draft for review purposes only and does not constitute Agency policy.
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This document is a draft for review purposes only and does not constitute Agency policy.
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