EPA/635/R-ll/012Ab
www.epa.gov/iris
United States
E n v i r o n m e n t a 1 Prot ec I i 0 n
Agency
Toxicological Review of Trimethylbenzenes
(CAS No. 25551-13-7, 95-63-6, 526-73-8, and 108-67-8}
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
Supplemental Information
June 2012
NOTICE
This document is an External Review draft. This information is distributed solely for the
purpose of pre-dissemination peer review under applicable information quality guidelines.
It has not been formally disseminated by EPA. It does not represent and should not be
construed to represent any Agency determination or policy. It is being circulated for review
of its technical accuracy and science policy implications.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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Toxicological Review ofTrimethylbenzene
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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Toxicological Review ofTrimethylbenzene
CONTENTS
APPENDIX A. HEALTH ASSESSMENTS AND REGULATORY LIMITS BY OTHER NATIONAL
AND INTERNATIONAL HEALTH AGENCIES A-l
APPENDIX B. INFORMATION IN SUPPORT OF HAZARD IDENTIFICATION AND DOSE-
REPONSE ANALYSIS B-l
B.I. PHYSICAL AND CHEMICAL PROPERTIES B-l
B.2. TOXICOKINETICS B-2
B.2.1. Absorption B-2
B.2.2. Distribution B-3
B.2.3. Metabolism B-4
B.2.4. Excretion B-9
B.3. PHYSIOLOGICALLY-BASED PHARMACOKINETIC MODELS B-9
B.3.1. Summary of Available PBPK models for 1,2,4-TMB B-9
B.3.2. 1,2,4-TMB PBPK Model Selection B-17
B.3.3. Details of Hissink et al. (2007) Model Analysis B-18
B.3.4. Summary of Available PBPK models for 1,3,5-TMB or 1,2,3-
TMB 48
B.4. HUMAN STUDIES B-49
B.5. ANIMAL TOXICOLOGY STUDIES B-63
B.6. HUMAN TOXICOKINETIC STUDIES B-134
B.7. ANIMAL TOXICOKINETIC STUDIES B-147
B.8. ANIMAL AND HUMAN TOXICOKINETIC STUDIES B-166
APPENDIX C. DOSE-RESPONSE MODELING FOR THE DERIVATION OF REFERENCE VALUES
FOR EFFECTS OTHER THAN CANCER AND CANCER RISK ESTIMATES C-l
C.I. BENCHMARK DOSE MODELING SUMMARY C-l
C.2. BENCHMARK DOSE MODELING SUMMARY - ALTERNATIVE
ANALYSIS WITH HIGH DOSES INCLUDED C-21
APPENDIX D. DOCUMENTATION OF IMPLEMENTATION OF THE 2011 NATIONAL
RESEARCH COUNCIL RECOMMENDATIONS D-l
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Toxicological Review ofTrimethylbenzene
APPENDIX E. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS AND
EPA'S DISPOSITION E-l
REFERENCES FOR APPENDICES R-l
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Toxicological Review ofTrimethylbenzene
TABLES
Table A-l. Other national and international health agency assessments forTMBs A-l
Table B-l. Physical properties and chemical identity of 1,2,4-TMB, 1,3,5-TMB, and 1,2,3-
TMB B-l
Table B-2. Measured and calculated partition coefficients for TMB isomers at 37°C B-12
Table B-3. PBPK model parameters for 1,2,4-TMB toxicokinetics in humans using the
Jarnberg and Johanson (1999)model structure B-13
Table B-4. Comparison of rat anatomical and physiological parameters in Hissink et al.
(2007) to those of Brown et al. (1997) B-20
Table B-5. Comparison of human anatomical and physiological parameters in Hissink et al.
(2007) to those of Williams and Leggett (1989) as reported by Brown et al. (1997)
B-22
Table B-6. Comparison of chemical-specific parameters in Hissink et al. (2007) to literature
data B-23
Table B-7. Parameter values for the rat and human PBPK models for 1,2,4 TMB used by EPA
B-29
Table B-8. Rat 1,2,4-TMB kinetic studies used in model development and testing B-30
Table B-9. Model simulated and experimental measured concentrations of 1,2,4 TMB in
male Wistar rats exposed to 1,2,4-TMB B-35
Table B-10. Model simulated and experimental measured concentrations of 1,2,4-TMB in
male Sprague-Dawley rats exposed to 100 ppm (492 mg/m3) 1,2,4-TMB (12 hr/d,
for 3 d) at the end of exposure or 12 hours after the last exposure B-36
Table B-ll. Model simulated and experimental measured concentrations of 1,2,4-TMB in
male Sprague-Dawley rats exposed to 1,2,4-TMB at the end of 12 hour exposure
B-37
Table B-12. Model simulated and experimental measured concentrations of 1,2,4-TMB in
male Sprague-Dawley rats exposed to 1,000 ppm (4,920 mg/m3) 1,2,4-TMB (12
hr/d, for 14 d) at the end of exposure B-38
Table B-13. Human kinetic studies of 1,2,4-TMB used in model validation B-39
Table B-14. Parameter sensitivity for venous blood 1,2,4-TMB concentration in rats exposed
to 1,2,4-TMB via inhalation B-44
Table B-15. Parameter sensitivity for steady-state venous blood 1,2,4-TMB concentration in
humans exposed to 1,2,4-TMB via inhalation B-46
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Table B-16. Characteristics and quantitative results for epidemiologic cross-sectional study of
exposure to 1,2,4-TMB. Battig et al. (1956b), as reviewed by Baettig et al. (1958)
B-49
Table B-17. Characteristics and quantitative results for epidemiologic cross-sectional study of
exposure to 1,2,4-TMB Billionnet et al. (2011) B-51
Table B-18. Characteristics and quantitative results for epidemiologic cohort study of
exposure to 1,2,4-TMB. Chen et al. (1999) B-53
Table B-19. Characteristics and quantitative results for controlled human exposure study of
exposure to 1,2,4-TMB in WS Lammers et al. (2007) B-56
Table B-20. Characteristics and quantitative results for epidemiologic cohort study of
exposure to 1,2,4-TMB. Lee et al. (2005) B-58
Table B-21. Characteristics and quantitative results for epidemiologic cross-sectional study of
exposure to 1,2,4-TMB Norseth et al. (1991) B-60
Table B-22. Characteristics and quantitative results for epidemiologic cross-sectional study of
exposure to 1,2,4-TMB Sulkowski et al. (2002) B-62
Table B-23. Characteristics and quantitative results for Baettig et al. (1958) B-63
Table B-24. Characteristics and quantitative results for Gralewicz et al. (1997a) B-67
Table B-25. Characteristics and quantitative results for Gralewicz et al. (1997b) B-71
Table B-26. Characteristics and quantitative results for Gralewicz and Wiaderna (2001)... B-73
Table B-27. Characteristics and quantitative results for Janik-Speichowicz (1998) B-76
Table B-28. Characteristics and quantitative results for Korsak et al. (1995) B-80
Table B-29. Characteristics and quantitative results for Korsak and Rydzynski (1996) B-83
Table B-30. Characteristics and quantitative results for Korsak et al. (1997) B-87
Table B-31. Characteristics and quantitative results for Korsak et al. (2000a) B-89
Table B-32. Characteristics and quantitative results for Korsak et al. (2000b) B-94
Table B-33. Characteristics and quantitative results for Lammers et al. (2007) B-99
Table B-34. Characteristics and quantitative results for Lutz et al. (2010) B-102
Table B-35. Characteristics and quantitative results for Maltoni et al. (1997) B-105
Table B-36. Characteristics and quantitative results for McKee et al. (2010) B-107
Table B-37. Characteristics and quantitative results for Saillenfait et al. (2005) B-110
Table B-38. Characteristics and quantitative results for Tomas et al. (1999a) B-115
Table B-39. Characteristics and quantitative results for Tomas et al. (1999b) B-117
Table B-40. Characteristics and quantitative results for Tomas et al. (1999c) B-118
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Table B-41. Characteristics and quantitative results for Wiaderna et al. (1998) B-121
Table B-42. Characteristics and quantitative results for Wiaderna et al. (2002) B-124
Table B-43. Characteristics and quantitative results for Wiglusz et al. (1975a) B-126
Table B-44. Characteristics and quantitative results for Wiglusz et al. (1975b) B-130
Table B-45. Characteristics and quantitative results for Jarnberg et al. (1996) B-134
Table B-46. Characteristics and quantitative results for Jarnberg et al. (1997a) B-138
Table B-47. Characteristics and quantitative results for Jarnberg et al. (1997b) B-140
Table B-48. Characteristics and quantitative results for Jarnberg et al. (1998) B-141
Table B-49. Characteristics and quantitative results for Jones et al. (2006) B-142
Table B-50. Characteristics and quantitative results for Kostrzewski et al. (1997) B-144
Table B-51. Characteristics and quantitative results for Dahl et al. (1988) B-147
Table B-52. Characteristics and quantitative results for Eide and Zahlsen etal. (1996)....B-148
Table B-53. Characteristics and quantitative results for Huo et al. (1989) B-149
Table B-54. Characteristics and quantitative results for Mikulski and Wiglusz (1975) B-151
Table B-55. Characteristics and quantitative results for Swiercz et al. (2002) B-152
Table B-56. Characteristics and quantitative results for Swiercz et al. (2003) B-154
Table B-57. Characteristics and quantitative results for Swiercz et al. (2006) B-156
Table B-58. Characteristics and quantitative results for Tsujimoto et al. (2000) B-159
Table B-59. Characteristics and quantitative results for Tsujimoto et al. (2005) B-160
Table B-60. Characteristics and quantitative results for Tsujino et al. (2002) B-161
Table B-61. Characteristics and quantitative results for Zahlsen et al. (1990) B-163
Table B-62. Characteristics and quantitative results for Zahlsen et al. (1992) B-165
Table B-63. Characteristics and quantitative results for Meulenberg and Vijverberg (2000)166
Table C-l. Model predictions (constant variance, high dose dropped) for increased latency
to paw-lick in male Wistar rats, 1,2,4-TMB (Korsak and Rydzynski, 1996) C-3
Table C-2. Model predictions (constant variance, high dose dropped) for decreased red
blood cells in male Wistar rats, 1,2,4-TMB (Korsak et al., 2000a) C-4
Table C-3. Model predictions (constant variance, high dose dropped) for decreased clotting
time in female Wistar rats, 1,2,4-TMB (Korsak et al., 2000a) C-5
Table C-4. Model predictions (modeled variance, high dose dropped) for decreased clotting
time in female Wistar rats, 1,2,4-TMB (Korsak et al., 2000a) C-5
Table C-5. Model predictions (constant variance) for decreased fetal weight in male
Sprague-Dawley rats, 1,2,4-TMB (Saillenfait et al., 2005) C-6
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Table C-6. Model predictions (constant variance) for decreased fetal weight in male
Sprague-Dawley rats, 1,2,4-TMB (Saillenfait et al., 2005) C-6
Table C-7. Model predictions (constant variance) for decreased fetal weight in female
Sprague-Dawley rats, 1,2,4-TMB (Saillenfait et al., 2005) C-8
Table C-8. Model predictions (constant variance) for decreased fetal weight in female
Sprague-Dawley rats. 1,2,4-TMB (Saillenfait et al., 2005) C-8
Table C-9. Model predictions (constant variance) for decreased maternal weight gain in
female Sprague-Dawley rats, 1,2,4-TMB (Saillenfait et al., 2005) C-10
Table C-10. Model predictions (constant variance) for increased latency to paw-lick in male
Wistarrats, 1,2,3-TMB (Korsak and Rydzynski, 1996) C-ll
Table C-ll. Model predictions (modeled variance) for increased latency to paw-lick in male
Wistarrats, 1,2,3-TMB (Korsak and Rydzynski, 1996) C-ll
Table C-12. Model predictions (modeled variance, high dose dropped) for increased latency
to paw-lick in male Wistar rats, 1,2,3-TMB (Korsak and Rydzynski, 1996) C-12
Table C-13. Model predictions (constant variance) for decreased segmented neutrophils in
male Wistar rats, 1,2,3-TMB (Korsak et al., 2000b) C-13
Table C-14. Model predictions (constant variance) for decreased segmented neutrophils in
female Wistar rats, 1,2,3-TMB (Korsak etal., 2000b) C-14
Table C-15. Model predictions (constant variance) for increased reticulocytes in male Wistar
rats, 1,2,3-TMB (Korsak et al., 2000b) C-15
Table C-16. Model predictions (constant variance) for decreased fetal weight in male
Sprague-Dawley rats, 1,3,5-TMB (Saillenfait et al., 2005) C-16
Table C-17. Model predictions (modeled variance) for decreased fetal weight in male
Sprague-Dawley rats, 1,3,5-TMB (Saillenfait et al., 2005) C-16
Table C-18. Model predictions (modeled variance) for decreased fetal weight in male
Sprague-Dawley rats, 1,3,5-TMB (Saillenfait et al., 2005) C-17
Table C-19. Model predictions (constant variance) for decreased fetal weight in female
Sprague-Dawley rats, 1,3,5-TMB (Saillenfait et al., 2005) C-17
Table C-20. Model predictions (modeled variance) for decreased fetal weight in female
Sprague-Dawley rats, 1,3,5-TMB (Saillenfait et al., 2005) C-18
Table C-21. Model predictions (modeled variance) for decreased fetal weight in female
Sprague-Dawley rats, 1,3,5-TMB (Saillenfait et al., 2005) C-18
Table C-22. Model predictions (constant variance) for decreased maternal weight gain in
female Sprague-Dawley rats, 1,3,5-TMB (Saillenfait et al., 2005) C-19
Table C-23. Model predictions (modeled variance) for decreased maternal weight gain in
female Sprague-Dawley rats, 1,3,5-TMB (Saillenfait et al., 2005) C-19
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Table C-24. Model predictions (constant variance) for increased latency to paw-lick in male
Wistarrats, 1,2,4-TMB (Korsak and Rydzyriski, 1996) C-22
Table C-25. Model predictions (modeled variance) for increased latency to paw-lick in male
Wistarrats, 1,2,4-TMB (Korsak and Rydzynski, 1996) C-22
Table C-26. Model predictions (constant variance) for decreased red blood cells in male
Wistar rats, 1,2,4-TMB (Korsak et al., 2000a) C-23
Table C-27. Model predictions (constant variance) for decreased clotting time in female
Wistar rats (Korsak etal., 2000a) C-24
Table C-28. Model predictions (modeled variance) for decreased clotting time in female
Wistar rats (Korsak etal., 2000a) C-24
Table C-29. Model predictions (constant variance) for decreased reticulocytes in female
Wistar rats, 1,2,4-TMB (Korsak et al., 2000a) C-25
Table C-30. Model predictions (modeled variance) for decreased reticulocytes in female
Wistar rats, 1,2,4-TMB (Korsak et al., 2000a) C-25
Table D-l. National Research Council recommendations that EPA is implementing in the
short term D-3
Table D-2. National Research Council recommendations that EPA is implementing in the
long-term (p. # in NRC report) D-9
FIGURES
Figure B-l. Metabolic scheme for 1,2,4-TMB B-7
Figure B-2. Metabolic scheme for 1,2,3-TMB B-8
Figure B-3. Metabolic scheme for 1,3,5-TMB B-8
Figure B-4. Physiological based toxicokinetic model for 1,2,4-TMB in humans B-ll
Figure B-5. Schematic of human model structure for 1,2,4-TMB using the NLE-based model
approach B-15
Figure B-6. Schematic of rat and human PBPK model structure B-16
Figure B-7. Simulated and measured blood concentrations of 1,2,4,-TMB in rats exposed to
600, 2,400, or 4,800 mg/m3 WS for 8 hours B-25
Figure B-8. Simulated and measured brain concentrations of 1,2,4-TMB in rats exposed to
600, 2,400, or 4,800 mg/m3 WS for 8 hours B-26
Figure B-9. Simulated and measured exhaled air concentrations of 1,2,4-TMB in three
volunteers exposed to 600 mg/m3 WS for4 hours B-27
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Figure B-10. Comparisons of model predictions to measured blood concentrations in rats
exposed to 1,2,4-TMB in WS (Hissink et al., 2007) (a) before and (b) after
numerical optimization B-31
Figure B-ll. Comparisons of model predictions to measured brain concentrations in rats
exposed to 1,2,4-TMB in WS (Hissink et al., 2007) using model parameters
optimized forfitto Hissink etal. (2007) rat blood data B-32
Figure B-12. Comparisons of model predictions to measured venous blood concentrations by
Swiercz et al. (2003) in rats repeatedly exposed to 1,2,4-TMB (a) before and (b)
after numerical optimization B-32
Figure B-13. Comparisons of model predictions to measured rat venous blood concentrations
by Swiercz et al. (2002) in acutely exposed rats (a) during and (b) after exposure.
B-34
Figure B-14. Comparisons of model predictions to measured human venous blood
concentrations of Kostrzewki et al. (1997) in human volunteers exposed to 154
mgl,2,4-TMB/m3for8hours B-40
Figure B-15. Comparisons of model predictions to measured human venous blood
concentrations of Jarnberg et al. (1998, 1997a; 1996) in volunteers exposed to 2
or 25 ppm (~10 or 123 mg/m3) 1,2,4-TMB for 2 hours while riding a bicycle (50
W) B-40
Figure B 16. Comparisons of model predictions to measured (a) human venous blood and (b)
end of exposure exhaled air 1,2,4-TMB in human volunteers exposed to 100 ppm
WS with 7.8% 1,2,4-TMB (38.4 mg/m31,2,4-TMB) (Hissink et al., 2007) B-41
Figure B-17. Time course of normalized sensitivity coefficients of moderately sensitive
chemical-specific parameters (response: venous blood concentration) in rats
exposed to (a) 25 ppm (123 mg/m3) or (b) 250 ppm (1,230 mg/m3) of 1,2,4-TMB
via inhalation for 6 hours (Swiercz et al., 2003; Swiercz et al., 2002) B-45
Figure B-18. Effect of route of exposure and dose rate on steady-state venous blood
concentration (t = 1,200 hr) for continuous human exposure to 1,2,4-TMB....B-48
Figure C-l. Plot of mean response by dose (mg/L 1,2,4-TMB) for increased latency to paw-
lick in male Wistar rats, with fitted curve for Exponential 4 model (BMR = 1 SD,
constant variance, high dose dropped). (Korsak and Rydzynski, 1996) C-3
Figure C-2. Plot of mean response by dose (mg/L 1,2,4-TMB) for decreased red blood cells in
male Wistar rats, with fitted curve for Linear model (BMR = 1 SD, constant
variance, high dose dropped). (Korsak et al., 2000a) C-4
Figure C-3. Plot of mean response by dose (mg/m31,2,4-TMB) for decreased fetal weight in
male Sprague-Dawley rats, with fitted curve for Linear model (BMR = 1 SD,
constant variance). (Saillenfait et al., 2005) C-7
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Figure C-4. Plot of mean response by dose (mg/m31,2,4-TMB) for decreased fetal weight in
male Sprague-Dawley rats, with fitted curve for Linear model (BMR = 5% RD,
constant variance). (Saillenfait et a I., 2005) C-7
Figure C-5. Plot of mean response by dose (mg/m3 1,2,4-TMB) for decreased fetal weight in
female Sprague-Dawley rats, with fitted curve for Linear model (BMR = 1 SD,
constant variance). (Saillenfait et a I., 2005) C-9
Figure C-6. Plot of mean response by dose (mg/m3 1,2,4-TMB ) for decreased fetal weight in
female Sprague-Dawley rats, with fitted curve for Linear model (BMR = 5% RD,
constant variance). (Saillenfait et a I., 2005) C-9
Figure C-7. Plot of mean response by dose (mg/m3 1,2,4-TMB) for decreased maternal
weight gain in female Sprague-Dawley rats, with fitted curve for Exponential 3
model (BMR= 1 SD, constant variance). (Saillenfait et al., 2005) C-10
Figure C-8. Plot of mean response by dose (mg/m3 1,2,3-TMB) for increased latency to paw-
lick in male Wistar rats, with fitted curve for Linear model (BMR = 1 SD, modeled
variance, high dose dropped). (Korsak and Rydzynski, 1996) C-12
Figure C-9. Plot of mean response by dose (mg/m3 1,2,3-TMB) for decreased segmented
neutrophils in male Wistar rats, with fitted curve for Exponential 2 model (BMR =
1SD, constant variance). (Korsak et al., 2000b) C-13
Figure C-10. Plot of mean response by dose (mg/m3 1,2,3-TMB) for decreased segmented
neutrophils in female Wistar rats, with fitted curve for Hill model (BMR = 1 SD,
constant variance). (Korsak et al., 2000b) C-14
Figure C-ll. Plot of mean response by dose (mg/m3 1,2,3-TMB) for increased reticulocytes in
male Wistar rats, with fitted curve for Linear model (BMR = 1 SD, constant
variance). (Korsak et al., 2000b) C-15
Figure C-12. Plot of mean response by dose (mg/m3 1,3,5-TMB) for decreased maternal
weight gain in female Sprague-Dawley rats, with fitted curve for Power model
(BMR = 1 SD, modeled variance). (Saillenfait et al., 2005) C-20
Figure C-13. Plot of mean response by dose (mg/L 1,2,4-TMB) for decreased red blood cells in
male Wistar rats, with fitted curve for Hill model (BMR = 1 SD, constant
variance). (Korsak et al., 2000a) C-23
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ABBREVIATIONS
Toxicological Review ofTrimethylbenzene
1,2,3-TMB 1,2,3-trimethylbenzene
1,2,4-TMB 1,2,4-trimethylbenzene
1,3,5-TMB 1,3,5-trimethylbenzene
AAQC Ambient air quality criterion
ABR amount of 1,2,4-TMB in the brain
ACGIH American Conference of
Governmental Industrial Hygienists
ADME Absorption, distribution, metabolism
and excretion
AEGL Acute exposure guideline limit
AIC Akaike Information Criterion
BAL bronchoalveolar lavage
BMD benchmark dose
BMDL lower confidence limit on the
benchmark dose
BMDS benchmark dose software
BMR benchmark response
BW body weight
CAS Chemical Abstracts Service
CI confidence interval
CMIX average of arterial and venous blood
concentrations
CNS central nervous system
CV concentration in venous blood
CVS concentration in venous blood exiting
slowly perfused tissues
CXEQ concentration in exhaled breath
DMBA dimethylbenzoic acid
DMHA dimethylhippuric acid
ECso half maximal effective concentration
EPA U.S. Environmental Protection
Agency
GD gestational day
HEC human equivalent concentration
i.p. intraperitoneal
IRIS Integrated Risk Information System
Km Michaelis-Menten constant
LOAEL lowest-observed-adverse-effect level
NCEA National Center for Environmental
Assessment
NIOSH National Institute for Occupational
Safely and Health
NOAEL No-observed-adverse-effect level
OMOE Ontario Ministry of the Environment
p probability value
PBPK physiologically based
pharmacokinetic (model)
POD point of departure
POI Point of impingement
QPC alveolar ventilation rate
QRTOTC sum of fractional flows to rapidly
perfused tissues, liver, and brain
QSTOTC sum of fractional flows to slowly
perfused tissues
RBC red blood cell
RDso 50% respiratory rate decrease
REL Recommended exposure limit
RfC reference concentration
RfD reference dose
ROS reactive oxygen species
SD standard deviation
SE standard error
TLV threshold limit value
TMB trimethylbenzene
TSCA Toxic Substances Control Act
TWA time-weighted average
UV ultraviolet
VLC volume of fat
Vmax Vi maximal enzyme rate
VOC volatile organic compound
W watt
WBC white blood cell
WS white spirit
X2 chi-squared
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APPENDIX A. HEALTH ASSESSMENTS AND
REGULATORY LIMITS BY OTHER NATIONAL AND
INTERNATIONAL HEALTH AGENCIES
Table A-l. Other national and international health agency assessments
for TMBs
Agency
National Institue for
Occupational Safety and
Health (NIOSH. 1992. 1988)
American Conference of
Governmental Industrial
Hygienists (ACGIH. 2002)
National Advisory Committee
for Acute Exposure Guideline
Levels for Hazardous
Substances (U.S. EPA, 2007)
Ontario Ministry of the
Environment (MOE, 2006)
Toxicity value
Recommended Exposure Limit (REL) for TMBs - 25 ppm (123 mg/m3)
time weighted average for up to a 10 hour work day and a 40 hour work
week, based on the risk of skin irritation, central nervous system
depression, and respiratory failure (Battig et al., 1956)
Threshold Limit Value (TLV) for VOC mixture containing 1,2,4-TMB and
1,3,5-TMB - 25 ppm (123 mg/m3) time weighted average for a normal 8-
hour work day and a 40-hour work week, based on the risk of irritation
and central nervous system effects (Battig et al.. 1956a)
Acute Exposure Guideline Level (AEGL)-l (nondisabling) - 180 ppm (890
mg/m3) to 45 ppm (220 mg/m3) (10 min to 8 hrs, respectively) (Korsak
and Rvdzvriski, 1996)
AEGL-2 (disabling) - 460 ppm (2,300 mg/m3) to 150 ppm (740 mg/m3)
(10 min to 8 hrs, respectively) (Gage, 1970)
For TMBs: 24 hr Ambient Air Quality Criterion (AAQC) - 0.3 mg/m3
based on CNS effects; half-hour Point of Impingement (POI) - 0.9 mg/m3
based on CNS effects (Wiaderna et al., 2002; Gralewicz and Wiaderna,
2001; Gralewicz et al., 1997a; Korsak and Rydzyriski, 1996)
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APPENDIX B. INFORMATION IN SUPPORT OF
HAZARD IDENTIFICATION AND DOSE-REPONSE
ANALYSIS
B.I. PHYSICAL AND CHEMICAL PROPERTIES
Table B-l. Physical properties and chemical identity of 1,2,4-TMB,
1,3,5-TMB, and 1,2,3-TMB
CAS Registry Number
Synonym(s)
Molecular formula
Molecular weight
Chemical structure
Melting point, °C
Boiling point, °C @ 760 mm
Hg
Vapor pressure, mm Hg @
25°C
Density, g/mL at 20 °C
Flashpoint, °C
Water solubility, mg/L at 25
°C
Other solubilities
Henry's law constant, atm-
ms/mol
Log KQW
Log Koc
Bioconcentration factor
Conversion factors
95-63-6
1,2,4-Trimethylbenzene,
pseudocumene,
asymmetrical
trimethylbenzene
108-67-8
1,3,5-Trimethylbenzene,
mesitylene, symmetrical
trimethylbenzene
526-73-8
1,2,3-Trimethylbenzene,
hemimellitene,
hemellitol, pseudocumol
C9H12
120.19
r
/
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Toxicological Review ofTrimethylbenzene
B.2. TOXICOKINETICS
1 There has been a significant amount of research conducted on the toxicokinetics of
2 1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB in experimental animals and humans. In vivo studies
3 have been conducted to evaluate the adsorption, distribution, metabolism and excretion
4 (ADME) of all isomers following exposure via multiple routes of exposure in rats (Swiercz
5 etal.. 2006: Tsujimoto etal.. 2005: Swiercz etal.. 2003: Swiercz etal.. 2002: Tsujino etal..
6 2002: Tsujimoto etal.. 2000: Eide and Zahlsen. 1996: Zahlsen etal.. 1990: Huoetal.. 1989:
7 Dahletal.. 1988: Mikulski and Wiglusz. 1975) and human volunteers (Tanasiketal.. 2008:
8 Tones etal.. 2006: Tarnbergetal.. 1997a: Tarnbergetal.. 1997b: Kostrzewski etal.. 1997:
9 larnberg et al.. 1996: Kostrewski and Wiaderna-Brycht. 1995: Fukaya et al.. 1994: Ichiba et
10 al.. 1992).
B.2.1. Absorption
11 Both humans and rats readily absorb 1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB into the
12 bloodstream following exposure via inhalation. Humans exposed to 25 ppm (1,2,3-TMB
13 mg/m3) 1,2,4-TMB, 1,2,3-TMB, or 1,3,5-TMB for 2 hours exhibited similar maximum
14 capillary blood concentrations (6.5 and 6.2 [iM, respectively), whereas absorption for 1,2,3-
15 TMB was observed to be higher (7.3 [iM) (larnberg etal.. 1998.1997a: larnberg etal..
16 1996). Kostrewski et al. (1997) observed equivalent maximal capillary blood
17 concentrations in humans exposed to 30.5 ppm (150 mg/m3) 1,2,4-TMB or 1,3,5-TMB for 8
18 hours (8.15 and 6.3 |iM, respectively). In the same study, human volunteers exposed to 100
19 mg/m3 (20.3 ppm) 1,2,3-TMB had capillary blood concentrations of 4.3 |iM. In humans
20 exposed to 25 ppm (123 mg/m3) 1,3,5-TMB for 4 hours, venous blood concentrations were
21 markedly lower (0.85 |iM), but this may be related to measurement of 1,3,5-TMB in the
22 venous blood (Jones etal.. 2006). 1,3,5-TMB has a higher blood:fatpartition coefficient
23 (230) than 1,2,4-TMB (173) or 1,2,3-TMB (164) (Tarnberg and Tohanson. 1999) and
24 therefore much of the 1,3,5-TMB absorbed into capillary blood may preferentially
25 distribute to adipose tissue before entering into the venous blood supply. Measurements of
26 respiratory uptake of 1,2,4-TMB, 1,2,3-TMB, or 1,3,5-TMB are fairly similar in humans (60
27 ± 3%, 48 ± 3%, and 55 ± 2%, respectively) and approximate equivalency is observed
28 between the respiratory uptake of 1,2,4-TMB between humans and rats (60 ± 3% and 44-
29 50%, respectively) fTarnberg etal.. 1996: Dahletal.. 19881
30 In rats, rapid absorption into the bloodstream was observed in many studies
31 following single exposures to 1,2,4-TMB, with maximal blood concentrations of 537, 221,
32 and 64.6 [iM observed after exposures to 1,000 ppm (4,920 mg/m3) for 12 hours, 450 ppm
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1 (2,214 mg/m3) for 12 hours, and 250 ppm (1,230 mg/m3) for 6 hours fSwiercz etal.. 2003:
2 Eide and Zahlsen. 1996: Zahlsen etal.. 19901 Zahlsen etal. (1990} observed a decrease in
3 blood concentrations of 1,2,4-TMB following repeated exposures, which they attribute to
4 induction of metabolizing enzymes; a similar decrease in 1,2,4-TMB blood concentrations
5 following repeated exposures was not observed in Swiercz et al. (2003). Using a 4-
6 comparment toxicokinetic model, Yoshida et al. (2010) estimated that a rat exposed to 50
7 [ig/m31,2,4-TMB for 2 hours would absorb 6.6 [ig/kg body weight. Using this same model,
8 the authors estimated that humans exposed to 24 [ig/m31,2,4-TMB for 2 hours would
9 absorb 0.45 [ig/kg body weight. 1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB have also been
10 observed to be absorbed and distributed via blood circulation following oral and dermal
11 exposures in rats (Tsujino etal.. 2002: Huo etal.. 1989). Lastly, calculatedblood:air
12 partition coefficients for 1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB (43.0, 66.5, and 59.1,
13 respectively) were similar in humans, indicating that the two isomers would partition
14 similarly into the blood (Jarnbergand Johanson. 1995). Additionally, the blood:air partition
15 coefficients between humans and rats were very similar for all three isomers: 1,2,4-TMB
16 (43.0 vs. 55.7), 1,2,3-TMB (66.5 vs. 62.6), and 1,3,5-TMB (59.1 vs. 57.7) fMeulenbergand
17 Vijverberg. 2000). This further indicates patterns of absorption would be similar across
18 species.
B.2.2. Distribution
19 No information exists regarding the distribution of any isomer in adult humans.
20 However, experimentally calculated tissue-specific partition coefficients were similar for
21 all three isomers across a number of organ systems (fat, brain, liver, muscle, and kidney)
22 fMeulenberg and Viiverberg. 20001 This strongly indicates that 1,2,4-TMB, 1,2,3-TMB, and
23 1,3,5-TMB can be expected to partition similarly into these various organ systems.
24 Trimethylbenzenes (unspecified isomer) have also been detected in cord blood, and
25 therefore can be expected to partition into the fetal compartment (Cooper et al.. 2001:
26 Dowty etal.. 1976). In rats, 1,2,4-TMB was observed to distribute widely to all examined
27 organ systems following oral exposure, with the highest concentrations found in the
28 stomach (509 [ig/g) and adipose tissue (200 [ig/g) (Huo etal.. 1989). Following inhalation
29 exposures, 1,2,4-TMB and 1,3,5-TMB were observed to distribute to all tissues examined,
30 with tissue-specific concentrations dependent on the external exposure concentration
31 (Swiercz etal.. 2006: Swiercz etal.. 2003: Eide and Zahlsen. 1996). 1,2,4-TMB distributed to
32 the adipose tissue to a much higher degree than to the brain, liver, or kidneys (Eide and
33 Zahlsen. 19961 Venous blood concentrations of 1,2,4-TMB and 1,3,5-TMB and liver
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Toxicological Review ofTrimethylbenzene
1 concentrations of 1,2,4-TMB were observed to be significantly lower in repeatedly exposed
2 animals versus animals exposed only once to higher concentrations (Swiercz et al.. 2006:
3 Swiercz et al.. 2003: Swiercz etal.. 2002}. Kidney concentrations of 1,3,5-TMB were
4 observed to be lower in repeatedly exposed animals versus animals exposed once, but only
5 at the lowest exposure concentration. The authors suggest that lower tissue concentrations
6 of TMB isomers observed in repeatedly-exposed animals is mostly likely due to induction
7 of metabolizing enzymes at higher exposure concentrations. This hypothesis is supported
8 by the observation of P-450 enzyme induction in the livers, kidneys, and lungs of rats
9 exposed to 1,200 mg/kg/day 1,3,5-TMB for 3 days (Pyvkko. 1980).
10 1,2,4-TMB was also observed to distribute to individual brain structures, with the
11 brainstem and hippocampus having the highest concentrations following exposure
12 [Swiercz etal.. 2003). Zahlsen et al. (1990) also observed decreasing blood, brain, and
13 adipose tissue concentrations following repeated exposures versus single day exposures in
14 rats exposed to 1,000 ppm (4,920 mg/m3). In the only study to investigate distribution
15 following dermal exposure, 1,2,4-TMB preferentially distributed to the kidneys [Tsujino et
16 al.. 2002). Concentrations in the blood, brain, liver, and adipose tissue were similar to one
17 another, but 1,2,4-TMB concentrations only increased in a dose-dependent manner in
18 adipose tissue, and continued to accumulate in that tissue following the termination of
19 exposure. Similar results were reported for 1,2,3-TMB and 1,3,5-TMB, but specific data
20 were not presented. Detailed information regarding the distribution of 1,2,3-TMB in rats
21 following inhalation or oral exposures is lacking. However, similar tissue-specific partition
22 coefficients for 1,2,3-TMB compared to 1,2,4-TMB and 1,3,5-TMB were similar across a
23 number of organ systems (Meulenbergand Vijverberg. 2000). indicating similar patterns of
24 distribution can reasonably be anticipated.
B.2.3. Metabolism
25 The metabolic profiles for each isomer were qualitatively similar between humans
26 and rats. In humans, both isomers are observed to be metabolized to benzoic and hippuric
27 acids. Approximately 22% of inhaled 1,2,4-TMB was collected as hippuric acid metabolites
28 in urine 24 hours after 2 hour exposures to 25 ppm (123 mg/m3) 1,2,4-TMB (Iamberg et
29 al.. 1997b). 3,4-dimethylhippuric acid (DMHA) comprised 82% of the dimethylhippuric
30 acids collected after exposure to 1,2,4-TMB, indicating that steric factors are important in
31 the oxidation and/or glycine conjugation of 1,2,4-TMB in humans. Approximately 11% of
32 inhaled 1,2,3-TMB was collected as hippuric acid metabolites (larnberg et al.. 1997b). As
33 with 1,2,4-TMB, steric influences seem to play an important role in the preferential
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Toxicological Review ofTrimethylbenzene
1 selection of which metabolites are formed: 2,3-DMHA comprised 82% of all hippuric acid
2 metabolites collected. Urinary hippuric acid metabolites for 1,3,5-TMB following the same
3 exposure protocol accounted for only 3% of inhaled dose. Greater amounts of urinary
4 benzoic and hippuric acid metabolites (73%} were observed after exposure to higher
5 amounts of 1,3,5-TMB (up to 30.5 ppm} for 8 hours (Kostrzewski etal.. 1997: Kostrewski
6 and Wiaderna-Brycht. 19951
7 Following occupational exposure to 1,2,4-TMB or 1,3,5-TMB, urinary benzoic acid
8 and hippuric acid metabolites were highly correlated with TMB isomer air concentrations
9 (Jones etal.. 2006: Fukaya etal.. 1994: Ichiba etal.. 1992}. Following oral exposures, the
10 total metabolism of the different isomers differs somewhat, with the total metabolism of
11 1,3,5-TMB being fairly complete (73%}, the total metabolism of 1,2,3-TMB being much less
12 (33.0%}, and the total metabolism of 1,2,4-TMB ranging from incomplete to almost totally
13 metabolized (37-86%} (Huo etal.. 1989: Mikulski and Wiglusz. 1975}. The major terminal
14 metabolites for 1,2,4-TMB and 1,3,5-TMB are dimethylhippuric acids (24-38% and 59%
15 total dose, respectively}. Dimethylhippuric acid metabolites represent a smaller fraction
16 (10.1%} of the metabolites produced following 1,2,3-TMB exposure.
17 Similar profiles in metabolism were observed in rabbits: DMBAs and DMHAs were
18 observed following oral exposure of rabbits to either 1,2,4-TMB or 1,3,5-TMB (Laham and
19 Potvin. 1989: Cerf etal.. 1980}. Specifically for 1,3,5-TMB, 68.5% of the administered oral
20 dose was recovered as the DMHA metabolite, with only 9% recovered as the DMBA
21 metabolite. Additionally, a minor metabolite not observed in rats, 5-methylisophthalic acid
22 was observed following exposure of rabbits (Laham and Potvin. 1989}. Additional terminal
23 metabolites for the three isomers include: mercapturic acids (~ 14-19% total dose},
24 phenols (~12% total dose}, and glucuronides and sulphuric acid conjugates (4-9% total
25 dose} for 1,2,4-TMB; mercapturic acids (~5% total dose}, phenols (
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Toxicological Review ofTrimethylbenzene
1 factors appear to be minimal regarding oxidation of the aromatic ring itself: the most
2 hindered phenol metabolites of 1,2,4-TMB and 1,2,3-TMB were either formed in equal or
3 greater proportions compared to less sterically hinder metabolites (Huo etal.. 1989}
4 (Tsujimoto etal.. 2005). The proposed metabolic schemes for 1,2,4-TMB, 1,2,3-TMB, and
5 1,3,5-TMB are shown in Figures B-l, B-2, and B-3.
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Toxicological Review ofTrimethylbenzene
2,4,5 -trimethy Iphenol
CH3
-CH3
2,3,5-trimethylphenol
2,4-dimethyl
benzyl
alcohol
CH2R
C\-\,
CH3
2,4-dimethyl-
benzyl
mercapturic acid
2,5-dimethyl-
benzvl alcohol
CH3
3,4-dimethyl-
benzyl
alcohol
' \
X' >*
CH3
2,5 -dimethy Ibenzy 1
CH2R
3,4-dimethyl-
benzyl
mercapturic acid
2,5-dimethyl
benzoic acid
2,4-dimethyl
benzoic acid
OH mercapturic acid
3,4-dimethyl
benzoic acid
2,4-dimethyl-
hippuric acid
Figure B-l. Metabolic scheme for 1,2,4-TMB.
3,4-dimethyl-
hippuric acid
O'
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Toxicological Review ofTrimethylbenzene
3,4,5-trimethyl-
phenol
1,2,3-trimethyl-
benzene
2,3,4-trimethyl-
phenol
•CH,
CH
•CH,
'CH,
CH2R
CH2OH
CH2OH
CH,
OH
CH2R
•CH,
2,6-dimethyl-
mercapturic acid
2,6-dimethyl-
benzyl alcohol
CH,
CH,
2,3-dimethyl- 2,3-dimethyl-
benzyl alcohol mercapturic acid
2,6-dimethyl-
hippuric acid
2,6-dimethyl-
benzoic acid
2,3-dimethyl-
benzoic acid
2,3-dimethyl-
CH hippuric acid
Figure B-2. Metabolic scheme for 1,2,3-TMB.
CH, CH
CH,
1,3.5-trimethyl-
benzene
CH,
3.5-dimethylbenzyl
alcohol
CH,
CH,
OH
OH
3.5-dimethylbenzoic
acid
H,
2,4,6-
trimethylphenol
3,5-dimethylhippurrc
°H
HN
acid
Figure B-3. Metabolic scheme for 1,3,5-TMB.
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B.2.4. Excretion
1 In humans at low doses (25 ppm [123 mg/m3]), half-lives of elimination from the
2 blood of all TMB isomers were split into four distinct phases, with the half-lives of the first
3 three phases being similar across isomers: 1,2,4-TMB (1.3 ± 0.8 min, 21 ± 5 min, 3.6 ± 1.1
4 hr), 1,2,3-TMB (1.5 ± 0.9 min, 24 ± 9 min, 4.7 ± 1.6 hr), and 1,3,5-TMB (1.7 ± 0.8 min, 27 ± 5
5 min, 4.9 ± 1.4 hr) (Tarnbergetal.. 1996). 1,3,5-TMB had a higher total blood clearance
6 value compared 1,2,4-TMB or 1,2,3-TMB (0.97 ± 0.06 L/hr/kg vs. 0.68 ± 0.13 or 0.63 ± 0.13
7 L/hr/kg, respectively}. The half-life of elimination for 1,3,5-TMB in the last and longest
8 phase is much greater than those for 1,2,4-TMB or 1,2,3-TMB (120 ± 41 hr vs. 87 ± 27 and
9 78 ± 22 hr, respectively}. Urinary excretion of unchanged parent compound was extremely
10 low (<0.002%} for all three isomers (Tanasiketal.. 2008: Tarnbergetal.. 1997b}. The half-
11 life of elimination of hippuric acid metabolites from the urine was also greater for 1,3,5-
12 TMB, compared to 1,2,4-TMB or 1,2,3-TMB (16 hr vs. 3.8-5.8 and 4.8-8.1 hr, respectively}
13 flarnbergetal.. 1997bl
14 Differences in the values of terminal half-lives may be related to interindividual
15 variation in a small sample population (n = 8-10} and difficulty measuring slow elimination
16 phases. All three isomers were eliminated via exhalation: 20-37% of the absorbed dose of
17 1,2,4-TMB, 1,2,3-TMB, or 1,3,5-TMB was eliminated via exhalation during exposure to 123
18 mg/m3 (25 ppm} for 2 hours (larnbergetal.. 1996} and elimination of 1,3,5-TMB via breath
19 was bisphasic with an initial half-life of 60 minutes, and a terminal half-life of 600 minutes
20 (Tones etal.. 2006}. Following exposure of rats to 25 ppm (123 mg/m3} 1,2,4-TMB or 1,3,5-
21 TMB for 6 hours, the terminal half-life of elimination of 1,3,5-TMB from the blood (2.7
22 hours} was shorter than that for 1,2,4-TMB (3.6 hours} (Swiercz et al.. 2006: Swiercz et al..
23 2002}. As dose increased, the half-lives for elimination from blood following single
24 exposures to 1,2,4-TMB (17.3 hours} became much longer than those for 1,3,5-TMB (4
25 hours}. This same pattern was observed for 4-week repeated exposures as well.
B.3. PHYSIOLOGICALLY-BASED PHARMACOKINETIC MODELS
B.3.1. Summary of Available PBPK models for 1,2,4-TMB
26 Jarnberg and Johanson (1999)
27 Jarnberg and Johanson (1999) describe a PBPK model for inhalation of 1,2,4-TMB in
28 humans. The model is composed of six compartments (lungs, adipose, working muscles,
29 resting muscles, liver, and rapidly perfused tissues) for the parent compound and one
30 (volume of distribution) for the metabolite, 3,4-DMHA (see Figure B-4). The lung
31 compartment includes lung tissue and arterial blood. Excretion of parent compound is
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1 assumed to occur solely by ventilation. As 1,2,4-TMB has a pronounced affinity to adipose
2 tissue, a separate compartment for fat is incorporated into the model. Remaining non-
3 metabolizing compartments are rapidly perfused tissues, comprising the brain, kidneys,
4 muscles, and skin.
5 Because previous experimental data was gathered during exercise (larnberg et al..
6 1997a: larnberg et al.. 1996). the muscle compartment was divided into two equally large
7 compartments, resting and working muscles. Two elimination pathways (a saturable
8 Michaelis-Menten pathway for all metabolites other than 2,4-DMHA [pathway I] and a first
9 order pathway [pathway II] for formation of 3,4-DMHA) from the hepatic compartment
10 were included. Metabolism was assumed to occur only in the liver compartment.
11 Tissue:blood partition coefficients of 1,2,4-TMB were calculated from experimentally
12 determined blood:air, water:air, and olive oil:air partition coefficients Qarnberg and
13 Tohanson. 1995) (Table B-2).
14 The model was used to investigate how various factors (work load, exposure level,
15 fluctuating exposure) influence potential biomarkers of exposure (end-of-shift and prior-
16 to-shift concentrations of parent compound in blood and 3,4-DMHA in urine). Biomarker
17 levels estimated at end-of-shift remained fairly constant during the week, whereas
18 biomarker levels prior-to-shift gradually increase throughout the week. This indicates end-
19 of-shift values represent the same day's exposures, whereas prior-to-shift values reflect
20 cumulative exposure during the entire work week. Increased work load increased uptake
21 of 1,2,4-TMB. For example, a work load of 150 W over an exposure period of 8 hours
22 increased the level of 1,2,4-TMB in the blood more than 2-fold, compared to levels of 1,2,4-
23 TMB in the blood after an 8 hour exposure at rest. Simulated 8-hour exposures at air levels
24 0 to 100 ppm (0 to 492 mg/m3) shows that overall metabolism is saturable, and that the
25 metabolic pathway yielding 3,4-dimethylbenzene becomes more important as exposure
26 concentrations increase.
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1
Lungs and arterial
blood
Rapidly perfused
tissues
Adipose tissue
Q
Working muscles
Resting muscles
Liver
Vmax/Kn
other | 3,4-dimethyl |
metabolites | hippuric acid |
I ^-T~'
urine
urine
C: concentration of 1,2,4-TMB; Cair: concentration in ambient air; Cart: concentration in arterial blood; Cven:
concentration in venous blood; 0^: alveolar ventilation; Qc0: cardiac output; Q,: blood flow to
compartment i (where i = rap = rapidly perfused tissues; f = adipose tissue; w = working muscles, r =
resting muscles, h = liver); Vmax: maximum rate of metabolism, pathway I; Km: Michaelis-Menten constant
for metabolic pathway I; CL1: intrinsic hepatic clearance of metabolic pathway II; ke: excretion rate
constant of 3,4-DMHA. Adapted from Jarnberg and Johanson (1999).
Figure B-4. Physiological based toxicokinetic model for 1,2,4-TMB in
humans.
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Table B-2. Measured and calculated partition coefficients for TMB
isomers at 37°C
Substance
1,3,5-TMB
1,2,4-TMB
1,2,3-TMB
Measured values3
P Saline:Air
n = 42
1.23(1.11-1.35)
1.61 (1.47-1.75)
2.73 (2.54-2.92)
P Oil:Air
n = 25
9,880 (9,620-10,140)
10,200 (9,900-10,400)
10,900 (10,500-11,300)
HU ma nP Blood: Air
n = 39
43.0 (40.8-45.2)
59.1 (56.9-61.3)
66.5 (63.7-69.3)
Calculated values
Human PBiood:Air
60.3
62.2
67.5
aMean values and 95%CI.
Calculated as (0.79 x p saimeiAir) + (0.006 x p oihAir); where 0.79 is the relative content of saline in blood and
0.006 is the relative content of fat in blood (Fiserova-Bergerova. 1983].
Adapted from Jarnberg and Johanson (19951.
1 Previously performed experimental human exposures to 1,2,4-TMB were used to
2 estimate the metabolic parameters and alveolar ventilation (Jarnberg et al.. 1997a:
3 Jarnberg et al.. 1996}. Individual simulated arterial blood concentrations and exhalation
4 rates of 1,2,4-TMB, as well as the urinary excretion rate of 3,4-DMHA, were simultaneously
5 adjusted to the experimentally obtained values by varying the alveolar ventilation at rest.
6 One individual's compound-specific and physiological parameters were then used for
7 subsequent model predictions (Table B-3).
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Table B-3. PBPK model parameters for 1,2,4-TMB toxicokinetics in
humans using the Jarnberg and Johanson (1999) mo del structure
Parameters
Body height (m)
Body weight (kg)
Vmax(u.mol/min)
Km (uM)
CL1 (L/min)
Elimination rate constant (min *)
Alveolar ventilation (L/min)
Rest
9.05
Both3
1.78
75.5
3.49
4.35
0.149
0.0079
SOW
20.2
Compartment volumes (L)
Lungs and arterial blood
Liver
Fat
Brain and kidneys
Working muscles
Resting muscles
1.37
1.51
25.0
1.49
16.6
16.6
Blood flows (L/min)
Cardiac output
Liver
Fat
Brain and kidneys
Working muscles
Resting muscles
5.17
1.67
0.55
1.86
0.55
0.55
9.16
Partition coefficients
Blood:air
Fatblood
Liverblood
Rapidly perfused tissues:blood
Muscle:blood
59
125
5
5
5
Parameters used for both working and resting conditions.
Adapted from Jarnberg and Johanson (1999).
Emond and Krishnan f20061
1 The Emond and Krishnan (2006} model was not developed specifically for
2 1,2,4-TMB, but rather to test a modeling concept. The PBPK model developed was to test
3 the hypothesis that a model could be developed for highly lipophilic volatile organic
4 chemicals (HLVOCs) using the neutral lipid-equivalent (NLE) content of tissues and blood
5 as the basis. This NLE-based modeling approach was tested by simulating uptake and
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Toxicological Review ofTrimethylbenzene
1 distribution kinetics in humans for several chemicals including a-pinene, d-limonene, and
2 1,2,4-TMB. The focus of this model review is to use of the model for the prediction of
3 1,2,4-TMB kinetics and distribution.
4 This model consisted of five compartments (see Figure B-5} with systemic
5 circulation, where the tissue volumes corresponded to the volumes of the neutral lipids
6 (i.e., their neutral lipid-equivalents}, rather than actual tissue volume as more commonly
7 found. NLE is the sum of the neutral (nonpolar} lipids and 30% of the tissue phospholipid
8 (fraction of phospholipids with solubility similar to neutral lipids} content. The model
9 describes inhalation of 1,2,4-TMB using a lumped lung/arterial blood compartment.
10 Clearance of 1,2,4-TMB is described in the model with exhalation, but more significantly
11 through first order hepatic metabolism. First-order metabolism is appropriate in the low
12 dose region (< 100 ppm [< 492 mg/m3]}, where metabolism is not expected to be
13 saturated.
14 In the study description, the mixed lung/arterial blood compartment is not a
15 standard structure for the lung/blood/air interface. The concentration in lung tissue is
16 assumed equal to alveolar blood, and the exhaled air concentration is equal to the
17 lung/blood concentration divided by the blood air partition coefficient. This approach is
18 appropriate, and appears to be accurately represented mathematically by the authors.
19 Physiological parameters appear to be within ranges normally reported. The
20 calculation of the NLE fraction is clearly explained and values used in the calculations are
21 clear and transparent. Other model parameters (e.g., alveolar ventilation, cardiac output,
22 blood flows, and volumes of compartments] were taken from Jarnberg and Johanson
23 (1999} and converted to the approximate NLE. Hepatic clearance rates were taken from
24 literature on in vivo human clearance calculations and then expressed in terms of NLE. The
25 NLE-based model was able to adequately predict human blood concentrations of 1,2,4-TMB
26 following inhalation of 2 or 25 ppm (9.8 or 123 mg/m3} for 2 hours without alteration to
27 model parameters obtained from literature.
This document is a draft for review purposes only and does not constitute Agency policy.
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V
F,
N
O
U
s
B
L
()
O
D
I
L
i
i
d
F
a
c
t
i
n
Adipose tissue
(Lipid Fraction) ^^^^^
Richly perfused tissues ^
(Lipid Fraction)
Resting muscles
(Lipid Fraction) ^^™^~
Working muscles ^
(Lipid Fraction)
Hepatic tissue
(Lipid Fraction)
1 » Metabolism
*
L
i
i
d
F
a
c
t
i
n
A
R
T
E
R
1
A
1,
B
I,
O
O
1)
Arrows represent blood flows, gas exchange, and metabolism as indicated. Source: Emond and Krishnan
(2006).
Figure B-5. Schematic of human model structure for 1,2,4-TMB using
the NLE-based model approach.
1 The PBPK model developed by Emond and Krishnan (2006} is used to test the
2 hypothesis that a model could be developed for HLVOCs using the NLE content of tissues
3 and blood as the basis. To test this NLE-based approach, the uptake and distribution
4 kinetics in humans for several chemicals including 1,2,4-TMB were simulated. The model
5 appeared to accurately reflect experimental data; however, a rodent model is needed for
6 this assessment for animal-to-human extrapolation and no known rodent NLE model for
7 1,2,4-TMB is available.
Hissinketal. (2007)
8 This model was developed to characterize internal exposure following white spirit
9 (WS] inhalation. Since WS is a complex mixture of hydrocarbons, including straight and
10 branched parrafins, two marker compounds were used including 1,2,4-TMB and n-decane.
11 The rat models were developed to predict the levels of 1,2,4-TMB and n-decane in blood
This document is a draft for review purposes only and does not constitute Agency policy.
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1 and brain, then the rat model was scaled allometrically to obtain estimates for human
2 blood following inhalation. Toxicokinetic data on blood and brain concentrations in rats of
3 two marker compounds, 1,2,4-TMB and n-decane, together with in vitro partition
4 coefficients were used to develop the model. The models were used to estimate an air
5 concentration that would produce human brain concentrations similar to those in rats at
6 the no-observed-effect-level (NOEL] for central nervous system (CNS] effects.
7 This is a conventional five compartment PBPK model for 1,2,4-TMB similar to
8 previously published models for inhaled solvents. The five compartments were: liver, fat,
9 slowly perfused tissues, rapidly perfused tissues, and brain (see Figure B-6}.
V
E
N
O
u
s
B
L
O
O
0
c,
Qc
c\
Q,
C',,
Q,
C\,
Q,,.
(",,„
Q,
V,,
Qi
<\i
V,,,.,, & K,,,
O,. C
Gas Exchange
Slowly perfused
Richly perfused
Brain
Fat
Liver
(mctaixslism)
Qc
A
R
T
E
R
I
A
L
B
L
O
O
D
Boxes represent tissue compartments, while solid arrows represent blood flows, gas exchange, and
metabolism as indicated. Source: Hissink et al. (2007).
Figure B-6. Schematic of rat and human PBPK model structure.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
1 All compartments are described as well mixed/perfusion limited. A lung
2 compartment is used to describe gas exchange. The liver was the primary metabolizing
3 organ where 1,2,4-TMB metabolism was described as saturable using Michaelis-Menten
4 kinetics. Since the brain is the target organ for CNS effects due to exposure to hydrocarbon
5 solvents, it was included as a separate compartment. For the rat, the authors reported that
6 Km and Vmax values were obtained by fitting predicted elimination time courses to observed
7 blood concentration profiles at three different exposure levels (obtained from the rat
8 exposure portion of the study}. For the human model, rat Vmax data was scaled to human
9 body weight (BW°-74) and Km values were used unchanged.
10 The model appears to effectively predict blood concentrations in rats and humans
11 and in the brains of rats following inhalation of WS. Changes to the rat model parameters to
12 fit the human data were as expected. The model is simple and includes tissues of interest
13 for potential dose metrics.
14 In rats, the model-predicted blood and brain concentrations of 1,2,4-TMB were in
15 concordance with the experimentally derived concentrations. In humans, experimental
16 blood concentrations of 1,2,4-TMB were well predicted by the model, but the predicted rate
17 of decrease in air concentration between 4-12 hours was lower compared to measured
18 values. The authors did not provide information on how model predictions compared to
19 data from animals or humans exposed to pure 1,2,4-TMB. Based on good model fits of
20 experimental data, the model was valid for the purpose of interspecies extrapolation of
21 blood and brain concentrations of 1,2,4-TMB as a component of WS.
B.3.2. 1,2,4-TMB PBPK Model Selection
22 All available 1,2,4-TMB PBPK models were evaluated for potential use in this
23 assessment. Of the three deterministic PBPK models available for 1,2,4-TMB [Hissinketal..
24 2007: Emond and Krishnan. 2006: Tarnbergand Tohanson. 1999). the Hissink et al. (2007)
25 model was chosen to utilize in this assessment because it was the only published 1,2,4-TMB
26 model that included parameterization for both rats and humans, the model code was
27 available, and the model adequately predicted experimental data in the dose range of
28 concern. The Hissink et al. (2007) model was thoroughly evaluated, including a detailed
29 computer code analysis (details follow in Section B.3.3).
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B.3.3. Details of Hissink et al. (2007) Model Analysis
B.3.3.1. Review and Verification of the Hissink et al. (200711,2,4-TMB PBPK Model
Verification of accuracy of the model code
1 In general, the model code and the description of the model in Hissink et al. (2007)
2 were in agreement. The one significant discrepancy was that the model code contained an
3 element that changed the metabolism rate (Vmax) during exposure in a manner that was not
4 documented in the paper. This additional piece of model code, when used in 8 hour rat
5 simulations with a body weight of 0.2095 kg, resulted in Vmax holding at 1.17 from the
6 beginning of exposure tot = 1 hr, then increasing linearly to 1.87 by the end of the
7 exposure and to 2.67 by the end of the post exposure monitoring period (t = 16 hrs, 8 hrs
8 after the end of exposure}. The published rat simulations, however, did not appear to be
9 entirely consistent with the inclusion of these Vmax adjustments, raising questions as to
10 whether the code that was verified was the code that was actually used in the final analyses
11 done for the published simulations. The impact of this deviation from the published Vmax
12 value is described below in regards to the verification of the Hissink et al. (2007) model.
13 Other minor issues were identified by examining the code and comparing it to the
14 model documentation in Hissink et al. (2007}. The code contained some elements that were
15 not necessary (e.g., i.v. dosing, repeated exposure, interruptions in daily exposure}, but
16 since these do not hinder proper functioning of the model, these elements were not
17 removed or modified. The mass balance equation omitted one term, the amount of 1,2,4-
18 TMB in the brain (ABR}; this term has been added. The coding for the blood flow was not
19 set up so as to ensure flow/mass balance. That is, values of sum of fractional flows to
20 rapidly perfused tissues, liver, and brain (QRTOTC} and sum of fractional flows to slowly
21 perfused tissues (QSTOTC} were selected such that their sum equals one, but if one value
22 were to be changed, the model code would not automatically compensate by changing the
23 other. Therefore, the code was modified so that QSTOTC = 1 - QRTOTC, to facilitate future
24 sensitivity analyses.
25 Human exhaled breath concentrations were compared to CXEQ (= CV/PB based on
26 the model code and consistent with the description of the experiment}, which would be
27 equivalent to the end-exhaled alveolar air after breath holding, but the method used to
28 calculate CXEQ was not noted in Hissink et al. (2007J. This is important because there can
29 be different definitions of exhaled breath depending on the measurement technique. For
30 example, mixed exhaled breath is typically calculated as 70% alveolar air and 30%
31 "inhaled" concentration, due to dead space.
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Toxicological Review ofTrimethylbenzene
1 Comparisons between the computer .m files and published descriptions [Hissink et
2 al.. 2007) indicated minor discrepancies and uncertainties in exposure concentrations and
3 body weight. Exposure concentrations in the simulations were set at the nominal exposure
4 levels, rather than analytically determined levels. The maximum deviation between the
5 nominal level and analytically determined levels occurred in the rat high exposure group,
6 with a nominal exposure of 4,800 mg/m3 WS (7.8% [38.4 mg/m3] 1,2,4-TMB) and mean
7 analytical concentrations ranging from 4,440 to 4,769 mg/m3—as much as 9.2% lower. Rat
8 body weights at time of exposure were reported as 242 to 296 g (Hissink et al.. 2007}. but
9 the .m files use values of 210.01, 204.88, and 209.88 gin the low-, mid-, and high-exposure
10 groups, respectively. Human volunteer body weights reportedly ranged from 69 to 82 kg,
11 and the text states that the fitted Vmax and Km were obtained for a 70 kg male [Hissink etal..
12 2007). but a body weight of 74.9 kg was used in the .m file. No changes to these parameters
13 were made in the model code, based on the assumption that additional data were available
14 to the model authors.
15 Measured human blood concentrations were compared to the average of arterial
16 and venous blood concentrations (CMIX), while the protocol states that blood was taken
17 from the cubital vein, so a more appropriate measure may have been venous blood exiting
18 the slowly perfused tissues compartment (CVS). This choice of dose metric is unlikely to
19 have contributed significantly to any errors in parameterizing the model (i.e., estimating
20 best-fit metabolism parameters) because the difference between the two values is
21 generally small. Revised model code and modeling results are provided on EPA's Health
22 Effects Research Online (HERO) database (U.S. EPA. 2011a).
Verification of model parameter plausibility
Anatomical and physiological parameters
23 The anatomical physiological parameters used by Hissink et al. (2007) were taken
24 from Arms and Travis (1988). but more current convention is to use the parameters in
25 Brown et al. (1997). Comparisons of the rat anatomical and physiological parameters in
26 these sources are found in Table B-4.
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Toxicological Review ofTrimethylbenzene
Table B-4. Comparison of rat anatomical and physiological parameters
in Hissink et al. (2007) to those of Brown et al. (1997)
Parameter
Alveolar ventilation rate (L/hr/kg0'7)
Total cardiac output (L/hr/kg ' )
Blood flow (% cardiac output)
Liver (total)
Fat
Brain
Rapidly perfused (total)
Adrenals
Heart
Kidneys
Lung
Slowly perfused (total)
Muscle
Skin
Total
Tissue volume (% body weight)
Liver
Fat
Brain
Rapidly perfused
Adrenals
Stomach
Small intestine
Large intestine
Heart
Kidneys
Lungs
Pancreas
Spleen
Thyroid
Slowly perfused
Muscle
Skin
Total
Hissink etal.(2007)a
20
20
25
9
1.2
49.8
15
100
4
7
0.72
4.28
75
91
nange irom crown ei
al. (1997)
12-54"
9.6-15
13.1-22.1
7
1.5-2.6
15.3-27.4
0.2-0.3
4.5-5.1
9.5-19
1.1-3
33.6
27.8
5.8
70.5-92.7
2.14-5.16
3.3-20.4
0.38-0.83
3.702-6.11
0.01-0.31
0.4-0.6
0.99-1.93
0.8-0.89
0.27-0.4
0.49-0.91
0.37-0.61
0.24-0.39
0.13-0.34
0.002-0.009
51.16-69.1
35.36-45.5
15.8-23.6
60.682-101.6
values in
agreement?
Yes
No
No
Acceptable0
No
No
No
Yes
Yes
Yes
Yes
Acceptable0
Values from Arms and Travis (1988).
"Assuming a standard 250 g rat.
°Hissink et al. (2007) value outside of literature range, but acceptable (see discussion in text).
1 Many disagreements in values were identified, particularly with respect to the blood
2 flows. In interpreting the blood flow percentages, it should be noted that the percentages
3 enumerated by Brown et al. (1997} do not sum to 100%, which is of course a physiological
4 requirement. Perfusion rates of various depots of fat may differ, so the single value or
5 fractional blood flow to fat given by Brown et al. (1997) of 7%, may be deemed sufficiently
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Toxicological Review ofTrimethylbenzene
1 uncertain that the Hissink et al. (2007) value of 9% is considered acceptable. Brown et al.
2 (1997) report substantially higher blood flow percentages to slowly perfused tissues (skin:
3 5.8% and muscle: 27.8%, for a total of 33.6%} than the value of 15% used by Hissink et al.
4 (2007}. The difference cannot be due to a smaller set of tissues being "lumped" into this
5 compartment, because Hissink et al. (2007} assign a larger volume fraction of tissue to this
6 compartment. Hissink et al. (2007} also assign a higher percentage of blood flow to the
7 liver than indicated by Brown et al. (1997J. Because no sensitivity analyses were conducted
8 by the authors, it is unclear what impact these discrepancies may have had on the
9 predicted 1,2,4-TMB kinetics and visual optimization of metabolism parameters.
10 Comparisons of the human anatomical and physiological parameters in Hissink et al.
11 (2007} and Brown et al. (1997} are found in Table B-5. In general, the agreement was
12 better for humans than it was for rats. Brown et al. (1997} propose a higher default body fat
13 percentage than was used by Hissink et al. (2007), but Hissink et al. (2007} used values
14 derived from measurements of the volunteers participating in the study. Because these
15 volunteers had relatively low percentages of body fat, it is appropriate that the volume of
16 slowly perfused tissue (including muscle} should be increased to compensate.
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Toxicological Review ofTrimethylbenzene
Table B-5. Comparison of human anatomical and physiological
parameters in Hissink et al. (2007) to those of Williams and Leggett
(1989) as reported by Brown et al. (1997)
Parameter
Alveolar ventilation rate (L/hr/kg ' )
Total cardiac output (L/hr/kg07)
Blood flow (% cardiac output)
Liver (total)
Fat
Brain
Rapidly perfused (total)
Adrenals
Heart
Kidneys
Lung
Thyroid
Slowly perfused (total)
Muscle
Skin
Total
Tissue Volume (% body weight)
Liver
Fat
Brain
Rapidly perfused
Adrenals
Stomach
Small intestine
Large intestine
Heart
Kidneys
Lungs
Pancreas
Spleen
Thyroid
Slowly perfused
Muscle
Skin
Total
nissirm ei ai.
(2007)a
20
20
nange irom crown
etal. (1997)
15
16
values in
agreement?
Acceptable
Acceptable
26
5
14
30
25
100
11-34.2
3.7-11.8
8.6-20.4
19.9-35.9
0.3
3-8
12.2-22.9
2.5
1.9-2.2
9-50.8
5.7-42.2
3.3-8.6
52.2-153.1
Yes
Yes
Yes
Yes
Yes
2.6
14.6
2
3
66.4
88.6
2.57
21.42
2
3.77
0.02
0.21
0.91
0.53
0.47
0.44
0.76
0.14
0.26
0.03
43.71
40
3.71
73.47
Yes
Acceptable
(measured)3
Yes
Acceptable
Acceptable
1
2
aThe Hissink et al. [2007] value differs from Brown et al. (1997), but is acceptable (see discussion in text).
Chemical-specific parameters
The chemical-specific model parameters, the partition coefficients, and the
metabolic parameters are summarized in Table B-6.
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Table B-6. Comparison of chemical-specific parameters in Hissink
fZOQT) to literature data
etal.
Parameter
Hissink et al. (2007)
Value
Technique
Literature
Value
Technique
values in
agreement?
Partition coefficients
Saline:Air
Olive oil:Air
Blood:Air- human
Blood:Air - rat
Rapidly perfused:Blood
Slowly perfused:Blood
Fat:Blood
Brain:Blood
Liver:Blood
3
13,200
85
148
2.53
1.21
62.7
2.53
2.53
In vitro
In vitro
In vitro
In vitro
Calculated
Calculated
Calculated
Calculated
Calculated
1.47-1.75"
9,900-
10,400a
59.6-61.33
—
—
-
63b
2b
—
In vitro
In vitro
In vitro
In vivo
In vivo
Acceptable
Acceptable
Acceptable
Yes
Acceptable
Metabolism
VmaxC-rat(mg/hr/kga7)
VmaxC- human (mg/hr/kg07)
Km - rat (mg/L)
Km- human (mg/L)
VmaxC/Km- human (L/hr/kg0'7)
3.5
3.5
0.25
0.25
14
Visual
optimization
Assumed
equal to rat
Visual
optimization
Assumed
equal to rat
Assumed
equal to rat
-
1.2-21°
-
0.42-4.0°
2.6-15°
Optimization
Optimization
Optimization
Yes
No
Yes
Jarnberg and Johanson (1995).
bZahlsen etal. (1990).
°Jarnberg and Johanson (1999).
1 Where data were available, the agreement is generally acceptable. While the rat-
2 derived Km is less than the lower 95% confidence interval value for the human Km, the
3 human VmaxC/Km ratio is in acceptable agreement. When considering sufficiently low
4 exposure concentrations, the performance of the Hissink et al. (2007} human model
5 metabolism parameters would be consistent with the Jarnberg and Johanson (1999) value.
Verification that the model can reproduce all figures and tables in the publication by
Hissink etal. f20071
6 The experimental data in Hissink et al. (2007} were estimated by use of Plot
7 Digitizer (version 2.4.1} to convert the symbols on the relevant figures into numerical
8 estimates. The model code provided (adapted for acslX}, with a variable value for Vmax, does
9 not appear to perfectly reproduce the rat simulations in Hissink et al. (2007} (Figures B-7a
10 and b and B-8a and b} (please note that the Hissink et al. (2007} figures have been
11 "stretched" to produce approximately the same x-axis scale found in the acslX figures}. It
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Toxicological Review ofTrimethylbenzene
1 appears to yield end-of exposure blood and brain concentrations that are about the same as
2 in the Hissink et al. (2007) simulations, but the post-exposure clearance appears faster in
3 EPA's calculations (see, for example, the 16 hr time points for the high exposures}. When
4 the simulations were run with Vmax constant (Figures B-7c and B-8c), as documented in
5 Hissink et al. (2007). the rat simulations yield higher blood and tissue concentrations than
6 depicted in Hissink et al. (2007). most notably at the high exposure concentration. Similar
7 results were obtained for the rat brain concentrations (Figure B-8). The human simulations
8 of blood and exhaled air appear to be faithfully reproduced by the model (Figure B-9). The
9 predicted brain concentration for humans exposed to 600 mg/m3 WS (45 mg/m31,2,4-
10 TMB) for 4 hours was reported as 721 ng/g (0.721 mg/L) in Hissink et al. (2007). whereas
11 the current simulation predicts a concentration of 0.818 mg/L.
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10000
0)
m
1000
100
10
(a)
8 12 16 20
Time (h)
Whle spirit exposure of rats to 0,05, 0.19, and 0.37 mg/L 1,2,4-TOB (Hissink et al., 2007)
(b)
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0
Time (hr)
White spirit exposure of rats to 0.05,0,19, and 0.37 mg/L 1,2,4-TW (Hissink et al., 2007)
0.01
(c)
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7,0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23,0 24.0
Time (hr)
(a) Hissink et al. (2007), Figure 2, lower panel (b) variable Vmax, (c) constant Vmax.
Figure B-7. Simulated and measured blood concentrations of 1,2,4,-TMB
in rats exposed to 600, 2,400, or 4,800 mg/m3 WS for 8 hours.
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Toxicological Review ofTrimethylbenzene
100000
•SJ 10000
i
1000
100
10
(a)
4 8 12 16 20
Time (h)
White spirit exposure of rats at 0,05, 0.19 and 0.37 mgA 1,2,4-TMB (Hissink etaL, 2007)
White spirit exposue of rate at 0.05, 0.19 and 0.37 mj/L 1,2,4-TMB (Hissink etaL, 2007)
10 14 16 IB 20 24
(a) Hissink et al. (2007). Figure 3, lower panel, (b) variable Vmax (c) constant Vmax.
Figure B-8. Simulated and measured brain concentrations of 1,2,4-TMB
in rats exposed to 600, 2,400, or 4,800 mg/m3 WS for 8 hours.
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Toxicological Review ofTrimethylbenzene
1000
0.01
(a)
8 12 16 20
Time (h)
White spirit exposure of humans at 45 mg/L (ICO ppm) (Hissink et al., 2007)
f' — t, cmix
D Ime2
(b)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 H 15 16 17 18 19 20 21 22 23 24
White spirit exposure of humans at45 mg/L (100 ppm)
•5
|
(c)
B D
•f — t, cxeq
^ D Ime2
10 12 14 16 18 20 22 24
(a) Hissink et al. (2007), Figure 4 (b) model simulation during exposure, and (c) model simulation after
exposure.
Figure B-9. Simulated and measured exhaled air concentrations of
1,2,4-TMB in three volunteers exposed to 600 mg/m3 WS for 4 hours.
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B.3.3.2. PBPKModel Optimization and Validation
Methods and Background
1 For all optimizations, the Nelder-Mead algorithm was used to maximize the log-
2 likelihood function (LLF]. A constant heteroscedasticity value of 2 (i.e., relative error
3 model] was assumed. Statistical significance of an increase in the LLF was evaluated for
4 95% confidence per Collins et al. (1999). All kinetic studies were conducted with adult
5 animals or adult human volunteers. In many cases, blood and tissue concentration data in a
6 numerical form were available from the literature (Swiercz et al.. 2003: Swiercz et al..
7 2002: Kostrzewski et al.. 1997: Eide and Zahlsen. 1996: Zahlsen etal.. 1992: Dahletal..
8 1988}. The 1,2,4-TMB blood, brain, and exhaled breath concentration data in Hissink et al.
9 (2007) were published in graphical format and a colleague of Dr. Hissink also provided
10 these in numerical form to Dr. Lisa Sweeney for use in this analysis.
11 Average estimates of the blood concentrations of 1,2,4-TMB (average and standard
12 deviation] in humans exposed only to 1,2,4-TMB as presented in graphs in Jarnberg et al.
13 (1998.1997a: 1996] were used in this evaluation. Estimates of the blood and tissue
14 1,2,4-TMB concentrations in rats presented in graphs in Zahlsen et al. (1990] were also
15 used in this evaluation. Prior to model optimization, physiological parameters were
16 modified from those in Hissink et al. (2007] to better reflect a more recent literature
17 compilation (Brown etal.. 1997] than the references cited by Hissink et al. (2007] (Table B-
18 7]. Where possible, study specific body weights and measured concentrations (rather than
19 nominal concentrations] have been used, as detailed in the .m files (U.S. EPA. 2011a]. For
20 the Zahlsen et al. (1990] 14-day study, body weights for exposures after the first exposure
21 were estimated based on European growth curves for male Sprague-Dawley rats (linear
22 regression of weights for weeks 6-9] (Harlan Laboratories. 2011].
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Table B-7. Parameter values for the rat and human PBPK models for
1,2,4 TMB used by EPA
Parameter
Body weight (kg)
Alveolar ventilation rate (L/hr/kg0'70)
Total cardiac output (L/hr/kg070)
RAT
0.230-0.390a
14
14
HUMAN (AT REST)
70
15
16
Blood flow (% of total cardiac output)
Liver
Fat
Brain
Rapidly perfused
Slowly perfused
17.6
9
2.0
37.8
33.6
17.5
8.5
11.4
37.7
24.9
Volume (% of body weight)
Liver
Fat
Brain
Rapidly perfused
Slowly perfused
4
7
0.57
4.43
75
2.6
21.42
2
3
59.58
Partition coefficients (dimensionless)
Blood: air
Rapidly perfused: blood
Slowly perfused: blood
Fat: blood
Brain: blood
Liver: blood
148
2.53
1.21
62.7
2.53
2.53
85
4.4
2.11
109
4.4
4.4
Liver metabolism
VmaxC(mg/h/kg0-70)
Km(mg/L)
4.17
0.322
1
2
Study specific.
Rat Model Optimization
The rat studies considered in model optimization and model testing (validation] are
summarized in Table B-8.
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Toxicological Review ofTrimethylbenzene
Table B-8. Rat 1,2,4-TMB kinetic studies used in model development
and testing
Reference
Hissink et al.
(2007)
Swiercz et
al. (2003)
Swiercz et
al. (2002)
Zahlsen et
al. (1990)
Zahlsen et
al. (1992)
Eide and
Zahlsen
(1996)
Dahletal.
(1988)
Strain
WAG/RijC
R/BR
(Wistar
derived)
Wistar
Wistar
Sprague-
Dawley
Sprague-
Dawley
Sprague-
Dawley
F344/N
Gender
Male
Male
Male
Male
Male
Male
Male
Nominal
concentration
102, 410, 820
ppm WS (7.8%
1,2,4-TMB [39.1,
157.3, 314.7
mg/m3])
25, 100, 250
(123, 492, 1,230
mg/m3)
25 100 250
(123, 492, 1,230
mg/m3)
1,000
(4,920 mg/m3)
100
492 mg/m3)
75, 150, 300, 450
369, 738, 1,476,
2,214 mg/m3)
100
(492 mg/m3)
Exposu re
regimen
8hr
6 hr/d,
5d/wk
4 wks
6hr
6hr
12 hr/d
14 d
12 hr/d
3d
12 hr
SOmin
1,2,4-TMB
measurement
Mixed blood
time course
Brain time
course
Venous blood
time course
Arterial blood,
liver, brain
Arterial blood
liver, brain
Venous blood
time course
Blood, brain,
perirenal fat on
days 1, 3, 7, 10,
and 14
Blood, brain,
liver, kidney,
and perirenal
fat at end of
exposures and
after 12 hr
recovery
Blood, brain,
liver, kidney,
and perirenal
fat
Inhalation
uptake
Use in model
evaluation
Optimization
(1,2,4-TMB in
mixture)
Testing
Optimization
(1,2,4-TMB
only)
Testing
Testing
Testing
Testing
Testing
Testing
Testing
Form of
comparison
Figure B-10
Figure B-ll
Figure B-12
Table B-9
Table B-9
Figure B-13
Table B-12
Table B-10
Table B-ll
Text
1 Values for VmaxC and Km were numerically optimized based on the fit of the model
2 predictions to the measured blood concentrations of 1,2,4-TMB of Hissink et al. (2007) for
3 rats exposed once to one of three concentrations of 1,2,4-TMB as a component of WS. The
4 optimized value of VmaxC was only modestly different from the value determined by Hissink
5 et al. (2007} (initial: 3.5 vs. optimized: 3.08 mg/hr/kg0-7} from visual optimization (with
6 slightly different physiological parameters], but the Km value differed by 5-fold (initial: 0.25
7 vs. optimized: 0.050 mg/L). The increase in the LLF from 42.6 to 58.2, with two adjustable
8 parameters, indicates that the improvement in fit (Figure B-10] is statistically significant.
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Toxicological Review ofTrimethylbenzene
1 The percentage of variation explained increased from 82.3 to 90.4%, and the fit by visual
2 inspection appears to be very good during exposure (modestly overpredicting] and
3 excellent in the post-exposure period. Using the optimized kinetic parameters, the rat brain
4 concentrations of 1,2,4-TMB were also well-predicted (Figure B-ll}.
"b.oi
(b)
White spirit exposure of rats to 0.05, 0.19, and 0.37 mg/L 1,2,4-TMB (Hissink et al., 2007)
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0
White spirit exposure of rats to 0.05, 0.19, and 0.37 mg/L 1,2,4-TMB (Hissink et al., 2007)
0.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0
Figure B-10. Comparisons of model predictions to measured blood
concentrations in rats exposed to 1,2,4-TMB in WS (Hissink et al.. 2007)
(a) before and (b) after numerical optimization.
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Toxicological Review ofTrimethylbenzene
White spirit exposure of rats at 0.05, 0.19 and 0.37 mg/L 1,2,4-TMB (Hissink et al., 2007)
„ 100
10 11 12 13 14 15 16
Time (hr)
Figure B-ll. Comparisons of model predictions to measured brain
concentrations in rats exposed to 1,2,4-TMB in WS (Hissink et al.. 2007)
using model parameters optimized for fit to Hissink et al. (2007) rat
blood data.
Venous blood 1,2,4-TMB in rats repeatedly exposed to 25, lOOor 250 ppm 1,2,4-TMB (Swiercz etal., 2003)
Venous blood 1,2,4-TMB in rats repeatedly exposed to 25, 100 or 250 ppm 1,2,4-TMB (Swiercz etal., 2003)
Figure B-12. Comparisons of model predictions to measured venous
blood concentrations by Swiercz et al. (2003) in rats repeatedly
exposed to 1,2,4-TMB (a) before and (b) after numerical optimization.
1 The VmaxC and Km values derived from optimization to the Hissink et al. (2007} rat
2 data were used as the starting values for optimizing fit to the venous blood data of Swiercz
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Toxicological Review ofTrimethylbenzene
1 et al. (2003). in which exposure was to 1,2,4-TMB (only] repeatedly for 4 weeks. Venous
2 blood samples were collected from the tail vein. The best fit parameters of VmaxC = 4.17
3 mg/hr/kg0-7 and Km= 0.322 mg/L produced an increase in the LLF from -28.1 to -15.6, a
4 statistically significant improvement, which increased the variation explained from 47.9 to
5 68.1% (Figure B-12}. The deviation between the model and experimental data is primarily
6 exhibeted on the high concentration data set. When this set is not considered, the percent
7 variation explained the remaining two sets is 94.5%. Optimization to the low and middle
8 concentrations alone (omitting the high concentration] does not substantially change the
9 parameters or increase the LLF (simulations not shown}. Optimization using the high
10 concentration alone yields VmaxC and Km estimates of 7.91 mg/hr/kg0-7 and 0.11 mg/L,
11 respectively, with 96.7 percent of variation explained (simulations not shown}.
12 Rat Model Validation
13 The parameters derived from the Swiercz et al. (2003} venous blood optimizations
14 were used to simulate other studies in which rats and humans (see below} were exposed to
15 1,2,4-TMB alone (without co-exposures}. The fit to the Swiercz etal. (2002} venous blood
16 data was very good (Figure B-13}. In fact, the fit to the acute, high-exposure blood
17 concentrations was superior to the fit to the repeated, high-exposure data (Figure B-12b}.
18 This may reflect adaptation (induction of metabolism} resulting from repeated, high
19 concentration exposures.
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Toxicological Review ofTrimethylbenzene
(a)
Venous blood 1,2,4-TMB during acute exposure to 25, 100, or 250 ppm 1,2,4-TMB (Swiercz et al., 2002)
o U.
Time (hr)
Venous blood 1,2,4-TMB after acute exposure to 25,100, or 250 ppm 1,2,4-TMB (Swiercz et al., 2002)
Figure B-13. Comparisons of model predictions to measured rat venous
blood concentrations by Swiercz et al. (2002) in acutely exposed rats
(a) during and (b) after exposure.
1 The model predictions of arterial blood and tissues in the repeated-exposure
2 Swiercz et al. (2003} study were not very accurate, considering that the venous blood data
3 from the same study were used for optimization (Table B-9}. The discrepancies between
4 seemingly contemporaneous venous and arterial blood measurements were noted by the
5 authors of the original study and may be due to collection delays (i.e., tail vein for venous
6 blood, decapitation for arterial samples}. The geometric mean error ratio (greater of
7 model/experiment or experiment/model} for these data was 2.8.
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Toxicological Review ofTrimethylbenzene
Table B-9. Model simulated and experimental measured concentrations
of 1,2,4 TMB in male Wistar rats exposed to 1,2,4-TMB
Exposure concentration
Model
(mg/L)
Experiment
(mg/L)a
Model:
Experiment ratio
Repeated exposure (Model t = 606 hr)
Arterial blood
Brain
Liver
25 ppm (123 mg/m3)
100 ppm (492 mg/m3)
250 ppm (1,230 mg/m3)
25 ppm (123 mg/m3)
100 ppm (492 mg/m3)
250 ppm (1,230 mg/m3)
25 ppm (123 mg/m3)
100 ppm (492 mg/m3)
250 ppm (1,230 mg/m3)
0.61
5.0
22.8
1.91
14.6
59.0
0.41
10.5
54.6
0.33
1.54
7.52
0.45
2.82
18.6
0.45
3.00
22.5
1.8
3.2
3.0
4.2
5.2
3.2
0.91
3.5
2.4
Acute exposure (Model t = 6 hr)
Arterial blood
Brain
Liver
25 ppm (123 mg/m3)
100 ppm (492 mg/m3)
250 ppm (1,230 mg/m3)
25 ppm (123 mg/m3)
100 ppm (492 mg/m3)
250 ppm (1,230 mg/m3)
25 ppm (123 mg/m3)
100 ppm (492 mg/m3)
250 ppm (1,230 mg/m3)
0.53
7.10
18.6
2.19
20.6
62.1
0.49
16.3
57.7
0.31
1.24
7.76
0.49
2.92
18.3
0.44
7.13
28.2
1.7
5.7
2.4
4.5
7.0
3.4
1.1
2.3
2.0
aData from Swiercz et al. (2003).
1 Zahlsen and co-workers [Eide and Zahlsen. 1996: Zahlsen etal.. 1992: Zahlsen et al..
2 1990} conducted studies in which male Sprague-Dawley rats were exposed to 1,2,4-TMB by
3 inhalation for 12 hr/d. For the studies conducted at concentrations similar to those in the
4 Swiercz studies (Tables B-ll and B-10), the model error was similar to that of the arterial
5 blood and tissue measurements in the Swiercz studies (geometric mean error of 3.3 for
6 Zahlsen et al. (1990). and 2.9 for Eide and Zahlsen (1996).
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Toxicological Review ofTrimethylbenzene
Table B-10. Model simulated and experimental measured
concentrations of 1,2,4-TMB in male Sprague-Dawley rats exposed to
100 ppm (492 mg/m3) 1,2,4-TMB (12 hr/d, for 3 d) at the end of
exposure or 12 hours after the last exposure
Venous blood
Brain
Liver
Kidney (compared to
rapidly perfused)
Fat
Day
1
2
3
Recovery"
1
2
3
Recovery"
1
2
3
Recovery"
1
2
3
Recovery"
1
2
3
Recovery"
Model
(mg/L)
8.52
8.71
8.72
1.08
22.6
23.1
23.1
0.46
18.2
18.7
18.7
0.077
22.6
23.1
23.1
0.46
491
503
504
29.1
Experiment
(mg/L)a
1.70
1.51
2.05
0.024
4.57
4.19
4.38
Nondetect
4.92
3.66
4.25
0.072
13.7
17.0
12.4
0.24
210
165
128
14.4
Model:
Experiment ratio
5.0
5.8
4.2
7.6
4.9
5.5
5.3
Not calculated
3.7
5.1
4.4
1.1
1.7
1.4
1.9
1.9
2.3
3.1
3.9
2.0
"Data from Zahlsen et al. (1992).
"Recovery period is designated as 12 hr after the last exposure.
1 There was essentially no difference in the measured venous blood concentration of
2 1,2,4-TMB in the Zahlsen et al. (1992} study at 100 ppm (492 mg/m3} and at 75 ppm (369
3 mg/m3} in the Eide and Zahlsen (1996} study((1.70 andl.69 mg/L, respectively}, so there
4 is evidently some inter-study variability or subtle differences in how the studies were
5 conducted, perhaps in the rapidity of sample collection. The Zahlsen et al. (1990} study,
6 which used a higher nominal concentration of 1,000 ppm (4,920 mg/m3}, exhibited greater
7 deviation between predicted and measured blood and tissue 1,2,4-TMB concentrations
8 (Table B-12}, which generally increased with a greater number of exposure days and then
9 plateaued (geometric mean errors of 2.7, 8.4,12.6,13.9, and 12.1 on exposure days 1, 3, 7,
10 10, and 14, respectively}.
11
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Toxicological Review ofTrimethylbenzene
Table B-ll. Model simulated and experimental measured
concentrations of 1,2,4-TMB in male Sprague-Dawley rats exposed to
1,2,4-TMB at the end of 12 hour exposure
Venous blood
Brain
Liver
Kidney (compared
to Rapidly
perfused)
Fat
Exposure concentration
75 ppm (369 mg/m3)
150 ppm (738 mg/m3)
300 ppm (1,476 mg/m3)
450 ppm (2,252 mg/m3)
75 ppm (369 mg/m3)
150 ppm (738 mg/m3)
300 ppm (1,476 mg/m3)
450 ppm (2,252 mg/m3)
75 ppm (369 mg/m3)
150 ppm (738 mg/m3)
300 ppm (1,476 mg/m3)
450 ppm (2,252 mg/m3)
75 ppm (369 mg/m3)
150 ppm (738 mg/m3)
300 ppm (1,476 mg/m3)
450 ppm (2,252 mg/m3)
75 ppm (369 mg/m3)
150 ppm (738 mg/m3)
300 ppm (1,476 mg/m3)
450 ppm (2,252 mg/m3)
Model
(mg/L)
4.21
17.8
48.3
78.6
11.5
46.6
125
203
7.39
42.2
120
198
11.5
46.6
125
203
255
987
2,636
4,276
Experiment
(mg/L)a
1.69
6.9
13.9
26.6
2.83
11.7
26.5
48.0
6.41
14.8
30.8
56.2
6.41
20.2
33.9
59.1
61.9
457
1,552
2,312
Model:
Experiment ratio
2.5
2.6
3.5
3.0
4.1
4.0
4.7
4.2
1.2
2.9
3.9
3.5
1.8
2.3
3.7
3.4
4.1
2.2
1.7
1.8
Data from Eide and Zahlsen (1996).
1 Dahl et al. (1988) exposed male F344 rats to 1,2,4-TMB at 100 ppm (492 mg/m3} for
2 80 minutes and monitored the total uptake. Under the conditions of the experiment, it was
3 determined that average rat took up 3.28 (trial 1} or 3.89 (trial 2} mg 1,2,4-TMB. In a model
4 simulation, the predicted uptake was 3.61 mg. Geometric mean model error for the two
5 trials was 1.2.
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Toxicological Review ofTrimethylbenzene
Table B-12. Model simulated and experimental measured
concentrations of 1,2,4-TMB in male Sprague-Dawley rats exposed to
1,000 ppm (4,920 mg/m3) 1,2,4-TMB (12 hr/d, for 14 d) at the end of
exposure
Venous blood
Brain
Fat
Day
1
3
7
10
14
1
3
7
10
14
1
3
7
10
14
Model
(mg/L)
181
293
372
395
399
465
747
946
1,005
1,014
9,919
17,328
22,323
23,763
23,961
Experiment
(mg/L)a
63.5
43.1
33.4
34.0
35.2
120
64.9
63.5
62.1
71.5
5,860
2,282
1,835
1,677
2,169
Model:
Experiment ratio
2.8
6.8
11.1
11.6
11.3
3.9
11.5
14.9
16.2
14.2
1.7
7.6
12.2
14.2
11.0
Data from Zahlsen et al. (1990).
Human Model Validation
1 Kinetic parameters derived from optimal fit for rat venous blood data (described
2 above] were tested for the applicability to human kinetics by comparison to studies in
3 which humans were exposed to 1,2,4-TMB alone or 1,2,4-TMB in co-exposures with WS
4 (Table B-13). The key data set for validation in humans was deemed to be Kostrzewski et
5 al. (1997} because these volunteers were exposed to 1,2,4-TMB alone (no co-exposure, as
6 in Hissink et al. (2007)) under sedentary conditions (i.e., level of effort was not elevated, as
7 in Jarnberg et al. (1998,1997a: 1996)).
8 Using the VmaxC and Km derived from the Swiercz et al. (2003) rat repeated exposure
9 data, the simulated blood concentration underestimated those measured during exposure
10 of human volunteers by Kostrzewski et al. (1997). then overpredicted blood concentrations
11 up to 7 hours post-exposure, and underpredicted subsequent measured blood
12 concentrations (Figure B-14). Of 21 blood measurements, only two differed from the
13 simulated value by more than a factor of 2 (maximum: 2.6), with a geometric mean
14 deviation of 1.5-fold between the simulated and measured values. The percent variation
15 explained was 69.74%. When Km was held constant and VmaxC was optimized (final value:
16 3.39 mg/hr/kg0-7), the improvement in fit was minimal (72.14% of variation explained),
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Toxicological Review ofTrimethylbenzene
1 and not statistically significant, so the rat-derived values were considered acceptable (see
2 the section regarding rat model optimization, page B-29).
Table B-13. Human kinetic studies of 1,2,4-TMB used in model
validation
Reference
Kostrzewski
et al. (1997)a
Jarnberg et al.
1997a; 1996)b
Hissink et al
(2007)°
Ethnicity
Not stated;
conducted
in Poland
Caucasian;
conducted
in Sweden
Not stated;
spoke Dutch
as "native
language"
Gender
Sex not
stated.
Assumed
male.
Male
Male
Nominal
concentration
30 ppm
(147.6 mg/m3)
2 and 25
(~10 and 123
mg/m3)
100 ppm WS
with 7.8% 1,2,4-
TMB
(-38.3 mg/m3
1,2,4-TMB)
Exposu re
regimen
8hr
2 hr at 50
W
(bicycle)
6hr
1,2,4-TMB
measurements
Venous blood
time course
Venous blood
and exhaled air
time course
Venous blood
and end exhaled
air time course
Use in
model
evaluation
Testing
Testing
(blood data
only)
Testing
Form of
comparison
Figure B-14
Figure B-15
Figure B-16
aFive volunteers, ages 24-37, with no known occupational exposure to 1,2,4-TMB. Height of 1.70 to 1.86 m and BW of
70-97 kg. The average of the high and low values for age, height, and weight plus assumed gender (male) were used to
calculate central tendency estimate of 22.44% for volume of body fat (VFC), per Deurenberg et al. (1991) (1991). QPC
estimated from the midpoint of the range for total ventilation (0.56 to 1 m3/hr), average of high and low body weights,
BW0'74 scaling, and an assumption that alveolar ventilation was 2/3 of total ventilation.
bTen volunteers, average age 35, range 26-48, with no known occupational exposure to solvents; volunteers were
instructed to avoid contact with organic solvent and to refrain from taking drugs or drinking alcoholic beverages for 2
days before exposure. Average BW 76.5 kg. Alveolar ventilation rate (QPC) estimated from the mean value for total
ventilation rate during exposure, average body weights, BW074scaling, and an assumption that alveolar ventilation was
2/3 of total ventilation. Digitized blood data (group averages) extracted from figures.
°Three volunteers, ages 23-26, BW 69-82 kg, mean body fat of 14.6% (skin caliper measurement); alcohol consumption
10-15 drinks/week (all subjects), one smoker (4 cigarettes per day).
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Toxicological Review ofTrimethylbenzene
Blood 1,2,4-TMB in human volunteers exposed to 154 mg/m3 1,2,4-TMB (Kostrzewski etal., 1997)
Y
\
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82 84 86 88 90 92 94 96
Time (hr)
Figure B-14. Comparisons of model predictions to measured human
venous blood concentrations of Kostrzewki et al. (1997) in human
volunteers exposed to 154 mg 1,2,4-TMB/m3 for 8 hours.
1 For comparisons between the Jarnberg et al. [1999: 1998.1997a: 1996) data and
2 the model, simulations were conducted with QPC (calculated as described in footnote to
3 Table B-13) at the elevated (working] level throughout the simulation, but with no other
4 adjustments made for exercise conditions. The model consistently underpredicted the
5 measured venous blood concentrations of 1,2,4-TMB (Figure B-15). At 25 ppm (123
6 mg/m3), blood concentrations were underpredicted by a factor of 2.1 to 3.5 during
7 exposure and by a factor of 1.04 to 1.5-fold in the post-exposure period, for a geometric
8 mean discrepancy of 1.7 for this concentration. At 2 ppm (~10 mg/m3}, blood
9 concentrations were underpredicted by factors of 1.7 to 2.7 during exposure and 1.01 to
10 1.2 in the post-exposure period, for a geometric mean discrepancy of 1.6 for this
11 concentration.
Blood concentrations of 1,2,4-TMB in volunteers exposed to 2 or 25 ppm 1,2,4-TMB (Jarnberg and coworkers)
Time (hr)
Figure B-15. Comparisons of model predictions to measured human
venous blood concentrations of Jarnberg et al. (1998.1997a: 1996) in
volunteers exposed to 2 or 25 ppm (~10 or 123 mg/m3) 1,2,4-TMB for 2
hours while riding a bicycle (50 W).
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Toxicological Review ofTrimethylbenzene
1 Comparisons of model predictions and experimental data were also made for the
2 human study described in Hissink et al. (2007) in which volunteers inhaled 100 ppm WS
3 with 7.8% 1,2,4-TMB (38.4 mg/m31,2,4-TMB) for 4 hours (Figure B-16). The agreement
4 between simulated and measured concentrations of 1,2,4-TMB in blood during exposure
5 was excellent. The agreement between the modeled and measured 1,2,4-TMB in end-
6 exhaled air during the post-exposure period was very good.
White spirit exposure of humans at 45 mg/L (100 ppm) (Hissink et al., 2007)
9
10
11
12
Ch D
rK
DOM:
(a)
567
Time (hr)
White spirit exposure of humans at 45 mg/L (100 ppm)
10 11 12 13 14
^ 1.0x10 E-2
dt
£
« 1.0x10 E-4
ra
S
I
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Toxicological Review ofTrimethylbenzene
1 predicting the kinetics of 1,2,4-TMB in test animals with no co-exposures. Another concern
2 was the potential for kinetic changes with repeated exposure. As the Swiercz et al. (2003)
3 rat kinetic study involved repeated exposure to 1,2,4-TMB without potentially confounding
4 co-exposures, and provides post-exposure venous blood time course data, it appears to be
5 the most suitable for describing kinetics relevant to chronic RfC and RfD development. The
6 VmaxC and Km values from the numerical optimization to the Hissink et al. (2007) rat data
7 were used as starting values for optimization of the fit to the Swiercz et al. (2003) venous
8 blood data. The improvement in fit for the low and middle concentrations (25 and 100 ppm
9 [123 and 492 mg/m3]] was apparent from careful visual inspection and was statistically
10 significant, and these values were used in subsequent validation simulations.
11 In general, the model simulations of venous blood concentrations in exposed Wistar
12 rats, uptake by F344 rats, and venous blood and exhaled breath of human volunteers were
13 acceptable. The measured Wistar rat arterial blood and tissue concentrations were
14 consistently overpredicted by the model, suggesting collection delays in the studies. The
15 model also consistently overpredicted the measured Sprague-Dawley rat tissue and blood
16 concentrations, including the "recovery" (12 hr post-exposure] samples, which should not
17 be subject to collection delays. Many of the "validation" comparisons were made at
18 exposure concentrations (250 ppm [1,230 mg/m3]or greater] for which the optimized
19 model did not provide accurate venous blood concentrations. It cannot be determined with
20 the available data whether the 2-3-fold differences between the model and Sprague-
21 Dawley rat blood concentrations at lower concentrations (75 and 150 ppm [369 and 738
22 mg/m3]] are due to methodological differences (e.g., in sample collections and analysis] or
23 true strain differences. Overall, we conclude that the optimized model produces acceptable
24 simulations of venous blood 1,2,4-TMB for chronic exposure to < 100 ppm (492 mg/m3] for
25 rats or < 30 ppm (147.6 mg/m3] for humans 1,2,4-TMB by inhalation. If rat exposures of
26 interest exceed 100 ppm (492 mg/m3], consideration should be given to reassessing model
27 validation at high concentrations using VmaxC and Km parameters optimized for repeated,
28 high concentration exposures [e.g., 250 ppm (1,230 mg/m3] from Swiercz et al.(2003]].
B.3.3.3. Sensitivity Analysis of Rat Model Predictions
29 The primary objective of the sensitivity analysis was to evaluate the ability of the
30 available data to unambiguously determine the values of both VmaxC and Km (i.e., parameter
31 identifiability]. Toward this end, sensitivity analyses were conducted using acslX. Because
32 the selected key data set was the venous blood concentrations in the Swiercz et al. (2003]
33 study, simulations were conducted to see how small changes in parameters changed the
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1 estimated venous blood concentrations under the conditions of this study, simulating the
2 first 12 hours (6 hrs exposure, 6 hrs post-exposure], conditions that are essentially
3 identical to those in Swiercz et al. (2002}. The evaluations were limited to the lowest (25
4 ppm [123 mg/m3]} and highest (250 ppm [1,230 mg/m3]} exposure concentrations. It
5 should be noted that after the optimization (Figure B-13b}, the agreement between the
6 model and the experimental data at the lower exposure concentration was superior to the
7 agreement at the high concentration, so the low concentration sensitivity analysis results
8 are somewhat more meaningful than the high concentration results. The results are
9 calculated as normalized sensitivity coefficients (NSC] (i.e., percent change in
10 output/percent change in input, calculated using the central difference method}.
11 The interpretation of the sensitivity analysis outputs focused on the times during
12 which blood concentrations were measured, so the sensitivity analyses for the first 15
13 minutes of exposure were not considered relevant. Parameters are grouped (Table B-14}
14 as relatively insensitive (maximum]NSC| < 0.2 for 0.25 hr < t < 12 hr}, moderately sensitive
15 (0.2 < maximum|NSC| < 1.0}, or highly sensitive (maximum|NSC| > 1.0}.
16 VmaxC/Km was identifiable from the data (as opposed to VmaxC and Km each being
17 identifiable}, one would expect that the NSC for these parameters would always be
18 opposite in sign, and equal in magnitude, which is not the case. We conclude that Km and
19 VmaxC are distinctly identifiable using the Swiercz et al. (2003: 2002} data.
20 While the focus of this sensitivity analysis was to evaluate the identifiability of
21 chemical-specific parameters from the available data, additional insights can be obtained
22 by considering the other "sensitive" parameters. Predicted blood concentrations were
23 sensitive to the value of QPC (ventilation rate}. If high concentrations produce a sedative
24 effect, decreases in ventilation could contribute to the model's greater over-prediction of
25 the experimentally measured values at high concentrations [e.g., as high as 1,000 ppm
26 (4,920 mg/m3}, in Zahlen et al. (1990}]. The accuracy of the predicted net uptake in the
27 Dahl et al. (1988} study indicates that, at 100 ppm (492 mg/m3}, the model value of QPC is
28 likely appropriate, since net uptake in this relatively short experiment (80 minutes} is
29 highly sensitive to the breathing rate (simulations not shown}. The fractional volumes of
30 the fat and slowly perfused tissues compartments are also moderately important
31 parameters (with time courses similar to those of the corresponding partition coefficients
32 shown in Figure B-15}. The volume of the fat compartment in particular is known to vary
33 with age and strain (Brown etal.. 1997}. so using the same value for all studies might have
34 an impact on the predicted kinetics.
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Table B-14. Parameter sensitivity for venous blood 1,2,4-TMB
concentration in rats exposed to 1,2,4-TMB via inhalation
Parameter
BW
CONC
QPC
VmaxC
Km
PB
PS
PR
PL
PBR
VFC
VSTOTC
VRTOTC
VLC
VBRC
QCC
QFC
QRTOTC
QLC
QBRC
Insensitive
(maximum |NSC| < 0.2)
H
L
L, H
L, H
L, H
L, H
L, H
L, H
H
L, H
Moderately sensitive
(0.2 < maximu m | NSC | <
1.0)
L, H
L, H
L
H
L, H
L, H
L, H
L, H
H
L, H
L, H
Highly sensitive (maximum
|NSC| >1.0)
L, H
L, H
L
L
L= low exposure concentration (25 ppm [123 mg/m ]), H = high exposure concentration (250 ppm [,1230
mg/m3]).
Body weight (BW), concentration of 1,2,4-TMB in the air (CONC), alveolar ventilation rate (QPC),
Michaelis-Menten maximum rate of metabolism (VmaxC), Michaelis-Menten constant: concentration
where Vm,ax is half-maximal (Vmax), blood:air partition coefficient (PB), fat:blood partition coefficient (PF),
slowly perfused:blood partition coefficient (PS), rapidly perfused:blood partition coefficient (PR),
liverblood partition coefficient (PL), brain:blood partition coefficient (PBR), volume of fat (VFC), volume
of slowly perfused tissues (VSTOTC), volume of rapidly perfused tissues (VRTOTC), volume of liver (VLC),
volume of brain (VBRC), cardiac output (QCC), blood flow to fat (QFC), blood flow to slowly perfused
tissues (QRTOTC), blood flow to liver (QLC), blood flow to brain (QBRC)
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I
c
V
n
w
O
2
-0.0
0.3
.1
°
E
o
Sensitivity analysis: rat CV, low concentration
exposure
(Swiercz et al.f 2002, 2003)
cv:km
cvivmaxc
cv:pb
cv:pf
cv:ps
Time (hr)
Sensitivity analysis: ratCV, high concentration
exposure
(Swiercz et al., 2002, 2003)
cv:km
cv:vmaxc
cv:pb
cv:pf
cv:ps
(b) Time(hr)
Figure B-17. Time course of normalized sensitivity coefficients of
moderately sensitive chemical-specific parameters (response: venous
blood concentration) in rats exposed to (a) 25 ppm (123 mg/m3) or (b)
250 ppm (1,230 mg/m3) of 1,2,4-TMB via inhalation for 6 hours
(Swiercz et al.. 2003: Swiercz et al.. 2002).
B.3.3.4. Sensitivity Analysis of Human Model Predictions
1 A sensitivity analysis for human model predictions to all parameters was conducted
2 for continuous inhalation exposures, and results are shown in Table B-15. The results are
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1 presented as normalized sensitivity coefficients (i.e., percent change in output/percent
2 change in input, calculated using the central difference method; NSC}. Similar to analyses
3 performed for the rat, parameters are noted as relatively insensitive (|NSC| < 0.2],
4 moderately sensitive (0.2 < |NSC| < 1.0], or highly sensitive (|NSC| > 1.0}. To bracket the
5 range of human equivalent concentrations (HECs], inhalation sensitivities were evaluated
6 at 10 and 150 ppm (49.2 and 738 mg/m3} concentration. The resulting coefficients
7 (Table B-15] are not surprising. The two fitted metabolic parameters, VmaxC and Km both
8 influence model predictions. The VmaxC sensitivity is higher at 150 ppm (738 mg/m3}
9 (|0.8873|] than at 10 ppm (49.2 mg/m3} (|0.238|) due to the slight metabolic saturation.
Table B-15. Parameter sensitivity for steady-state venous blood 1,2,4-
TMB concentration in humans exposed to 1,2,4-TMB via inhalation
Parameter
BW
CONC
QPC
VmaxC
Km
PB
PS
PR
PL
PBR
VFC
VSTOTC
VRTOTC
VLC
VBRC
QCC
QFC
QRTOTC
QLC
Insensitive
(maximum | NSC | < 0.2)
L, H
L, H
L, H
L, H
L, H
L, H
L, H
L, H
L, H
L, H
L, H
L, H
L, H
L, H
L, H
Moderately sensitive
(0.2 < maximu m | NSC | <
1.0)
L
L, H
L, H
L, H
L, H
Highly sensitive
(maximum | NSC | > 1.0)
H
L = low exposure concentration (10 ppm [49.2mg/m3]), H = high exposure concentration (150 ppm [738
mg/m3]).
Body weight (BW), concentration of 1,2,4-TMB in the air (CONC), alveolar ventilation rate (QPC),
Michaelis-Menten maximum rate of metabolism (VmaxC), Michaelis-Menten constant: concentration
where Vm,ax is half-maximal (Vmax), blood:air partition coefficient (PB), fat:blood partition coefficient (PF),
slowly perfused:blood partition coefficient (PS), rapidly perfused:blood partition coefficient (PR),
liverblood partition coefficient (PL), brain:blood partition coefficient (PBR), volume of fat (VFC), volume
of slowly perfused tissues (VSTOTC), volume of rapidly perfused tissues (VRTOTC), volume of liver (VLC),
volume of brain (VBRC), cardiac output (QCC), blood flow to fat (QFC), blood flow to slowly perfused
tissues (QRTOTC), blood flow to liver (QLC), blood flow to brain (QBRC)
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B.3.3.5. Modification of the Hissink et al. (2007) model to include oral route of
exposure
1 For derivation of an oral RfD, the updated 1,2,4-TMB PBPK model based on Hissink
2 et al. (2007} was further modified by adding code for continuous oral ingestion. It was
3 assumed that 100% of the ingested 1,2,4-TMB is absorbed by constant infusion of the oral
4 dose into the liver compartment. There were no oral data available to calibrate the model
5 for oral absorption and no data were available evaluate the model predictions following
6 oral ingestion either. Thus, the assumption that 100% of the dose would enter the liver is a
7 common assumption.
8 The contribution of the first-pass metabolism in the liver for oral dosing was
9 evaluated by simulating steady state venous blood levels (at the end of 50 days continuous
10 exposure] for a standard human at rest (70 kg] for a range of concentrations and doses. For
11 ease of visual comparison (Figure B-18], concentrations were converted to daily doses
12 based on the amount of 1,2,4-TMB inhaled, as computed by the model. (An inhaled
13 concentration of 0.001 mg/L [0.20 ppm (0.98 mg/m3}] is equivalent to an inhaled dose of
14 0.12 mg/kg/day.} At both very low and very high daily doses by inhalation or oral dosing,
15 steady state CV is essentially linear with respect to the daily dose, but with different
16 CV/dose ratios and a transition zone between 1 and 100 mg/kg/day. At low daily doses,
17 equivalent inhalation doses result in steady state blood concentrations 4-fold higher than
18 an equivalent oral dose due to the hepatic first-pass effect. The first-pass effect becomes
19 insignificant with respect to steady-state venous blood concentrations for daily doses in
20 excess of -50 mg/kg/day.
21
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00
J_
o
TJ
°ro
0.1
0.01
•Oral
Inhalation
0.1
10
100 1000 10000 100000
Daily dose (mg/kg/d)
Figure B-18. Effect of route of exposure and dose rate on steady-state
venous blood concentration (t = 1,200 hr) for continuous human
exposure to 1,2,4-TMB.
B.3.3.6. Conclusions
1 Several changes were made to the model for use in this assessment: (1} Updated
2 physiological parameters were implemented [Brown etal.. 1997): (2} Hepatic metabolism
3 was revised to omit variation over time and new VmaxC and Km values were estimated
4 through numerical optimization; and (3} An oral dosing component was added to the
5 model as constant infusion into the liver compartment. The values were optimized to
6 Hissink et al. (2007) data and resulted in a VmaxC of 4.17 mg/hr/kg0-7 and Km of 0.322 mg/L.
7 In addition, the model was tested for its ability to predict published rat data resulting from
8 exposure to 1,2,4-TMB alone [Swiercz etal.. 2003: Swiercz etal.. 2002: Eide and Zahlsen.
9 1996: Zahlsen etal.. 1992: Zahlsen etal.. 1990: Dahl etal.. 1988). Using the optimized
10 values, the model adequately predicted the data and lower concentrations. Human data
11 fHissink etal.. 2007: larnbergand lohanson. 1999: larnberg etal.. 1998.1997a:
12 Kostrzewski etal.. 1997: larnberg et al.. 1996) were also utilized to validate model
13 predictions.
B.3.4. Summary of Available PBPK models for 1,3,5-TMB or 1,2,3-TMB
14 There are currently no available PBPK models for rodents or humans for either
15 1,3,5-TMB or 1,2,3-TMB.
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B.4. HUMAN STUDIES
Table B-16. Characteristics and quantitative results for epidemiologic
cross-sectional study of exposure to 1,2,4-TMB. Battig et al. (1956b). as
reviewed by Baettig et al. (1958)
Study (location)
Outcome assessment
• Transportation plant in Switzerland
• Survey was conducted to investigate the
CNS, respiratory, hematological effects of
long-term TMB exposure
• Additional information on working history,
personal history, and psychiatric health was
collected
POPULATION CHARACTERISTICS
Exposed population
Referent or control description
• 27 TMB-exposed workers that worked primarily in
the painting shop of the transportation plant
• 10 unskilled workers from the same plant
that were not exposed to TMB vapors.
Exposure assessment
Statistical analysis
• Exposure level: 10-60 ppm (49.2-295 mg/m ) in
working rooms
• Exposure duration: approximately 10 years
• Compounds to which study participants were
exposed: Fleet-X DV-9, a solvent that contained
1,2,4-TMB and 1,3,5-TMB (50% and 30%,
respectively) for approximately 10 years. Fleet-X
DV-99 also potentially contained 1,2,3-TMB and
numerous methylethylbenzenes.
• No statistical analyses were reported.
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RESULTS
Exposure subgroup
• Increased self reports of vertigo, headaches, and drowsiness during work.
• Increased presence of chronic asthmatic bronchitis, anemia, and altered blood clotting characteristics
(e.g., increased clotting time and tendency to hemorrhage).
• Increased vitamin C deficiency was observed in controls, but the authors attribute this to nutritional
deficiencies in this population.
Effect estimate ( 95% Cl)
:igure 1. Clinical findings obtained from workers exposed to TMB compared to unskilled worker controls not
exposed to TMB.
Painters n=27
Unskilled workers n=10
Subjective Asthmatic Anemia
complaints bronchitis <4.5x10(6>
voiced during RBC/uL
work
Tendency Vitamin C
to deficiency
hemorrhage
Source: Reproduced with permission of Springer-Verlag (Baettig et al. 1958)
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Table B-17. Characteristics and quantitative results for epidemiologic
cross-sectional study of exposure to 1,2,4-TMB Billionnet et al. (2011)
Study (location)
• Random selection of dwellings throughout
France
Outcome assessment
• Standardized, self-administered questionnaire
was completed by participants to determine
number and severity of respiratory effects,
particularly asthma and rhinitis.
• Additional information on daily habits, smoking
status, and sociodemographic variables was
collected.
• Diagnosis of rhinitis or asthma was not
confirmed by a physician.
POPULATION CHARACTERISTICS
Exposed population
• 1,612 individuals living in 567 dwellings, aged
15 or older.
• Surveys were conducted and air samples were
collected over a period of one week.
Exposure assessment
• Exposure level: For 1,2,4-TMB, exposure varied
from undetectable to 111.7 ng/m3, with
median concentration 4.0 ng/m .
• Exposure duration: Not reported; reported
measurements represent the means of one
week of monitoring.
Referent or control description
• The study cohort was also used as the control
group. Dwellings with low levels of individual
volatile organic compound (VOCs) were used
as controls for that particular compound.
Statistical analysis
• Pollutant correlations tested by Spearman's
rank correlation coefficient.
• Generalized estimating equation approach
used to adjust for correlations between
individuals within same dwelling.
• Global VOC score was created to address
exposure to multiple pollutants.
• All models were adjusted for age, sex, and
smoking status.
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RESULTS
Exposure subgroup
• Statistically significant increase in odds ratios for asthma following 1,2,4-TMB exposure.
• No statistically significant increase in odds ratio for rhinitis and 1,2,4-TMB exposure.
Effect estimate ( 95% Cl)
F
f
igure 1. Odds ratios for asthma and asthma/rhinitis and exposure to 1,2,4-TMB. For all models, data was adjusted
or confounders.
Odd si
Odds ratio for asthma
according to adjusted
marginal model
Odds ratio for asthma
25th vs. 75th
p ere entiles
Odds ratio for asthma
95th vs. 75th
p ere entiles
Ratios for Asthma Associated with 1,2,4-TMB Exposure
4*L
Q
O.
1
A
V
01234567
Odds Ratio
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Table B-18. Characteristics and quantitative results for epidemiologic
cohort study of exposure to 1,2,4-TMB. Chen et al. (1999)
Study (location)
• Dockyard in Scotland, United Kingdom
Outcome assessment
• Survey was conducted to determine mortality,
symptoms, and risks of paint exposure.
• Additional information on age, education,
smoking, alcohol consumption, and personality
was collected.
POPULATION CHARACTERISTICS
Exposed cohort
• 1292 TMB-exposed males who worked as
painters in a dockyard for at least 1 yr between
1950 and 1992.
• Follow up period extended from 1960 through
1994
Exposure assessment
• Exposure level: Specific concentrations not
discussed
• Exposure duration: at least 1 yr; range 1-41
years
• Compounds to which study participants were
exposed: white spirit (1,2,4-TMB), xylene, TMB
(unspecified), n-butanol, trichlorethylene,
naptha, and cumene.
Referent or control description
• 953 individuals matched by age and selected
from lists of patients of local primary care
physicians.
Statistical analysis
• Intra-cohort proportional mortality ratios were
calculated, as were standardized mortality
ratios for comparison with all Scottish males.
95% confidence intervals calculated assuming
a Poisson distribution.
• X2test used to assess differences in
neuropsychological symptoms between
painters and non-painters.
• Brestow-Cox model used to adjust for
covariates including educational level,
smoking, alcohol consumption, and social
conformity.
• Log-regression model used for case-control
study.
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RESULTS
Exposure subgroup
• Increased prevalence rate ratios for neuropsychological symptoms amongst painters.
• Rate ratios increased significantly with increasing number of years of exposure, even after adjustment for
possible confounders.
• Multivariate-adjusted odds ratios within nested case-control analysis showed same relationship.
Effect estimate ( 95% Cl)
Figure 1. Unadjusted and adjusted prevalence rate ratios for neuropsychological symptoms in dockyard painters
vs. controls. With increasing years of exposure, rate ratios were found to increase. Symptoms included difficulty in
buttoning and unbuttoning, trembling hands, or unsteadiness in arms or legs. For trend in unadjusted rate ratios,
p<0.00001.
Unadjusted a
Neuropsychc
UNADJUSTED
1-4 Years Exposure
5-9 Years Exposure
10-14 Years Exposure
15-41 Years Exp o sur e
ADJUSTED
1-4 Years Exposure
5-9 Years Exposure
10-14 Years Exposure
15-41 Ye ars Exp o sur e
nd Adjusted Prevalence Rate Ratios for
•logical Symptoms in Dockyard Painters
A
V
V
O,
1
O
A
v
A,
V
A
V
A
V
01234567
Prevalence Rate Ratio
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Figure 2. The effect of elapsed time since cessation of painting on all symptoms. Values reported are prevalence
rate ratios for painters vs. non-painters. No significant decrease in risk with increasing post-exposure time was
found.
Prevalence Rate Ratios for Neuropsychological Symptoms in
Dockyard Painters Following Cessation of Exposure
Active Painters A
1-10 years post-exposure
11-18 years post-exposure
>19 years post-exposure
234
Prevalence Rate Ratio
Figure 3. The effect of exposure duration on odds ratio for neuropsychological symptoms. With increasing years of
exposure, odds ratios were found to increase.
Odds Ratios for Neuropsychological Symptoms in Dockyard Painters
1-4 Years Exposure
5-9 Years Exposure
10-14 Years Exposure
15-41 Years Exposure
2 4
10 12 14 16 IS 20 22 24 26
Odds Ratio
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Table B-19. Characteristics and quantitative results for controlled
human exposure study of exposure to 1,2,4-TMB in WS Lammers et al.
fZOQT)
Study design
Species Sex N Exposure route Dose range Exposure duration
Humans M 12 Inhalation 57 or 570 mg/m 4 hrs
Additional Study details
• Human volunteers were exposed to 57 or 570 mg/m3 during two test sessions separated by 1 wk, each lasting
4 hrs.
• Several tests were conducted to evaluate impact of WS on CNS. These included tests of observation, reaction
time, and hand-eye coordination.
• In humans, attention deficit was observed following WS inhalation.
• The study protocol was approved by the TNO's Institutional Review Board
Observation
Fatigue (scale score)
Pre-test
Ihr
3 hrs
Post-test
Vigor (scale score
Pre-test
Ihr
3 hrs
Post-test
Hand-eye coordination test
(pixels in InMAE)
Pre-test
Ihr
3 hrs
Post-test
Finger tapping test (no. of
taps in 30 seconds)
Pre-test
Ihr
3 hrs
Post-test
Test scores (mean ± SD) at various time points in humans exposed to 57 or 570
mg/m3 WS for 4 hrs
57 mg/m3
570 mg/m3
Mood and affect
1.11 ±0.04
1.06 ± 0.03
1.21 ±0.12
1.38 ±0.15
3.35 ±0.20
3.58 ±0.16
3.27 ±0.20
2.98 ±0.23
1.11 ±0.05
1.17 ±0.09
1.29 ±0.13
1.51 ±0.23
3.53 ±0.09
3.23 ±0.20
3.32 ±0.22
3.05 ±0.22
Psychomotor skills (hand-eye coordination and finger tapping)
1.69 ±0.05
1.56 ±0.05
1.64 ± 0.05
1.62 ± 0.04
201 ±7
205 ±5
202 ±8
198 ±7
1.67 ± 0.04
1.64 ± 0.04
1.63 ± 0.04
1.55 ±0.06
203 ±6
194 ±6
196 ±6
200 ±6
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Reaction time test (latency,
ms)
Pre-test
0.25 hrs
Ihr
2.25 hrs
3 hrs
Post-test
Color word vigilance test
(latency, ms)
Pre-test
Ihr
3 hrs
Post-test
-— 220 -,
LU •""
(0
't'
0 210
i
& 200-
(A
O
co
'» 190
2
i_
<\
— o — placebo tapping test with the dominant
—A— White Spirit hand at different time points during
and after exposure.
pre-test 1-hr 3-hr post -test
time of testina
Health Effect at LOAEL
n/a
NOAEL
n/a
LOAEL
n/a
Comments: Exposure to 1,2,4-TMB was via WS, which is comprised of additional substances. LOAEL and NOAEL for
1,2,4-TMB alone cannot be extracted from this study because other constituents of the WS mixture may confound
results.
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Table B-20. Characteristics and quantitative results for epidemiologic
cohort study of exposure to 1,2,4-TMB. Lee et al. (2005)
Study (location)
Outcome assessment
• A shipyard in Ulsan, Korea
• Various neurobehavioral parameters were
measured with computer-based
neurobehavioral assessments.
• Measured parameters included simple
reaction time, symbol digit substitution, and
finger tapping speed.
• Additional information on occupational
history, medical history, age, work duration,
education level, alcohol use, and smoking
status.
POPULATION CHARACTERISTICS
Exposed population
Referent or control description
• 180 shipyard workers exposed to mixed organic
solvents.
• Workers were exposed generally during painting
activities within the shipyard.
• 60 Shipyard workers that were not exposed
to mixed organic solvents were used as the
referent group
Exposure assessment
Statistical analysis
• Data on exposure was collected from 61 workers
who wore passive dosimeters on 3 work days.
• Average Exposure duration: 16.5±9 years in
exposed workers.
• A cumulative exposure index was calculated
for each worker.
• Student t-test was used to determine
statistical significance of results in exposed
workers compared to non-exposed workers.
RESULTS
Exposure Subgroup
• Exposed workers showed significant alterations to symbol digit distribution, dominant hand finger tap
rate, and non-dominant hand finger tap rate.
• Work duration was also found to influence symbol digit substitution
Observation
Results of Neurobehavioral Test of Study Subjects
Unadjusted Mean ±Std Dev
Painters
Controls
p-value
Adjusted Mean (S.E.)
Painters
Controls
p-value
Simple Reaction
Time
297.2±70.0
292.2±95.0
0.671
296.0(5.9)
295.8(10.9)
0.992
Symbol Digit
Substitution
3233.2±998.9
2,693.8±711.8
0.000
3,156.6
(67.7)
2,691.6
(124.3)
0.000
Finger tap speed
DHb
62.6±8.2
66.4±9.7
0.000
63.0 (0.6)
65.5(1.2)
0.046
Finger tap speed
NDHC
55.9±8.0
60.2±9.7
0.000
56.1(0.7)
60.3(1.2)
0.003
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Observation
Simple Reation
Time
Symbol Digit
Substitution
Finger Tap
Speed DH
Finger Tap
Speed NDH
Neurobehavioral Test Results by Duration of Work, Adjusted for Age and Education
<10 Working Years (S.E.)
n = 48
297.8 (20.4)
2,972.1(282.5)
64.8 (2.3)
57.6 (2.4)
10-20 Working Years (S.E.)
n = 41
297.9 (11.2)
3,033.8 (155.1)
63.9 (1.3)
56.3 (1.3)
>20 Working Years (S.E.)
n = 91
292.3 (11.6)
3,452.4 (160.7)*
61.3(1.3)**
55.2 (1.3)
Adjusted for age and education
bFinger tapping speed of dominant hand
°Finger tapping speed of non-dominant hand
*, **p< 0.05, p = 0.052
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-20. Characteristics and quantitative results for epidemiologic
cross-sectional study of exposure to 1,2,4-TMB Norseth etal. (1991)
Study (location)
• Norway
Outcome assessment
• Symptoms were recorded via a standard
questionnaire on the last day of monitoring.
• Monitoring of organic compounds was
conducted for 5 days in workers who were
divided into subsets based on their level of
exposure.
• Asphalt, weather, and traffic density data was
recorded daily.
POPULATION CHARACTERISTICS
Exposed population
• In the first group, 79 workers were divided into
groups of 5 or 6 based on their exposure level.
• A second group of 254 (of which the initial
group of 79 was representative) workers
completed questionnaires about symptoms.
Exposure assessment
• Mean concentration of 1,2,4-TMB was 0.015
ppm (0.074 mg/m3), with range between 0 and
0.122 (0 - 0.60 mg/m3) ppm.
• Mean concentration of 1,3,5-TMB was 0.0014
ppm (0.0069 mg/m3), with range between 0
and 0.011 (0-0.054 mg/m3) ppm.
• Exposure duration: Not reported;
measurements represent the means of five
days of monitoring.
Referent or control description
• A group of 247 maintenance workers who
were not exposed to asphalt. The group was
given a questionnaire similar to the exposed
group.
Statistical analysis
• Exact two-sided Fisher-Irving test was used to
analyze differences in symptom frequency.
• Mean difference between groups calculated
via two-sided Wilcoxon rank-sum test with a
significance level of 5%.
• Spearman's correlation coefficient used to
estimate correlation between symptoms and
possible confounders.
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Toxicological Review ofTrimethylbenzene
RESULTS
Exposure subgroup
• An increase in number of several symptoms was associated with asphalt exposure when asphalt-exposed
road workers were compared with workers not exposed to asphalt.
• 1,2,4-TMB was found to increase number of symptoms, while no similar correlation was found for 1,3,5-
TMB.
Effect estimates3
Observation
Abnormal fatigue
Reduced appetite
Laryngeal/pharyngeal
irritation
Eye irritation
Other, unspecified symptom
Symptoms associated with asphalt exposure in exposed and non-exposed hroups
of workers*
Days with
symptom
Asphalt workers
(n = 79)
Asphalt workers
(n = 254)
Non-asphalt
workers (n = 247)
Symptoms of asphalt exposure
None
1-2
3-5
None
1-2
3-5
None
1-2
3-5
None
1-2
3-5
None
1-5
64.6
21.5
13.9
86.1
12.7
1.3
63.3
21.5
15.2
54.4
22.8
22.1
91.1
8.9
75.2
14.6
10.2
89.8
7.5
2.8
74.0
15.4
10.6
68.9
22.4
8.7
85.4
14.6
84.6
9.7
5.7
95.1
4.1
0.8
83.0
11.7
5.3
85.4
10.5
4.1
92.3
7.7
aFor correlation between symptom sum and 1,2,4-TMB exposure, r = 0.31, p<0.01.
*AII differences between asphalt workers (n = 254) and non-asphalt workers (n = 247) were statistically significant
(p<0.05).
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-21. Characteristics and quantitative results for epidemiologic
cross-sectional study of exposure to 1,2,4-TMB Sulkowski etal. (2002)
Study (location)
• A factory in which paints and varnishes are
produced
Outcome assessment
• Hearing examinations were carried out in an
"audiobus," a motor vehicle equipped with
soundproof cabin and diagnostic tools.
• Several tests were conducted on subjects,
including air and bone pure tone audiometry,
impedance audiometry with tympanometry,
acoustic reflex threshold measurement, and
otoacoustic emissions.
• Electronystagmographic tests were conducted
in an outpatient clinical setting.
POPULATION CHARACTERISTICS
Exposed population
• 61 factory workers in direct contact with
solvent vapors.
• Job titles included resin synthesis analyzers,
dry component mixers, mill operators,
dispenser operators, colorists, and product
packers.
Exposure assessment
• Data on exposure was collected from 61
workers who wore passive dosimeters on 3
work days.
• Average Exposure duration: 15. 8±9.1 years.
Referent or control description
• 40 non-exposed workers from the same
factory.
Statistical analysis
• Statistical methods utilized included student t-
test, calculation of means, and linear
regression analysis.
RESULTS
Exposure Subgroup
• 47.5% of exposed individuals and 5% of the control population exhibited symptoms of vestibular
dysfunction, as indicated by decreased duration, amplitude and slow-phase angular velocity of induced
nystagmus.
• High frequency hearing loss as indicated by pure tone audiometry was detected in 42% of exposed
individuals versus 5% of the control population.
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Toxicological Review ofTrimethylbenzene
B.5. ANIMAL TOXICOLOGY STUDIES
Table B-22. Characteristics and quantitative results for Baettig et al.
(1958)
Study design
Species
Rats
Sex
M
N
8 rats per
dose
Exposure route
i.p. injection
Dose range
0, 200, 500, and 1,700 ppm
(0, 984, 2,460, 8,364
mg/m3) TMB mixture.
Exposure duration
4 mos; 8 hrs/d, 5/wk
Additional study details
• Mixture of 1,2,4-, 1,2,3-, and 1,3,5-TMB were tested for their effects on growth, (as measured by body
weight), behavior, food intake, red blood cell count, and hemoglobin concentration, and various histological
parameters.
• Rat behavior was assessed qualitatively.
• TMB mixture (i.e., Fleet-X DV-99) was the same as assessed in the occupational exposure study.
• Study was translated from German to English prior to receipt by EPA.
340
330
o 320
~ 310
I) 300
•| 290
a 280
% 270
o 260
< 250
240
230
220
Control rats
Exposed rats
Days of Exposure
1 Dec 1 Jan 1 Feb 1 Mar
Dates in Treatment
Figure 2. Effect of long-term exposure to trimethylbenzene (about 1,700 ppm [8,364 mg/m3]) on the growth of rats.
Open circles: Average body weights of the exposed rats. Closed circles: Average weights of the control rats. Hatched
[and dotted] area[s]: Double square deviation from the mean values plotted.
Source: Reproduced with permission of Springer-Verlag (Baettig et al. 1958)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
90
80
70
fee
"1 50
1 4°
^ 30
20
10
•s.
wiiiimiTimu] HT
•».
iMMi^i^m
1
1
Ut" ":T ' r"
™nnilllllll[lJI
N
V
I | Exposed rats
[
Figure 3. Behavior of
Tfj..^ the relative number of
• H '. Zethlenene
•DH • AD=OOB tnmethyiben ene-
^•-^i." -1 • ^xpn^d rat« (pxpn^iir^1
x A about 1,700 ppm [8,364
V. ^ -^ mg/m3]).
\ v Source: Reproduced
>k with permission of
N. Springer-Verlag (Baettig
\ etal. 1958)
Control rats
Days of exposure
ate: 1 Nov 1 Dec
Month
November
December
January
February
March
April
Uan 1Feb 1 Mar 1 April
Number of days
exposed per
month
5
14
20
17
15
13
Average daily food intake
(g/lOOgbw per month) Difference Difference
Control
Rats
5.32
5.46
5.19
4.80
4.73
Exposed (absolute) (%)
Rats
2.42 -3.10 -56.13
5.07 -0.93 -7.16
6.16 +0.97 +15.60
5.46 +0.66 +12.09
4.80 +0.07 +1.46
4.32
Table 1. Average intake of food by the rats during experimental exposure to TMB mixture
Source: Reproduced with permission of Springer-Verlag (Baettig et al. 1958)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
60
t 50
1 40
1. 30
o
•4->
I 20
*.»
1 10
o>
Q_
Date
Figure 4. Behavior
1,700 ppm [8,364
Source: Reproduct
| | Exposed rats
- Illllllllllll Control rats
--
.^ ^ ^ -
" ^pBiiim
rrrrrTrmTmmrm^
IH
/-'\
,' p = 0.05
V
»••*
I pllplllw^1
Days of exposure
: 1 Nov 1 Dec 1 Jan 1 Feb 1 Mar 1 April
of the relative number of neutrophil leukocytes in trimethylbenzene exposed rats (exposure: about
mg/m3]).
?d with permission of Springer-Verlag (Baettig et al. 1958)
Month
November
December
January
February
March
April
Average intake of
Number of days drinking water
exposed per (g/lOOgbw rat/month)
month Control
rats
5 9.21
14 9.71
20 9.38
17 7.78
15 7.12
13
Exposed
rats
10.55
17.18
22.31
15.92
14.16
15.66
Difference Difference
[aDsoiutej [ voj
+ 1.34 +12.70
+7.47 +43.47
+ 12.93 +57.91
+8.14 +51.13
+7.04 +49.70
Table 2. Average intake of drinking water by rats during experimental exposure to 1MB.
Source: Reproduced with permission of Springer-Verlag (Baettig et al. 1958)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
1.08
^ 1.07
— 1.06
CD
.2 1.05
o
•^ 1 .04
2
ra
j= 1.03
CD
& 1.02
1.01
> k
\
s \
"*** \
: i .A
I s W^ - = ^^^^
-»
12345
Time in hours after dilution test
Figure 5. Specific gravity of spontaneous and dilution urines in TMB-exposed rats (exposure: about 1,700 ppm [8,364
mg/m3]).
Source: Reproduced with permission of Springer-Verlag (Baettig et al. 1958)
Urinary
phenol
fraction
Total
Free
Bound
Total
Free
Bound
Total
Free
Bound
Intensity of
exposure
(ppm)
1700
1700
1700
500
500
500
200
200
200
Duration of
exposure
(days)
15
15
15
21
21
21
10
10
10
Duration of
exposure, in days
to significant
increase of phenol
excretion
4
8
4
8
8
21
10
10
Not increased
Time in days to
normalization of phenol
excretion after
discontinuation of
exposure
10
3
9
6
1
1
1
1
-
Table 3. Effect of TMB inhalation on urinary phenol excretion in the rat.
Source: Reproduced with permission of Springer-Verlag (Baettig et al. 1958)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Health Effect at LOAEL
Increased urinary excretion of free
and total phenols
NOAEL
0 ppm
LOAEL
200 ppm (984 mg/m3)
Comments: Battig et al. (1956a) is published in German. However, Baettig et al. (1958) presents an English-
translation of the results originally presented in Battig et al. (1956a). As such, a separate study summary table is not
provided for Battig et al. (1956a). or of the eight rats in the long-term inhalation experiment died and were
subsequently replaced within the first 2 weeks. Behavioral changes were assessed qualitatively. The substance to
which rats were exposed was comprised of a mixture of all three TMB structural isomers and may have also contained
methylethylbenzene structural isomers. Authors make a statement implying that dose was not consistent throughout
experiment.
Table B-24. Characteristics and quantitative results for Gralewicz et al.
f!997al
Study design
Species
Wistar rats
Sex
M
N
15 rats
per dose
Exposure route
Inhalation (6 h/d, 5
ds/wk)
Dose range
0, 25, 100, or 250 ppm (0,
123, 492, or 1,230 mg/m3)
1,2,4-TMB
Exposure duration
4 wks
Additional study details
• Animals were exposed to 1,2,4-TMB in 1.3 m3 dynamic inhalation exposure chambers for 6 hrs/d, 5 d/wk for 4
wks. Food and water was provided ad libitum.
• Animals were randomized and assigned to the experimental groups.
• Rats were tested with a variety of behavioral tests, including radial maze performance, open field activity,
passive avoidance, active two-way avoidance, and shock-induced changes in pain sensitivity.
• Tests were performed on days 14-54 following exposure.
• Rats displayed decreased performance on several tests at the 100 ppm and 250 ppm (492 and 1,230 mg/m3)
exposure levels.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
120
*» too
tn
.—
£ SO
o
*>
*_ 60
o
1 40
I 20
O
20
i a
8. 16
•= 1*
a
2 12
•5 10
i. 8
1 «
z 4
2
O
7
TMBO TMB25 TMB1OO TMB2SO
TMS25
TMB1OO TMB25O
Figure 1. A comparison of spontaneous locomotor
(upper diagram), exploratory (middle diagram, and
grooming (lower diagram) activity of rats in an open
field during a 5-min observation period. The test was
performed 25 days after a 4-week exposure to 1MB. The
bars represent group means and SE (n = 15 for each
group). *p<0.05 compared with TMBO group (0 ppm
control group).
TMBO
TMB25
TMB1OO TMB2SO
160 r
140
120 -
100 -
S 80
- trial 1
ITTI1 - trial 2
- trial 3 (shock)
- trial 4
- trial 5
- trial 6
Figure 2. Diagrams illustrating the effect of a 4-
week exposure to 1,2,4-TMB on the step-down
passive avoidance learning in rats. The test was
performed on days 35-45 after exposure. Trials
1, 2, and 3 were performed at 24-hr intervals.
The step-down response was punished by a 10-
s foot shock only in trial 3. Trials 4, 5, and 6
were performed 24 hr, 3 days, and 1 days after
trial 3, respectively. The maximum step-down
latency was 180 s. The bars represent group
means and SE (n = 15 for each group).
***p<0.001 compared with respective data
from group TMBO (0 ppm control group).
TMBO TMB25
TMBtOO TM8250
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
60
•—x
S 50
•*-*>"
>. 40
u
c
I 30
£ 20
1
o 10
0
o
o
300
250
200
150
100
a
o 50
T
i
^
T
*#*
I
- L1
- L2
TMBO TMB25 TMB100 TMB250
***
T
Figure 3. Hot plate behavior tested in rats on day 50
(trials 1 and 2) and day 51 (trial 3) after 4-week
exposure to 1,2,4-TMB. Bars represent group means
and SE (n = 15 for each group).
Upper diagram: a comparison of the latency of the
paw-lick response to a thermal stimulus (54.5°C) on day
50. LI: paw-lick latency in trial 1 performed before a 2
min intermittent foot shock. L2: paw-lick latency in trial
2 performed several seconds after the foot shock.
***p<0.001 compared with LI in the same group.
Lower diagram: A comparison of the change in the
paw-lick latency noted 24 hrs after foot shock (trial 3).
***p<0.001, **p<0.01 when compared to TMBO (0
ppm control group).
TMBO TMB25 TMB100 TMB250
- block 3
- block 4
- block 5
~ block 6
Figure 4. A comparison of the
active avoidance performance
increment during a single 30-trial
training session in consecutive
groups of rats. The testing was
performed on day 54 after 4-week
exposure to 1,2,4-TMB. Bars
represent the percentage (group
mean and SE, n = 15 for each
group) of avoidance response in
successive five-trial blocks. No
avoidance response was noted in
any group during the first 10 trials
and therefore blocks 1 and 2 were
omitted in the analysis.
TMBO
TMB25
TMB100
TMB250
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Health Effect at LOAEL
NOAEL
LOAEL
Open field grooming
significantly increased, lower
than expected step down
latency
25 ppm (123 mg/m
100 ppm (492 mg/m
Comments: CNS disturbances were observed up to 2 months after termination of exposure, indicating the persistence
of effects after the metabolic clearance of 1,2,4-TMB from the test animals. Duration of exposure only 4 weeks.
Generally, short-term exposure studies have limited utility in quantitation of human health reference values.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-25. Characteristics and quantitative results for Gralewicz et al.
fl997b_l
Study design
Species
Wistar rats
Sex
VI
N
9 rats per
dose
Exposure route
Inhalation (6 hr/d, 5
d/wk)
Dose range
0, 25, 100, or 250 ppm (0,
123, 492, or 1,230 mg/m3)
1,2,4-TMB
Exposure duration
4 wks
Additional study details
• Animals were exposed to 1,2,4-TMB in 1.3 m3 dynamic inhalation exposure chambers for 6 hrs/d, 5 d/wk for 4
wks. Food and water was provided ad libitum.
• Animals were randomized and assigned to the experimental groups.
• Rats were tested to determine whether exposure to 1,2,4-TMB altered the pattern of occurrence of spike
wave discharges (SWD).
• Rats exposed to 1,2,4-TMB at 100 or 250 ppm (492 or 1,230 mg/m3) did not show an increase in SWD activity.
Rats exposed to 0 or 25 ppm (0 or 123 mg/m3) 1,2,4-TMB showed progressively decreasing levels of SWD
activity.
90
75
so
45
30
o
"ST
^ 20 h
T
j& r
I
I
before exposure
24r H after «xp.
3Q days after exp.
LLLll 12O days after exp.
TMBO TMB25 TMB100 TMBS50
Figure 1. Diagrams showing the effect
of a 4-week inhalation exposure to
1,2,4-TMB on the contribution of
transitional (upper diagram, high
arousal (middle diagram), and slow-
wave sleep (lower diagram)) states in
the rat EEG during successive 1-hour
recording periods. The bars represent
group means and SE.
TMBO TMB25 TMB10O TMB25O
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
140 p T
.
-c 120
-*"••*
I KL-
J ^
? ^
i
5 /^
"S f/ V
^
^
5 ^S
•\ y v
..
T
|
rfl 1
0
1 ^
^
I ^T
} S
i
^
iZiZJDeiurc ejipvourc
^324 h after exp.
Figure 2. Diagram showing
the effect of a 4-week
inhalation exposure to 1,2,4-
TMB on the SWD burst
gg£j30 days after exp. occurrence (upper diagram)
Oj A
5 ^
B ^
I i
Bl ^
5 /,
S ^
Q /i
6 p;^
R v .
DUI 120 days after exp. and on the Percent
contribution of SWD activity
within TRANS state (lower
* - P<0.5 eompared diagram) during successive 1-
to the preexposure . .. . . ,-.
f * hour recording periods. The
value . .
bars represent group means
*
KIT
x 1
J
$(
^8 1
TMBO TMB25 TMB100 TMB250
^ 45
^ 40
01 35
t—
c
£ 30
"i
25
o
'•^ 20
-Q
^
O
O
Q
5
C
r
1
-
-
T
T
ii
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I
&
A
%
j_
T
I
1
X
8 T
8
1
5 ^S,
i
*
-
T T
I I
L
X M
8 /
II \
X ^ "
0 ^
1 i
i
T ^
1- ^
¥l( t
9 Y
X f/
8 ^
i2_ 2
AT
UI
>al
^?s
$X
ov
So
M> ~~ " "
TMBO TMB25 TMB100 TMB250
Health Effect at LOAEL
Decreased spike-wave discharges
NOAEL
25 ppm (123 mg/m3)
and SE. * denotes p<0.05 in
comparison to the
preexposure value in the
same group.
LOAEL
100 ppm (492 mg/m3)
Comments: CNS disturbances were observed up to 4 months after termination of exposure, indicating the persistence
of effects after the metabolic clearance of 1,2,4-TMB from the test animals. Duration of exposure only 4 weeks.
Generally, short-term exposure studies have limited utility in quantitation of human health reference values.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-26. Characteristics and quantitative results for Gralewicz and
Wiaderna (2001)
Study design
Species
Wistar rats
Sex
M
N
10 or 11
rats per
dose
Exposure route
Inhalation (6 hr/d, 5
ds/wk)
Additional study details
Dose range
0 or 100 ppm (0 or 492
mg/m3) 1,2,3-, 1,2,4-, or
1,3,5-TMB
Exposure duration
4 wks
• Animals were exposed to 1,2,3-, 1,2,4- or 1,3,5-TMB in 1.3 m3 dynamic inhalation exposure chambers for 6
hrs/d, 5 d/wk for 4 wks. Food and water was provided ad libitum.
• Animals were randomized and assigned to the experimental groups.
• Rats were tested with a variety of behavioral tests, including radial maze performance, open field activity,
passive avoidance, active two-way avoidance, and shock-induced changes in pain sensitivity.
• Tests were performed starting 2 wks post-exposure.
• 1,2,3-, 1,2,4-, and 1,3,5-TMB-exposed rats showed alterations in performance
in spontaneous locomotor
activity, passive avoidance learning, and paw-lick latencies.
Hi- day 1
MR- day z Figure
t=l- day 3 °
i.B
§ 1.6
6 1.4
a
£ 1.8
C
I 1'°
£ 0.8
a
•g 0.6
jj 0.4
6
9 0.2
0 0
1. Radial maze performance of rats exposed for 4 weeks to
m- day 4 m-xylene or a TMB isomer at a concentration of 100 ppm (492
ay mg/m3 . The test (one trial a day) was performed on days 14-18
.
-
-
1 II
|
= ''
l\
E:
||
T
T after exposure. The diagrams illustrate the number of perseveration
=
(upper
i
]{
diagram) and omission (lower diagram) errors in successive
daily trials.
Denotation:
Control- sham exposed group (n=10),
XYL- m-xylene exposed group (n=ll),
PS- 1,2,4-TMB exposed group (n=ll),
MES- 1,2,3-TMB exposed group (n=ll),
control XYL PS MES HM HM- hemimellitene exposed group (n=ll).
5.5
5.0
o 4.5
» 4.0
2 3-5
Jj 3.0
o 2.5
0 2.0
| 1-5
g 1.0
55 0.5
0 0
r Bars represent group means and SE.
-
-
-
' L
- I
-
_
a
s
i
L
i
i
~
E |
- !
1
:
:
:
:
I
[
E
:
=
-
II
j
E
= ;
_
Control XYi PS MES HM
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
DO
BO
rt
•»••<
m
m
O
t-,
<0
120
100
80
60
40
20
* — p<0.05 compared
to control
o — p<0.05 compared
to PS and MES
Figure 2. A comparison of open-
field locomotor activity in sham-
exposed and solvent-exposed rats.
The test was performed on day 25
after a 4-week exposure to m-
xylene or a TMB isomer at
concentration of 100 ppm (492
mg/m3). Bars represent group
means and SE.
Control XYL PS MES HM
Figure 3. Diagram illustrating the effect of a 4-week inhalation exposure to m-xylene or a TMB isomer at concentration
of 100 ppm (492 mg/m3) on the step-down response latency in the passive avoidance test. The test was performed on
days 39-48 after exposure. Trials 1, 2, and 3 were performed at 24 h intervals. The step-down response was punished
by a 10 s footshock in trial 3 only.
- trial
- trial
160
140
•o
r-
o 120
o
0
M
c 100
o
o 80
?
j 60
o
•o
I
a 40
20
n
-
-
'.
~
.
\
d, j
|f:
wX
III
bd
[ |"'"j' ,'['"
MM
tf
{
.
.
• .
T
.
:
+
;
I
ffi ''•
|}:
":
i-
^^
1S2S
*
1
2
trial 3 (shock)
trial 4
- trial 5
- trial 6
— p<0.05 compared
to control
Trials 4, 5, and 6 were performed
24 hr, 3 days, and 7 days after
trial 3, respectively. The maximum
time of staying on the platform was
180 s. Bars represent means and
SE.
Control XYL PS MES
HM
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
60
50
40
30
2 20
I
*
a
o_
10
LI
_
nrrn-
F=l-
L3
p<0.05 compared
to control
I
Figure 4. A comparison of sham-exposed and
solvent-exposed rats with respect to the latency
of the paw-lick response to heat (54.5°C) before
(LI), several seconds after (L2), and 24 hr after a
2 min intermittent footshock. The test was
performed on days 50 and 51 after a 4-week
inhalation exposure to m-xylene or a TMB
isomer at a concentration of 100 ppm
(492 mg/m3). Bars represent group means and
SE.
Control XYL
PS
MBS
HM
45
40
a 35
a 30
o
25
20
15
10
« - p<0.05 compared
to control
Figure 5. Active avoidance learning in rats
after a 4-week inhalation exposure to m-
xylene or a TMB isomer at a concentration
of 100 ppm (492 mg/m3). In one massed-
trial session (inter-trial interval 20-40 s;
maximum number of trials 60) the rats
learned to shuttle between two
neighboring compartments in order to
avoid a footshock. The test was
performed on day 54-60 after exposure.
Bars represent group means and SE of the
number of trials.
Control XYL
PS
MES
HM
Health Effect at LOAEL
NOAEL
LOAEL
Deleterious effects on
locomotor activity, passive
avoidance learning, and paw-
lick latencies
n/a
100 ppm (492 mg/m ) 1,2,3-TMB,
1,2,4-TMB, or 1,3,5-TMB
Comments: CNS disturbances were observed up to 2 months after termination of exposure, indicating the persistence
of effects after the metabolic clearance of 1,2,4-TMB from the test animals. Duration of exposure only 4 weeks.
Generally, short-term exposure studies have limited utility in quantitation of human health reference values.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-27. Characteristics and quantitative results for Janik-
Speichowicz (1998)
Study Design
Species
Balb/c Mice
Sex
M&
F
N
4 or 5
mice/
dose
group
Exposure route
I.P. injection
Dose range
0, 1470, 2160, and 2940
mg/kg body weight
Exposure duration
Single exposure, or 2 i.p.
injections spaced out over 24
hours
Additional study details
• Animals were given one or two injections of i.p. injections of 1,2,3-TMB.
• Animals were randomized and assigned to the experimental groups.
• Most deaths occurred within the first 2 d following single injections.
• LD50 was determined to be 3,670 mg/kg for males and 2,700 mg/kg for females.
• Micronuclei and chromatid exchange assays were conducted on extracted bone marrow to assess
genotoxicity.
• Multiple indicators of genotoxicity were used, giving adequate evidence to assess the genotoxic potential of
acute exposure to 1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB .
Figure 1. Dose-related increase in the number of His+ revertants for 1,2,3-TMB in S. typhimurium strains
TA102
TA100
TA98
TA97a
I
dose [/ul/plate]
-S9 +S9
• 20
1200 1000 800 600 400 200
-S9 Revertants / plate
200
400
+S9
* - mutagenic effect (a 2-fold or greater increase in the number of revertants
per plate, as compared with the solvent control number)
Spontaneous revertants: TA97a 129+10 (-39): 141±17 (+S9);
TA98 23+2 (-39); 3S±6 !+S9);
TA100 126±4 (-S9); 119±5 (+S9);
TA102 282±33 (-S9); 315±32 (+S9)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Observation
TA97a (-S9)
TA97a (+S9)
TA98 (-S9)
TA98 (+S9)
TA100(-S9)
TA100(+S9)
TA102(-S9)
TA102(+S9)
Observation
TA97a (-S9)
TA97a (+S9)
TA98 (-S9)
TA98 (+S9)
TA100(-S9)
TA100(+S9)
TA102(-S9)
TA102(+S9)
Observation
Males 30 h harvest time
Males 48 h harvest time
Males 72 h harvest time
Females 30 h harvest time
Females 48 h harvest time
Females 72 h harvest time
Males 30 h harvest time
Males 48 h harvest time
Males 72 h harvest time
Females 30 h harvest time
Females 48 h harvest time
Females 72 h harvest time
Exposure to 1,2,4-TMB (u.g or u.L)
100
0 (Solvent
control)
212±7 126±13
145±5 141+12
24±3 23±3
31±3 31±5
123±71 125±41
25±4 21±10
258±6 280±12
294±11 315±14
1
148±23
152±7
24±3
35±4
138±15
126±62
290±33
279±24
5
158±10
168±8
29±5
28±1
148±18
125±5
262±16
276±11
10
165±8
176±21
41±7
29±4
143±9
112±4
273±20
276±11
20
141±25
155±20
27±8
30±3
124±7
108±3
214±8
236±32
30
115±3
106±7
TOXa
29±6
118±4
110±4
TOX
TOX
Exposure to 1,3,5-TMB (u.g or u.L)
100
0 (Solvent
control)
127±15 131±10
183±6 157±19
22±4 22±4
30±3 32±5
138±13 143±15
142±10 138±82
263±23 60±12
337±13 336±23
1
141±13
180±26
27±3
31±4
143±4
137±3
268±17
347±34
5
149±29
196±16
28±5
35±5
152±8
147±29
280±19
334±30
10
139±17
155±30
25±2
31±2
140±26
139±16
261±25
353±11
20
129±13
137±29
37±5
39±5
154±14
131±10
238±5
340±37
30
125±8
138±20
23±5
28±2
130±7
108±11
198±2
324±10
40
NTb
128±11
TOX
31±1
TOX
115±6
NT
NT
Exposure to 1,2,3-TMB (mg/kg body weight)
0
1470
2160
% of Polychromatic Erythrocytes with
--
0.18±009
--
--
0.20±0.08
--
0.17±0.06
0
0
17±0.05
17±0.05
--
--
--
Micronuclei
--
--
--
0
0
0
22±0.09
20±0.08
20±0.14
2940
(±SD)
0.22±0.07
0.21±0.10
0.21±0.11
--
--
--
Ratio of polychromatic to normochromatic erythrocytes
--
0.81
--
--
0.95
--
0.82
0.45
0.50
--
--
--
--
--
--
0.90
0.84
0.78
0.85
0.72
0.62
--
--
--
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Observation
Males 30 h harvest time
Males 48 h harvest time
Males 72 h harvest time
Females 30 h harvest time
Females 48 h harvest time
Females 72 h harvest time
Males 30 h harvest time
Males 48 h harvest time
Males 72 h harvest time
Females 30 h harvest time
Females 48 h harvest time
Females 72 h harvest time
Observation
Males 30 h harvest time
Males 48 h harvest time
Males 72 h harvest time
Females 30 h harvest time
Females 48 h harvest time
Females 72 h harvest time
Males 30 h harvest time
Males 48 h harvest time
Males 72 h harvest time
Females 30 h harvest time
Females 48 h harvest time
Females 72 h harvest time
Exposure to 1,2,4-TMB (mg/kg body weight)
0
2000
3280
4000
% of Polychromatic Erythrocytes with Micronuclei (± SD)
--
0.18±0.07
--
--
0.23±0.05
--
0.15±0.10
0.18±0.10
0.20±0.08
--
--
--
--
--
--
0.23±0.5
0.18±0.05
0.13±0.05
0.23±0.10
0.16±0.8
0.16±0.07
--
--
--
Ratio of polychromatic to normochromatic erythrocytes
--
0.95
--
--
0.95
--
1.18
1.02
1.02
--
--
--
--
--
--
0.98
1.01
0.85
1.16
0.74
0.68*
--
--
--
Exposure to 1,3,5-TMB (mg/kg body weight)
0
1800
2960
3600
% of Polychromatic Erythrocytes with Micronuclei (± SD)
--
0.21+0.08
--
--
0.20±0.08
--
0.20±0.00
0.17±0.09
0.17±0.09
--
--
--
--
--
--
0.17±0.09
0.20±0.00
0.22±0.05
0.24±0.11
0.17±0.05
0.14±0.05
--
--
--
Ratio of polychromatic to normochromatic erythrocytes
--
0.61
--
--
0.60
--
0.62
0.56
0.58
--
--
--
--
--
--
0.51
0.60
0.58
0.40*
0.33
0.42*
--
--
--
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Toxicological Review ofTrimethylbenzene
Figure 1. Sister chr
15
~- 7 1
LU
O
V)
6
5
4
3
2
omatid exchanj
ft
V
solvent
control
O hemimellitene
O pseudocumene
V mesitylene
V ® V - signi
as po
Health Effect at LOAEL
Significant increase in SCE
induction relative to control
ies induced in bone marrow cells of \mp:Balb/c mice.
1
ti
' T :
IT T
T I 1 T T
1 I °
: \ 1 T * *
(i1!
J- 1 !
900 1800 270
c e
i i
t t
y y
2940
0 3600
ficant difference vs. control at p
-------
Toxicological Review ofTrimethylbenzene
Table B-28. Characteristics and quantitative results for Korsak et al.
f!9951
Study design
Species
IMP:DAKWistar
rats and Balb/C
mice
Sex
M
N
8-
10/dose
Exposure route
Inhalation
Dose range
250-2000 ppm (1,230-
9840 mg/m3) 1,2,4-TMB
Exposure duration
4 hrs - neurotoxicity tests
6 minutes - respiratory tests
Additional study details
• Animals were exposed to 1,2,4-TMB in a dynamic inhalation chamber (1.3 m3 volume) with 12-15 air
changes/hr.
• Mean initial body weights were 250-300 g for rats and 23-30 g for mice; animals were housed in wire mesh
stainless steel cages, with food and water provided ad libitum.
• Animals were randomized and assigned to the experimental groups. Before rotarod experiment, rats were
trained, and only rats that balanced for 2 minutes on 10 consecutive days were used.
• Rotarod, hot plate, and respiratory tests were conducted to measure effects on neuromuscular activity, pain
sensitivity, and respiratory rate respectively.
Figure 1. Rotarod performance of rats exposed to 1,2,4-TMB (i.e., pseudocumene). Rats were exposed to vapors of
solvent for 4 hrs. Rotarod performance was tested immediately after termination of exposure. Each point represents
probit of failures on rotarod in a group of 10 rats.
§ 8-
B
is fri
2
. 5-
0)
CO
§ 4^
ex
en
£ 3H
£050=4693 mg/m3
(954 ppm)
1000
100000
10000
Concentration of pseudocumene, mg/m3
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Figure 2. Hot-plate behavior in rats exposed to 1,2,4-TMB (i.e., pseudocumene). Rats were exposed to vapors of
solvent for 4 hrs. Hot-plate behavior was tested immediately after termination of exposure. Each point represents the
mean value of separate measurements of latency over the control in 10 rats.
ss
"2
•E
o
o
0)
§
Q
%
_l
1 V
65-
60-
55-
50-
45-
40-
35-
30-
oc
a
ECgo = 5682 mg/m3
(11 55 ppm) /''
/
y
f»*
/'
j/f
/
a
jT
a S**
1000
Concentration of pseudocumene, mg/m 3
10000
Figure 3. Time-response relationship for the effect of 1,2,4-TMB (i.e., pseudocumene) on respiratory rate in mice. Each
point represents the mean value in 8-10 mice. After termination of 6 min exposure recovery of respiratory rate was
observed.
2
c
o
u
o
a?
a
tn
&
— 1244 mg/m (253 ppm)
2686 mg/m3(S46 ppm)
-*- S18S msr/m3U054 ppm)
6391 ma/m3(1299 ppm)
9465 mg/m3(192e ppm)
10
12
This document is a draft for review purposes only and does not constitute Agency policy.
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-------
Toxicological Review ofTrimethylbenzene
Figure 4. Respiratory rate of mice exposed to 1,2,4-TMB (i.e., pseudocumene) in 8-10 mice. The decrease of
respiratory rate observed in the 1st minute of exposure was taken for consideration. The regression line was
determined by the least squares procedure.
100-
o
Q.
CD
•
of
1
•a
90-
80-
70-
60-
50-
40-
30-
20-
D
= 2844 mg/m3
(578 ppm)
1fooo
10000
Concentration of pseudocumene, mg/m3
Health Effect at LOAEL
NOAEL
LOAEL
Decreased respiration rate,
impaired rotarod test
performance, decreased pain-
response time
n/a
n/a
Comments: No values are provided for dose-specific responses, and NOAEL and LOAEL cannot be determined.
Exposures were of an acute duration, and therefore not suitable for reference value derivation. However,
qualitatively, this study provided evidence of CNS disturbances that, when considered together with short-term and
subchronic neurotoxicity studies, demonstrate that TMB isomers perturb the CNS of exposed animals. The respiratory
effects in mice also qualitatively support respiratory effects observed in rats exposed subchronically to 1,2,4-TMB and
1,2,3-TMB.
This document is a draft for review purposes only and does not constitute Agency policy.
B-82 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofTrimethylbenzene
Table B-29. Characteristics and quantitative results for Korsakand
Rydzynski (1996)
Study design
Species
IMP: Wistar
rats
Sex
M
N
9-10/
dose
(1,2,4-
TMB)
10-30/
dose
(1,2,3-
TMB)
Exposure route
Inhalation (4 hrs or
6h/d, 5 d/wk, for 3
mos)
Dose range
Acute exposure: 250-2,000
ppm 1,230 - 9840 mg/m3)
1,2,3-, 1,2,4-, or 1,3,5-TMB
Subchronic exposure: 0,
123, 492, or 1,230 mg/m3
Exposure duration
4 hrs or 3 mos
Additional study details
• Animals were exposed to either 1,2,3-, 1,2,4-, or 1,3,5-TMB in a dynamic inhalation chamber (1.3 m3 volume)
with 16 air changes/hr.
• Mean initial body weights were 250-300 g; rats were housed in wire mesh stainless steel cages, with food and
water provided ad libitum.
• Animals were randomized and assigned to the experimental groups.
• Rotarod and hot plate tests were conducted to measure effects on neuromuscular function and pain
sensitivity respectively.
Rotarod performance was tested immediately after termination of exposure.
Normal neuromuscular function was indicated by the rats' ability to remain on a rod rotating at 12rpm for 2
minutes.
Hot-plate behavior was tested immediately after termination of exposure.
Latency of 60 seconds was considered as 100% inhibition of pain sensitivity.
Authors investigated the effects of exposure to 1,2,3-, 1,2,4- and 1,3,5- 1MB on rotarod test performance and
pain-sensing response two weeks after the termination of exposure.
This document is a draft for review purposes only and does not constitute Agency policy.
B-83 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Figure 1. Rotarod performance of rats exposed to 1,2,3-TMB (hemimellitene), 1,2,4-TMB (pseudocumene), or 1,3,5-
rMB (mesitylene). Rats were exposed to solvent vapors for 4 hrs. Rotarod performance was tested immediately after
termination of exposure. Each point represents probit of failures on rotarod in a group of 10 rats. Normal
neuromuscular function was indicated by the rats' ability to remain on a rod rotating at 12 rpm for 2 mins. The rotating
rod was suspended 20 cm above metal bars connected to a 80 V/2 mA power source.
of
H
a.
e
tfl
o
.*;
W
o
Q.
g
PSEUDOCUMENE
ECM = 4693 mg/m3
(954 ppm)
1000
10000
100000
8
7-
6
5-
4
3-
MES1TYLENE o
,/
.6
O/
ECM=4738mg/m3
(963 ppm)
1000
10000
100000
s w
2
O- 5-
S
HEMIMELLITENE
ECW= 3779 mg/m3
(768 ppm)
1000
10000
Concentration, mg/m3
100000
Source: Reproduced from Korsak and Rydzyriski (1996)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Figure 2. Hot-plate behaviors in rats exposed to 1,2,3-TMB (hemimellitene), 1,2,4-TMB (pseudocumene), or 1,3,5-TMB
(mesitylene). Hot-plate behavior was tested immediately after termination of exposure. Each point represents the
mean value of separate measurements of latency in 10 rats. Latency of 60 sec was considered as 100% inhibition of
pain sensitivity.
Effects of exposure to trimethythenzene isomers
I 65_
1. 60-
i
1 55^
f
:l 45
1 40-
C 35
£ '
10
100-
^
.2 90-
IB
> 80-
Q-
•§ 70
as 6°-
.& so-
'S 40-
H
c
•^ 30-
in ^"
"1 10-
£
0
HEMIMELLITENE
/
ECM = 41 72 mg/rrT / °
(848 ppm) c,/
df
j'
^ /'"
/'
f
.*
/
./•'
,'
/
D'
^
1000
Concentration, mg/m1
ource: Reproduced from Korsak and Rydzyriski (1996)
10000
This document is a draft for review purposes only and does not constitute Agency policy.
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-------
Toxicological Review ofTrimethylbenzene
40
35
S
= ™
% of failure V.offai
8fe • w ^ |
_ O 0 O o 0 C
Pseiidocumene ^^^
/ /-^
*^— !T
i
0 4 °"Ur°8
Hemimellltene
/-^^
|
0 4 CKP°-urcB
^\
A
•X" control
, -^25 ppm
TlHOO ppm
X -m- 250 ppm
13 15 weeks
-i
•X1 control
•JIM 00 ppm
M -•• 250 ppm
13 15 weeks
Observation
Control
25 ppm (100 mg/m3)
100 ppm (492 mg/m3)
250 ppm (1,230 mg/m3)
250 ppm (1,230 mg/m3) 2 wks after termination of exposure
Health Effect at LOAEL
Decreased pain sensitivity
Figure 3. Rotarod performance of rats exposed to
1,2,3-TMB (hemimellitene) or 1,2,4-TMB
(pseudocumene) at concentrations of 25, 100, and
250 ppm (123, 492, 1,230 mg/m3) . Rats were
exposed to vapors of solvents for 6 hr/d, 5 ds/wk, 3
mos. Statistical significance marked by asterisks,
p<0.005.
Source: Reproduced from Korsak and Rydzyriski
(1996)
Latency of the paw-lick response, sec
1,2,4-TMB 1,2,3-TMB
15.4 ±5.8 9.7 ±2.1
18.2 ±5.7 11.8 ±3.8*
27.6 ±3.2** 16.3 ±6.3***
30.1 ±7.9** 17.3 ±3.4**
17.3 ±3.9 11.0 ±2.4
NOAEL LOAEL
n/a for 1,2,3-TMB 25 ^J1" ^ ^
25 ppm (123 mg/m3) for 1,2,3-TMB
19ATMR 100 ppm (492 mg/m3) for
'' 1,2,4-TMB
Comments: Although rotarod data are useful in providing a qualitative description of neuromuscular impairment
following 1,2,4-TMB or 1,2,3-TMB exposure, in comparison to effects on pain sensitivity, the data are not considered as
robust regarding suitability for derivation of reference values. Namely, data are presented as dichotomized values
instead of a continuous measurement of latency. The acute exposures were not suitable for reference value
derivation. However, qualitatively, effects observed following acute exposures provided evidence of CNS disturbances
that, when considered together with subchronic neurotoxicity tests, demonstrate that TMB isomers perturb the CNS of
exposed animals. It is unclear whether the latency to pawlick and rotarod tests were performed sequentially in the
same cohort of animals.
*, ** statistically significant from controls at p < 0.05 and p < 0.01, respectively.
*** Level of significance not reported in Table 1 from Korsak and Rydzyriski (1996), however the results of an ad-hoc t-
test (performed by EPA) indicated significance at p < 0.01
This document is a draft for review purposes only and does not constitute Agency policy.
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-------
Toxicological Review ofTrimethylbenzene
Table B-30. Characteristics and quantitative results for Korsak et al.
f!9971
Study design
Species
MP:DAK
i/Vistar rats
and Balb/C
nice
Sex
vl
N
^cute -
S/dose
subchronic
- 6-7/dose
Exposure route Dose range
^cute -Inhalation, 6 ^cute - 250-2000 ppm
minutes [1,230 - 9840 mg/m3) 1,2,4-
Jubchronic 0 FMB, 1,2,3-TMB, or 1,3,5-
nhalation,6 hr/d, 5 TMB
d/wk Jubchronic - 0, 123, 492,
1,230 mg/m3 1,2,4-TMB
Exposure duration
Acute -6 minutes
Subchronic-90 d
Additional study details
• Animals were exposed to 1,2,4-TMB in a dynamic inhalation chamber (1.3 m3 volume) with 12-15 air
changes/hr.
• Rats weighed 250-300 g and were housed in stainless steel wire mesh cages, with food and water provided
ad libitum.
• Rats were anesthetized 24 hrs after termination of exposure, and bronchoalveolar (BAL) fluid was collected
from lung lavage.
• All rats exposed to 1,2,4-TMB survived until the end of exposure and no clinical observations of toxicological
significance were reported.
Observation
Exposure concentration (mg/m3)
0
123
492
1,230
Body weight (mean ± SD)
Body weight g)
411 ±28
383 ± 25
409 ± 56
416 ± 27
BAL cell counts (mean ± SD)
Total cells (106/cm3)
Macrophages (106/cm3)
Polymorphonuclear leucocytes
(106/cm3)
Lymphocytes
(106/cm3)
Cell viability (%)
1.93 ±0.79
1.83 ± 0.03
0.04 ± 0.02
0.06 ±0.01
98.0 ± 1.7
5.82 ±1.32***
3.78 ±0.8
1.54 ±0.7
0.5 ±0.2
95.5 ±1.6
5.96 ±2.80**
4.95 ±0.2**
0.52 ±0.6
0.5 ±0.4
95.3 ±3.5
4.45 ± 1.58*
3.96 ±0.3**
0.21 ±0.3
0.2 ±0.1
95.3 ±3.1
BAL protein levels and enzyme activities (mean ±SD)
Total protein
(mg/mL)a
Mucoproteins (mg/mL)a
Lactate dehydrogenase (mU/mL)a
Acid phosphatase mU/mL)a
0.19 ±0.04
0.16 ±0.03
34.2 ± 8.52
0.87 ±0.20
0.26 ±0.07*
0.14 ±0.02*
92.5 ±37.2***
1.28 ±0.37*
0.26 ±
0.06*
0.13 ±0.02
61.3 ±
1.52 ±
22.9*
0.42*
0.24 ±0.08
0.12 ±0.02
53.8 ±28.6
1.26 ±0.22*
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Toxicological Review ofTrimethylbenzene
10 11 12 13
Figure 1. Time-response relationship for
the effect of 1,2,4-TMB
(i.e., pseudocumene) on respiratory rate
in mice. Each point represents the mean
value in 8-10 mice. After termination of
6 min exposure recovery of respiratory
rate was observed.
Source: Reproduced from Korsak et al.
(1997)
Health Effect at LOAEL
NOAEL
LOAEL
Increased Total BAL cells
n/a
123 mg/m3
Comments: The observed markers of inflammation are coherent with the observed respiratory irritative effects
observed in mice exposed to 1,2,4-TMB acute (i.e., 6 min). The authors did not report at which dose groups the
numbers of polymorphonuclear leucocytes and lymphocytes were significantly elevated relative to control.
a Jonckheere's test for trend: total protein, p = 0.0577; mucroprotein, p = 0.3949; lactate dehydrogenase, p = 0.2805;
acid phosphatase, p = 0.0164.
*, **, *** statistically significant from control at p < 0.05, 0.01, and 0.001, respectively.
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Toxicological Review ofTrimethylbenzene
Table B-31. Characteristics and quantitative results for Korsak et al.
fZOQQal
Study design
Species Sex N Exposure route Dose range Exposure duration
IMP: Wistar M 10/dose Inhalation (6 hr/d, 5 0, 123, 492, 1,230 mg/m3 90 d
rats and F d/wk)
Additional study details
• Animals were exposed to 1,2,4-TMB in a dynamic inhalation chamber (1.3 m3 volume) with 16 air changes/hr.
• Mean initial body weights were 213 ± 20 for males and 160 ± 11 for females; rats were housed in
polypropylene cages with wire-mesh covers (5 animals/cage), with food and water provided ad libitum.
• Animals were randomized and assigned to the experimental groups.
• Hematological parameters were evaluated prior to exposure and 1 wk prior to termination of exposure, and
for the 1230 mg/m3 exposure group, also evaluated two weeks after termination of exposure; blood clinical
chemistry parameters were evaluated 18 hrs after termination of exposure (animals were deprived of food for
24 hrs).
• Necropsy was performed on all animals. Pulmonary lesions were graded using an arbitrary scale: 1 = minimal,
2 = mild, 3 = moderate, 4 = marked.
Observation
Terminal body weight (g)
Absolute organ weight (g)
Lungs
Liver
Spleen
Kidney
Adrenals
Testes
Heart
Relative organ weight (g)
Lungs
Liver
Spleen
Kidney
Adrenals
Testes
Heart
Terminal body weight (g)
Absolute organ weight (g)
Lungs
Exposure concentration (mg/m3)
0
123
492
1,230
Body and Organ weights (mean ± SD)
Males
368 ± 22
1.78 ±0.28
10.27 ± 1.82
0.68 ±0.08
2.06 ±0.13
0.048 ± 0.007
3.72 ±0.35
0.90 ± 0.04
0.496 ± 0.056
2.896 ±0.456
0.189 ±0.011
0.588 ±0.029
0.011 ±0.003
1.041 ± 0.076
0.252 ±0.013
390 ± 26
1.83 ±0.25
11.43 ± 1.05
0.85 ±0.19*
2.24 ±0.15
0.046 ± 0.0050
3.90 ±0.38
0.94 ± 0.06
0.475 ± 0.056
2.894 ±0.427
0.220 ± 0.041
0.585 ± 0.022
0.010 ± 0.000
1.020 ± 0.079
0.239 ± 0.020
399 ± 22
2.93 ±0.26*
10.78 ±1.33
0.79 ±0.09
2. 14 ±0.15
054 ±0.011
4.03 ±0.27
0.94 ± 0.08
0.586 ±0.115
2.990 ±0.465
0.210 ±0.018
0.587 ± 0.065
0.022 ± 0.024
1.067 ±0.102
0.249 ± 0.014
389 ± 29
1.78 ±0.36
10.86 ± 2.04
0.72 ±0.08
2.18 ±0.16
0.047 ± 0.005
3.87 ±0.24
0.96 ±0.07
0.477 ± 0.080
2.901 ±0.479
0.200 ± 0.018
0.586 ± 0.040
0.011 ±0.003
1.039 ± 0.077
0.258 ± 0.020
Females
243 ± 16
1.29 ±0.18
243 ± 19
1.32 ±0.12
230 ± 14
1.25 ±0.13
229 ±21
1.23 ±0.11
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Toxicological Review ofTrimethylbenzene
Liver
Spleen
Kidney
Adrenals
Ovaries
Heart
Relative organ weight (g)
Lungs
Liver
Spleen
Kidney
Adrenals
Ovaries
Heart
Observation
Hematocrit (%)
Hemoglobin (g/dL)
RBCs (x 103/mm3)c
WBCs (x 103/mm3)d
Rod neutrophil (%)
Segmented neutrophil (%)
Eosinophil (%)
Lymphocyte (%)
Monocyte (%)
Lymphoblast (%)
Myelocyte (%)
Erythroblase (%)
Reticulocyte (%)
Platelet (x 103/mm3)
Clotting time (sec)
Hematocrit (%)
Hemoglobin (g/dL)
RBCs (x 103/mm3)c
WBCs (x 103/mm3)d
Rod neutrophil (%)
Segmented neutrophil (%)
Eosinophil (%)
Lymphocyte (%)
6.48 ±1.02 6.54 ±0.69
0.59 ±0.08 0.61 ±0.11
1.55 ±0.12 1.50 ±0.14
0.065 ± 0.007 0.070 ± 0.008
0.09 ±0.02 0.09 ±0.01
0.66 ±0.07 0.64 ±0.05
0.555 ±0.058 0.581 ±0.040
2.770 ±0.222 2.881 ±0.309
0.255 ±0.025 0.266 ±0.031
0.667 ±0.030 0.661 ±0.047
0.0028 ± 0.006 0.031 ± 0.006
0.043 ± 0.008 0.041 ± 0.006
0.284 ± 0.023 0.283 ± 0.025
5.81 ±0.83 6.72 ±1.34
0.49 ±0.06* 0.52 ±0.08
1.38 ±0.11* 1.44 ±0.19
0.066 ±0.010 0.061 ±0.013
0.09 ±0.27 0.09 ±0.02
0.61 ±0.07 0.63 ±0.06
0.596 ±0.051 0.569 ±0.053
2.758 ±0.223 3.078 ± 0.434
0.237 ±0.036 0.24 ±0.033
0.660 ± 0.042 0.662 ± 0.036
0.032 ± 0.006 0.029 ± 0.006
0.045 ± 0.013 0.047 ± 0.009
0.291 ±0.025 0.289 ±0.015
Exposure concentration (mg/m3)
0
123
492
1,230
l,230a
Trend
testb
Hematological parameters (mean ± SD)
Males
49.9 ±1.9
15.1 ±1.1
9.98 ±1.68
8.68 ±2.89
0.0 ± 0.0
24.1 ±9.2
1.2 ± 1.7
73.5 ± 10.3
1.1 ±1.3
0.0 ±0.0
0.0 ±0.0
0.0 ± 0.0
3.1 ±2.3
294 ± 46
43 ±19
50.4 ±2.0
15.6 ±0.9
9.84 ± 1.82
8.92 ± 3.44
0.4 ±0.5
19.7 ±6.5
1.2 ± 1.0
76.2 ±7.1
2.5 ±2.1
0.0 ±0.0
0.0 ±0.0
0.0 ± 0.0
2.3 ±1.4
293 ± 73
41 ±17
50.0 ±1.9
15.4 ±0.9
8.50 ±1.11
8.30 ± 1.84
0.2 ±0.4
20.7 ±7.7
0.4 ±0.6
76.8 ±8.5
2.3 ±2.2
0.0 ±0.0
0.2 ±0.4
0.0 ± 0.0
2.8 ±2.1
359 ± 46
37 ±13
50.6 ± 1.5
15.4 ±0.6
7.70 ±1.38**
15.89 ±5.74**
0.9 ±1.5
18.9 ± 10.8
1.7 ±1.4
75.8 ±16.0
1.8 ±2.5
0.8 ±1.3
0.0 ± 0.0
0.0 ±0.0
3.1 ±2.5
335 ± 80
33 ±7
50.1 ±1.1
16.0 ± 1.0
7.61 ±1.6
7.11 ±2.1
0.7 ±0.8
29.4 ±6.4
1.5 ± 1.5
65.4 ±8.9
2.7 ±2.5
0.3 ±0.9
0.0 ±0.0
0.0 ± 0.0
6.4 ±3.2
386 ± 70
56 ±21
0.2993
0.2112
0.0004
0.0019
0.0589
0.0730
0.2950
0.1297
0.3818
0.1387
0.4046
0.5000
0.4900
0.0741
0.1457
Females
46.0 ±1.6
14.5 ±0.9
8.22 ±1.16
7.50 ±1.31
1.4 ± 1.6
22.8 ±6.5
1.2 ±0.6
73.2 ±7.9
46.6 ±2.7
13.8 ±1.3
7.93 ± 2.04
6.76 ±2.95
0.5 ±0.7
15.5 ±7.9
16 ± 1.6
79.4 ± 8.4
47.0 ±2.7
14.4 ± 0.9
8.51 ±1.13
9.55 ±4.48
0.4 ±0.5
20.7 ±7.5
1.1 ±1.7
75.5 ±7.4
46.5 ±4.1
14.2 ± 0.9
7.71 ±1.58
9.83 ±3.74
0.4 ±0.9
17.4 ±9.3
1.2 ±2.1
78.8 ±11.6
45.8 ±1.3
14.9 ±0.9
6.99 ± 1.8
7.11 ±2.4
0.5 ±0.7
20.5 ±9.5
2.0 ±1.7
74.1 ±9.5
0.2336
0.3461
0.1891
0.0307
0.3270
0.1868
0.1051
0.2140
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Toxicological Review ofTrimethylbenzene
Monocyte (%)
Lymphoblast (%)
Myelocyte (%)
Erythroblase (%)
Reticulocyte (%)
Platelet (x 103/mm3)
Clotting time (sec)
Observation
AST(U/dL)e
ALT(U/dL)f
ALP (U/dL)g
SDH (U/dL)h
GGT full/ml)'
Bilirubin (mg/dL)
Total cholesterol (mg/dL)
Glucose (mg/dL)
Total protein (g)
Albumin (g)
Creatinine (mg/dL)
Urea (mg/dL)
Calcium (mg/dL)
Phosphorus (mg/dL)
Sodium (mmol/L)
Potassium (mmol/L)
Chloride (mmol/L)
1.2 ± 1.3
0.0 ±0.0
0.0 ±0.0
0.0 ± 0.0
3.5 ±2.6
306 ± 34
30 ±10
2.6 ±2.8
0.1 ±0.3
0.0 ±0.0
0.0 ± 0.0
1.7 ±2.0
234 ± 50*
23 ±4
1.3
0.5
0.5
0.0
1.8
±1.7
±1.5
±1.5
±0.0
±0.9
303 ± 48
19
±5**
1.5 ±0.8 1.5 ±1.4
0.7 ±1.1 0.8 ±1.3
0.1 ±0.3 0.1 ±0.3
0.0 ±0.0 0.0 ±0.0
1.0 ±0.6* 5.8 ±3.6
325 ± 57 349 ± 77
22 ±7* 48 ± 19
0.4156
0.1361
0.3189
0.5000
0.0137
0.1542
0.0034
Exposure concentration (mg/m3)
0
123
492
1,230
Trend
testb
Clinical chemistry parameters (mean ± SD)
Males
138.7 ± 20.6
51.7 ±5.9
80.4 ± 12.0
6.6 ±1.4
0.22 ± 0.44
1.027 ±0.193
63.6 ± 13.0
141.9 ±23.9
5.43 ± 1.00
3.25 ±0.60
0.506 ± 0.099
54.2 ± 8.6
10.4 ± 0.5
6.27 ±0.49
139.0 ± 1.4
4.87 ±0.36
106.6 ± 1.2
141.3 ±21.0
48.3 ±7.8
86.2 ±22.0
8.1 ±0.8**
0.20 ± 0.42
0.974 ±0.338
69.1 ±12.0
163.8 ±29.7
5.47 ± 1.39
3.45 ±0.56
0.437 ±0.138
48.8 ±8.3
10.8 ±0.5
6.50 ±0.57
1393 ±
1.3
4.97 ±0.34
106. 1±
1.7
134.
49
5 ±27.0
7 ±9.1
84.9 ±21.0
7.8 ±1.0*
0.20 ± 0.42
1.106 ±0.289
72.4 ±14.9
157
9 ±23.2
5.34 ±1.29
3.41 ±0.83
0.510 ±0.150
47
10
6 ±3.4
7 ±0.8
6.49 ±0.61
139
.6 ± 1.4
4.97 ±0.25
106
.3 ±1.5
138 ±35.0
46.8 ±5.1
90.5 ± 19.0
8.0 ±1.1**
0.20 ± 0.42
0.932 ±0.175
70.6 ± 19.5
162.2 ±28.9
5.82 ± 1.49
3.53 ±0.66
0.490 ±0.178
49.0 ±8.7
10.8 ±0.7
6.46 ±0.78
139.0 ±1.4
4.83 ± 0.40
106.7 ± 1.2
0.2223
0.0637
0.1518
0.0083
0.4700
0.2594
0.0920
0.0876
0.3242
0.2279
0.3982
0.1145
0.2449
0.1580
0.4950
0.2907
0.4353
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Toxicological Review ofTrimethylbenzene
AST(U/dL)e
ALT(U/dL)f
ALP (U/dL)g
SDH (U/dL)h
GGT full/ml)'
Bilirubin (mg/dL)
Total cholesterol (mg/dL)
Glucose (mg/dL)
Total protein (g)
Albumin (g)
Creatinine (mg/dL)
Urea (mg/dL)
Calcium (mg/dL)
Phosphorus (mg/dL)
Sodium (mmol/L)
Potassium (mmol/L)
Chloride (mmol/L)
Observation
Proliferation of peribronchial
lymphatic tissue (0-4)k
Formation of
lymphoepithelium in bronchii
(0-4)
Bronchitis and
bronchopneumonia (0-4)
Interstitial lymphocytic
infiltration (0-3)
Alveolar macrophages (0-3)
Cumulative score of all
individuals
Females
139.4 ± 16.6
49.8 ±6.3
41.2 ±7.8
5.9 ±1.5
0.20 ± 0.42
0.745 ± 0.342
64.5 ± 11.9
118.2 ±28.8
6.91 ±0.53
3.42 ±0.24
0.655 ±0.135
52.7 ±7.8
10.5 ± 0.6
4.75 ±0.54
137.9 ± 1.7
4.54 ±0.22
104.9 ± 2.0
136.7 ±27.1
51.4 ±8.2
37.2 ±6.8
7.3 ±1.7
0.30 ± 0.48
0.690 ±0.396
65.7 ±12.8
138.8 ±38.5
7.44 ±0.89
3.46 ±0.27
0.553 ±0.104
49.6 ±6.7
10.8 ±0.8
5.05 ±0.70
138.0 ± 1.8
4.39 ±0.61
105.5 ± 1.3
145.5 ± 22.7
50.4 ± 9.0
39.8 ±11.0
7.1 ±1.8
0.10 ±0.32
0.743 ± 0.248
64.1 ±10.8
104.5 ± 23.8
7.08 ±0.35
3.61 ±0.26
0.629 ±0.153
52.8 ±10.5
10.6 ± 0.5
5.34 ±0.74
137.8 ±2.5
4.51 ±0.26
105.9 ± 1.6
141.4 ±15.6
55.1 ±9.5
49.8 ±15.5
7.0 ±1.6
0.44 ± 0.53
0.642 ± 0.257
62.5 ±7.6
129.9 ±39.7
6.94 ±0.64
3.42 ±0.15
0.577 ±0.133
52.2 ±11.8
10.8 ±0.6
4.90 ± 1.01
138.2 ±2.2
4.46 ±0.25
106.4 ± 1.8
0.2118
0.1844
0.1740
0.0637
0.2821
0.3092
0.4775
0.4838
0.4036
0.2408
0.1641
0.4718
0.3011
0.4050
0.3628
0.4108
0.0601
Exposure concentration (mg/m3)
[Dose Group ID]
0
[1]
123
[2]
492
[3]
1,230
[4]
Comparison to
controls'
Trend
testb
Males
16.01
18.1
19.0
14.8
14.1
13.9
15.6
15.6
18.3
18.4
14.8
15.1
30.6
27.9
26.1
26.9
24.1
29.1
17.4
18.2
16.5
19.4
26.4
21.3
1-3*
1-3*
1-4*
1-3*
0.13
22
0.49
0.12
0.002
0.02
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Toxicological Review ofTrimethylbenzene
Proliferation of peribronchial
lymphatic tissue (0-4)k
Formation of
lymphoepithelium in bronchii
(0-4)
Bronchitis and
bronchopneumonia (0-4)
Interstitial lymphocytic
infiltration (0-3)
Alveolar macrophages (0-3)
Cumulative score of all
individuals
Health Effect at LOAEL
Increased pulmonary lesions,
decreased RBCs, and
increased WBCs in males
Females
19.4
18.3
19.0
15.8
19.7
16.8
21.7
20.1
22.9
14.5
14.9
15.3
21.2
25.1
19.0
21.5
16.6
21.3
NOAEL
123 mg/m3
17.5
16.1
19.0
29.2
29.8
27.3
1-4*
ns
ns
0.36
0.48
0.48
0.0017
0.03
0.01
LOAEL
492 mg/m3
Comments: The observed inflammatory lesions are coherent with observations of increased inflammatory cell
populations in bronchoalveolar lavage fluid in Korsak et al. (1997). The authors did not report the incidences of
pulmonary lesions, but rather the results of the Kruskall-Wallis test. This makes it difficult to interpret the dose-
response relationship and limits analysis of these endpoints to the NOAEL/LOAEL method for determining a POD,
rather than using BMD modeling.
aEffects measured in rats exposed to 1,230 mg/m3 2 wks after termination of exposure.
p-value reported from Jonckheere's trend test
cred blood cells, dwhite blood cells, easpartate aminotransferase, falanine aminotransferase, 8alkaline phosphatase,h
sorbitol dehydrogenase, 'y-glutamyltransferase,
J Reports the results of pair-wise statistical significance of exposure groups compared to controls (i.e., 1-3 would
indicate that the 492 mg/m3 was statistically significantly different from controls)
kgrading system (0-4, 0-3; see Additional study details above)
1 results presented as ranges of the Kruskal-Willistest.
*, ** Statistically significant from controls at p < 0.05 and 0.01, respectively.
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Toxicological Review ofTrimethylbenzene
Table B-32. Characteristics and quantitative results for Korsak et al.
fZOQQbl
Study design
Species Sex
IMP: Wistar M&F
rats
N Exposure route Concentration range Exposure duration
10/dose, Inhalation (6 hr/d, 5 0, 123, 492, 1,230 mg/m3 90 d
20 in d/wk) 1,2,3-TMB
1,230
mg/m3
group
Additional study details
• Animals were exposed to 1,2,3-TMB in a dynamic inhalation chamber (1.3 m3 volume) with 16 air changes/hr.
• Mean initial body weights were 290 ± 25 g for males and 215 ± 13 g for females; rats were housed in
polypropylene cages with wire-mesh covers (5 animals/cage), with food and water provided ad libitum.
• Animals were randomized and assigned to the experimental groups.
• Hematological parameters were evaluated prior to exposure and 1 wk prior to termination of exposure, and
for the 1230 mg/m3 exposure group, also evaluated two weeks after termination of exposure; blood clinical
chemistry parameters were evaluated 18 hrs after termination of exposure (animals were deprived of food for
24 hrs).
• Necropsy was performed on all animals.
• Pulmonary effects were graded using an arbitrary scale: 0 = normal status, 1 = minimal, 2 = mild, 3 =
moderate, 4 = marked.
Observation
Terminal Body weight
(g)
Absolute organ weight (g)
Lungs
Liver
Spleen
Kidney
Adrenals
Testes
Heart
Relative organ weight (g)
Lungs
Liver
Spleen
Kidney
Adrenals
Testes
Heart
Exposure concentration (mg/m3)
0
123
492
1,230
Body and organ weights (mean ± SD)
Males
390 ± 35
1.90 ±0.22
8.28 ±0.97
0.71 ±0.06
2.34 ±0.27
0.059 ± 0.012
3.78 ±0.44
1.04 ±0.13
0.510 ±0.071
2.208 ±0.163
0.190 ±0.019
0.623 ± 0.049
0.016 ± 0.003
1.014 ± 0.087
0.277 ±0.027
408 ± 50
1.86 ±0.26
8.83 ± 1.40
0.12 ±0.10
2.29 ±0.23
0.061 ±0.016
3.69 ±0.24
0.98 ±0.11
0.479 ± 0.026
2.271 ±0.129
0.187 ±0.015
0.594 ± 0.029
0.016 ± 0.003
0.961 ±0.091
0.252 ±0.018
404 ± 33
1.99 ±0.37
9.05 ±0.99
0.82 ±0.11
2.48 ±0.25
0.061 ±0.013
3.71 ±0.36
1.08 ±0.13
0.504 ± 0.082
2.287 ±0.115
0.207 ±0.021
0.629 ±0.033
0.015 ± 0.003
0.941 ±0.063
0.274 ± 0.032
413 ± 46
1.88 ±0.34
9.54 ±1.50
0.79 ±0.20
2.50 ±0.25
0.061 ±0.012
3.91 ±0.12
1.15 ±0.19
0.468 ± 0.073
2.414 ±0.214*
0.203 ± 0.058
0.637 ±0.060
0.016 ± 0.003
1.002 ±0.106
0.284 ± 0.026
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Toxicological Review ofTrimethylbenzene
Terminal Body weight (g)
Absolute organ weight (g)
Lungs
Liver
Spleen
Kidney
Adrenals
Ovaries
Heart
Relative organ weight (g)
Lungs
Liver
Spleen
Kidney
Adrenals
Ovaries
Heart
Observation
Hematocrit (%) Males
Hematocrit (%) Females
Hemoglobin (g/dL) Males
Hemoglobin (g/dL) Females
RBCs (x 103/mm3)c Males
RBCs (x 103/mm3)c Females
WBCs (x 103/mm3)d Males
WBCs (x 103/mm3)d Females
Rod neutrophil (%) Males
Rod neutrophil (%) Females
Segmented neutrophil (%)
Males
Segmented neutrophil (%)
Females
Eosinophil (%) Males
Eosinophil (%) Females
Lymphocyte (%) Males
Lymphocyte (%) Females
Monocyte (%) Males
Monocyte (%) Females
Females
268 ± 18
1.62 ±0.15
6.05 ± 0.42
0.63 ±0.05
1.58 ±0.16
0.080 ± 0.014
0.12 ±0.03
0.74 ±0.05
0.651 ±0.053
2.434 ±0.143
0.257 ±0.027
0.639 ±0.076
0.032 ± 0.005
0.051 ±0.014
0.298 ±0.016
262 ±21
1.55 ±0.33
5.85 ±0.47
0.61 ±0.10
1.53 ±0.12
0.082 ± 0.010
0.12 ±0.03
0.71 ±0.50
0.637 ±0.122
2.400 ± 0.088
0.249 ± 0.032
0.628 ± 0.024
0.034 ± 0.004
0.050 ± 0.014
0.291 ±0.012
263 ± 14
1.47 ±0.18
5.94 ±0.51
0.57 ±0.05*
1.54 ±0.10
0.083 ±0.011
0.13 ±0.02
0.75 ±0.06
0.604 ± 0.049
2.448 ±0.190
0.234 ±0.19
0.638 ±0.032
0.034 ± 0.005
0.056 ± 0.006
0.309 ± 0.024
259 ± 23
1.51 ±0.16
6.05 ± 0.44
0.56 ±0.06*
1.62 ±0.16
0.075 ±0.015
0.14 ±0.04
0.73 ±0.08
0.639 ± 0.076
2.555 ±0.214
0.237 ±0.022
0.686 ±0.058
0.032 ± 0.008
0.060 ± 0.018
0.307 ± 0.026
Exposure concentration (mg/m3)
0
123
492
1,230
1230a
Trend
testb
Hematological parameters (mean ± SD)
46.4 ± 1.6
42.7 ±2.2
16.4 ± 1.0
13.9 ±0.7
9.49 ± 2.03
8.03 ± 1.11
10.09 ± 2.23
10.71 ±4.28
0.8 ± 1.0
0.4 ±0.8
24.8 ±4.5
23.1 ±6.1
1.3 ± 1.4
1.4 ± 1.0
71.2 ±5.0
73.2 ±7.9
1.9 ± 1.6
2.0 ±2.0
45.8 ±2.6
45.0 ± 2.4
17.6 ± 1.6
15.1 ±1.0*
10.25 ± 1.29
8.73 ± 1.24
9.38 ±3.29
9.54 ±2.37
1.0 ±1.1
0.6 ±0.6
25.4 ±5.8
19.7 ±3.4
0.8 ± 1.0
0.6 ±0.6
71.6 ±6.8
77.5 ±4.9
1.3 ± 1.4
1.6 ±1.6
45.7 ± 1.3
41.8 ±1.6
17.6 ±0.8
14.6 ±0.6
10.11 ±1.27
7.79 ± 1.57
7.71 ±3.45
13.02 ± 3.07
0.4 ±0.5
1.1 ±1.4
20.7 ±5.8
16.4 ±4.2*
0.8 ±1.1
0.7 ±0.8
75.4 ±4.7
80.4 ±5.1
2.3 ± 20
1.1 ±1.3
45.5 ±2.1
41.5 ± 24
15.0 ±1.2
14.7 ±0.9
8.05 ± 1.38*
7.27 ± 1.32
9.03 ±275
13.01 ±4.53
0.5 ±0.6
0.4 ±0.8
17.7 ±8.3*
11.9 ±7.1**
0.6 ±0.8
0.8 ±0.9
79.3 ±
78.0**
84.0 ±
78.0**
1.6 ±22
2.1 ±1.7
43.5 ± 26
41.7 ± 20
ND
ND
8.6 ± 1.5
6.6 ±1.8
6.3 ±4.6
62 ±2.5
5.2 ±3.0
1.8 ±2.2
27.5 ±9.2
19.6 ±8.3
0.6 ±0.6
0.7 ±0.8
63.7 ±11.3
75.7 ±9.9
3.1 ±3.7
1.3 ±1.8
0.1615
0.0198
0.0688
0.0748
0.0011
0.0185
0.1661
0.0189
0.1878
0.4711
0.0032
0.0000
0.1439
0.2778
0.0015
0.0003
0.3014
0.2426
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Lymphoblast (%) Males
Lymphoblast (%) Females
Myelocyte (%) Males
Myelocyte (%) Females
Erythroblast (%) Males
Erythroblast (%) Females
Reticulocyte (%) Males
Reticulocyte (%) Females
Platelet (x 103/mm3) Males
Platelet (x 103/mm3) Females
Clotting time (sec) Males
Clotting time (sec) Females
Observation
AST (U/dL)e Males
AST (U/dL)e Females
ALT (U/dL)f Males
ALT (U/dL)f Females
ALP (U/dL)g Males
ALP (U/dL)g Females
SDH (U/dL)h Males
SDH (U/dL)h Females
GGT (uAJ/ml)1 Males
GGT (uAJ/ml)1 Females
Bilirubin (mg/dL) Males
Bilirubin (mg/dL) Females
Total cholesterol (mg/dL)
Males
Total cholesterol (mg/dL)
Females
Glucose (mg/dL) Males
Glucose (mg/dL) Females
Total protein (g) Males
Total protein (g) Females
Albumin (g) Males
Albumin (g) Females
Creatinine (mg/dL) Males
Creatinine (mg/dL) Females
Urea (mg/dL) Males
Urea (mg/dL) Females
Calcium (mg/dL) Males
0.0 ± 0.0
0.0 ±0.0
0.0 ±0.0
0.0 ± 0.0
0.0 ±0.0
0.0 ± 0.0
2.8 ±1.3
2.6 ±0.9
262 ± 51
224 ± 68
29.7 ±8.6
27.2 ±2.8
0.0 ± 0.0
0.0 ±0.0
0.0 ±0.0
0.0 ± 0.0
0.0 ±0.0
0.0 ± 0.0
2.1 ±1.7
4.6 ±2.5*
266 ± 70
290 ± 70
23.0 ± 10.0
25.0 ±9.4
0.2 ±0.6
0.1 ±0.3
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
3.8 ±2.1
5.2 ± .50*
257 ± 81
249 ± 53
37.9 ±9.9
23.8 ±9.5
0.2 ±0.6 0.0 ±0.0
0.3 ±0.7 0.0 ±0.0
0.0 ±0.0 0.0 ±0.0
0.5 ±0.2 0.0 ±0.0
0.0 ±0.0 0.0 ±0.0
0.1 ±0.3 0.0 ±0.0
4.5 ±1.8* 6.9 ±3.1**
4.4 ±3.0 6.8 ±3.5
242 ± 76 277 ± 80
204 ± 44 258 ± 45
29.2 ±15.6 21.7 ±5.4
25.1 ±12.1 25.9 ±8.0
0.2911
0.1403
0.5000
0.3963
0.5000
0.2995
0.0017
0.0459
0.1708
0.0329
0.4650
0.3479
Exposure concentration (mg/m3)
0
123
492 1,230
Trend
testb
Clinical chemistry parameters (mean ± SD)
107.8 ± 14.2
96.1 ±9.4
41.3 ±2.0
39.7 ±3.5
70.5 ± 15.2
21.5 ±2.7
1.6 ±0.7
1.7 ±0.7
0.77 ±0.66
0.55 ±0.72
0.600 ±0.516
0.911 ±0.348
63.1 ±10.1
60.1 ±12.2
95.5 ±13.1
115.9 ±8.5
7.84 ±0.13
8.24 ± 1.24
3.15 ±0.73
3.22 ± 1.28
41.24 ± 8.94
62.54 ± 10.66
38.7 ±4.5
42.0 ±5.5
10.6 ± 0.6
102.9 ±
96.9 ±
40.7 ±
39.5 ±
15.1
9.9
3.1
6.4
70.6 ± 11.7
25.8 ±
8.4
2.3 ±1.3
1.9 ±0.9
0.77 ±0.97
0.44 ±1.01
0.600 ±0.516
1.161 ±0.469
62.2 ±11.6
62.4 ±15.3
110.8 ±
121.0 ±
14.7
17.5
8.02 ± 0.50
8.36 ±1.14
3.15 ±1.33
3. 17 ±1.03
41.35 ± 11.28
61.60 ±
38.1 ±
7.07
9.1
43.5 ±4.4
10.7 ± 0.8
103
117
41
36
6 ± 14.5
1±23.9
5 ±5.5
2 ±3.3
66.5 ± 10.8
31.1 ±8.6*
2.5 ±0.9
1.5 ±0.7
0.40 ±0.51
0.66 ±1.11
0.800 ± 0.422
0.930 ± 0.463
64.5 ± 16.2
62
100
3 ±7.7
2 ± 15.2
109.2 ± 5.8
7.76 ±0.27
8.65 ±0.84
3.08 ± 1.30
2.58 ±1.28
40.79 ± 9.30
67.11 ±10.86
36
40
10
9 ±4.1
0±4.3
8 ±0.7
119.6 ±27.3
104.6 ± 15.7
45.5 ±5.6
30.5 ±9.9**
63.7 ±15.7
30.5 ±9.9*
2.7 ±0.7*
1.8 ±1.0
0.50 ±0.75
0.30 ±0.48
0.625 ±0.518
0.976 ±0.421
65.0 ±9.1
64.4 ±14.1
114.5 ± 20.6
109.8 ± 10.8
8.04 ±0.59
8.62 ±0.96
2.95 ±1.12
3.60 ±1.17
43.61 ±13.10
59.71 ±7.51
41.7 ±7.5
39.0 ±29
10.9 ± 0.5
0.2223
0.2118
0.0637
0.1844
0.1518
0.1740
0.0083
0.0637
0.4700
0.2821
0.2594
0.3092
0.0920
0.4775
0.0876
0.4838
0.3242
0.4036
0.2279
0.2408
0.3982
0.1641
0.1145
0.4718
0.2449
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Calcium (mg/dL) Females
Phosphorus (mg/dL) Males
Phosphorus (mg/dL) Females
Sodium (mmol/L) Males
Sodium (mmol/L) Females
Potassium (mmol/L) Males
Potassium (mmol/L) Females
Chloride (mmol/L) Males
Chloride (mmol/L) Females
Observation
Proliferation of peribronchial
lymphatic tissue (0-3)kMales
Proliferation of peribronchial
lymphatic tissue (O-S)Females
Formation of
lymphoepithelium in bronchii
(0-3) Males
Formation of
lymphoepithelium in bronchii
(0-3) Females
Goblet cells (0-3) Males
Goblet cells (0-3) Females
Interstitial lymphocytic
infiltration (0-3) Males
Interstitial lymphocytic
infiltration (0-3) Females
Alveolar macrophages (0-3)
Males
Alveolar macrophages (0-3)
Females
Bronchitis and broncho-
pneumonia (0-4) Males
Bronchitis and broncho-
pneumonia (0-4) Females
Cumulative score of all
individual Males
Cumulative score of all
individual Females
Health Effect at LOAEL
Pulmonary lesions
11.1 ±0.8
8.60 ±0.95
6.56 ±0.70
143.9 ±2.1
144.0 ± 1.5
4.70 ±0.35
4.52 ±0.41
107.3 ± 2.3
108.1 ±3.2
11.7 ±0.3
8.26 ±0.60
6.25 ±1.17
144.1 ±1.5
143.8 ±1.3
4.45 ±0.28
4.51 ±0.43
107.7 ±4.3
108.1 ±1.5
11.8 ±0.2 11.8 ±0.7 0.3011
9.19 ±0.88 9.41 ±0.55 0.1580
6.41 ± 1.02 7.18 ± 1.09 0.4050
143.9 ± 25 144.8 ± 24 0.4950
142.7 ±1.3 143.8 ±1.4 0.3628
4.75 ±0.37 4.97 ±0.56 0.2907
4.28 ±0.41 4.37 ±0.34 0.4108
106.8 ± 1.8 106.5 ± 1.9 0.4353
107.1 ±1.3 107.2 ±23 0.0601
Exposure concentration (mg/m )
[Dose group ID]
0
[1]
2.01 (23.4)m
24 (22.8)
1.5 (23.9)
1.8 (27.9)
1.8 (18.6)
1.3 (11.9)
0.4 (18.0)
1.2 (23.7)
0.9 (17.9)
1.5(26.1)
0.5(20.1)
0.2 (17.6)
7.1(19.8)
8.4 (24.9)
123
[2]
1.2 (11.5)
1.3(12.1)
0.9 (14.9)
0.7(11.1)
1.5 (14.5)
1.6 (16.9)
0.1(14.1)
0.6 (15.3)
0.9 (17.9)
1.1(21.1)
0.2 (16.6)
0.4 (22.5)
4.8(11.2)
5.7 (13.5)
492
[3]
1.8(22.0)
1.5(16.4)
1.0(16.0)
1.1(16.9)
2.5(28.5)
2.0(23.1)
0.4(18.0)
0.8(17.9)
1.2(22.6)
0.5(17.8)
0.8(23.8)
0.2(17.5)
7.7(24.2)
6.5(16.8)
NOAEL
492
mg/m3
1230
[4]
2.0(23.5)
L3 (22.3)
1.5(25.7)
1.5(23.8)
1.8(18.2)
2.4 (28.4)
1.5(31.0)
1.1(22.9)
1.2(21.7)
0.7 (14.8)
0.7(19.5)
0.6(21.8)
8.7(25.8)
8.2 (24.6)
Comparison to
controls'
1-2*
1-2**; 1-3
1-3*; 1-4**
1-3*; 1-4**
1-4*
1-2*
Trend
testb
p = 0.2
p = 0.2
p = 0.3
p = 0.3
p = 0.18
p = 0.001
p = 0.006
t
p=0.4
p = 0.15
p = 0.01
p = 0.3
p = 0.3
p = 0.01
p = 0.4
LOAEL
1230 mg/m3
Comments: The observed inflammatory lesions are coherent with observations of increased inflammatory cell
populations in bronchoalveolar lavage fluid due to 1,2,4-TMB exposure in Korsak et al. (1997). The authors did not
report the incidences of pulmonary lesions, but rather the results of the Kruskall-Wallis test. This makes it difficult to
interpret the dose-response relationship and limits analysis of these endpoints to the NOAEL/LOAEL method for
determining a POD, rather than using BMD modeling.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
aEffects measured in rats exposed to 1,230 mg/m3 2 wks after termination of exposure.
p-value reported from Jonckheere's trend test
cred blood cells, dwhite blood cells, easpartate aminotransferase, falanine aminotransferase, galkaline phosphatase,h
sorbitol dehydrogenase, 'y-glutamyltransferase,
J Reports the results of pair-wise statistical significance of exposure groups compared to controls (i.e., 1-3 would
indicate that the 492 mg/m3 was statistically significantly different from controls)
kgrading system (0-4, 0-3; see Additional study details above)
mean
m results presented as ranges of the Kruskal-Willis test.
*, ** Statistically significant from controls at p < 0.05 and 0.01, respectively.
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Toxicological Review ofTrimethylbenzene
Table B-33. Characteristics and quantitative results for Lammers et al.
fZOQT)
Study design
Species Sex N Exposure route Dose range Exposure duration
WAG/RijCR/BR M 8 /group Inhalation (8 r
Wistar rats for 3 consecut
days)
/day 0, 600, 2,400, or 4,800 3 d
ive mg/m3l,2,4-TMB (as a
constituent of WS)
Additional study details
• Rats were exposed to 1,2,4-TMB as a constituent of WS at concent
3 d. Several tests were conducted to evaluate impact of WS on CNS
spontaneous motor activity and learned visual discrimination.
• White spirit was found to affect performance and learned behavior
Observation
•ations of 0, 600, 2,400, or 4,800 mg/m3 for
. These included tests of observation,
in rats.
Functional observations and physiological parameters in rats following exposure to
WS (exposure concentration mg/m )
0
600
2,400
4,800
Functional observation battery (mean ± SD)
Gait score3
Before first 8 hr
exposure
After first 8 hr
exposure
After third 8 hr
exposure
1.00 ± 0.00
1.00 ± 0.00
1.00 ± 0.00
1.00 ± 0.00
1.00 ± 0.00
1.00 ± 0.00
1.00 ± 0.00
1.13 ±0.13
1.00 ± 0.00
1.00 ± 0.00
1.25 ±0.16
1.00 ± 0.00
Click response15
Before first 8 hr
exposure
After first 8 hr
exposure
After third 8 hr
exposure
2.13 ±0.13
2.88 ±0.13
2.13 ±0.13
2.63 ±0.18
2.50 ±0.19
3.25 ±0.31*
2.38 ±0.18
2.75 ±0.37
2.88 ±0.23
2.50 ±0.19
2.63 ±0.18
2.75 ±0.25
Physiological parameters (mean ± SD)
Body weight (g)
Before first 8 hr
exposure
After first 8 hr
exposure
After third 8 hr
exposure
270.0 ±2.61
279.7 ±2.53
280.9 ± 2.68
269.2 ±2.48
277.7 ±3.11
278.4 ± 2.44
273.3 ±3.52
278.0 ±3.21**
275.9 ±2.83***
272.8 ±2.20
273.8 ±2.51***
268.5 ±2.67***
Body temperature (°C)
Before first 8 hr
exposure
After first 8 hr
exposure
After third 8 hr
exposure
37.60 ±0.34
36.41 ±0.05
36.60 ±0.10
37.33 ±0.39
36.25 ±0.12
36.44 ±0.17
37.49 ±0.39
36.16 ±0.11
36.25 ±0.05
37.29 ±0.37
35.95 ±0.21
36.11 ±0.09**
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
cr 300° i
CO
1
~+
£ 2500-
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ra
0)
•^ 2000-
S
o
u
§ 1500
*Ml
_w
5
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Figu
-o— control url l
-^*— 600mg/m3 dur
— • — 2400 mg/m3 asse
— a — 4800 mg/m3
^\T**
pre-test 1 x 8 hr 3 x 8 hr
time of testing
Observation
Lever response latency (sec)
Before first 8 hr
exposure
After first 8 hr
exposure
After second 8 hr
exposure
After third 8 hr
exposure
One day after third 8
hr exposure
Number of lever response
latencies <2 sec
Before first 8 hr
exposure
After first 8 hr
exposure
After second 8 hr
exposure
After third 8 hr
exposure
One day after third 8
hr exposure
Number of lever response
latencies >6 sec
Before first 8 hr
exposure
After first 8 hr
exposure
re 1. Effects of WS
otal distance run
ng motor activity
'ssment in rats.
Visual discrimination performance in rats exposed to WS for 3 consecutive days
(exposure concentration in mg/ms)c
0
1.93 ±0.34
2.44 ±0.56
2.17 ±0.41
3.21 ±1.22
2.27 ±0.52
68.00 ± 5.46
70.38 ±2.93
70.62 ± 3.60
71.50 ±3.38
72.50 ±3.58
3.88 ±0.90
5.00 ±1.10
600
2.09 ±0.24
2.66 ±0.29
2.32 ±0.29
2.68 ±0.41
1.93 ±0.16
67.38 ±2.58
61.88 ±3.92
68.00 ± 3.81
66.38 ±3.34
69.75 ±2.90
5.25 ±0.84
7.62 ±1.83
2,400
1.70 ±0.15
3.24 ±0.21
2.10 ±0.18
3.86 ±0.65
1.88 ±0.16
77.12 ±4.32***
58.75 ±2.58***
69.00 ±2.98***
63.75 ±5.04***
73.38 ±2.93***
3.25 ±0.45*
11. 12 ±0.85*
4,800
2.29 ±0.31**
12.00 ±2.37**
4.88 ±1.53**
6.31 ±1.35**
2.34 ±0.31**
71.25 ±4.00**
45.62 ±4.87**
61.50 ±5.00**
55.62 ±5.12**
64.88 ±4.23**
5.62 ±0.92**
25.75 ±5.05**
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
After second 8 hr
exposure
After third 8 hr
exposure
One day after third 8
hr exposure
Drink response latency (sec)
Before first 8 hr
exposure
After first 8 hr
exposure
After second 8 hr
exposure
After third 8 hr
exposure
One day after third 8
hr exposure
Health Effect at LOAEL
n/a
4.38 ±0.96
7.38 ±2.07
4.62 ±1.31
0.35 ± 0.04
0.37 ± 0.04
0.36 ± 0.04
0.38 ±0.05
0.36 ±0.03
5.62 ±0.78
6.88 ±1.16
4.38 ±1.07
0.29 ±0.03
0.31 ±0.03
0.28 ±0.03
0.32 ±0.04
0.31 ±0.02
NOAEL
n/a
5.00 ±0.65*
10.88 ±1.96*
3.75 ±0.70*
0.36 ±0.03
0.39 ±0.02
0.33 ±0.02
0.39 ±0.02
0.34 ± 0.02
12.25 ±3.80**
17.50 ±2.76**
6.50 ±1.86**
0.32 ±0.02
0.52 ± 0.04
0.39 ± 0.04
0.43 ± 0.07
0.33 ± 0.04
LOAEL
n/a
Comments: Exposure to 1,2,4-TMB was via WS, which is comprised of additional substances. LOAEL and NOAEL cannot
be extracted from this study because other constituents of the WS mixture may confound results.
aGait score indicates the severity of gait changes and is scored as 1 (normal) to 4 (severely abnormal).
bClick response was scored as 0 (no reaction) to 5 (exaggerated reaction).
°Data for parameters that did not show statistically significant group differences are not shown; statistical analysis:
repeated measures ANCOVA + pairwise group comparisons.
*,**,*** Statistically significant from controls at p < 0.05, p < 0.01, and p < 0.001 respectively.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-34. Characteristics and quantitative results for Lutz et al.
fZOlQl
Study design
Species
Wistar rats
Sex
M
N
6-8 rats
per dose
Exposure route
Inhalation (6 hr/d, 5
d/wk)
Dose range
0, 25, 100, or 250 ppm (0,
123, 492, or 1,230 mg/m3)
1,2,3- or 1,2,4-TMB
Exposure duration
4 wks
Additional study details
• Animals were exposed to 1,2,3- or 1,2,4-TMB in 1.3 m3 dynamic inhalation exposure chambers for 6 hrs/d,
5 days/wk for 4 wks. Food and water was provided ad libitum.
• Animals were randomized and assigned to the experimental groups.
• Behavioral sensitivity to amphetamine was measured via test of open-field locomotor activity.
• Differences were observed between 1,2,3- and 1,2,4-TMB exposed rats, with 1,2,3-TMB-exposed rats
displaying greater amphetamine sensitization than 1,2,4-TMB exposed rats.
Session 1
(before AMPH sensitization)
Session 2
(after AMPH sensitization)
40-
5x2.5 mg/kg AMPH
Control
] Block 1
HEM HEM
25 ppm 100 ppm
^1 Block 2
HEM
250 ppm
Control
HEM
25 ppm
BlockS
Block 4
Blocks
HEM HEM
100 ppm 250 ppm
BlockS
Block 1 — control (prcinjcction) activity, block 2 — activity after the SAL injection, blocks 3, 4,5 and 6 — activity during successive 30 min sections
after AMPH (0.5 mg/kg) injection.
ANOVA: group effects: F (3.24) =9.80; P = 0.0002; session effects: F (1.24) =34.22; P = 0.0000; interaction: F (3.24) =20.64; P = 0.0000.
* P < 0.05 — compared to post SAL measurement.
"* P < 0.05 — compared to control 0 in the same session.
*** P < 0.05 — compared to corresponding measure before sensitization.
The bars represent mean values and SEM of the ambulatory activity (distance in metres) in successive 30 min blocks in the rats exposed
to hemimellitene on the locomotor response to AMPH challenge before (session 1) and 14 days after (session 2) a repeated
(2.5 mg/kg, I/day x 5 days) AMPH treatment.
Figure 1. Diagram illustrating the effect of prior exposure to 1,2,3-TMB on the locomotor response (all measurements)
to the amphetamine challenge before (session 1) and 14 days after (session 2) a repeated (2.5 mg/kg, I/day x 5 day)
amphetamine treatment.
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Toxicological Review ofTrimethylbenzene
Figure 2. Diagram illustrating the effect of prior exposure to 1,2,3-TMB on the locomotor response (pooled
measurements) to the amphetamine challenge before (session 1) and 14 days after (session 2) a repeated
amphetamine treatment (2.5 mg/kg, 1/d x 5 d).
Session 1
(before AMPH sensitization)
Session 2
(after AMPH sensitization)
11
»240-|
j
'200
160
120
5x2.5 mg/kg AMPH
HEM
25 ppm
HEM HEM
100 ppm 250 ppm
Control HEM HEM HEM
25 ppm 100 ppm 250 ppm
* P < 0.05 — compared to control. "' P < 0.05 — compared to corresponding measure before sensitization.
Bars represent mean values and SEM of the cumulated locomotor activity (distance in metres) during the 2-hour measurement
following AMPH (0.5 mg/kg) challenge.
Figure 3. Diagram illustrating the effect of prior exposure to 1,2,4-TMB on the locomotor response (all measurements)
to the amphetamine challenge before (session 1) and 14 days after (session 2) a repeated (2.5 mg/kg, I/day x 5 days)
amphetamine treatment. Remaining notations are the same as in Figure 1.
Session 1
(before AMPH sensitization)
Session 2
(after AMPH sensitization)
40-
5x2.5 mg/kg AMPH
Control
Block 1
PS
25 ppm
PS
100 ppm
PS
250 ppm
Control
PS
25 ppm
PS
100 ppm
PS
250 ppm
Block 2 | | Block3
Block 4 | | Block 5
Block6
ANOVA: group effects: F (3.25) =8.90; P = 0.004. Session effects: F (1.25) =30.91; P = 0.0000. Interaction: F (3.25) =29.48; P = 0.0000.
* P < 0.05 — compared to post SAL measurement.
** P < 0.05 — compared to control 0 in the same session.
*** P < 0.05 — compared to corresponding measure before sensitization.
The bars represent mean values and SEM of the ambulatory activity (distance in metres) in successive 30 min blocks.
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Toxicological Review ofTrimethylbenzene
Figure 4. Diagram illustrating the effect of prior exposure to 1,2,4-TMB on the locomotor response (pooled
measurements ) to amphetamine challenge before (session 1) and 14 days after (session 2) a repeated amphetamine
treatment (2.5 mg/kg, 1/d x 5 d).
8 a
; 280-
}
J
?240-
I
I
200-
160"
120-
80-
40 -
Session 1
(before AMPH sensitization)
Session 2
(after AMPH sensitization)
5x2.5 mg/kg AMPH
J_
J_
Control
PS
25ppm
PS PS
100 ppm 250 ppm
Control
PS PS PS
25 ppm 100 ppm 250 ppm
* P < 0.05 — compared to control. ** P < 0.05 — compared to corresponding measure before sensitization.
Bars represent mean values and SEM of the cumulated locomotor activity (distance in metres) during the 2-hour measurement
following AMPH (0.5 mg/kg) challenge.
Health Effect at LOAEL
Increased sensitivity to
amphetamine as measured by
open-field locomotion
NOAEL
0 ppm
LOAEL
25 ppm (123 mg/m3) 1,2,4-TMB or
1,2,3-TMB
Comment: This study observed increased amphetamine sensitization, particularly in rats exposed to 100 ppm (492
mg/m3) 1,2,3-TMB, and provided evidence for differences in toxicity between different TMB isomers. Control group
for 1,2,4-TMB also showed statistically significant increase in locomotor activity after receiving amphetamine
treatment.
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Toxicological Review ofTrimethylbenzene
Table B-35. Characteristics and quantitative results for Maltoni et al.
(1997)
Study design
Species Sex N Exposure route Dose range Exposure duration
Sprague- M 50 males, Stomach tube (in 0 or 800 mg/kg BW 4 d/wk for 104 wks
Dawley rats: 50 females olive oil) 1,2,4-TMB
CRC/BT per group
Additional study details
• Rats were exposed to 1,2,4-TMB for 2 years via stomach tube administration 4 d/wk.
• Animals were 7 wks old at start of experiments.
• Systematic necropsy was conducted upon animal death.
• A slight increase in total number of tumors was detected amongst males and females, and an increase in the
number of head cancers in males was also observed.
Observation
Long-term carcinogenicity of 1,2,4-TMB
0 mg/kg
800 mg/kg
Total number of tumors
Males
Total benign and
malignant tumors
Malignant tumors
No. malignant
tumors/100 rats
54.0
24.0
26.0
62.0
26.0
34.0
Females
Total benign and
malignant tumors
Malignant tumors
No. malignant
tumors/100 rats
70.0
22.0
22.0
66.0
24.0
32.0
Both sexes
Total benign and
malignant tumors
Malignant tumors
No. malignant
tumors/100 rats
62.0
23.0
24.0
64.0
25.0
33.0
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Toxicological Review ofTrimethylbenzene
Head cancers
Males
Zymbal gland cancer
Ear duct cancer
Neuroesthesio-
epitheliomas
Oral cavity cancers
Total head cancers
2.0
-
-
-
2.0
4.0
2.0
2.0
2.0
10.0
Females
Zymbal gland cancer
Ear duct cancer
Neuroesthesioepi-
theliomas
Oral cavity cancers
Total head cancers
2.0
2.0
-
2.0
6.0
2.0
-
4.0
-
6.0
Both sexes
Zymbal gland cancer
Ear duct cancer
Neuroesthesio-
epitheliomas
Oral cavity cancers
Total head cancers
Health Effect at LOAEL
Various malignant and non-
malignant cancers
2.0
1.0
-
1.0
4.0
NOAEL
n/a
3.0
1.0
3.0
1.0
8.0
LOAEL
800 mg/kg
Comments: Neuroesthesioepithelioma is uncommon in Sprague-Dawley rats, although there were increases in the
number of neuroesthesioepithelioma in both males and females. Only one dose level was tested (800 mg/kg), making
any determination of dose-response impossible. Statistical significance of data not provided, although post-hoc
statistical tests performed by EPA failed to observe any statistical increase in tumors.
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Toxicological Review ofTrimethylbenzene
Table B-36. Characteristics and quantitative results for McKee et al.
fZOlQl
Study design
Species Sex
Wistar rats M
N Exposure route Dose range Exposure duration
8 rats per Inhalation (
group
D, 125, 1,250, or 5,00
mg/m3 1,2,4-TMB
D 8 hrs/d for 3 consecutive days
Additional study details
• Animals were exposed to 1,2,4-TMB for 8 hrs/d for 3 d in modified H1000 inhalation chambers.
• Animals were randomized and assigned to the experimental groups.
• Test on neurobehavioral effects were conducted prior to, during, and after exposure period.
• Motor activity was affected on the third day of exposure in the highest exposure group, although brain
concentrations of 1,2,4-TMB were lower than on previous days.
Observation
Forelimb grip strength (g)
One-day pre-exposure
First 8 hr exposure
Third 8 hr exposure
Total distance traveled (cm)
One-day pre-exposure
First 8 hr exposure
Third 8 hr exposure
Number of movements
One-day pre-exposure
First 8 hr exposure
Third 8 hr exposure
Observation
Exposure concentration 1,2,4-TMB (mg/m3)
0
125
1,250
5,000
Results of functional and motor activity observations
1,107 ± 41.2
1,064 ± 39.9
908 ±56.1
1,065 ± 52.3
814 ±91.7*
847 ± 64.3
1,223 ± 25.9
1,059 ± 59.8
956 ±67.7
1,090 ± 47.0
1,023 ± 55.7
1,156 ±68.7*
3,773 ± 120
2,479 ± 110
2,459 ± 118
3,598 ± 301
3,048 ± 257
2,740 ± 226
3,543 ± 167
2,125 ± 171
1,967 ± 316
3,575 ± 119
1,897 ± 200
1,172 ± 226*
1,054 ±31
697 ± 29
687 ± 31
999 ± 80
848 ± 66
744 ± 56
990 ± 44
600 ± 48
541 ± 82
998 ± 32
529 ± 53
329 ±61*
Exposure concentration 1,2,4-TMB (mg/m3)
0
125
1,250
5,000
Visual discrimination performance testing (means ± SD)
Trials3
One-day pre-exposure
First 8 hr exposure
Third 8 hr exposure
One-day post-exposure
100 ± 0.0
100 ± 0.0
100 ± 0.0
100 ± 0.0
100 ± 0.0
100 ± 0.0
100 ± 0.0
100 ± 0.0
100 ± 0.0
100 ± 0.0
100 ± 0.0
100 ± 0.0
100 ± 0.0
99. 13 ±0.88
100 ± 0.0
100 ± 0.0
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Toxicological Review ofTrimethylbenzene
Percentage reinforcements obtained15
One-day pre-exposure
First 8 hr exposure
Third 8 hr exposure
One-day post-exposure
99.88 ±0.13
100 ± 0.0
99.63 ±0.26
99.63 ±0.26
99.88 ±0.13
100 ± 0.0
99.63 ±0.26
99.88 ±0.13
99.88 ±0.13
99.38 ±0.63
99.63 ±0.38
99.88 ±0.13
100 ± 0.0
99.74 ±0.17
100 ± 0.0
100 ± 0.0
Discrimination ratio0
One-day pre-exposure
First 8 hr exposure
Third 8 hr exposure
One-day post-exposure
0.81 ±0.84
0.86 ±0.02
0.89 ± 0.02
0.87 ±0.03
0.84 ± 0.03
0.91 ±0.03
0.88 ±0.03
0.89 ± 0.03
0.83 ± 0.02
0.91 ±0.01
0.94 ±0.01
0.92 ±0.02
0.83 ±0.03
0.95 ±0.01*
0.95 ±0.02
0.88 ±0.03
Percentage inter-trial intervals responded tod
One-day pre-exposure
First 8 hr exposure
Third 8 hr exposure
One-day post-exposure
12.88 ± 2.00
12.50 ±2. 12
12.00 ±1.65
10.88 ± 1.39
10.13 ± 1.56
8.88 ±2.03
8.88 ±2.24
10.63 ± 1.81
10.75 ± 1.94
11.50 ±2.60
8.25 ±1.71
11.25 ±0.92
10.38 ± 1.84
10.19 ± 1.28
5.75 ±1.39
8.50 ± 1.40
Repetitive errors6
One-day pre-exposure
First 8 hr exposure
Third 8 hr exposure
One-day post-exposure
8.25 ±3.71
2.00 ±0.50
2.63 ± 1.70
4.75 ±2.81
7.63 ±1.70
3.25 ± 1.47
4.75 ±1.81
2.75 ± 1.35
10.75 ±2.73
4.63 ± 1.58
3.00 ±0.78
4.63 ± 3.09
7.25 ±1.75
1.88 ±0.67
1.25 ±0.73
4.13 ± 1.38
Repetitive inter-trial responses
One-day pre-exposure
First 8 hr exposure
Third 8 hr exposure
One-day post-exposure
3.63 ± 1.02
6.13 ±1.73
7.25 ± 1.24
6.63 ±1.94
5.88 ± 1.33
3.88 ±1.22
3.25 ±0.88
2.88 ±0.83
7.25 ± 1.93
5.63 ± 1.97
2.25 ±1.52*
5.13 ± 1.54
3.25 ± 1.35
8.38 ±2.50
1.63 ±0.98*
2.63 ±0.68
Trial response latency8
One-day pre-exposure
First 8 hr exposure
Third 8 hr exposure
One-day post-exposure
1.83 ±0.18
1.70 ±0.18
1.91 ±0.23
1.68 ±0.16
2.25 ±0.55
2.38 ±0.43
2.69 ±0.69
2.70 ±0.60
2.06 ± 0.40
2.52 ±0.40
2.75 ±0.94
2.18 ±0.73
2.28 ±0.43
3.91 ±0.73*
1.82 ±0.13
1.45 ± 0.06
Standard deviation of response latency
One-day pre-exposure
First 8 hr exposure
Third 8 hr exposure
One-day post-exposure
2.16 ±0.38
2.06 ±0.38
2.74 ±0.71
1.84 ±0.38
3.82 ±1.57
3.64 ±1.32
4.03 ± 1.50
5.95 ± 2.40
3.33 ± 1.42
4.19 ± 1.65
5.25 ± 3.04
5.88 ±4.21
4.65 ±2.23
7.33 ±3.43
2.34 ±0.40
1.81 ±0.38
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Toxicological Review ofTrimethylbenzene
Latency <2 sec
One-day pre-exposure
First 8 hr exposure
Third 8 hr exposure
One-day post-exposure
61.75 ±4.55
68.50 ± 3.84
70.38 ±4.34
69.38 ±2.98
70.13 ±2.23
69.75 ±3.75
64.13 ±4.35
67.63 ±3.20
67.75 ±66.88
65.76 ±3.13
74.88 ±1.75
78. 13 ±3.05
66.88 ±3.22
52.13 ±3.96
79.00 ± 2.32
78.00 ± 2.34
Latency >6 sec1
One-day pre-exposure
First 8 hr exposure
Third 8 hr exposure
One-day post-exposure
3.38 ±0.71
3.88 ±0.58
4.25 ±0.98
2.13 ±0.67
5.38 ±1.48
5.00 ±1.69
5.63 ±2.44
6.00 ±1.68
4.63 ± 1.15
6.00 ± 1.34
5.63 ± 1.92
3.38 ± 1.40
4.00 ± 1.05
10.63 ± 1.80*
3.13 ±0.61
1.88 ±0.35
Drink response latency1
One-day pre-exposure
First 8 hr exposure
Third 8 hr exposure
One-day post-exposure
Health Effect at LOAEL
n/a
0.29 ±0.01
0.26 ±0.01
0.30 ± 0.02
0.27 ±0.01
0.32 ± 0.02
0.30 ± 0.02
0.32 ±0.03
0.34 ± 0.03
NOAEL
n/a
0.38 ±0.03*
0.43 ± 0.03*
0.37 ±0.02
0.36 ±0.03
0.33 ± 0.02
0.49 ± 0.03*
0.34 ±0.03
0.30 ±0.02
LOAEL
n/a
Comments: This study observed alterations in a number of parameters, including forelimb grip strength, total distance
traveled, number of movements, and several visual discrimination performance tests. LOAEL and NOAEL cannot be
determined because a dose-response relationship was not apparent. Statistically significant results occurred in a low
exposure group and not others, while forelimb grip was found to be significantly increased in the highest exposure
group on day 3. Acute duration of exposure (exposure on 3 consecutive days). Generally, acute exposure studies have
limited utility in quantitation of human health reference values.
aTotal number of trials completed during each session, maximum = 100.
bl\lumber of reinforcements obtained divided by the number of reinforcements delivered (xlOO).
cNumber of correct trial responses divided by the number of trial responses.
dThe number of inter-trial intervals in which at least 1 response was made divided by the total number of ITI (xlOO).
eThe total number of incorrect trial responses following an initial incorrect response.
fThe total number of ITI responses following an initial ITI response.
gThe latency (seconds) to make a correct trial response.
hThe number of responses within 2 seconds.
'The number of responses taking more than 6 seconds.
JThe mean latency (seconds) to obtain reinforcement.
*Statistically significant from controls at p < 0.05.
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Toxicological Review ofTrimethylbenzene
Table B-37. Characteristics and quantitative results for Saillenfait et al.
f20Q51
Study design
Species
Sprague-
Dawley rats
Sex
F&
M
N Exposure route
24 dams Inhalation (6 h/d
per dose GD 6-20)
Dose range Exposure duration
0, 100, 300, 600, 900 ppm Gestational days 6-20
(0, 492, 1,476, 2,952, or
4,428 mg/m3) 1,2,4-TMB; 0,
100, 300, 600, 1,200 ppm (0,
492, 1,476, 2,952, or 5,904
mg/m3) 1,3,5-TMB
Additional study details
• Animals were exposed to 1,2,4- or 1,3,5-TMB in 200 L glass/steel inhalation chambers for 6 hrs/d starting on
GD 6 and ending on GD 20.
• Animals were randomized and assigned to the experimental groups.
• After GD 20, dams were sacrificed and weighed, as were their uteri and any fetuses.
• Decreases in maternal body weight and fetal toxicity were observed.
Observation
No. treated
No. (%) pregnant at
euthanization
No. deaths
Body weight
Body weight
g) on day 6
change (g)
Days 0-6
Days 6-13
Days 13-21
Days 6-21
Corrected weight gain3
Food consumption (g/day)
Days 0-6
Days 6-13
Days 13-21
Days 6-21
Exposure concentration to 1,3,5-TMB
0 ppm
100 ppm
(492mg/m3)
300 ppm
(l,476mg/m3)
600 ppm
(2,952 mg/m3)
1,200 ppm
(5,904 mg/m3)
Maternal parameters
24
21(87.5)
0
274 ± 17g
24
22 (91.7)
0
273 ± 16
24
21 (87.5)
0
274 ± 21
24
17 (70.8)
0
270 ± 17
24
18 (75.0)
0
275 ± 14
31 ±11
25 ±12
110 ± 14
135 ± 15
29 ±14
31 ±8
29 ±4
109 ± 10
138 ± 11
30 ±9
31 ±7
23 ±6
95 ±21*
118 ± 24*
20 ±12
29 ±8
16 ±8**
80 ±20**
95 ±24**
7 ±20**
28 ±8
10 ±7
63 ±26**
73 ±28**
-12 ± 19**
22 ±2
22 ±2
26 ±2
24 ±2
22 ±3
22 ±2
25 ±2
24 ±2
22 ±2
20 ±1*
24 ±2*
22 ±2*
22 ±2
18 ±2**
21 ±3**
20±2**
23 ±2
17 ±2**
19 ±3**
18 ±2**
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Observation
All litters"
No. of corpora lutea per dam
Mean no. of implantation
sites per litter
Mean % post-implantation loss
per litter0
Mean % dead fetuses per
litter
Mean % resorption sites per
litter
Live littersd
Mean no. of live fetuses per
litter
Mean % male fetuses per
litter
Fetal body weight (g)
All fetuses
Male fetuses
Female fetuses
Observation
Total no. fetuses examined
(litters)
External
Visceral
Skeletal
Malformations
Diaphragmatic hernia
Multiple skeletal
malformations6
External variations
Club foot (bilateral)
Visceral variations
Dilated renal pelvis
Distended ureter
Skeletal variations
Fifth sternebrae incomplete
ossification or unossifiedf
Exposure concentration to 1,3,5-TMB
0 ppm
100 ppm
(492mg/m3)
300 ppm
(l,476mg/m3)
600 ppm
(2,952 mg/m3)
1,200 ppm
(5,904 mg/m3)
Gestational parameters
21
15.3 ± 1.5g
14.9 ±1.5
4.8 ±4.2
0.0 ± 0.0
4.8 ±4.2
21
14.1 ±1.6
49.3 ± 13.5
22
15.4 ±1.7
14.9 ±1.8
3.9 ±4.3
0.0 ± 0.0
3.9 ±4.3
22
14.3 ± 1.7
48.2 ± 16.3
21
15.5 ± 1.7
14.5 ± 3.4
6.8 ±8.5
0.0 ±0.0
6.3 ±6.5
21
13.4 ± 3.4
52.1 ±18.1
17
14.9 ±2.1
13.0 ±5.1
1.6 ±3.7
0.0 ±0.0
1.6 ±3.7
17
12.8 ±5.0
51.1 ±20.9
18
15.2 ±1.5
13.6 ±3.7
4.4 ±6.9
0.0 ± 0.0
4.4 ±6.9
18
13.1 ±3.7
48.5 ± 18.2
5.64 ±0.35
5.80 ±0.41
5.50 ±0.32
5.61 ±0.24
5.76 ±0.27
5.47 ±0.21
5.43 ± 0.45
5.50 ±0.31
5.27 ±0.47
5.36 ±0.68
5.39 ±0.55*
5.18 ±0.68
4.98 ±0.56**
5.10 ±0.57**
4.81 ±0.45**
Exposure concentration to 1,3,5-TMB
0 ppm
100 ppm
(492mg/m3)
300 ppm
(l,476mg/m3)
600 ppm
(2,952 mg/m3)
1,200 ppm
(5,904 mg/m3)
Fetal variations and malformations
297 (21)
149 (21)
148 (21)
314 (22)
157 (22)
157 (22)
282 (21)
141 (20)
141 (21)
217 (17)
109 (15)
108 (17)
236 (18)
118 (18)
118 (18)
0
KD
0
0
KD
0
0
KD
0
0
0
0
KD
0
0
0
0
0
0
0
2(2)
12(9)
0
14(8)
5(4)
18(8)
0
5(3)
2(2)
11(6)
2(2)
2(2)
7(4)
7(5)
12(7)
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Toxicological Review ofTrimethylbenzene
Fourth sternebrae, split
Cervical rib, rudimentary
Fourteenth rib,
supernumerary
Thoracic vertebra centra,
incomplete ossification
Observation
No. treated
No. (%) pregnant at
euthanization
No. deaths
Body weight (g) on day 6
Body weight change (g)
Days 0-6
Days 6-13
Days 13-21
Days 6-21
Corrected weight gain3
Food consumption (g/day)
Days 0-6
Days 6-13
Days 13-21
Days 6-21
0
2(2)
11(8)
10(5)
0
0
9(6)
8(6)
0
5(5)
11(6)
10(7)
0
5(3)
15(8)
9(7)
KD
2(2)
17(8)
9(7)
Exposure concentration to 1,2,4-TMB
0 ppm
100 ppm
(492mg/m3)
300 ppm
(l,476mg/m3)
600 ppm
(2,952 mg/m3)
900 ppm
(4,428 mg/m3)
Maternal parameters
25
24 (96.0)
0
271 ± 18g
24
22 (91.7)
0
272 ± 21
24
22 (91.7)
0
272 ± 22
24
22 (91.7)
0
275 ± 19
24
24 (100)
0
269 ± 18
27 ±8
27 ±8
105 ± 28
131 ± 33
29 ±12
28 ±6
27 ±6
98 ±16
124 ± 18
31 ±14
28 ±7
26 ±6
100 ± 20
126 ± 24
27 ±12
28 ±12
19 ±8**
97 ±17
116 ± 23
15 ± 17**
24 ±8
14 ±12**
82 ± 14**
95 ±19**
0±14**
23 ±2
21 ±3
26 ±3
24 ±3
23 ±2
20 ±2
25 ±2
23 ±2
23 ±2
20 ±2
24 ±2
22 ±2
23 ±3
18 ±2**
23 + 3**
21 ±3**
23 ±3
17 ±2**
22 + 3**
20±2**
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Observation
All litters"
No. of corpora lutea per dam
Mean no. of implantation
sites per litter
Mean % post-implantation loss
per litter0
Mean % dead fetuses per
litter
Mean % resorption sites per
litter
Live littersd
Mean no. of live fetuses per
litter
Mean % male fetuses per
litter
Fetal body weight (g)
All fetuses
Male fetuses
Female fetuses
Observation
Total no. fetuses examined
(litters)
External
Visceral
Skeletal
Malformations
Diaphragmatic hernia
Multiple skeletal
malformations6
External variations
Club foot (bilateral)
Visceral variations
Dilated renal pelvis
Distended ureter
Skeletal variations
Third sternebrae,
incomplete ossification
Fifth sternebrae
incomplete ossification or
unossifiedf
Exposure concentration to 1,2,4-TMB
0 ppm
100 ppm
(492mg/m3)
300 ppm
(l,476mg/m3)
600 ppm
(2,952 mg/m3)
900 ppm
(4,428 mg/m3)
Gestational parameters
24
15.4±2.1g
14.2 ±3.3
10.0 ±22.1
0.0 ± 0.0
10.0 ±22.1
23
13.9 ±2.5
46.6 ±17.1
22
15.2 ± 1.3
13.7 ±2.9
8.6 ±8.9
0.3 ± 1.5
8.3 ±9.1
22
12.5 ±3.0
46.0 ±14.1
22
15.2 ±2.1
14.1 ±3.2
5.8 ±6.8
0.0 ±0.0
5.8 ±6.8
22
13.3 ±3.2
49.9 ± 13.4
22
15.8 ±1.7
14.9 ± 2.4
5.0 ±5.7
0.0 ±0.0
5.0 ±5.7
22
14.1 ±2.3
46.2 ± 15.4
24
15.7 ±2.5
15.0 ±2.4
5.4 ±6.7
0.0 ± 0.0
6.4 ±6.7
24
14.3 ±2.6
50.4 ± 16.2
5.71 ±0.34
5.86 ±0.34
5.57 ±0.33
5.64 ±0.31
5.79 ±0.30
5.51 ±0.31
5.56 ±0.47
5.72 ±0.49
5.40 ± 0.45
5.40 ±0.39*
5.55 ±0.48*
5.28 ±0.40*
5.60 ±0.40**
5.20 ±0.42**
4.92 ±0.40**
Exposure concentrations to 1,2,4-TMB
0 ppm
100 ppm
(492mg/m3)
300 ppm
(l,476mg/m3)
600 ppm
(2,952 mg/m3)
900 ppm
(4,428 mg/m3)
Fetal variations and malformations
319 (23)
160 (23)
159 (23)
275 (22)
137 (22)
138 (22)
293 (22)
147 (22)
146 (22)
310 (22)
155 (22)
155 (22)
342 (24)
171 (24)
171 (24)
0
0
0
0
KD
0
0
KD
KD
0
3(3)
0
0
0
0
3(3)
7(4)
3(3)
5(3)
3(3)
8(5)
3(3)
8(5)
3(2)
2(2)
0
KD
KD
0
0
4(4)
0
5(4)
0
6(6)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Extra ossification site
Cervical rib, rudimentary
Fourteenth rib,
supernumerary
Thirteenth rib, short
(unilateral)
Thoracic vertebral centra,
incomplete ossification
Health Effect at LOAEL
Maternal toxicity: decrease in
maternal body weight and
food consumption
Developmental toxicity:
significant reduction in fetal
body weight
0
KD
25 (10)
KD
8(6)
KD
2(2)
13(8)
0
4(4)
0
0
18 (12)
0
7(4)
NOAEL
Maternal toxicity: 300 ppm (1,476 mg/m3)
for 1,3,5-TMB and 1,2,4-TMB
Fetal toxicity: 300 ppm (1,476 mg/m3) for
1,2,4- and 1,3,5-TMB
0
3(2)
21 (10)
0
6(6)
0
2(2)
34 (16)
0
7(5)
LOAEL
Maternal toxicity: 600 ppm (2,952 mg/m3)
for 1,3,5-TMB and 1,2,4-TMB
Fetal toxicity: 600 ppm (2,952 mg/m3) for
1,2,4- and 1,3,5-TMB
Comments: This study observed alterations in a number of maternal and fetal parameters, including decreased
maternal and fetal weight. Values reported by authors can be used to determine NOAEL and LOAEL There was no
investigation of pre-implantation developmental toxicity due to 1,2,4-TMB or 1,3,5-TMB exposure. 1,2,3-TMB maternal
or developmental toxicity not investigated.
Body weight gain during GD 6-21 minus gravid uterine weight.
blncludes all animals pregnant at euthanization.
cResorptions plus dead fetuses.
Includes all animals with live fetuses at euthanization.
eRunt showing skeletal alterations including missing ribs, missing thoracic vertebrae, incomplete ossification of
sternebrae and skull bones.
fUnossified = alizarine red S negative.
gMean ±SD.
*, ** Statistically significant from controls at p < 0.05 and 0.01, respectively.
This document is a draft for review purposes only and does not constitute Agency policy.
B-114 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Table B-38. Characteristics and quantitative results for Tomas et al.
f!999al
Study design
Species Sex N Exposure rout
WAG/Rij M 6 rats per Oral (gavage, i
Rats dose oil)
Additional study details
• 1,2,3-, 1,2,4-, and 1,3,5-TMB were testec
before and after oral administration (in c
• Solvent concentration in peripheral bloc
• All three TMB isomers were found to cai
450 oil TOLUENE
i
£300- 0.002 mol/kg J 0.0
i*. fc ft T . \_
s, s, s2 s, s, s, sz s3 s0 s,
HEMIMELLITENE
?300-
o; 0L
E 150 _
450-i PSEUDOCUMENE
1"300 - T
I150' 1 . * fl i '
i^n if]
dsn— i
MESITYLENE
!"300-
-i 150 - ^ • * *
ir1! 1 •
0 «i rn 1 III gi r=n
e Dose range
n olive 0, 2, 8, or 32 mmol/kg BW
(240, 960, 3,840 mg/kg
BW). 1,2,3-, 1,2,4-, and
1,3,5-TMB
Exposure duration
Acute
for their effects on electrocortical arousal by an electrocardiogram
)live oil) of 0, 0.002, 0.008, or 0.032 mol/kg BW of each isomer.
id was determined via head space gas chromatography.
ise a slight increase in locomotor activity.
Figu
ofh
)8 mol/kg 0.032 mol/kg folk
T and
. . B.i. dos
* * W * * * i
m mol
Sc c c c c
2 °3 °0 °1 °2 °3
Sou
al.(
m '* • •
* * m ^* *
,. irtn
s .
* * 1 • * *
re 1. Changes in total duration
igh-voltage spindle episodes
iwing acute exposure to toluene
1,2,3-, 1,2,4-, or 1,3,5-TMB at
2S of 0.002, 0.008, and 0.032
/kg.
rce: Reproduced from Tomas et
1999a)
S0 - preinjection - ^ * . p
-------
Toxicological Review ofTrimethylbenzene
40 -1 TOLUENE
^ T T 0.002 mol/kg
^2°~ i^nn i *
till
T 0.008 mol/kg 0.032 mol/kg
i 1
1; * ; 1; ; ;
s, s, s2 s3 s, s, s2 s3 s0 s, s2 s3 s, s, s2 s3
40 -i
m HEMIMELLITENE
f 20 -
1 W* *1 I; 1 ; |i; ;
So c c
» S1 S2 °3
PSEUDOCUM
40-
U)
I20' 1 ;n
"I f " T
o HOU
S, S, 8, S3
MESITYLENE
i ": idB
S0 - preinjection - ^
S, - 20 min postinjection - 1 \
S2 - 40 min postinjection - 1 i
S3- 60 min postinjection - 1 I
Health Effect at LOAEL
Abnormal electrocortical
stimulation
s, s, s2 s3 s0 s, s2 s3
ENE
I ]
* * I.
Figure 2. Changes in number
of high-voltage spindle
episodes following acute
exposure to toluene and
123- 124- and 1 3 5-TMB
0.032 mol/kg.
Source: Reproduced from
Tomasetal. (1999a)
s, s, s2 s3 s, s, s2 s3
\ m
i . • i i
li*l iL* *
s, s, s2 s3 s, s, s2 s3
* - p<0.001 compare to oil group
•- p<0.001 compare to control
measurement
NOAEL
n/a
LOAEL
2 mmol/kg 1,2,3-TMB, 1,2,4-TMB, and
1,3,5-TMB
Comments: Exposures were of an acute duration, and therefore not suitable for reference value derivation. However,
qualitatively, this study provided evidence of CNS disturbances that, when considered together with short-term and
subchronic neurotoxicity studies, demonstrate that TMB isomers perturb the CNS of exposed animals.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-39. Characteristics and quantitative results for Tomas et al.
fl999b_l
Study design
Species
WAG/Rij rats
Sex
M
N
10 rats per
dose
Exposure route
Oral (in olive oil)
Dose range
0, 8, 16, or 32 mmol/kg
BW (960, 1,920, or 3,850
mg/kg BW) 1,2,4-TMB,
1,2,3-TMB, or 1,3,5-TMB
Exposure
duration
Acute
Additional study details
• 1,2,3-, 1,2,4-, and 1,3,5-TMB were tested for their effects on locomotor activity by an open field test
following oral administration (in olive oil) of 0, 8,16, or 32 mmol/kg BW of all isomers.
• All three TMB isomers were found to cause a slight increase in locomotor activity.
1000
s-
10
O.OO8 mol/kg
- p<0.0001 compare to time point 1, 2
injection
I I I I I I I ,J
234
Time points
0.015 mol/kg
* - p
-------
Toxicological Review ofTrimethylbenzene
Table B-40. Characteristics and quantitative results for To mas et al.
f!999cl
Study design
Species
Wistar rats
Sex
M
N
4 rats per
dose
Exposure route
i.p. injection
Dose range
6.6 mmol/kg BW 1,2,3-,
1,2,4-, and 1,3,5-TMB
Exposure duration
Acute
Additional study details
• 1,2,3-, 1,2,4-, and 1,3,5-TMB were tested for their effects on the CNS by monitoring evoked hippocampal and
cortical activity following i.p. injection of 6.6 mmol/kg BW of any isomer.
• Solvent concentration in peripheral blood was determined via head space gas chromatography.
• Significant differences in hippocampal and cortical activity occurred following injection.
TOLUENE
MES1TYLENE
PSEUDOCUMENE
HEMIMELLITENE
Figure 1. Amplitude
abnormalities of the
cortical Nl wave 30
and 60 min after i.p.
solvent injection.
Source: Reproduced
from Tomas et al.
(1999c)
TOLUENE
MESITYLENE
PSEUDOCUMENE
HEMIMELLITENE
Figure 2. Amplitude
abnormalities of the
cortical P1-N1 wave
30 and 60 minutes
after i.p. solvent
injection.
Source: Reproduced
(Tomas etal. 1999)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
TOLUENE
MESITYLENE
PSEUDOCUMENE
HEMIMELLITENE
Figure 3. Amplitude
abnormalities of the
hippocampal Nl wave 30
and 60 min after i.p.
solvent injection.
Source: Reproduced from
Tomasetal. (1999c)
0
5
10
15
20
25
30
35
40
45
50
TOLUENE
MESITYLENE
PSEUDOCUMENE
HEMIMELLITENE
T
T
Q 30 min
n 60 min
T
Figure 4. The effect of
i.p. solvent injection on
the cortical EEG in the
13-20.75 Hz frequency
band.
Source: Reproduced
from Tomas et al.
(1999c)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
TOLUENE
MESITYLENE
PSEUDOCUMENE HEMIMELLITENE
60 -
50 -
40 -
30 -
20 -
10 -
% 0
E*JA _-
30 n
^60n
T
1
-_-_-_-_-_-
im
im
T
T
I
_-_-_-_-_-_
\
1
"_-_-_-_-_-
T
—
-----------
—
-----------
---"„----
—
------------
—
_-_-_-_-_-_
T
Figure 5. The effect of
i.p. solvent injection on
the hippocampal EEG in
the 1-3.75 Hz
frequency band.
Source: Reproduced
from Tomas et al.
(1999c)
TOLUENE
MESITYLENE
PSEUDOCUMENE HEMIMELLITENE
u
10-
20-
1A
30
40-
CA _
wv
fin-"
-:-:-:-:-:-:
-----------
::::::::::::
::::::::::::
„"„"„"„"„"„"
-:-:-:-:-:-:
L----------I
-----------
-----„-„-„-
i
J_
Q"i(\
Q60
i
mm
min
1
:-:-:-:-:-:
„---„-„-„-„
::::::::::::
:::::::::::
~_~_~_~_~_~
:-:-:-:-:-:•
.---------.-
_-_-_-_-_-_•
1
1
i
1
_i_
i
-:-:-:-:-:-:
-„-----„---
-I-I-I-I-I-!
-I-I-I-I-I-!
-:-:-:-:-:-:
-----------
I
1
1
1
_L
i
:-:-:-:-:-:
„---„-„-„-„
:::::::::::
:::::::::::
:-:-:-:-:-;
:-:-:-:-:-:
-----------
-:-:-:-:-:-
„-„-„-„-„-„
-:-:-:-:-:-
,
1
1
i
Figure 6. The effect of
i.p. solvent injection on
the hippocampal EEG in
the 7-9.75 Hz
frequency band.
Source: Reproduced
from Tomas et al.
(1999c)
Health Effect at LOAEL
n/a (acute exposure study, one
dose level)
NOAEL
n/a
LOAEL
6.6 mmol/kg 1,2,3-TMB, 1,2,4-TMB,
and 1,3,5-TMB
Comments: Unable to quantify dose-response relationship from data because only one dose group used. Exposures
were of an acute duration, and therefore not suitable for reference value derivation. However, qualitatively, this study
provided evidence of CNS disturbances that, when considered together with short-term and subchronic neurotoxicity
studies, demonstrate that TMB isomers perturb the CNS of exposed animals.
This document is a draft for review purposes only and does not constitute Agency policy.
B-120 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofTrimethylbenzene
Table B-41. Characteristics and quantitative results for Wiaderna et al.
f!9981
Study design
Species
Sex
N
Exposure route
Dose range
Exposure duration
Wistar rats
M
13 or 14
rats/ dose
Inhalation (6 h/d, 5
d/wk)
0 or 25,100, or 250 ppm
(0, 123, 492, or 1,230
mg/m3) 1,2,3-TMB
4 wks
Additional study details
• Animals were exposed to 1,2,3-TMB in 1.3 m3 dynamic inhalation exposure chambers for 6 hrs/d, 5 d/wk for 4
wks. Food and water was provided ad libitum.
• Animals were randomized and assigned to the experimental groups.
• Rats were tested with a variety of behavioral tests, including radial maze performance, open field activity,
passive avoidance, and active two-way avoidance.
• Tests were performed on days 14-18 following exposure.
• Neurobehavioral effects were observed at 25 and 100 ppm (123 and 492 mg/m3) concentrations, but not at
250 ppm (1,230 mg/m3).
300
250
200
o
"is 15°
•o
100
50
0
3.0
2.5
2.0
o
S> 1,5
1 .0
0.5
O.O
1
day 2
day 3
day 4
day
HMO
HM25 HM100 HM250
Figure 1. Radial maze performance of rats
exposed for 4 weeks to 1,2,3-TMB. The test
(one trial a day) was performed on days 14-18
after exposure. Upper diagram: changes in
trial duration, i.e., the time of successive eight
arm entries, during successive days of training.
Lower diagram: number of perseveration
errors in successive daily trials.
Denotation of groups: HMO-sham exposed
group (n = 13), HM25, HM100, HM250-groups
exposed to 1,2,3-TMB at concentrations of 25
ppm (123 mg/m3, n = 13), 100 ppm (492
mg/m3, n = 14), and 250 ppm (1,230 mg/m3, n
= 13) respectively. Bars represent group
means and standard error.
* p < 0.05 compared to trial 1 in the same
group.
HMO
HM25
HM100 HM250
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Toxicological Review ofTrimethylbenzene
100 r
Figure 2. A comparison of spontaneous locomotor
(upper diagram), exploratory (middle diagram), and
grooming (lower diagram) activity of rats in an open
field during a 5-min observation period. The test was
performed 25 days after a 4-week exposure to 1,2,3-
TMB. Denotation of groups as in Figure 1 (above).
The bars represent group means and SE.
group HMO
group HM25
group HM100
o
c
V
c
s
o
TJ
I
Cl-
140
120
100
80
60
40
20
trial
trial
trial
trial
trial
(shock)
- trial 6
Figure 3. Diagrams illustrating the effect of a
4-week exposure to 1,2,3-TMB on the step-
down passive avoidance learning in rats. The
test was performed on days 39-48 after
exposure. Trials 1, 2, and 3 were performed
at 24 hr intervals. The step-down response
was punished by a 10 sec footshock in trial 3
only. Trials 4, 5, and 6 were performed 24 hr,
3 d, and 7 d after trial 3, respectively. The
maximum step-down latency was 180 sec.
Denotations of groups as in Figure 1 (above).
The bars represent group means and SE
*, *** p < 0.05 and p < 0.001, compared with
respective data from control group.
HMO
HM25
HM100
HM250
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Toxicological Review ofTrimethylbenzene
L2/L1
£52 L3/LI
Figure 4. Hot-plate behavior tested in rats on day 50 (trials
1 and 2) and day 51 (trial 3) after a 4-week exposure to
1,2,3-TMB. Denotation of groups as in Figure 1 (above).
The bars represent group means and SE. Upper diagram: A
comparison of the latency of the paw-lick response to a
thermal stimulus (54.5°C) on day 50. Ll-paw-lick latency in
trial 1 performed before a 2 min intermittent footshock.
L2-paw-lick latency in trial 2 performed several seconds
after the footshock. L3-paw-lick latency in trial 3 performed
24 hr after the footshock
* p < 0.05 compared to L2/L1 of the same group.
50
40
3O
30
10
50
•C 40
B
I 30
3 20
n
-2 10
O
1OO
s
o
3 80
BO
4O
2O
O
Training
m
m
m
m
HUO H1IE6 HHinn HMS50
Retraining
Figure 5. Active avoidance learning and retention in rats after a 4-week
exposure to 1,2,3-TMB. Upper and middle diagrams: comparisons of the
number of trials to attain an avoidance criterion (four avoidance
responses during five successive trials) during the training (upper diagram
and retraining (middle diagram) session). Lower diagram: a retention
score calculated according to the formula: %Ret = (1 - Resc/Tesc) x 100,
where Resc and Tesc are numbers of escape responses during retraining
and training, respectively. Denotation of groups as in Figure 1 (above).
The bars represent group means and SE.
* p < 0.05 compared to control group.
1IHO HUES H1I100
Retention
1
Health Effect at LOAEL
NOAEL
LOAEL
Impaired learning of passive
avoidance
n/a
25 ppm (123 mg/m3
Comments: CNS disturbances were observed up to 2 months after termination of exposure, indicating the persistence
of effects after metabolic clearance of 1,2,3-TMB from the test animals. No effects were observed in the 250 ppm
(1,230 mg/m3) exposure group. Duration of exposure only 4 weeks. Generally, short-term exposure studies have
limited utility in quantitation of human health reference values.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-42. Characteristics and quantitative results for Wiaderna et al.
f20Q21
Study design
Species
LOD: Wistar
rats
Sex
MM
N
12 rats
per dose
Exposure route
Inhalation (6 hr/d, 5
d/wk)
Dose range
0 or 25, 100, or 250 ppm
(0, 123, 492, or 1,230
mg/m3) 1,2,3-TMB
Exposure duration
4 wks
Additional study details
• Animals were exposed to 1,3,5-TMB in 1.3 m3 dynamic inhalation exposure chambers for 6 hrs/d, 5 d/wk for 4
wks. Food and water was provided ad libitum.
• Animals were randomized and assigned to the experimental groups.
• Rats were tested with a variety of behavioral tests, including radial maze performance, open field activity,
passive avoidance, active two-way avoidance, and shock-induced changes in pain sensitivity.
• 1,3,5-TMB-exposed rats showed alterations in performance in spontaneous locomotor activity, active and
passive avoidance learning, and paw-lick latencies.
* p< 0.001 compared to MESO
n trial 1
n trial 2
• try 3
Q trial 4
n trial 5
• trial 6
Figure 1. Passive avoidance. The
comparison of the time of staying
on the platform in the consecutive
test trials. The test was performed
between days 35 and 45 after the
exposure to 1,3,5-TMB. Leaving the
platform in trial 3 was punished by
an electric shock. Trials 1, 2, 3, and
4 were performed at 24 hr
intervals, while trials 5 and 6 were
effected 3 and 7 days after trial 3,
respectively.
The bars represent group means
andSE.
MESO
MES25
MES10Q
MES250
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Figure 2. Hot plate. The comparison
60i
50-
S40
&
i
aiSO
1?
|20-
10
Q
* p < 0.02 compared to MESO and MES25 in the same trial of latency of the reaction (paw-lick)
to the thermal stimulus before (LI),
immediately after (L2) and 24 hr
after (L3) intermittent 2 min
T
1
_L
i
i
electric shock in rats exposed to
I-J-! 1,3,5-TMB. The test was performed
on days 50 and 51 after the
r nL1 exposure.
DL2 The bars represent group means
~\ andSE
MESO MES25 MES100 MES250
35
in
ou
1 25
b
Q nn
"*~^ £U
i
B 15
o>
.D
1 10
3 IVJ
5
Q
nnn ._„ Figures. Active avoidance. The
*p< 0.02 compared to MESO . ,tu
^ comparison of the rat groups
* * T exposed to 1,3,5-TMB for the
1 number of trials (attempts)
1
I
required to reach the avoidance
criterion (4 shock avoidances) in 5
consecutive trials (attempts) during
the training session. The test was
performed on day 54 (training) and
day 60 (retraining) after the
exposure.
The bars represent group means
andSE.
MESO MES25 MES100 MES250
Health Effect at LOAEL NOAEL LOAEL
Shorter retention of passive . „ .„__ . 3.
. . _. H n/a 25 ppm 123 mg/m
avoidance reaction
Comments: This study observed alterations in a number of behavioral tests. Values reported by authors can be used
to determine LOAEL and NOAEL. CNS disturbances observed up to 2 months after termination of exposure, indicating
the persistence of effects following metabolic clearance of 1,3,5-TMB from the test animals.Unable to quantify dose-
response relationship from data because responses either equal at all exposure concentrations or elevated only at one
exposure concentration. Duration of exposure only 4 weeks. Generally, short-term exposure studies have limited utility
in quantitation of human health reference values.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-43. Characteristics and quantitative results for Wiglusz et al.
fl975al
Study design
Species Sex
Wistar rats M
N
5-8 per
dose
Exposure route Dose range Exposure duration
Inhalation 0, 1.5, 3.0, or 6.0 mg/L (0, Acute study: 6 hrs
1,500, 3,000, or 6,000 Short-term study: 6 hrs/d, 6
mg/m3) 1,3,5-TMB d/wk for 5 wks
Additional study details
• Male Wistar rats were exposed in a short-term study to 0, 1.5, 3.0, or 6.0 mg/L 1,3,5-TMB.
• In a separate chronic study, male Wistar rats were exposed to 3.0 mg/L 1,3,5-TMB for 6 hrs/d, 6 d/wk, for 5
wks.
• Rats weighed 240-280 g and were housed in stainless steel wire mesh cages, with food and water provided
ad libitum.
• Blood samples were collected for 3 days before exposure then on days 1, 7, 14, and 28.
Observation
Day 0
Day 1
Day 7
Day 14
Day 28
Day 0
Day 1
Day 7
Day 14
Day 28
Day 0
Day 1
Day 7
Day 14
Day 28
1,3,5-TMB exposure concentration (mg/L)— hematological parameters
following single 6 hour exposure
0
1.5
3.0
6.0
Hemoglobin in g% (mean ± SD)
14.1 ±1.3
-
-
15.1 ±0.8
14.8 ± 0.5
15.2 ±0.3
-
14.0 ± 0.5
14.6 ±0.5
14.9 ±0.7
15.0 ±0.8
14.8 ±1.0
13.5 ±0.5
13.6 ±0.6
13.6 ±0.8
14.2 ±1.1
13.9 ±2.1
13.5 ±0.8
13.1 ±0.4
14.8 ±0.4
Million erythrocytes per mm3 serum (mean ± SD)
4.91 ±0.19
-
-
5.37 ±0.90
5.17 ±0.18
5.35 ±0.09
-
5. 18 ±0.18
4.99 ±0.11
5.26 ±0.07
4.96 ±0.15
5.32 ±0.02
4.93 ±0.16
5.09 ±0.10
5.12 ±0.10
5.51 ±0.17
5.31 ±0.11
4.89 ±0.17
4.77 ±0.10
5.20 ±0.27
Thousand leukocytes per mm3 serum (mean ±SD)
11.08 ±3.14
-
-
8.0 ±2.16
6.83 ± 1.27
12.26 ±3.50
-
11.70 ±2.97
12.06 ± 3.33
11.50 ± 10.48
13.01 ±3. 10
11.38 ±1.37
11.66 ±1.50
11.70 ±1.05
11.96 ±1.16
8.90 ± 3.88
8.24 ±3.88
12.32 ±5.01
10.68 ±1.21
9.92 ±2.42
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Day 0
Day 1
Day 7
Day 14
Day 28
Day 0
Day 1
Day 7
Day 14
Day 28
Day 0
Day 1
Day 7
Day 14
Day 28
Day 0
Day 1
Day 7
Day 14
Day 28
Day 0
Day 1
Day 7
Day 14
Day 28
Percent segmented neutrophilic granulocytes (mean ± SD)
8.5 ±4.1
--
--
10.6 ± 2.5
15.6 ±6.3
13.5 ±3.6
--
20.2 ± 6.04
12.2 ±5.9
12.5 ±6.4
18.5 ±2.3
22.5 ±5.4
31.3 ± 10.3
30.1 ±6.2
35.0 ±6.7
16.6 ±2.8
53.6 ±22.5
26.7 ±12.5
20.6 ±23.7
15.8 ±3.8
Percent bacciliform neutrophilic granulocytes (range)
0.6 (0-1)
-
-
0.0
0.0
0.0
--
0.0
0.16 (0-1)
1 (0-2)
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Percent acidophilic granulocytes (mean ± SD)
1.1 ±0.7
-
-
2.8 ±1.3
4.1 ±2.9
2.6 ±1.9
-
1.1 ±1.1
5.1 ±3.2
3.1 ±1.7
0.5 ±0.5
0.0
3.1 ±0.5
4.8 ±1.0
6.0 ±4.1
1.8 ±1.7
0.14 ±0.3
0.0
2.6 ±2.6
2.2 ±2.8
Percent lymphocyte (mean ± SD)
88.6 ± 4.4
-
-
85.4 ± 1.5
78.6 ±8.3
82.8 ±4.13
-
77.6 ±4.8
82.0 ±3.8
81.8 ±7.6
67.8 ±2.3
73.3 ±5.4
65.0 ±7.9
64.3 ±5.8
57.1 ±4.1
79.4 ±4.3
44.0 ±21.3
71.2 ±12.5
75.0 ±23.0
81.2 ±5.8
Percent monocyte (mean ± SD)
1.6 ±0.8
-
-
0.5 ±0.4
1.6 ± 1.0
1.0 ±0.6
-
0.8 ±1.1
0.6 ±0.5
1.6 ± 1.0
1.1 ±0.9
1.1 ±0.4
0.3 ±0.5
0.3 ±0.8
1.6 ±1.2
2.2 ± 1.0
2.3 ±1.8
1.7 ± 1.9
1.2 ±0.4
1.0 ±0.8
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
'It of neuotrophilic granulocytes .
QK. <^j rw
3 o s
% of flcutrophiltc granulocytes
\* SV S S
- N
•fci
*— ""
•
— — ^— control
mesltylene
—
"~~~---, w , _.. — -' ~
Figure 1. Percentage
of segmented
neutrophilic
• f.Omgli granu|ocytes after 6
,3.0mgli hrs exposure to
'.6.0mgft 1/3>TMB.
— -*
-* — — «
<3 2 7 /•# i/ay^ after exposure ZQ '
— — contra
mestty.
—~- — • '
x
X
§ i OLl i I i iiiii _i_ri i £ i lit
I
lene 3.0mg/t
i 1 I B
Figure 2.
Percentage of
segmented
neutrophilic
granulocytes
during
exposure to
1,3,5-TMB 3.0
mg/L for 6
hrs/d, 6 d/wk,
for 5 wks.
d * 7 < ** days of exposure 2B 1
Observation
Control group
1,3,5-TMB group
Control group
1,3,5-TMB group
Control group
1,3,5-TMB group
Control group
1,3,5-TMB group
Hematological parameters during 5 week exposure to 1,3,5-TMB (means ± SD)
Day 0 Day 1 Day 7
Hemoglobin in g%
13.0 ±4.7 14.6 ±2.5 14.6 ± 2.5
14.6 ±0.7 15.5 ±0.6 14.8 ±1.1
Day 14
15.6 ±3.2
14.5 ±0.9
Day 28
14.2 ± 5.0
13.8 ±0.5
Million erythrocytes per mmBSerum
5.42 ±0.78 6.12 ±04 6.40 ±0.25
6.08 ±1.18 6.35±0.38 6.11 ±0.63
6.46 ±0.39
5.74 ±1.1
6.18 ±0.61
5.05 ±2.2
Thousand leukocytes per mm3 Serum
10.63 ±4.27 13.66 ±2.91 11.13 ±2.52
13.76 ±3.70 11.43 ±4.0 9.53 ± 2.55
14.53 ± 2.64
12.23 ± 4.04
11.46 ± 2.74
13.40 ±5. 18
% Segmented neutrophilic Granulocytes
17.1 ±11.9 14.5 ±8.1 12.1 ±2.5
14.0 ± 5.0 17.0 ± 9.4 16.6 ± 5.0
13.6 ±6.3
21.5 ±7.4
15.6 ±3.2
18.4 ± 8.6
% Bacciliform neutrophilic granulocytes
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Control group
1,3,5-TMB group
Control group
1,3,5-TMB group
Control group
1,3,5-TMB group
Control group
1,3,5-TMB group
0.83 (1-2)
0.6
(1-2)
0.66 (1-2)
0.4 (0-1)
1.33 (1-3)
1 (1-2)
1.33 (1-2)
1.8 (2-5)
1.0 (0-1)
1.4 (1-2)
% Acidophilic granulocytes
1(1-4)
1.5
(1-3)
2.1(1-4)
1.0 (1-3)
3.3 (1-7)
0.8 (1-2)
1.8 (1-4)
1.0 (1-2)
1.6 (1-4)
0.8 (0-1)
% Lymphocyte
79.6 ± 11.7
79.8 ±5.5
81.6 ±8.6
81.0 ±7.7
81.8 ±4.7
80.5 ±6.5
81.1 ±5.2
74.0 ±9.4
80.0 ± 2.4
77.2 ±8.4
% Monocyte
1.1
0.6
Health Effect at LOAEL
Increase in percent segmented
neutrophilic granulocytes
(1-3)
(1-3)
1.0 (0-2)
0.8 (1-2)
1.5 (1-4)
0.8 (1-2)
NOAEL
1.5 mg/L
1.0 (1-2)
1.3 (1-3)
1.5 (1-3)
2.7 (2-4)
LOAEL
3.0 mg/L
Comments: This study slight increases in percent segmented neutrophilic granulocytes on day 14 of the short-term
exposure study. Authors do not report statistical significance of results. Only one dose group used in chronic study.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-44. Characteristics and quantitative results for Wiglusz et al.
fl975bl
Study design
Species Sex
Wistar rats M
N
6/dose
Exposure route Dose range Exposure duration
Inhalation 0, 0.3, 1.5, or 3.0 mg/L (0, Acute study: 6 hrs
300, 1,500, or 3,000 Short-erm study: 6 hrs/d, 6
mg/m3+) 1,3,5-TMB d/wk for 5 wks
Additional study details
• Male Wistar rats were exposed in a short-term study to 0, 0.3, 1.5, or 3.0 mg/L 1,3,5-TMB.
• In a separate chronic study, male Wistar rats were exposed to 3.0 mg/L 1,3,5-TMB for 6 hrs/d, 6 d/wk, for 5
wks.
• Rats weighed 240-280 g and were housed in stainless steel wire mesh cages, with food and water provided
ad libitum.
• Blood samples were collected for 3 days before exposure then on days 1, 7, 14, and 28.
Observation
Day 0
Day 2
Day 7
Day 14
Day 28
Day 0
Day 2
Day 7
Day 14
Day 28
Day 0
Day 2
Day 7
Day 14
Day 28
1,3,5-TMB exposure concentration (mg/L)— hematological parameters
following single 6 hour exposure (means ± SE)
0
0.3
1.5
3.0
Aspartate amino transferase activity
79.0 ±7.9
81.8 ±6.2
82.2 ±4.3
82.6 ±8.5
79.6 ±7.6
78.0 ±7.7
90.0 ±5.7
76.8 ±4.2
73.0 ±4.2
72.6 ±7.2
75.3±7.3
71.8±3.3
71.2±2.2
76.3±6.7
84.2±7.9
81.6 ±4.2
74.6 ±4.5
84.1 ±5.6
76.1 ±3.9
79.5 ± 10.6
Alanine amino transferase activity
34.0 ± 4.5
34.0 ± 4.6
31.0 ±3.1
32.0 ±3.2
34.0 ± 3.8
35.6 ±4.1
308 ± 2.7
37.5 ±5.6
31.4 ±2.5
31.3 ±5.2
32.6 ±4.5
30.6 ± 8.3
29.3 ±4.5
34.6 ±5.3
30.4 ± 9.4
29.1 ±3.6
26.5 ±1.2
39.5 ±3.0
36.3 ±1.7
39.3 ±2.7
Alkaline phosphatase activity
28.6 ±9.6
27.8 ±5.1
31.8 ±5.8
27.0 ±4.7
30.5 ±3.2
30.9 ±3.3
26.0 ±7.2
28.1 ±5.9
33.6 ±2.4
28.0 ±6.9
27.4 ±6.4
29.7 ±2.6
32.8 ±1.8
28.9 ±5.2
23.0 ±4.7
37.3 ±5.6
30.5 ±6.5
58.7 ±8.9*
42.1 ±2.9
-
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
«3
.;; ta>
. control
mesitt/len'e 0.3
mesitylene 1.5
mesitylene 3.0 mg\l
Figure 1. Serum
activity of
aspartate amino
transferase after
6 hrs exposure
to 1,3,5-TMB;
values are
expressed in %
of initial values.
o-
a 2
I :
H
(fays after exposure
— control
— —- mesitylcne 0.3 mgfl
mesltyleae, 1.5 my/£
mesitylene 3.0 mgfi
§
{20
90
t-f efay? after exposure
Figure 2.
Serum activity
of alanine
amino
transferase
after 6 hrs
exposure to
1,3,5-TMB;
values are
expressed in
% of initial
values.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
S
{20
-SO
o'
control
mesitt/lene 0,3 mg/l
mesitylene l.o mg/l
mesitylene J.o mgjl
f\gure3. Serum
activity of alkaline
phosphatase after
6 hrs exposure to
1,3,5-TMB; values
are expressed in %
of initial values.
0 2
14 days after exposure 2&
1
Observation
Hematological parameters during 5 week exposure to 1,3,5-TMB (means ± SD)
DayO
Dayl
Day 3
Day?
Day 14
Day 28
Aspartate amino transferase activity
Control group
89.5 ±2.3
74.5 ±6.9
79.6 ±10.5 83.2 ±10.6
83.5 ±7.3
82.2 ±6.3
1,3,5-TMB group
72.0 ±5.1
70.8 ±5.2
81.3 ±9.1
80.0 ±6.3
93.4 ± 1.4*
79.6 ±9.4
Alanine amino transferase activity
Control group
34.0 ±4.1
33.8 ±5.0
35.6 ±2.6
30.5 ±4.9
30.0 ±4.5
35.6 ±4.6
1,3,5-TMB group
34.8 ±3.6
28.0 ±6.32
3.33 ±3.8
35.1 ±3.9
36.4 ±4.0
36.5 ±5.0
Ornithite carbamyl transferase activity
Control group
2.7 ±0.2
2.6 ±0.2
3.1 ±0.2
2.8 ±0.1
2.6 ±0.3
3.6 ±0.3
1,3,5-TMB group
2.6 ±0.4
2.5 ±0.6
3.8 ±0.4
3.5 ±0.2
2.6 ±0.2
3.7 ±0.4
Alkaline phosphatase activity
Control group
27.8 ±4.0
28.8 ±3.8
28.5 ±6.8
26.5 ±3.9
27.2 ±8.8
25.8 ±3.0
1,3,5-TMB group
32.4 ±1.8
23.6 ±3.6
22.2 ±3.6
30.2 ±6.9
25.6 ±5.9
32.6 ±4.8
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
co/zfrol
3.O myft
Figure 4. Serum
activity of
aspartate amino
transferase during
exposure to 1,3,5-
TMB at 3.0 mg/L
for 6 hrs/d, 6 d/wk,
for 5 wks; values
are expressed in %
of initial values.
ofay* of exposure
ltd
ts
;C
^
. 60
,'.-'•' •— control
/ ••*
rn e$itt/(ene 3.0
Figure 5. Serum
activity of alanine
amino transferase
during exposure to
l,3,5-TMBat3.0
mg/L for 6 hrs/d, 6
d per wk, for 5 wks;
values are
expressed in % of
initial values.
14 cfat/s of exposure
toe
control
Figure6. Serum
activity of alkaline
phosphatase during
exposure to 1,3,5-TMB
at 3.0 mg/L for 6 hrs/d,
6 d/wk, for 5 wks;
values are expressed in
% of initial values.
-M-U-
0 / 3
e/ays of
Health Effect at LOAEL
NOAEL
LOAEL
Increase in alkaline phosphatase
activity
1.5 mg/L
3.0 mg/L
dLUVIiy
Comments: This study observed increases in alkaline phosphatase activity on day 7 of the short-term exposure study
Only one dose group used in chronic study. Data not recorded daily; significant gaps exist between sampling days.
*Statistically significant in relation to initial values (p < 0.05).
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
B.6. HUMAN TOXICOKINETIC STUDIES
Table B-45. Characteristics and quantitative results for Jarnberg et al.
f!9961
Study design
Species
Caucasian
humans
Sex N Exposure route Dose range Exposure duration
2 ppm and 25 ppm (~10 _ , , ,,
9 per . , , . .-.,., / 3>.. -. -. 2 hrs exposure, followed by 4 hrs
M . K Inhalation and 123 mg/m 1,2,3-, ,_
dose observation
1,2,4-, or 1,3,5-TMB
Additional study details
• Caucasian males were exposed tc
1,3,5-TMB in an inhalation chaml
• Study subjects were asked to per
generating 50 W power during 2
• 1,2,3-, 1,2,4-, and 1,3,5-TMB con
chromatography.
• No significant irritation or CNS ef
• Results imply extensive depositio
• Exhalation accounted for 20-37//
for <0.002%.
• The study was approved by the R
) 2 ppm (~10 mg/m3 1,2,4-TMB and 25 ppm (123 mg/m3) ]
jerfor 2 hrs.
Form light cycling to simulate a work environment, with pa
hr exposure.
:entrations in exhaled air, blood, and urine were determine
:ects were observed.
n in adipose tissue.
> of absorbed amount while urinary excretion of unchangec
egional Ethical Committee at the Karolinska Institute
L,2,3-, 1,2,4-, or
rticipants
;d via gas
i TMBs accounted
Respiratory uptake and urinary excretion of TMB isomers following 2 hour inhalation exposure (mean ± 95%CI)
Exposure
Respiratory uptake (%)a
Net respiratory
uptake (%)b
Respiratory uptake (mmol)a
Net respiratory
uptake (mmol)
Respiratory excretion (%)c
Net respiratory
excretion (%)d
Urinary excretion (%)e
25 ppm (123
mg/m3)l,2,3-
TMB
56 ±4
48 ±3
1.4 ±0.1
1.2 ±0.1
37 ±9
28 ±8
0.0023 ± 0.0008
25 ppm (123
mg/m3)l,3,5-
TMB
62 ±3
55 ±2
1.6 ±0.1
1.4 ±0.1
25 ±6
16 ±4
0.0016 ± 0.0015
25 ppm (123
mg/m3) 1,2,4-
TMB
64 ±3
60 ±3
1.6 ±0.1
1.5 ±0.1
20 ±3
14 ±2
0.0010 ± 0.0004
2 ppm (~10
mg/m3) 1,2,4-
TMB
63 ±2
61 ±2
0.16 ±0.01
0.15 ±0.01
15 ±5
9±4
0.0005 ± 0.0002
Kinetic values of TMB isomers following 2 hour inhalation exposure (mean ± 95%CI)
Kinetic parameter
Total calculated blood clearance
(L/hr/kg)f
Total apparent calculated blood
clearance (L/hr/kg)g
Exhalatory blood clearance (L/hr/kg)
Metabolic blood clearance (L/hr/kg)f
1st Phase half-life (min)
2nd Phase half-life (min)
3rd Phase half-life (min
4th Phase half-ife (min)
AUC (u.M x hrs)
Volume of distribution (L/kg)
Mean residence time (hrs)
25 ppm (123
mg/m3)l,2,3-
TMB
0.63 ±0.13
0.54 ±0.11
0.23 ±0.07
0.39 ±0.11
1.5 ±0.9
24 ±9
4.7 ± 1.6
78 ±22
32 ±6
30 ±6
57 ±22
25 ppm (123
mg/m3)l,3,5-
TMB
0.97 ±0.16
0.86 ±0.12
0.24 ±0.10
0.72±0.11
1.7 ±0.8
27 ±5
4.9 ± 1.4
120 ± 41
22 ±4
39 ±8
42 ±11
25 ppm (123
mg/m3) 1,2,4-
TMB
0.68 ±0.13
0.63 ±0.11
0.14 ±0.04
0.54 ±0.10
1.3 ±0.8
21 ±5
3.6 ±1.1
87 ±27
35 ±10
38 ±11
69 ±32
2 ppm (~10
mg/m3) 1,2,4-
TMB
0.87 ±0.37
0.82 ±0.32
0.14 ±0.10
0.74 ±0.29
1.4 ± 1.8
28 ±14
5.9 ±2.5
65 ±20
3.6 ±2.0
28 ±3
47 ±22
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Figure 1. Concentration of 1,2,4-TMB in capillary blood during and after 2 hr exposure to 25 ppm (123 mg/m3) 1,2,4-
TMB (mean values ± 95% Cl).
124TMB in blood
4 -
3-
2 '
1 -
0
60 120 180 240
Time (min)
300
360
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Toxicological Review ofTrimethylbenzene
Figure 2. Concentration of 1,3,5-TMB in capillary blood during and after 2 hr exposure to 25 ppm (123 mg/m3) 1,3,5-
TMB (mean values ± 95% Cl).
135TMB in blood
9 -
a -
y -
180 24O
Time (mi n)
Figure 3. Concentration of 1,2,3-TMB in capillary blood during and after 2 hr exposure to 25 ppm (123 mg/m3) 1,2,3-
TMB (mean values ± 95% Cl).
123TMB in blood
9-
8 -
7 -
6 -
5-
4 -
3 -
2 -
1
O
6O 120 180 24O
Time (min)
300
36O
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Figure 4. Concentration of 1,2,4-TMB in capillary blood from 10 subjects exposed to 2 and 25 ppm (~10 and 123
mg/m3) of 1,2,4-TMB (mean values ± 95% Cl)
124TMB in blood
G/M)
1 -
0.1;
O.OT
60
120
180
Time (min)
240
300
360
Comments: Exposure duration possibly not sufficient to detect metabolic changes. Metabolites not measured.
aPercent of dose calculated as net uptake + amount cleared by exhalation during exposure .
bPercentage of dose calculated as net uptake.
°During and post-exposure, percentage of the respiratory uptake.
dPost-exposure, percentage of net respiratory uptake.
ePost-exposure, percentage of respiratory uptake.
'Calculated from respiratory uptake.
Calculated from net respiratory uptake.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-46. Characteristics and quantitative results for Jarnberg et al.
f!997al
Study design
Species
Caucasian
Human
Sex
M
N
9
Exposure route
Inhalation
Dose range
11 mg/m3 1,2,4-TMB
Exposure duration
2hrs
Additional study details
• Nine Caucasian males were exposed to 11 mg/m31,2,4-TMB alone or 11 mg/m31,2,4-TMB as a component of
300 mg/m3 WS.
• Exposure lasted 2 hrs, during which study subjects were required to cycle producing 50 W continuously to
simulate a work environment.
• Gas chromatography was used to measure 1,2,4-TMB levels in air.
• HPLC was used to measure urinary metabolites.
• Irritation was not reported amongst subjects at these exposure levels.
• The study was approved by the Regional Ethical Committee at the Karolinska Institute and was only
performed after informed consent.
Figure 1. Mean (± SD) capillary blood concentration of 1,2,4-TMB during and after exposure to 1,2,4-TMB alone and
1,2,4-TMB as a component of WS.
1,2,4-TMB in blood (\M)
Exposure to white spirit
Exposure to 1,2,4-TMB
60 120
Time (min)
180
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Results from 2 hour exposure to 1,2,4-TMB alone or 1,2,4-TMB as a component of WS (mean ±SD)
Exposure
Net respiratory uptake (mmol)
AUC (u.M x min), 0-3 hr
Half-life of 3,4-DMHA (hr)
Excretion of 3,4-DMHA (%d), Q-6 hr
1,2,4-TMB alone
0.15 ±0.01
53 ±4
3.7±0.4b
11 ±2
1,2,4-TMB in WS
0.14 ±0.02
86 ±9
3.0 ±0.7
18 ±3
p-value
0.5a
<0.0001a
0.2°
0.007°
Figure 2. Urinary excretion rate of 3,4-dimethylhippuric acid against the midpoint time of urine collection in 9 male
volunteers exposed to 11 mg/m3of 1,2,4-TMB, either alone or as a component of WS (mean ± 95% Cl).
Urinary excretion rate
of 3,4-DMHA (|imol/h)
61
Exposure to white spirit
Exposure to 1,2,4-TMB
4 8 12 16
Time (h from onset of exposure)
20
Comments: Metabolites (DMBAs) measured in urine. Exposure duration possibly not sufficient to detect other
metabolic changes. Only one exposure group; multiple concentrations not tested.
Student's t-test
b Recalculated for 9 subjects form a 120 mg/m3 exposure to 1,2,4-TMB
c Analysis of variance
d 5 of net respiratory uptake
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-47. Characteristics and quantitative results for Jarnberg et al.
fl997b_l
Study design
Species Sex N Exposure route Dose range Exposure duration
25 ppm (123 mg/m3) 1,2,3-
Caucasian M 1Q inhalation 1MB, 1,2,4-TMB, or 1,3,5- 2 hrs
Humans JMB
Additional study details
• Ten males were exposed to 25 ppm (123 mg/m3) 1,2,3-TMB, 1,2,4-TMB or 1,3,5-TMB for 2 hrs or 2 ppm (~10
mg/m3 1,2,4-TMB for 2 hrs.
• Study subjects were asked to perform light cycling to simulate a work environment, with participants
generating 50 W power during 2 hr exposure.
• Isomers of all DMHA metabolites in urine were detected via HPLC.
• Approximately 22% of inhaled 1,2,4-TMB, 11% of inhaled 1,2,3-TMB, and 3% of inhaled 1,3,5-TMB was found
to be excreted as DMHAs in urine within 24 hrs following exposure.
• The study was approved by the Regional Ethical Committee at the Karolinska Institute and only with the
informed consent of the subjects and according to the 1964 Declaration of Helsinki
Half-times of urinary excretion rate, recoveries, and rates of urinary DMHA isomer excretion (mean ± 95% Cl)
Exposure
1,2,3-TMB
1,2,3-TMB
1,2,4-TMB
1,2,4-TMB
1,2,4-TMB
1,3,5-TMB
Isomer
2,3-DMHA
2,6-DMHA
3,4-DMHA
2,4-DMHA
2,5-DMHA
3,5-DMHA
Half-time (hr)
4.8 ±0.8
8.1 ±1.5
3.80 ±0.4
5.8 ±0.9
5.3 ±1.5
16 ±6
Urinary recovery %
(24 hrs)
9±3
2±2
18 ±3
3 ±0.8
<1±0.2
3±2
Excretion rate,
|ig/min, 0-24 hrs
19 ±3
4.2 ± 1.7
44 ±6
8.2 ±1.4
1.6 ±0.5
8.9 ±2.1
Comments: Metabolites (DMBAs) measured in urine. Exposure duration possibly not sufficient to detect metabolic
changes associated with longer time points. Toxicokinetics studied at only one concentration.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-48. Characteristics and quantitative results for Jarnberg et al.
f!9981
Study design
Species
Caucasian
humans
Sex
M
N
9
subjects
Exposure route
Inhalation
Dose range
2 ppm (~10 mg/m3) 1,2,4-
TMB, 2 ppm (~10 mg/m3)
in WS, 25 ppm (123
mg/m3) 1,2,4-TMB
Exposure duration
2 hrs exposure, followed by 6 hrs
observation
Additional study details
• Caucasian males were exposed to 2 ppm (~10 mg/m3) 1,2,4-TMB, 2 ppm (~10 mg/m3) in WS, 25 ppm (123
mg/m3) 1,2,4-TMB in an inhalation chamber for 2 hrs.
• Study subjects were asked to perform light cycling to simulate a work environment.
• 1,2,4-TMB concentration was determined via gas chromatography.
• DMHA metabolites were measured with HPLC.
• Blood levels of 1,2,4 TMB and its urinary metabolites were found to be higher in the WS exposure group
suggesting that components of WS could interfere with TMB metabolism.
• No significant irritation or CNS effects were observed.
• The study was approved by the Regional Ethics Committee of the Karolinska Institute and was only performed
after informed consent.
Kinetic results following 2 hour inhalation exposure to 1,2,4-TMB and 1,2,4-TMB in WS—mean values (95% Cl)
Kinetic parameter
Actual [TMB] (ppm)
Respiratory uptake (mmol)a
Net respiratory uptake
AUCb|00d (u.M x min)
Total blood clearance (L/min)
Metabolic blood clearance (L/min)
Exhalatory blood clearance (L/min)
Mean residence time (hr)
Volume of distribution, steady state (L)
Half-life in blood, TMB, 1st phase (min)
Idem, TMB, 2nd phase (hr)
Half-life in urine, 3,4-DMHA (hr)
Urinary recovery, 3,4-DMHA (%)b, 0-6 hr
Idem (%)b, 0-22 hR
2 ppm (~10 mg/m3)
group
2.22(2.13-2.31)
0.16(0.14-0.18)
0.15(0.14-0.16)
95 (54-137)
2.09 (1.52-2.66)
1.71(1.15-2.26)
0.39 (0.28-0.50)
4.6 (-1.3-10.5)
293 (69-517)
3.9 (1.4-6.4)
4.3 (-0.5-9.0)
NDC
11 (9-13)
ND
2 ppm (~10 mg/m3)
inWS
2.26 (2.20-2.32)
0.16(0.14-0.18)
0.14(0.12-0.16)
157 (136-178)*
1.06 (0.89-1.23)**
0.79 (0.62-0.96)*
0.28 (0.20-0.36)
4.8(2.1-7.5)
271 (139-403)
5.9(3.1-8.7)
4.8(2.1-7.5)
3.0 (2.3-3.7)
18( 15-21) *
27 (23-31)
25 ppm (123
mg/m3) alone
23.9(22.7-25.1)
1.73 (1.61— .85)
1.52 (1.37-1.67)
1286 (1131-1441)
1.38(1.23-1.53)*
1.06 (0.87-1.25)*
0.32 (0.24-0.40)
3.8(1.8-5.8)
294 (165-423)
6.1(5.3-6.9)
4.0(2.2-5.8)
3.8(3.4-4.2)
14 (12-16)
18 (15-21)
Comments: Multiple exposure concentrations were tested and multiple tissues were analyzed. Study of 1,2,4-TMB as
a component of WS. Toxicokinetics of 1,2,3- and 1,3,5-TMB not studied.
aNet respiratory uptake + amount cleared by exhalation during exposure.
b% of net respiratory uptake.
°Not determined.
*p < 0.05, **p < 0.01, compared to 2 ppm (~10 mg/m3) alone by repeated measures ANOVA
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-49. Characteristics and quantitative results for Jones et al.
f20Q61
Study design
Species
Human
Sex
M/F
N
2 per sex
Exposure route
Inhalation
Dose range
25 ppm (1,2,3-TMB
mg/m3) 1,3,5-TMB
Exposure duration
4hrs
Additional study details
• Two males and two females were exposed to 25 ppm (1,2,3-TMB mg/m3) 1,3,5-TMB in an inhalation chamber
for 4 hrs.
• 1,3,5-TMB concentration in exhaled air, venous blood, and urine was determined via gas chromatography.
• No significant irritation or CNS effects were observed during the inhalation study, although one volunteer was
treated with a 2 cm2gauze patch soaked with liquid 1,3,5-TMB and reported mild itching, erythema, and
oedema where gauze contacted skin.
• Authors conclude that urinary DMBA and breath TMB are suitable markers of TMB exposure, and that
repeated exposures during work week can result in significant accumulation in tissues.
• The study was approved by the Health and Safety Executive's Research Ethics Committee
Figure 1. Mean ± SD urinary total DMBAs. Black and grey arrows represent 24 and 48 hrs respectively, following a
single 4 hr exposure to 25 ppm (1,2,3-TMB mg/m3) 1,3,5-TMB.
.- BO.O i
~ 50.0 H
i
^ 400 H
Jz
a 300
cc
^ 100 H
Time
50
100
Time (hours)
150
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Figure 2. Mean ± SD blood levels of 1,3,5-TMB during and after 4 hr exposure to 25 ppm (1,2,3-TMB mg/m ) 1,3,5-
TMB.
J 0.40 -
CO
0.20 -\
0.00 I
2 3 4
Time (hours)
Figure 3. Mean ± SD breath levels of 1,3,5-TMB during and after 4 hr exposure to 1,3,5-TMB.
IBOn
<~' Exposure Trre
o 120 -
E M
£ 100-
CD
eo
15
£ 40 -
CD
33 4
0
10 IS
Time (hours)
23
25
Comments: Metabolite (DMBA) concentration measured in urine. Subjects tested included males and females. Small
number of study subjects (n = 4). Exposure duration possibly not sufficient to detect metabolic changes. Other
metabolites not measured.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-50. Characteristics and quantitative results for Kostrzewski et
al. f!9971
Study design
Species Sex N
Human M/F 5
Exposure route Dose range Exposure duration
Between 5 and 150 mg/m
Inhalation 1,2,4-TMB, 1,3,5-TMB, and 4 or 8 hrs
1,2,3-TMB
Additional study details
• Five humans were exposed to 1,2,4-TMB, 1,3,5-TMB, and 1,2,3-TMB at concentrations between 5 and 150
mg/m3.
• Exposure durations were either 4 or 8 hrs.
• TMBs were measured in blood and urinevia gas chromatography.
• DMBA excretion was found to follow an open, two-compartment model.
1,2
Sampling time
(hrs)
0
0.25
.50
1
2
4
8
0.05
.10
.15
.25
.50
1
2
4
6
8
25
32
49
56
73
3-, 1,2,4-, and 1,
3,5-TMB concentration in blood before, during, and after exposure
1,2,3-TMB
Blood
concentr-
ation
(ug/dm3)
0
259
290
295
380
341
520
261
277
287
277
—
204
133
85
65
64
54
29
19
21
14
SD
0
94.5
91.54
57.11
93.17
186.94
129.42
50.36
57.89
38.18
35.47
—
17.78
38.55
8.96
23.69
11.59
14.57
3.51
13.01
11.31
3.50
1,2,4-TMB
Blood
concentr-
ation
(ug/dm3)
0
194
460
533
730
810
979
580
496
447
387
246
131
101
85
63
69
54
48
46
31
26
SD
0.00
19.80
57.36
46.61
128.89
112.40
171.12
36.2
85.03
106.69
65.83
128.54
19.87
14.17
13.65
11.03
7.09
3.74
10.24
9.98
9.32
9.49
1,3,5-TMB
Blood
concentr-
ation
(ug/dm3)
0
181
308
355
482
603
751
434
388
309
298
247
190
121
94
76
74
45
44
42
42
-
SD
0.00
25.01
5.29
44.80
201.57
184.13
122.87
36.40
64.16
38.78
65.48
34.00
41.13
24.60
16.52
25.81
20.16
13.93
20.19
7.93
9.81
-
Excretion rate [V, mg/hr) of dimethylbenzoic acid (DMBA) in urine during and after exposure to 1,2,3-TMB, 1,2,4-
TMB, or 1,3,5-TMB
1,2,3-TMB exposure
Sampling time (hr) 2,3-DMBA 2,6-DMBA
V (mg/hr) SD V (mg/hr) SD
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Toxicological Review ofTrimethylbenzene
0
0-2
2-4
4-6
6-8
8-10
10-12
12-14
14-16
16-23
23-27
27-31
31-35
35-39
39-47
47-51
51-55
55-59
59-63
63-71
71-75
75-79
79-83
83-87
87-95
Sampling time (hr)
0
0-2
2-4
4-6
6-8
8-10
10-12
12-14
14-16
16-23
23-27
27-31
31-35
35-39
39-47
47-51
51-55
55-59
59-63
63-71
71-75
75-79
0.000
3.518
10.745
16.594
23.468
16.874
14.769
11.929
7.715
3.976
1.876
1.822
1.471
2.292
1.388
1.125
1.543
1.505
1.154
0.535
0.802
0.999
0.886
0.349
0.365
0.000
0.852
1.856
5.028
5.291
2.353
1.964
2.070
2.236
0.782
0.213
0.893
0.551
0.998
0.660
0.414
0.468
0.683
0.481
0.119
0.383
0.712
0.343
0.165
0.163
0.000
0.099
0.097
0.146
0.202
0.160
0.150
0.161
0.129
0.110
0.067
0.079
0.081
0.143
0.102
0.109
0.172
0.139
0.055
0.031
0.053
0.059
0.086
0.046
0.000
0.000
0.097
0.084
0.039
0.070
0.004
0.035
0.048
0.038
0.042
0.021
0.052
0.055
0.032
0.037
0.041
0.058
0.050
0.063
0.030
0.001
0.030
0.078
0.050
0.000
1,2,4-TMB exposure
2,4- and 2,5-DMBA
V(mg/hr)
0.000
6.632
12.931
21.148
29.263
16.616
15.619
17.328
13.832
7.023
4.052
2.570
2.209
1.211
1.262
1.174
0.370
0.928
1.591
0.948
1.122
0.748
SD
0.000
3.069
4.315
7.067
9.240
11.451
2.935
2.218
2.176
2.565
0.674
0.760
0.666
1.075
0.256
0.459
0.228
0.327
1.162
0.276
0.049
0.441
3,4-DMBA
V(mg/hr)
0.000
19.949
22.731
26.906
35.346
12.082
6.198
6.029
4.415
2.520
1.870
2.005
1.523
1.247
0.957
0.953
0.659
0.936
1.286
0.869
0.851
0.422
SD
0.000
5.489
4.536
6.525
11.017
10.205
2.325
2.135
1.372
1.043
0.525
0.460
0.610
0.895
0.099
0.623
0.231
0.515
0.391
0.141
0.246
0.231
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Toxicological Review ofTrimethylbenzene
79-83
83-87
87-95
Sampling time (hr)
0
0-2
2-4
4-6
6-8
8-10
10-12
12-14
14-16
16-23
23-27
27-31
31-35
35-39
39-47
47-51
51-55
55-59
59-63
63-71
71-75
75-79
79-83
83-87
87-95
1.082 0.733
„
„
0.744 0.328
„
„
1,3,5-TMB exposure
3,5-DMBA
l/(mg/hr)
0.000
3.538
8.854
12.334
19.204
19.413
23.535
22.460
16.941
10.790
6.908
6.558
3.983
3.946
3.110
3.244
2.343
3.669
2.436
1.600
1.025
1.044
0.750
—
-
SD
0.000
0.833
2.955
3.905
6.092
6.329
7.606
3.254
4.350
3.116
2.691
3.657
2.367
2.073
0.838
1.140
1.355
1.882
1.303
1.305
0.639
0.825
0.645
—
-
Comments: Metabolites (DMBAs) measured in urine. Toxicokinetics studied over a range of exposures. Exposure
duration possibly not sufficient to detect other metabolic changes. Only one study subject per exposure group.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
B.7. ANIMAL TOXICOKINETIC STUDIES
Table B-51. Characteristics and quantitative results for Dahl etal.
f!9881
Study Design
Species
F344 Rats
Sex
M
N
2 rats
Exposure route
Inhalation
Dose range
l-5000ppm 1,2,4-TMB
Exposure duration
80 minutes per day for 5
consecutive days
Additional study details
• Male F344 rats weighing between 264 and 339 g were housed in polycarbonate cages for the duration of the
experiment.
• Vapors were pumped into exposure chamber at flow rate of 400ml/min past the nose of each rat in the nose-
only exposure tube.
• The amount of absorbed hydrocarbon vapor was calculated from the flow rate and the output from the nose-
only tube as measured by gas chromatography every minute during each 80 minute exposure.
• Concentrations were increased each day. Days 1-5 concentrations were Ippm, lOppm, lOOppm, lOOOppm,
and SOOOppm respectively.
• 1,2,4-TMB uptake in one rat was observed to be 11.5±2 nmol/kg/min/ppm. For the second rat, uptake was
observed to be 15.7±2.4 nmol/kg/min/ppm.
Comments: Study duration was short term (5 days). Reported values for uptake represent averages of uptake
throughout experiment, despite the widely differing doses administered. This makes it difficult to quantify dose-
specific uptake. Statistical power is limited because only two rats were used.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-52. Characteristics and quantitative results for Eide and
Zahlsen etal. (1996)
Study design
Species
Sprague-
Dawley rats
Sex
M
N
4 per
dose
Exposure route
Inhalation
Dose range
0, 75, 150, 300, 450 ppm
(0, 369, 738, 1,476, or
2,214 mg/m3) 1,2,4-TMB
Exposure duration
12 hr exposures in inhalation
chamber
Additional study details
• Male Sprague-Dawley rats were exposed to 75,150, 300, or 450 ppm (0, 369, 738,1,476, or 2,214 mg/m3)
1,2,4-TMB in an inhalation chamber for 12 hrs.
• Food and water was give ad libitum except during exposure, and animal weight ranged between 200 g and
250 g prior to exposure.
• Hydrocarbon concentration tissue concentrations were determined via head space gas chromatography.
Daily mean concentrations did not vary by more than ±5.3% from nominal concentrations.
• 1,2,4-TMB was found in higher concentrations in blood than n-nonane and trimethylcyclohexane.
Tissue 1,2,4-TMB concentrations following 12 hour 1,2,4-TMB inhalation exposure
Exposure
75ppm (369 mg/m3)
150 ppm (738 mg/m3)
300 ppm (1,476 mg/m3)
450 ppm (2,214 mg/m3)
Blood
(nmol/kg)
14.1
57.5
115.5
221.3
Brain (umol/kg)
23.6
97.5
220.9
400.2
Liver (umol/kg)
53.4
123.1
256.3
468.6
Kidneys
(u,mol/kg)
53.4
168.5
282.4
492.5
Fat (u,mol/kg)
516
3806
12930
19270
Comments: Fat was analyzed and shown to retain higher concentrations of 1,2,4-TMB than all other tissues. Multiple
exposure concentrations were tested and multiple tissues were analyzed. No data on urinary elimination. No data on
metabolites of 1,2,4-TMB.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-53. Characteristics and quantitative results for Huo etal.
f!9891
Study design
Species Sex N Exposure route Dose range Exposure duration
0.08 mmol/kg, 0.8
Wistar rats M . ra S per Oral, in olive oil mmol/kg, 0.49 u.Ci/kg 3, 6, 12, and 24 hrs
d°Se 1,2,4-TMB
Additional study details
• Single doses of 14C labeled 1,2,4-TMB administered orally to rats.
• Tissues were analyzed at 3, 6, 12, and 24 hr time points for the tissue distribution study and continuously for
24 hrs in the metabolism study.
• Percent 1,2,4-TMB distributed to individual tissues determined via liquid scintillation counter, concentration
of metabolites analyzed via gas chromatography.
• 1,2,4-TMB was distributed widely throughout the body, though particularly high levels were found in adipose
tissue.
• Over 99% of radio-labeled material was recovered from urine within 24 hrs.
• Three most common metabolites were 3,4-DMHA (30.2%), 2,4-DMBA (12.7%), and 2,5-DMBA (11.7%).
Tissue distribution and urinary excretion following single oral dose of 14C- 1,2,4-TMB
% Dose of radioactivity in tissue and urine (mean ± SD for three rats)
Tissue/Urine
Liver
Kidney
Lung
Heart
Testis
Spleen
Brain
Stomach
Intestine
Serum
Muscle
Skin
Adipose Tissue
Urine
3 hrs
2.76 ±0.39
0.56 ±0.11
0.10 ±0.03
0.03 ±0.01
0.09 ± 0.04
0.03 ± 0.02
0.08 ± 0.04
2.39 ± 1.47
2.96 ± 1.82
0.67 ±0.14
2.38 ±0.23
3.99 ±1.51
28.05 ± 9.28
15.0 ±1.1
6 hrs
2.69 ±0.60
0.52 ±0.12
0.06 ± 0.03
0.01
0.12 ±0.03
0.03 ±0.01
0.03 ± 0.02
1.33 ±0.98
3.33 ±1.31
0.57 ±0.09
1.88 ± 1.63
2.29 ±0.98
26.31 ±18.18
32.6 ±7.9
12 hrs
1.54 ±0.38
0.14 ±0.10
0.03 ± 0.03
-
0.04 ± 0.04
0.01 ±0.01
0.03 ± 0.03
0.09 ± 0.06
1.39 ± 1.03
0.26 ±0.15
0.64 ±0.10
0.16 ±0.25
4.97 ±0.97
50.7 ±7.9
24 hrs
0.13 ±0.04
0.06 ± 0.05
0.01 ±0.01
-
-
-
-
0.04 ± 0.03
0.25 ±0.35
0.12 ±0.21
-
-
0.67 ±0.15
99.8 ±4.1
Concentration (|ig/g) radioactive material in tissue (mean ± SD)
Tissue
Liver
Kidney
Lung
Heart
Testis
Spleen
Brain
Stomach
Intestine
Serum
3 hrs
72 ±9
68 ±16
17 ±9
8±2
8±4
11 ±5
11 ±5
509 ±313
35 ±22
17 ±3
6 hrs
81 ±20
60 ±13
12 ±6
2±1
11 ±2
13 ±5
6±2
263 ±218
47 ±17
15 ±1
12 hrs
45 ±12
17 ±12
4±4
-
3±4
5±5
4±4
18 ±11
21 ±15
6±3
24 hrs
5±2
7±6
2±4
-
-
-
-
10 ±7
4±6
3±6
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Muscle
Skin
Adipose Tissue
6±1
20 ±7
200 ± 64
5±4
12 ±4
193 ± 125
1±0
1±1
33 ± 8 5 ± 1
Urinary metabolites of 1,2,4-TMB 24 hours after single oral dose in rats (values ± SD)
Metabolite
2,3,5-AND2,4,5-TMPa
2,3,6-TMP
Total phenols
2,4-DMBOHb
2,5-DMBOH
3,4-DMBOH
Total alcohols
2,4-DMBAc
2,5-DMBA
3,4-DMBA
Total benzoic acids
2,4-DMHAd
2,5-DMAH
3,4-DMHA
Total hippuric acids
Total metabolies
%Dose (0.08 mmol/kg) in urine
Free
all rats
2.6 ±1.2
--
2.7 ±1.1
0.1 ±0.1
0.1 ±0.0
-
0.2 ±0.1
0.8 ±0.1
0.5 ±0.0
0.2 ±0.1
1.5 ±0.1
5.0 ±1.9
0.5 ±0.2
27.3 ±8.4
32.7 ±10.5
37.1 ±11.4
Conjugated
all rats
5.1 ±1.4
3.9 ±0.7
9.0 ±2.0
12.5 ±2.6
11.6 ±2.7
1.9 ±0.9
26.0 ±5.5
5.2 ±2.0
3.1 ±1.3
0.7 ±0.2
8.9 ±3.4
2.0 ±1.0
0.3 ±0.3
3.3 ±1.2
5.6 ±2.3
49.5 ± 13.0
Total
all rats
7.7 ±2.2
4.0 ±0.6
11.8 ±2.9
12.7 ±2.6
11.7 ±2.7
1.9 ±0.8
26.3 ±5.4
6.0 ±2.0
3.6 ±1.3
0.8 ±0.2
10.4 ± 3.3
7.0 ±2.6
0.8 ±0.3
30.2 ± 9.4
37.9 ±12.1
86.4 ± 23.0
%Dose (0.8 mmol/kg) in urine
Free
Ratl
2
0
2
0
0
.5
.1
.6
.1
1
-
0
0
0
0
1
3
0
.1
.8
3
1
.2
.3
.2
23.1
26.6
30.4
Rat 2
1.5
0.4
1.9
0.4
0.2
0.1
0.7
2.5
1.2
0.2
3.9
2.7
0.1
17.9
20.8
27.2
Conjugated
Ratl
4.3
2.1
6.3
11.5
8.7
0.9
21.1
6.8
3.5
0.5
10.8
4.8
0.5
15.6
20.9
59.1
Rat 2
2.0
1.5
3.5
7.2
8.7
0.8
16.8
1.5
2.1
0.2
3.8
1.2
0.1
7.1
8.4
32.4
Total
Ratl
6.7
2.1
8.8
11.6
8.8
0.9
21.2
7.6
3.9
0.5
11.9
8.1
0.7
38.7
47.5
89.5
Rat2
3.5
1.8
5.3
7.6
8.9
0.9
17.5
4.0
2.3
0.4
6.7
3.7
0.2
25.0
28.9
58.4
Comments: Many tissues examined for radioactive and metabolite content. Multiple metabolites measured. Small
numbers of rats per dose group, particularly for the 0.8 mmol/kg group (n = 2). Time points only extend to 24 hours.
'trimethylphenol, dimethylbenzoic alcohol, cdimethylbenzoic acid, dimethylyhippuric acid.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-54. Characteristics and quantitative results for Mikulski and
Wiglusz (1975)
Study design
Species
Wistar rats
Sex
M
N
9 rats/dose
Exposure route
Unspecified
Dose range
1.2 g/kg BW 1,2,3-, 1,2,4-
, and 1,3,5-TMB
Exposure duration
48hrs
Additional study details
• Rats weighing between 210 and 350 g were with treated with 1,2,3-, 1,2,4-, or 1,3,5-TMB at 1.2g/kg body
weight.
• In one experiment, urine was collected every 4 hrs over a period of 3 d.
• In a second experiment, metabolites were collected from rats were treated with mesitylene (1,3,5-TMB),
pseudocumene (1,2,4-TMB), or hemimellitene (1,2,3-TMB).
• Phenobarbital was found to inhibits the metabolism of TMBs to dimethylhippuric acids
Urinary excretion of glycine, glucuronic, and sulphuric acid conjugates of TMBs
Not treated
1,3,5-TMB
1,2,4-TMB
1,2,3-TMB
% of dose (mean ±SD)
Glycine
conjugates
59.1 ±5.2
23.9 ±2.3
10.1 ±1.2
Glucuronides
4.9 ± 1.0
4.0 ±0.5
7.9 ±1.3
Organic sulphates
9.2 ±0.8
9.0 ±2.1
15.0 ±3.5
Total
73.2
36.9
33.0
Treated with Phenobarbital
1,3,5-TMB
1,2,4-TMB
1,2,3-TMB
35.1 ±3.4
30.6 ±2.5
5.7 ±1.1
9.8 ±1.3
12.2 ±2.8
11.3 ±2.0
8.1 ±1.4
17.4 ±3.6
22.3 ±3.0
53.0
60.2
39.3
Comments; Kinetic data for all three TMB isomers and their metabolites were included in study. However, the
authors did not report method for dosing.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table B-55. Characteristics and quantitative results for Swiercz etal.
f20Q21
Study design
Species Sex N Exposure route Dose range Exposure duration
25, 100, or 250 ppm (123,
,m.P' M 4/dose Inhalation 492, 1.230 mg/m3) 1,2,4- 6 hrs
Wistarrats JMB
Additional study details
• Two males and two females were exposed to 25, 100, or 250 ppm (123, 492, 1.230 mg/m3) 1,2,4-TMB in an
inhalation chamber for 6 hrs.
• 1,2,4-TMB concentration was determined via gas chromatography.
• Blood samples were taken from the tail vein at various timepoints up to 6 hrs after start of exposure.
• The half-life of 1,2,4-TMB elimination was found to increase with increasing exposure.
Air concentrations of 1,2,4-TMB and body mass of rats (means ± SD)
Biological material
Blood during 6 hr exposure
Blood after 6 hr exposure
Urine after 6 hr exposure
1,2,4-TMB nominal
concentration
25 ppm (123 mg/m3)
100 ppm (492 mg/m3)
250 ppm (1,230 mg/m3)
25 ppm (123 mg/m3)
100 ppm (492 mg/m3)
250 ppm (1,230 mg/m3)
25 ppm (123 mg/m3)
100 ppm (492 mg/m3)
250 ppm (1,230 mg/m3)
1,2,4-TMB actual
concentration (ppm)
25 ±2
109 ± 10
262 ±21
26 ±3
101 ±3
238 ±9
27 ±3
98 ±3
240 ±7
Rat body weight (g)
200 ± 10
228 ± 10
190 ± 12
349 ±6
333 ± 18
336 ±5
355 ± 10
338 ± 10
330 ± 12
Blood 1,2,4-TMB concentration during 6 hour inhalation exposure (mean ± SD)
Time
15 (min)
30
45
1 (hrs)
2
3
4
5
6
1,2,4-TMB concentration
25 ppm
(123 mg/mg3)
0.22 ±0.07
0.33 ±0.08
0.49 ±0.16
0.53 ±0.14
0.73 ±0.16
0.80 ±0.17
0.72 ±0.15
0.79 ±0.22
0.94 ±0.16
100 ppm
(492 mg/mg3)
1.12 ±0.80
1.99 ± 1.09
3.56 ±0.49
4.29 ±0.60
5.10 ±0.34
6.22 ±0.70
7.40 ± 1.05
7.72 ± 1.48
8.32 ± 1.34
250 ppm
1,230 mg/mg3)
4.02 ± 0.85
4.87 ± 1.61
6.97 ± 1.22
8.67 ±0.54
14.5 ±2.6
17.8 ±1.6
20.0 ±0.5
23.3 ±2.6
23.6 ±1.8
This document is a draft for review purposes only and does not constitute Agency policy.
B-152 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Blood concentrations of 1,2,4-TMB following 6 hour exposure (mean ± SD)
Time
3 (min)
15
30
45
1 (hrs)
2
3
4
5
6
1,2,4-TMB concentration
25 ppm
(123 mg/mg3)
0.68 ±0.09
0.47 ± 0.04
0.40 ± 0.05
0.36 ± 0.04
0.34 ± 0.03
0.23 ± 0.04
0.17 ±0.04
0.12 ±0.02
0.10 ±0.02
0.08 ± 0.02
100 ppm
(492 mg/mg3)
4.44 ± 1.54
3.72 ±0.96
2.98 ±0.88
2.89 ±0.86
1.79 ±0.49
1.25 ±0.33
0.88 ±0.29
0.61 ±0.20
0.41 ±0.14
0.33 ±0.06
250 ppm
1,230 mg/mg3)
20.9 ± 4.03
20.7 ±5.13
17.1 ±4.71
15.9 ±5.74
14.9 ± 3.77
10.2 ± 3.04
8.05 ± 2.25
6.13 ± 1.64
3.98 ±0.43
3.20 ±0.52
Dimethylbenzoic acid (DMBA) urine concentrations after 6 hour exposure to 1,2,4-TMB (mean ± SD)
1,2,4-TMB
25 ppm (123 mg/m3)
100 ppm (492 mg/m3)
250 ppm (1,230 mg/m3)
2,5-DMBA(mg/L)
23.6 ±8.6
54.0 ± 5.4
109.4 ±71.1
2,4-DMBA(mg/L)
37.6 ± 12.9
130.9 ±22.1
308.8 ±220.1
3,4-DMBA(mg/L)
79.9 ±33.3
200.8 ± 25.8
571.8 ±381.6
Comment: Metabolites (DMBAs) measured in urine. Appropriate number of animals per dose group (n = 4).
Exposure duration possibly not sufficient to detect other metabolic changes.
This document is a draft for review purposes only and does not constitute Agency policy.
B-153 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Table B-56. Characteristics and quantitative results for Swiercz etal.
f20Q31
Study design
Species Sex N Exposure route Dose range Exposure duration
25, 100, or 250 ppm (123,
Wistar rats M 4/dose Inhalation 492, 1.230 mg/m3) 1,2,4- 6hrsor4wks
TMB
Additional study details
• Male Wistar rats were exposed to either 25, 100, or 250 ppm (123, 492, 1.230 mg/m3) pseudocumene (1,2,4-
TMB) in an inhalation chamber for either 6 hrs or 4 wks.
• Rats were sacrificed following exposure period and tissues were analyzed 1,2,4-TMB content via gas
chromatography.
• Venous elimination was found to follow an open two-compartment model.
• Within brain structures, the brainstem was found to contain the highest levels of 1,2,4-TMB.
Air concentrations of 1,2,4-TMB in inhalation chamber and body weight (mean ± SD)
Biological material
Arterial blood and brain
structure from rats after 6
hrs
Arterial blood and brain
structure from rats after 4
wks
Liver, lung, and brain
homogenate after 6 hrs
Liver, lung, and brain
homogenate after 4 wks
Venous blood collected
following 4 wk exposure
1,2,4-TMB nominal
concentration in inhaled
air
25 ppm (123 mg/m3)
100 ppm (492 mg/m3
250 ppm (1,230 mg/m3
25 ppm (123 mg/m3)
100 ppm (492 mg/m3
250 ppm (1,230 mg/m3
25 ppm (123 mg/m3)
100 ppm (492 mg/m3
250 ppm (1,230 mg/m3
25 ppm (123 mg/m3)
100 ppm (492 mg/m3
250 ppm (1,230 mg/m3
25 ppm (123 mg/m3)
100 ppm (492 mg/m3)
250 ppm (1,230 mg/m3
1,2,4-TMB actual
concentration in inhaled
air (ppm)
21 ±2
116 ±5
215 ± 15
24 ±3
99 ±7
249 ± 19
28 ±1
123 ±9
256 ±7
25 ±2
103 ±8
249 ± 13
24 ±3
99 ±7
249 ± 19
Rat body weight (g)
219 ± 13
180 ± 28
220 ± 24
327 ±21
295 ±31
268 ±21
227 ± 15
246 ± 11
228 ± 12
310 ± 10
328 ± 23
320 ± 20
321 ±6
300 ± 22
373 ± 48
Venous blood 1,2,4-TMB concentrations after 4 week inhalation exposure
Time
3 (min)
15
30
45
l(hr)
2
3
4
5
6
1,2,4-TMB concentration mean ± SD
25 ppm
(123 mg/mg3)
0.56 ±0.18
0.43 ±0.10
0.33 ±0.03
0.28 ±0.05
0.22 ±0.02
0.17 ±0.06
0.11 ±0.04
0.07 ± 0.04
0.07 ±0.01
0.06 ± 0.02
100 ppm
(492 mg/mg3)
4.06 ± 0.46
3.73 ±1.21
3.02 ± 1.43
2.86 ±0.89
2.62 ±0.82
1.83 ±0.17
0.88 ±0.24
0.64 ±0.21
0.39 ±0.11
0.37 ±0.14
250 ppm
1,230 mg/mg3)
13.77 ±3.34
11.82 ± 3.05
8.28 ± 2.07
7.21 ±1.84
6.27 ± 1.72
4.50 ± 1.04
3.17 ±0.76
1.73 ±0.37
1.30 ±0.22
1.25 ±0.22
This document is a draft for review purposes only and does not constitute Agency policy.
B-154 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Liver, lung, and brain homogenates and arterial blood 1,2,4-TMB concentrations following inhalation exposure
(mean ±SD)
Exposure
Blood 6 hrs (mg/L)
Blood 4 wks (mg/L)
Brain 6 hrs (mg/kg)
Brain 4 wks (mg/kg)
Liver 6 hrs (mg/kg)
Liver 4 wks (mg/kg)
Lung 6 hrs (mg/kg)
Lung 4 wks (mg/kg)
25 ppm
(123 mg/mg3)
0.31 ±0.12
0.33 ±0.11
0.49 ± 0.06
0.45 ± 0.05
0.44 ± 0.01
0.45 ±0.15
0.43 ±0.11
0.47 ± 0.20
100 ppm
(492 mg/mg3)
1.24 ±0.41
1.54 ±0.32
2.92 ±0.73
2.82 ± 0.40
7.13 ±1.31
3.00 ± 0.49*
4. 14 ±0.54
3.74 ±0.82
250 ppm
1,230 mg/mg3)
7.76 ± 1.64
7.52 ±2.11
18.34 ± 1.92
18.63 ±4.27
28. 18 ±5.34
22.47 ±4. 10
18.90 ± 3.72
22.47 ±4. 10
1,2,4-TMB in various brain structures following 1,2,4-TMB inhalation exposure
Brain structure (time)
Brain stem (6 hrs)
Temporal cortex (6 hrs)
Hippocampus (6 hrs)
Cerebellum (6 hrs)
Brain stem (4 wks)
Temporal cortex (4 wks)
Hippocampus (4 wks)
Cerebellum (4 wks)
1,2,4-TMB concentration (mg/kg), mean ± SD
25 ppm
(123 mg/mg3)
0.54 ±0.11
0.31 ±0.06*
0.28 ±0.09*
0.32 ±0.09*
0.38 ±0.23
0.25 ±0.07
0.41 ±0.27
0.33 ±0.05
100 ppm
(492 mg/mg3)
3.38 ±0.84
2.30 ±0.71
1.89 ±0.29*
1.99 ± 0.40*
2.33 ± 1.24
2.03 ±0.66
3.03 ± 0.48
3.20 ± 0.40
250 ppm
1,230 mg/mg3)
26.91 ±5.33
13.54 ±2.33*
12.99 ±2.18*
12.91 ±2.05*
21.95 ±3.81
15.71 ±3.54
12.44 ± 2.63*
10.85 ± 2.47*
Comments: Adipose tissue was not examined for 1,2,4-TMB content. Metabolite concentration was not measured. No
control group.
P < 0.05 in comparison to brainstem
This document is a draft for review purposes only and does not constitute Agency policy.
B-155 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Table B-57. Characteristics and quantitative results for Swiercz etal.
f20Q61
Study design
Species Sex N Exposure route Dose range Exposure duration
IMP-WIST 25, 100, or 250 ppm (123,
M 5/dose Inhalation 492, 1.230 mg/m ) 1,3,5- 6hrsor4wks
Wistarrats JMB
Additional study details
• Male Wistar rats were exposed to either 0, 25, 100, or 250 ppm (123, 492, 1.230 mg/m3) mesitylene (1,3,5-
TMB) in an inhalation chamber for either 6 hrs or 4 wks.
• Rats were sacrificed following exposure period and tissues were analyzed for 1,3,5-TMB content via gas
chromatography.
• 1,3,5-TMB was found in the lungs in greater quantities following repeated exposures at 100 ppm (492 mg/m3)
and 250 ppm (1.230 mg/m3).
Air concentrations of 1,3,5-TMB in inhalation chamber and body weight (mean ± SD)
Biological material
Liver, lung, and kidney
homogenates after 6 hr
exposure
Liver, lung, and kidney
homogenates after 4 wk
exposure
Blood collected after 6 hr
exposure
Blood collected after 4 wk
exposure
Urine collected after 6 hr
exposure
Urine collected after 4 wk
exposure
1,3,5-TMB nominal
concentration in inhaled
air
Control
25 ppm (123 mg/m3)
100 ppm (492 mg/m3
250 ppm (1,230 mg/m3)
Control
25 ppm (123 mg/m3)
100 ppm (492 mg/m3
250 ppm (1,230 mg/m3)
Control
25 ppm (123 mg/m3)
100 ppm (492 mg/m3)
250 ppm (1,230 mg/m3)
Control
25 ppm (123 mg/m3)
100 ppm (492 mg/m3)
250 ppm (1,230 mg/m3)
Control
25 ppm (123 mg/m3)
100 ppm (492 mg/m3)
250 ppm (1,230 mg/m3)
Control
25 ppm (123 mg/m3)
100 ppm (492 mg/m3)
250 ppm (1,230 mg/m3)
1,3,5-TMB actual
concentration in inhaled
air (ppm)
0
25 ±2
97 ±14
254 ± 20
0
23 ±2
101 ±8
233 ± 16
0
24 ±2
101 ±7
240 ± 22
0
23 ±2
101 ±8
233 ± 16
0
25 ±2
102 ± 10
238 ± 27
0
25 ±2
102 ± 10
238 ± 27
Rat body weight (g)
246 ±9
254 ± 11
242 ± 14
249 ±7
331 ±17
311 ±26
320 ± 38
328 ±21
251 ±7
250 ±5
239 ±7
249 ± 10
310 ±9
307 ± 15
310 ± 33
309 ± 19
280 ±9
278 ± 10
335 ± 15
273 ± 18
310 ± 10
295 ± 15
331 ± 19
320 ± 28
This document is a draft for review purposes only and does not constitute Agency policy.
B-156 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Concentrations of 1,3,5-TMB in various tissues after exposure to 1,3,5-TMB (mean ± SD)
1,3,5-TMB exposure
duration and target
concentration
6 Mrs— 25 ppm
(123 mg/m3)
6 Mrs— 100 ppm
(492 mg/m3)
6 Mrs— 250 ppm
(1,230 mg/m3)
4Wks-25ppm
(123 mg/m3)
4 Wks-100 ppm
(492 mg/m3)
4Wks-250ppm
(1,230 mg/m3)
Liver (u.g/g
tissue)
0.30 ±0.07
3.09 ±0.50
17.00 ± 6.08
0.22 ±0.01
3.01 ±0.58
12.98 ±4. 16
Lung (|ig/g tissue)
0.31 ±0.12
2.87
17.36
0.42
1.99
11.20
±0.57
±5.56
±0.12
±0.75
±3.61
Kidney (u.g/g tissue)
4.49 ± 1.93
13.32 ±2.58
31.80 ± 9.44
1.73 ±0.30*
15.61 ±2.14
35.97 ±8.53
Blood (u.g/g tissue)
0.31 ±0.12
3.06 ±0.65
13.36 ± 1.54
0.31 ±0.08
2.30 ±0.52
7.55 ±1.43**
Concentrations of 3,5-DMBA in various tissues after exposure to 1,3,5-TMB (mean ± SD)
1,3,5-TMB exposure
duration and target
concentration (ppm)
6 Mrs— 25 ppm
(123 mg/m3)
6 Mrs— 100 ppm
(492 mg/m3)
6 Mrs— 250 ppm
(1,230 mg/m3)
4Wks-25ppm
(123 mg/m3)
4 Wks— 100 ppm
(492 mg/m3)
4 Wks-250 ppm
(1,230 mg/m3)
Liver (u.g/g
tissue)
12.62 ± 1.62
26.05 ±2.77
36.92 ±1.61
6.52 ±0.67**
21.67 ±3.14**
53.07 ±5.41**
Lung (|ig/g tissue)
2.87
5.50
13.39
3.69
8.90 ±
±0.55
±0.55
±1.90
±1.21
0.98**
19.79 ±2.70**
Kidney (u.g/g tissue)
8.77 ±0.99
27.01 ±9.86
60.91 ±19.78
11.06 ±4.33
31.03 ± 18.56
82. 10 ±14.48
Urine (mg/18 hrs)
0.52 ±0.03
3.66 ±0.57
10.99 ± 3.90
0.83 ±0.15*
4.36 ±0.86
11.92 ±3.05
Venous blood 1,3,5-TMB concentration following 6 hr 1,3,5-TMB inhalation exposure
Time
25 ppm
(123 mg/mg3)
3(min) 0.31 ±0.12
15 0.26 ±0.13
30 0.15 ±0.04
45 0.10 ±0.03
1 (hrs) 0.06 ± 0.02
2 0.04 ± 0.02
3 ND***
4 ND
5 ND
6 ND
1,3,5-TMB (u.g/mL)
100 ppm 250 ppm
(492 mg/mg3) 1,230 mg/mg3)
3.06 ±0.65 13.36 ±1.54
2.51 ±0.17 13.05 ±1.61
2.35 ±0.57 12.06 ±1.23
1.41 ±0.27 10.53 ±1.71
1.35 ±0.30 8.85 ±0.90
1.34 ±0.39 6.14 ±0.53
0.79 ±0.30 4.54 ±0.67
0.57 ±0.14 3.49 ±1.16
0.38 ±0.14 2.31 ±0.67
0.20 ± 0.04 0.76 ± 0.06
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Venous blood 1,3,5-TMB concentration following 4 wk 1,3,5-TMB inhalation exposure
Time
3 (min)
15
30
45
1 (hrs)
2
3
4
5
6
1,3,5-TMB (u,g/mL)
25 ppm
(123 mg/mg3)
0.31 ±0.08
0.26 ±0.03
0.19 ±0.02
0.17 ±0.03
0.12 ±0.03
0.05 ±0.01
ND
ND
ND
ND
100 ppm
(492 mg/mg3)
2.30 ±0.52
1.83 ± 0.47
1.57 ±0.39
1.41 ±0.13
1.33 ±0.15
0.95 ±0.22
0.72 ±0.17
0.41 ±0.11
0.39 ±0.05
0.29 ±0.13
250 ppm
1,230 mg/mg3)
7.55 ± 1.43
6.51 ±1.50
4.56 ±0.98
3.65 ±0.62
3.69 ± 1.25
3.14 ±0.64
2.28 ±0.19
1.74 ±0.17
1.23 ±0.34
1.14 ±0.20
Comments: Kinetics of 1,3,5-TMB elimination are reported and discussed in detail. Extensive analysis of 3,5-DMBA.
Adipose tissue was not examined for 1,3,5-TMB content.
P < 0.05 in comparison to brainstem
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Table B-58. Characteristics and quantitative results for Tsujimoto etal.
fZOQQl
Study design
Species Sex N
SlcWistar r/1 A .
M 4perdc
rats
Exposure route
>se i.p. in corn oil
Dose range Exposure duration
0, 0.3, 1, and 3 mmol/kg
BW 1,2,4-TMB
Additional study details
• Groups of four male Wistar rats dosed with 0, 0.3, 1, or 3 mmol/kg BW 1,2,4-TMB.
• Urine samples collected for 2 d.
• HPLC used to quantify amount of dimethylbenzyl mercapturic acid in urine.
Urinary excretion of dimethylbenzyl mercapturic acid in 1,2,4-TMB treated rats
0.3
1.0
3.0
% of dose ± SD
0-24 hr
14.0 ± 1.2
19.4 ±1.8
16.7 ±6.2
24-48 hr Total
ND 14.0 ±1.2
ND 19.4 ±1.8
2.5 ±1.6 19.2 ±4.8
Comments: This study observed a marked decrease in dimethylbenzyl mercapturic acid excretion between 24 and 48
hours following exposure. Authors do not report specific speciation data for 2,4-, 2,5-, or 3,4-dimethylbenzyl
mercapturic acid.
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Table B-59. Characteristics and quantitative results for Tsujimoto etal.
f20Q51
Study Design
Species
Sex
Exposure route
Dose range
Exposure duration
Wistar
rats
M
4 per dose
i.p. in corn oil
0, 0.3,1, and 3 mmol/kg
BW given 1,2,3- or 1,3,5-
TMB
2d
Additional study details
• Groups of four male Wistar rats were given 1,2,3- or 1,3,5-TMB intraperitoneally in doses of 0, 0.3,1, or 3
mmol/kg BW.
• Urine samples collected for 2 days, then analyzed for trimethylphenols (IMP) via GC-MS
Urinary excretion (% of dose ± SD) of phenolic metabolites in 1,2,3-TMB treated rats
Dose
(mmol/kg)
2,3,4-Trimethylphenol
0-24 hr
24-48 hr
Total
3,4,5-Trimethylphenol
0-24 hr
24-48 hr
Total
0.3
5.90 ±2.62
0.46 ± 0.34
6.36 ±2.92
ND
ND
ND
1.0
7.93 ± 5.00
0.35 ±0.16
8.28 ±4.85
<0.24
ND
<0.24
3.0
6.20 ± 3.45
0.57 ±0.34
6.77 ±3.60
<0.19
<0.04
<0.19
Urinary excretion (% of dose ± SD) of phenolic metabolites in 1,3,5-TMB treated rats
2,4,6-Trimethylphenol
Dose (mmol/kg)
0-24 hr
24-48 hr
Total
0.3
7.04 ± 1.24
0.53 ±0.29
7.57 ±0.99
1.0
4.39 ±0.61
0.51 ±0.12
4.90 ±0.64
3.0
3.32 ±0.58
0.82 ±0.34
4.14 ±0.67
Comments: This study observed a marked decrease in TMP excretion between 24 and 48 hours following exposure.
This study does not include data for 1,2,4 TMB and phenolic metabolites. Variation between rats (high standard
deviation) within exposure groups.
ND-not detected
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Table B-60. Characteristics and quantitative results for Tsujino et al.
f20Q21
Study design
Species
Wistar rats
Sex
M
N
3 for Experiment 1, 36 for
Experiment 3 (shown below in
Figure 3)
Exposure route
Dermal (via
saturated cotton)
Dose range
1 mL kerosene
Exposure duration
0, 1, 3, or 6 hrs
Additional study details
• In first experiment, rats were dermally exposed to kerosene on a saturated, sealed piece of cotton for 1 hr to
analyze TMB and aliphatic hydrocarbon (AHC) dermal absorption.
• In second experiment, 44 rats were divided into four groups which varied by exposure duration, post-
exposure time, and/or exposure either before or after death.
• TMBs were detected at greater levels than AHCs, and were only detected in traces following post-mortem
exposure.
• Trace concentrations of TMBs following post-mortem exposure suggest TMB must circulate in blood before
being distributed to organs.
1 hr exposure and ratio of TMBs to internal standard (o-xylene d10) (mean ± SD)
Tissue source
Blood
Brain
Lung
Liver
Spleen
Kidney
Muscle
Adipose
OVERALL
Post-mortem samples spiked with
kerosene (positive control)
3.6 ±1.6
3.6 ±1.6
1.2 ±0.5*
1.1 ±0.5
0.7 ±0.3
1.0 ± 0.4
1.2 ±0.5*
0.9 ±0.3*
1.4 ±0.3***
Post-mortem samples following dermal
exposu re
0.4 ±0.4
0.14 ±0.05*
0.09 ± 0.03
0.3 ±0.09**
0.1 ±0.04
0.5 ±0.1**
0.09 ± 0.02
0.15 ±0.07
0.21 ±0.05*
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1,2,4-TMB in Various Tissues following 1 hr Exposure and Ante vs. Post-Mortem Exposure
Figure 1. 1,2,4-TMB levels in rats immediately after 1 hour of dermal exposure to kerosene are compared
between ante-mortem (group I) and post-mortem (group IV) groups. Data represent mean ± SE. The data
were analyzed using two-way ANOVA (* p < 0.05, ** p < 0.01)
600
• Antemortem exposure
0 Postmortem exposure
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Table B-61. Characteristics and quantitative results for Zahlsen et al.
(1990)
Study design
Species
Sprague-
Dawley rats
Sex
M
N
24
Exposure route
Inhalation
Dose range
1,000 ppm (4,920 mg/m3)
1,2,4-TMB
Exposure duration
12 hr exposures on days 1, 3, 7,
10, and 14
Additional Study details
• Male Sprague-Dawley rats were exposed to 1,000 ppm (4,920 mg/m3) 1,2,4-TMB in an inhalation for 12 hrs
on days 1, 3, 7,10, and 14.
• Food and water was given ad libitum except during exposure, and animal weight ranged between 150 g and
200 g prior to exposure on day 1.
• Hydrocarbon concentration in blood was determined via head space gas chromatography. Daily mean
concentrations did not vary by more than ±10% from nominal concentrations.
• Multiple exposures to 1,2,4-TMB resulted in decreases in blood concentrations following subsequent
exposures, possibly due to the induction of metabolic enzymes that play a role in the metabolism of 1,2,4-
TMB.
Figure 1. Blood concentrations (+SD) of n-nonane, 1,2,4-TMB, and 1,2,4-trimethylcyclohexane following 12 hr
exposures on days 1, 3, 7,10, and 14.
700r
5 10
TIME OF EXPOSURE (days)
*TMB
n~C9
*TMCH
15
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Figure 2. Brain concentrations (+SD) of n-nonane, 1,2,4-TMB, and 1,2,4-trimethylcyclohexane following 12 hr
exposures on days 1, 3, 7,10, and 14.
n-C9
TMCH
TMB
o
0
5 10
TIME OF EXPOSURE (days)
Figure 3. Perirenal fat concentrations (+SD) of n-nonane, 1,2,4-TMB, and 1,2,4-trimethylcyclohexane following 12 hr
exposures on days 1, 3, 7,10, and 14.
_ 70000r
o>
I 60000 -
J? 50000-
z 40000-
R 300OO-
UJ
o
g
o
20000h
10OOO
5 10
TIME OF EXPOSURE (days)
15
Brain:blood and fat:blood TMB distribution after 12 hr exposure at end of day 14
Compound
Brain:bloodTMB ratio
Fat:blood TMB ratio
Concentration ratio
2.0
63
Comments: Perirenal fat was analyzed and shown to retain higher concentrations of 1,2,4-TMB than blood. Exposure
was not continuous (only occurred on days 1, 3, 7,10, and 15). Only one exposure concentration (1,000 ppm [4,920
mg/m3]) was tested, and there were no control groups.
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Table B-62. Characteristics and quantitative results for Zahlsen et al.
f!9921
Study Design
Species Sex N Exposure route Dose range Exposure duration
Sprague- M 4/time Inhalation 100 ppm C9 armoate 12 hours/day for 3 days
Dawley rats point
Additional study details
• Food and water was given ad libitum, except during exposure.
• Rats weighed between 150-200g and were between 40 and 50 days of age.
• 4 rats were housed in each cage, and each exposure chamber contained 4 cages; 16 rats were present at the
beginning of exposure.
• At each time point, 4 rats were sacrificed and their tissues analyzed for C9 aromate presence
Observation
Blood Day 1
Blood Day 2
Blood Day 3
Blood Reca
Brain Day 1
Brain Day 2
Brain Day 3
Brain Rec
Liver Day 1
Liver Day 2
Liver Day 3
Liver Reca
Kidney Day 1
Kidney Day 2
Kidney Day 3
Kidney Reca
Fat Day 1
Fat Day 2
Fat Day 3
Fat Reca
C9 Aromate Concentration in Rat Tissues at Various Time Points (MeaniS.D)
100 ppm C9 Exposure Group
14.2±0.7
12.6±0.9
17.1±2.2
0.2±0.1
38.1±1.5
34.9±3.9
36.5±2.2
nd
41.0±4.5
30.5±3.4
35.4±2.4
0.6±0.1
113.8±26.5
142.0±35.2
103.6±18.8
2.0±0.3
1741±329
1375±88
1070±93
120±52
Comments: Data was collected immediately following exposure and 12 hours following exposure, providing insight
into metabolic clearance and excretion. Study duration was short term (5 days), making it difficult to determine if
tissue concentration changes following chronic exposure.
Rec=After 12 hour recovery
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B.8. ANIMAL AND HUMAN TOXICOKINETIC STUDIES
Table B-63. Characteristics and quantitative results for Meulenberg and
Vijverberg (2000)
Study Design
Species Sex
Rat and F &
Human M
N Exposure route Dose range
Varies n/a not given
Exposure duration
not given
Additional study details
• Authors examined partition coefficients for many volatile organic compounds from multiple studies.
• 1,2,3-, 1,2,4-, and 1,3,5-TMB were among the volatile organic compounds considered for review.
• Partition coefficients for blood, fat, brain, liver, muscle, and kidney were reported for both rats and humans.
Observation
p
roil:air
n
rsaline:air
Blood
Fat
Brain
Liver
Muscle
Kidney
Blood
Fat
Brain
Liver
Muscle
Kidney
Partition Coefficients for 1,2,3-, 1,2,4- and 1,3,5-TMB
1,2,3-TMB
1,2,4-TMB
1,3,5-TMB
Reported and predicted partition coefficients For oil, saline, and air
10,900a
2.73a
10,200a
1.61a
9,880a
1.23a
Reported and predicted PtiSsue:air values for various human tissues
66.5a
4879b
220
306
155
122
59.1a
4566
206
286
144
114
43a
4423
199
277
140
110
Reported and predicted Ptissue:air values for various rat tissues
62.6
6484
591
288
111
1064
55.7
6068
552
269
104
995
55.7
5878
535
260
100
963
Comment: This study evaluated a number of parameters, presenting predicted partition coefficients for blood, fat,
brain, liver, muscle, and kidney tissue in both humans and rats. Reported values based on single trial.
aAveraged values as reported by Jarnberg and Johanson (1995).
bAII other values predicted by Meulenberg and Vijverberg (2000).
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APPENDIX C DOSE-RESPONSE MODELING FOR THE
DERIVATION OF REFERENCE VALUES FOR EFFECTS
OTHER THAN CANCER AND CANCER RISK
ESTIMATES
C.I. BENCHMARK DOSE MODELING SUMMARY
1 This appendix provides technical detail on dose-response evaluation and
2 determination of points of departure (POD] for relevant neurological, respiratory,
3 hematological, and developmental toxicity endpoints. The endpoints were modeled using
4 the U.S. EPA's Benchmark Dose Software (BMDS}. For every continuous endpoint, BMDS
5 continuous models were fitted to the data. Model parameters were estimated using the
6 maximum likelihood method. Model fit was assessed following the draft Benchmark Dose
1 Technical Guidance Document [U.S. EPA. 2000) as follows. For each model, first the
8 homogeneity of the variances (the "constant variance" case] was tested using a likelihood
9 ratio test (BMDS Test 2}. If Test 2 was not rejected (x2 p-value > 0.10], the model was fitted
10 to the data under the constant variance case. If Test 2 was rejected (x2 p-value < 0.10], the
11 variance was tested as a power function of the mean (the "modeled variance" case] using a
12 likelihood ratio test (BMDS Test 3}. If Test 3 was not rejected (x2 p-value > 0.10], the model
13 was fitted to the data under the modeled variance case. For fitting models in either the
14 constant variance or modeled variance case, models were tested for adequacy of fit to the
15 means using a likelihood ratio test (BMDS Test 4, with x2 p-value < 0.10 indicating
16 inadequate fit}.
17 Other factors were used to assess the model fit, such as scaled residuals, graphical
18 fit, and adequacy of fit in the low-dose region and near the benchmark response (BMR}. For
19 the continuous endpoints (latency to paw-lick, decreased RBC, decreased reticulocytes,
20 decreased clotting time, decreased fetal weight, and decreased maternal weight change], a
21 BMR equal to a change in the meal response equivalent to 1 standard deviation of the
22 estimated mean was chosen as the response level. A BMR equal to a change in the meal
23 response equivalent to 1 standard deviation is recommended as the response level for
24 endpoints for which no data exist as to what level of response to consider adverse (U.S.
25 EPA. 2000}. In addition to this a BMR of 5% relative deviance was also used as a response
26 level for the decreased fetal weight endpoints. As a decrease of 10% body weight is often
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1 used as a biologically significantly response level for adult animals, a 5% decrease in body
2 weight was determined as biologically significant for prenatal rats.
3 For each endpoint, the best-fit model was selected from among the models
4 exhibiting adequate fit. For each model, the BMDL was calculated using the profile
5 likelihood method, where the BMDL refers to the 95% lower confidence limit on the
6 benchmark does (BMD}. If the BMDL estimates were "sufficiently close," that is, differed by
7 at most 3-fold, the model selected was the one that yielded the lowest Akaike Information
8 Criterion (AIC} value. If more than one model had the lowest AIC, BMDL values from these
9 models were averaged to obtain a POD. If the BMDL estimates were not sufficiently close,
10 the lowest BMDL was selected as the POD. When two models are displayed on the same
11 row, this indicates that these models returned the same modeling results. This happens
12 when a more complex model reverts to a simpler form. For example, a polynomial 3°
13 model can revert to a polynomial 2° form if the beta3 coefficient is not estimated. When
14 models in this case are selected as the best-fit model, the most simple form (i.e., poly 2°
15 instead of poly 3°} is selected as the best-fit model.
16 Below are tables summarizing the modeling results for the modeled endpoints. The
17 following parameter restrictions were applied:
18 • for multistage models, beta restricted to > 0;
19 • for the polynomial models, betarestricted to > 0; and
20 • for the Hill and continuous power models, power restricted to > 1.
21 For all endpoints from Korsak et al. (2000a: 1997} and Korsak and Rydzynski
22 (1996}. external exposure concentrations were first converted into the internal dose metric
23 of weekly average venous blood concentration (mg/L}, and these dose metrics were used
24 as the dose inputs for BMD modeling. Due to PBPK model insufficiency at the high dose (i.e.,
25 estimating higher internal blood metrics compared to observed blood data}, all high doses
26 were dropped prior to modeling (see Dose-Response Analysis section in Volume 1 for more
27 detail}. Section C.2 is included for comparison at the end of this appendix that includes
28 BMD modeling results when the high doses were not dropped. All modeling results (i.e.,
29 BMDs and BMDLs} for the Korsak studies are provided in mg/L. As a PBPK model was not
30 applied to the endpoints from Saillenfait et al. (2005J. modeling results for these endpoints
31 are provided in mg/m3. Additionally, as no PBPK model was available for 1,2,3-TMB, all
32 endpoints from Korsak et al. (2000b) are provided in mg/m3.
33 Comprehensive modeling results for all endpoints are provided on EPA's Health
34 Effects Research Online (HERO} database fU.S. EPA. 2011bl
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Table C-l. Model predictions (constant variance, high dose dropped) for
increased latency to paw-lick in male Wistar rats, 1,2,4-TMB (Korsak
and Rydzynski. 1996)
Model"
Exponential 2
Exponential 3
Exponential 4b
Linear
Polynomial 2°
Polynomial 3°
Power
Good ness-of -fit
p-value
0.5045
n/a
0.6236
AIC
122.2153
123.7699
122.010727
BMD1SD
(mg/L)
0.42102
0.233402
0.354545
BMDL1SD
(mg/L)
0.328286
0.0864608
0.259068
Basis for Model
Selection
Of the models that
provided an adequate
fit and a valid BMDL
estimate, the
Exponential 4 model
was selected based on
lowest BMDL
Constant variance case presented (Test 2 p-value = 0.169). Selected model in bold; scaled residuals for selected
model for concentrations 0, 0.1272, and 0.8666 mg/L were 6.09 x 10"08, -1.09 x 10"08, and -3.65 x 10"08
respectively.
bAlthough a goodness-of-fit p-value was not calculated for the Exponential 4 model (due to estimated model
parameters = dose groups), inspection of scaled residuals and visual fit indicated appropriate model fit.
30
25
20
15
10
Exponential Model 4 with 0.95 Confidence Level
Exponential
BMDL
BMD
0.1
0.2
0.3
0.4 0.5
dose
0.6
0.7
0.8
0.9
14:44 03/12 2012
Figure C-l. Plot of mean response by dose (mg/L 1,2,4-TMB) for
increased latency to paw-lick in male Wistar rats, with fitted curve for
Exponential 4 model (BMR = 1 SD, constant variance, high dose
dropped). (Korsak and Rydzynski. 1996)
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Table C-2. Model predictions (constant variance, high dose dropped) for
decreased red blood cells in male Wistar rats, 1,2,4-TMB (Korsak et al..
2000a1
Model"
Exponential 2
Exponential 3b
Exponential 4
Linear
Polynomial 2°b
Polynomial 3°
Power
Good ness-of -fit
p-value
0.8653
n/a
0.8653
0.8864
n/a
AIC
59.81949
61.79073
59.81949
59.811121
61.790726
BMD1SD
(mg/L)
0.847227
0.870338
0.847227
0.851043
0.869761
BMDL1SD
(mg/L)
0.467889
0.469066
0.184658
0.499419
0.5002
Basis for Model
Selection
Of the models that
provided an adequate
fit and a valid BMDL
estimate, the Linear
model was selected
based on lowest AIC
Constant variance case presented (Test 2 p-value = 0.2848). Although Test 1 p-value (0.091) was greater than
0.05, visual inspection of the dose-response curve indicates that responses do differ between dose groups.
Selected model in bold; scaled residuals for selected model for concentrations 0, 0.1339, and 0.8671 mg/L were
-0.0916, 0.108, and -0.0167 respectively.
bAlthough a goodness-of-fit p-value was not calculated for the Exponential 3, polynomial, or power models
(due to estimated model parameters = dose groups), inspection of scaled residuals and visual fit indicated
appropriate model fit.
Linear Model with 0.95 Confidence Level
11.5
11
10.5
10
9.5
8.5
7.5
Linear
BMDL
BMD
0.1
0.2
0.3
0.4 0.5
dose
0.6
0.7
0.8
0.9
09:31 04/19 2012
Figure C-2. Plot of mean response by dose (mg/L 1,2,4-TMB) for
decreased red blood cells in male Wistar rats, with fitted curve for
Linear model (BMR = 1 SD, constant variance, high dose dropped).
(Korsak et al.. 2000a)
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Table C-3. Model predictions (constant variance, high dose dropped) for
decreased clotting time in female Wistar rats, 1,2,4-TMB (Korsaketal..
2000a1
Model3
Exponential 2
Exponential 3
Exponential 4b
Linear
Polynomial 2°
Polynomial 3°
Power
Good ness-of -fit
p-value
0.0676
n/a
0.05648
AIC
151.6841
150.3436
151.99019
BMD1SD
(mg/L)
0.624689
0.118085
0.69465
BMDL1SD
(mg/L)
0.35101
0.0006662
0.441274
Basis for Model
Selection
No model selected as
Test 2 p-value was <
0.10
Constant variance case presented (Test 2 p-value = 0.008489). This p-value indicates that a constant variance
model does not adequately describe the observed variances. BMDS recommends using a non-homogenous
variance model.
bp-value not reported due to estimated model parameters = dose groups
Table C-4. Model predictions (modeled variance, high dose dropped) for
decreased clotting time in female Wistar rats, 1,2,4-TMB (Korsaketal..
2000a)
Model"
Exponential 2
Exponential 3
Exponential 4b
Linear
Polynomial 2°
Polynomial 3°
Power
Good ness-of -fit
p-value
0.00949
n/a
0.007771
AIC
150.0056
145.2775
150.362869
BMD1SD
(mg/L)
0.829105
0.154524
0.866447
BMDL1SD
(mg/L)
0.456483
0.000850437
0.533906
Basis for Model
Selection
No model selected as
the only appropriate
fitting model
(Exponential)
returned an
implausibly low BMDL
estimate.
aModeled variance case presented (Test 3 p-value = 0.1159).
bA goodness-of-fit p-value was not calculated for the Exponential 4 model (due to estimated model parameters
= dose groups), inspection of scaled residuals and visual fit indicated appropriate model fit. However, this
model returned an unreasonably low BMDL value. Therefore, this endpoint cannot be modeled in BMDS and
the NOAEL/LOAEL approach is recommended.
This document is a draft for review purposes only and does not constitute Agency policy.
C-5 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Table C-5. Model predictions (constant variance) for decreased fetal
weight in male Sprague-Dawley rats, 1,2,4-TMB fSaillenfait et al., 20051
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5
Hill
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p- value
0.5714
0.8333
0.5714
0.5459
0.5588
0.6217
0.8828
0.9521
0.8432
AIC
-84.27301
-83.91341
-84.27301
-81.91341
-81.936294
-84.509084
-84.028802
-84.179982
-83.937043
BMD1SD
(mg/m )
2,803.48
3,440.45
2,803.48
3,440.45
3,440.86
2,839.22
3,398.61
3,444.47
3,440.84
BMDL1SD
(mg/m3)
2,139.69
2,348.58
2,052.08
2,348.58
2,367.37
2,201.74
2,382.65
2,408.2
2,368.19
Basis for Model
Selection
Of the models that
provided an adequate
fit and valid BMDL
estimate, the linear
model was selected
based on the lowest
AIC (BMDLs differed
by less than 3-fold).
Constant variance case presented (Test 2 p-value = 0.1008), selected model in bold; scaled residuals for
selected model for concentrations 0, 492,1,471, 2,913, and 4,408 mg/m3 were -0.336, -0.324, 0.486, 0.906,
-0.694, respectively.
Table C-6. Model predictions (constant variance) for decreased fetal
weight in male Sprague-Dawley rats, 1,2,4-TMB (Saillenfait et al.. 2005)
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5
Hill
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p-value
0.5714
0.8333
0.5714
0.5459
0.5588
0.6217
0.8828
0.9521
0.8432
AIC
-84.27301
-83.91341
-84.27301
-81.91341
-81.936294
-84.509084
-84.028802
-84.179982
-83.937043
BMD5%
(mg/m )
2,009.49
2,861.09
2,009.49
2,861.09
2,857.59
2,057.05
2,798.98
2,841.49
2,857.43
BMDL5%
(mg/m3)
1,577.44
1,716
1,427.9
1,716
1,749.71
1,640.07
1,760.54
1,777.39
1,750.98
Basis for Model
Selection
Of the models that
provided an adequate
fit and valid BMDL
estimate, the linear
model was selected
based on the lowest
AIC (BMDLs differed
by less than 3-fold).
aConstant variance case presented (Test 2 p-value = 0.1008), selected model in bold; scaled residuals for
selected model for concentrations 0, 492,1,471, 2,913, and 4,408 mg/m3 were -0.336, -0.324, 0.486, 0.906,
-0.694, respectively.
This document is a draft for review purposes only and does not constitute Agency policy.
C-6 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Linear Model with 0.95 Confidence Level
5.8
5.6
5.4
Linear
BMDL
BMD
500
1000
1500
2000 2500
dose
3000
3500
4000
4500
10:32 04/19 2012
Figure C-3. Plot of mean response by dose (mg/m31,2,4-TMB) for
decreased fetal weight in male Sprague-Dawley rats, with fitted curve
for Linear model (BMR = 1 SD, constant variance). (Saillenfaitetal..
2005)
Linear Model with 0.95 Confidence Level
5.8
5.6
5.4
Linear
BMDL
BMD
500
1000
1500
2000 2500
dose
3000
3500
4000
4500
10:35 04/19 2012
Figure C-4. Plot of mean response by dose (mg/m31,2,4-TMB) for
decreased fetal weight in male Sprague-Dawley rats, with fitted curve
for Linear model (BMR = 5% RD, constant variance). (Saillenfait et al..
2005)
This document is a draft for review purposes only and does not constitute Agency policy.
C-7 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Table C-7. Model predictions (constant variance) for decreased fetal
weight in female Sprague-Dawley rats, 1,2,4-TMB (Saillenfaitetal..
20051
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5
Hill
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p- value
0.5056
0.654
0.5056
0.3568
0.3698
0.5547
0.7252
0.832
0.6693
AIC
-101.6488
-101.1358
-101.6488
-99.13583
-99.180649
-101.899075
-101.342513
-101.617243
-101.182018
BMD1SD
(mg/m )
2,650.97
3,312.88
2,650.97
3,312.88
3,311.58
2,692.29
3,258.79
3,322.13
3,311.53
BMDL1SD
(mg/m3)
2,044.51
2,212.4
1,947.94
2,212.4
2,241.33
2,108.65
2,264.38
2,306.76
2,242.38
Basis for Model
Selection
Of the models that
provided an adequate
fit and valid BMDL
estimate, the linear
model was selected
based on the lowest
AIC (BMDLs differed
by less than 3-fold).
Constant variance case presented (Test 2 p-value = 0.3936), selected model in bold; scaled residuals for
selected model for concentrations 0, 492, ,1471, 2,913, and 4,408 mg/m3 were 0.39, -0.187, -0.566, 0.519,
-0.158, respectively.
Table C-8. Model predictions (constant variance) for decreased fetal weight in
female Sprague-Dawley rats. 1,2,4-TMB (Saillenfaitetal.. 2005)
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5
Hill
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p-value
0.5056
0.654
0.5056
0.3568
0.3698
0.5547
0.7252
0.832
0.6693
AIC
-101.6488
-101.1358
-101.6488
-99.13583
-99.180649
-101.899075
-101.342513
-101.617243
-101.182018
BMD5%
(mg/m3)
1,951.39
2,778.64
1,951.39
2,778.64
2,773.5
2,001.36
2,703.42
2,764.88
2,773.32
BMDL5%
(mg/m3)
1,549
1,662.76
1,398.32
1,662.76
1,702.36
1,612.89
1,718.54
1,746.99
1,703.72
Basis for Model
Selection
Of the models that
provided an adequate
fit and valid BMDL
estimate, the linear
model was selected
based on the lowest
AIC (BMDLs differed
by less than 3-fold).
Constant variance case presented
selected model for concentrations
-0.158, respectively.
(Test 2 p-value = 0.3936), selected model in bold; scaled residuals for
0, 492, 1,471, 2,913, and 4,408 mg/m3 were 0.39, -0.187, -0.566, 0.519,
This document is a draft for review purposes only and does not constitute Agency policy.
C-8 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Linear Model with 0.95 Confidence Level
5.8
5.6
5.4
5.2
4.8
10:09 04/19 2012
500 1000 1500
2000 2500
dose
3000 3500 4000 4500
Figure C-5. Plot of mean response by dose (mg/m31,2,4-TMB) for
decreased fetal weight in female Sprague-Dawley rats, with fitted curve
for Linear model (BMR = 1 SD, constant variance). (Saillenfaitetal..
2005)
Linear Model with 0.95 Confidence Level
5.8
5.6
5.4
5.2
4.8
Linear
BMDL
BMD
10:15 04/19 2012
500 1000 1500 2000 2500 3000 3500 4000 4500
dose
Figure C-6. Plot of mean response by dose (mg/m31,2,4-TMB) for
decreased fetal weight in female Sprague-Dawley rats, with fitted curve
for Linear model (BMR = 5% RD, constant variance). (Saillenfait et al..
20051
This document is a draft for review purposes only and does not constitute Agency policy.
C-9 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Table C-9. Model predictions (constant variance) for decreased
maternal weight gain in female Sprague-Dawley rats, 1,2,4-TMB
(Saillenfait et al.. 2005)
Model"
Exponential 2b
Exponential 3
Exponential 4b
Exponential 5
Hill
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p- value
< 0.0001
0.7552
< 0.0001
0.4537
0.593
0.1319
0.7004
0.7393
AIC
1025.385
717.3518
773.2296
719.3518
719.075964
720.406291
717.502596
717.394507
BMD1SD
(mg/m3)
3.67497
2,821.1
Not Computed
2,821.1
2,781.23
2,009.47
2,888.45
2,821.04
BMDL1SD
(mg/m3)
Bad Completion
2,247.99
0
2,247.99
2,161.92
1,649.63
2,132.32
2,129.53
Basis for Model
Selection
Of the models that
provided an adequate
fit and valid BMDL
estimate, the
Exponential 3 model
was selected based on
the lowest AIC (BMDLs
differed by less than 3-
fold).
Constant variance case presented (Test 2 p-value = 0.4284). Selected model in bold; scaled residuals for
selected model for concentrations 0, 492,1,471, 2,913, and 4,408 mg/m3 were -0.1845, 0.5186, -0.4013,
0.1315, -0.2808, respectively.
bThe Exponential 2 and 4 models did not return BMD and/or BMDL values and were excluded from further
consideration.
Exponential Model 3 with 0.95 Confidence Level
40
30
20
10
-10
11:00 04/19 2012
500
1000
1500
2000 2500
dose
3000
3500
4000
4500
Figure C-7. Plot of mean response by dose (mg/m31,2,4-TMB) for
decreased maternal weight gain in female Sprague-Dawley rats, with
fitted curve for Exponential 3 model (BMR = 1 SD, constant variance).
(Saillenfait et al.. 2005)
This document is a draft for review purposes only and does not constitute Agency policy.
C-10 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Table C-10. Model predictions (constant variance) for increased latency
to paw-lick in male Wistar rats, 1,2,3-TMB (Korsak and Rydzynski.
19961
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5b
Hillb
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p- value
0.005704
0.5461
n/a
n/a
0.01728
AIC
262.2082
254.2393
255.8749
255.874906
259.991214
BMD1SD
(mg/m3)
700.938
192.288
201.187
185.863
577.555
BMDL1SD
(mg/m3)
566.333
107.132
111.315
110.398
442.59
Basis for Model
Selection
No model selected as
Test 2 p-value was <
0.1..
Constant variance case presented (Test 2 p-value = 0.0.0001146). This p-value indicates that a constant
variance model does not adequately describe the observed variances. BMDS recommends using a non-
homogenous variance model.
bp-value not reported due to estimated model parameters = dose groups
Table C-ll. Model predictions (modeled variance) for increased latency
to paw-lick in male Wistar rats, 1,2,3-TMB (Korsak and Rydzynski.
1996)
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5b
Hillb
Linear
Polynomial 2°
Polynomial 3°b
Power
Goodness-of-fit
p-value
<0.0001
0.301
n/a
n/a
0.0003247
AIC
259.5324
241.4193
242.5858
265.438765
254.414778
BMD1SD
(mg/m )
496.844
86.2091
113.028
334.7333
319.651
BMDL1SD
(mg/m3)
329.318
46.7265
51.9836
Not calculated
195.989
Basis for Model
Selection
No model selected as
Test 3 p-value was <
0.1.
aModeled variance case presented (Test 3 p-value = 0.07076). This p-value indicates that a modeled variance
model does not adequately describe the observed variances.
bp-value not reported due to estimated model parameters = dose groups
°The 3rd degree polynomial model failed to converge.
This document is a draft for review purposes only and does not constitute Agency policy.
C-ll DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Table C-12. Model predictions (modeled variance, high dose dropped)
for increased latency to paw-lick in male Wistar rats, 1,2,3-TMB
(Korsak and Rydzynski. 1996)
Model3
Exponential 2
Exponential 3
Exponential 4b
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p- value
0.07449
n/a
0.2016
AIC
203.2651
202.0839
201.714812
BMD1SD
(mg/m3)
192.144
104.546
152.065
BMDL1SD
(mg/m3)
131.627
52.5736
97.1911
Basis for Model
Selection
Of the models that
provided an adequate
fit and valid BMDL
estimate, the linear
model was selected
based on the lowest
AIC (BMDLs differed
by less than 3-fold).
aModeled variance case presented (Test 3 p-value = 0.5008). Selected model in bold; scaled residuals for
selected model for concentrations 0,123, and 492 mg/m3 were -0.102, 0.319, and -0.354, respectively.
bAlthough a goodness-of-fit p-value was not calculated for the Exponential 4 model (due to estimated model
parameters = dose groups), inspection of scaled residuals and visual fit indicated appropriate model fit.
Linear Model with 0.95 Confidence Level
22
20
18
16
14
12
10
13:05 03/29 2012
100
200
300
400
500
dose
Figure C-8. Plot of mean response by dose (mg/m31,2,3-TMB) for
increased latency to paw-lick in male Wistar rats, with fitted curve for
Linear model (BMR = 1 SD, modeled variance, high dose dropped).
(Korsak and Rydzynski. 1996)
This document is a draft for review purposes only and does not constitute Agency policy.
C-12 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Table C-13. Model predictions (constant variance) for decreased
segmented neutrophils in male Wistar rats, 1,2,3-TMB (Korsaketal..
2000b1
Model3
Exponential 2
Exponential 3
Exponential 4
Exponential 5b
Hillb
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p- value
0.7155
0.4482
n/a
n/a
0.6711
AIC
189.1052
191.0108
192.4867
192.486705
189.233222
BMD1SD
(mg/m3)
915.77
814.879
547.805
564.348
979.089
BMDL1SD
(mg/m3)
534.809
261.734
137.551
Not calculated
632.777
Basis for Model
Selection
Of the models that
provided an adequate
fit and valid BMDL
estimate, the
Exponential 2 model
was selected based on
the lowest AIC (BMDLs
differed by less than 3-
fold).
Constant variance case presented (Test 2 p-value = 0.2692). Selected model in bold; scaled residuals for
selected model for concentrations 0,123, 492 and 1,230 mg/m3 were -0.16, 0.16, -0.-1.94 x 10"07, and 0.-4.06 x
10"08, respectively.
bA goodness-of-fit p-value was not calculated for the Exponential 5 or Hill models, inspection of scaled residuals
indicated appropriate model fit, however, inspection of visual fit indicated uncertain dose-response
characteristics, and therefore, these models were excluded from consideration.
Exponential Model 2 with 0.95 Confidence Level
30
25
20
10
09:14 04/19 2012
1200
Figure C-9. Plot of mean response by dose (mg/m31,2,3-TMB) for
decreased segmented neutrophils in male Wistar rats, with fitted curve
for Exponential 2 model (BMR = 1 SD, constant variance). (Korsaketal..
2000b1
This document is a draft for review purposes only and does not constitute Agency policy.
C-13 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Table C-14. Model predictions (constant variance) for decreased
segmented neutrophils in female Wistar rats, 1,2,3-TMB (Korsaketal..
2000b1
Model3
Exponential 2
Exponential 3
Exponential 4
Exponential 5
Hill
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p- value
0.6401
0.5208
0.5692
0.4533
AIC
177.6514
179.1714
179.083138
178.341743
BMD1SD
(mg/m3)
517.048
365.397
337.442
645.521
BMDL1SD
(mg/m3)
334.805
134.354
99.2111
465.309
Basis for Model
Selection
Of the models that
provided an adequate
fit and valid BMDL
estimate, the Hill
model was selected
based on the lowest
BMDL (BMDLs differed
by more than 3-fold).
aConstant variance case presented (Test 2 p-value = 0.09252). Although this p-value is less than 0.10, it indicates
a marginal fit at the 95% confidence level, and therefore a constant variance is determined to adequately fit the
observed variance data. Selected model in bold; scaled residuals for selected model for concentrations 0,128,
523, and 1,269 mg/m3 were 0.209, -0.412, 0.312, and -0.108, respectively.
Hill Model with 0.95 Confidence Level
25
20
15
10
Hill
BMDL
BMD
0
09:03 04/19 2012
200
400
600
dose
800
1000
1200
Figure C-10. Plot of mean response by dose (mg/m31,2,3-TMB) for
decreased segmented neutrophils in female Wistar rats, with fitted
curve for Hill model (BMR = 1 SD, constant variance). (Korsaketal..
2000b1
This document is a draft for review purposes only and does not constitute Agency policy.
C-14 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Table C-15. Model predictions (constant variance) for increased
reticulocytes in male Wistar rats, 1,2,3-TMB (Korsaketal.. 2000b)
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5b
Hill'
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p- value
0.2733
0.1397
n/a
n/a
0.3105
AIC
89.08418
90.67033
91.37006
91.370061
88.828645
BMD1SD
(mg/m3)
1112.25
900.404
540.186
554.848
1025.1
BMDL1SD
(mg/m3)
806.744
308.017
140.925
Not calculated
652.898
Basis for Model
Selection
Of the models that
provided an adequate
fit and valid BMDL
estimate, the Linear
model was selected
based on the lowest
AIC (BMDLs differed
by less than 3-fold).
Constant variance case presented (Test 2 p-value = 0.5223). Selected model in bold; scaled residuals for
selected model for concentrations 0,128, 523 and 1,269 mg/m3 were 0.555, -1.14, 0.793, and -0.212,
respectively.
bA goodness-of-fit p-value was not calculated for the Exponential 5 or Hill models, inspection of scaled residuals
indicated appropriate model fit, however, inspection of visual fit indicated uncertain dose-response
characteristics, and therefore, these models were excluded from consideration.
Linear Model with 0.95 Confidence Level
Linear
BMDL
BMD
200
400
600
dose
800
1000
1200
09:52 04/19 2012
Figure C-ll. Plot of mean response by dose (mg/m31,2,3-TMB) for
increased reticulocytes in male Wistar rats, with fitted curve for Linear
model (BMR = 1 SD, constant variance). (Korsaketal.. 2000b)
This document is a draft for review purposes only and does not constitute Agency policy.
C-15 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Table C-16. Model predictions (constant variance) for decreased fetal
weight in male Sprague-Dawley rats, 1,3,5-TMB (Saillenfait et al.. 2005)
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5
Hill
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p- value
0.6927
0.6981
0.397
0.4094
0.6496
AIC
-66.94125
-65.6776
-63.67902
-63.715888
-66.753074
BMD1SD
(mg/m3)
3,396.62
2,604.81
2,603.37
2,572.4
3,513.03
BMDL1SD
(mg/m3)
2,560.01
1,341.07
1,341.3
1,274.69
2,694.51
Basis for Model
Selection
No model selected as
Test 2 p-value was <
0.10
Constant variance case presented (Test 2 p-value = 0.002368), this p-value indicates that a constant variance
model does not adequately describe the observed variances. BMDS recommends using a non-homogenous
variance model.
Table C-17. Model predictions (modeled variance) for decreased fetal
weight in male Sprague-Dawley rats, 1,3,5-TMB (Saillenfait et al.. 2005)
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5
Hill
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p-value
0.5214
0.4304
0.3877
0.4276
0.4791
AIC
-73.29149
-71.85947
-70.79949
-65.644335
-73.066751
BMD1SD
(mg/m3)
2,523.27
2,041.7
2,044.66
2,407.38
2,636.36
BMDL1SD
(mg/m3)
1,779.29
1,125.34
1,237.6
1,295.43
1,890.46
Basis for Model
Selection
No model selected as
Test 3 p-value was <
0.10
Modeled variance case presented (Test 3 p-value = 0.06027, except the Hill model, for which Test 3 p-value =
0.00544). This p-value indicates that a modeled variance model does not adequately describe the observed
variances. Therefore, this endpoint cannot be modeled in BMDS and the NOAEL/LOAEL approach is
recommended.
This document is a draft for review purposes only and does not constitute Agency policy.
C-16 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofTrimethylbenzene
Table C-18. Model predictions (modeled variance) for decreased fetal
weight in male Sprague-Dawley rats, 1,3,5-TMB (Saillenfait et al.. 2005)
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5
Hill
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p- value
0.5214
0.4304
0.3877
0.4276
0.4791
AIC
-73.29149
-71.85947
-70.79949
-65.644335
-73.066751
BMD5%
(mg/m3)
2,187.66
1,781.91
1,872.45
1,652.76
2,282.12
BMDL5%
(mg/m3)
1,645.82
1,025.78
1,137.83
793.582
1,744.39
Basis for Model
Selection
No model selected as
Test 3 p-value was <
0.10
aModeled variance case presented (Test 3 p-value = 0.06027, except the Hill model, for which Test 3 p-value =
0.00544). This p-value indicates that a modeled variance model does not adequately describe the observed
variances. Therefore, this endpoint cannot be modeled in BMDS and the NOAEL/LOAEL approach is
recommended.
Table C-19. Model predictions (constant variance) for decreased fetal
weight in female Sprague-Dawley rats, 1,3,5-TMB (Saillenfait et al..
2005)
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5
Hill
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p-value
0.9112
0.7655
0.7656
0.9085
AIC
-61.96218
-59.96227
-59.962704
-61.950195
BMD1SD
(mg/m3)
3,581.71
3,573.06
3,569.61
3,676.95
BMDL1SD
(mg/m3)
2,669
1,915.99
1,865.62
2,794.36
Basis for Model
Selection
No model selected as
Test 2 p-value was <
0.10
aConstant variance case presented (Test 2 p-value < 0.0001), this p-value indicates that a constant variance
model does not adequately describe the observed variances. BMDS recommends using a non-homogenous
variance model.
This document is a draft for review purposes only and does not constitute Agency policy.
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Table C-20. Model predictions (modeled variance) for decreased fetal
weight in female Sprague-Dawley rats, 1,3,5-TMB (Saillenfaitetal..
20051
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5
Hill
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p- value
0.01931
0.05097
0.5334
0.4769
0.0148
0.01552
AIC
-67.53742
-69.49883
-73.06401
-59.505126
-67.061071
-67.061071
BMD1SD
(mg/m3)
2692.79
1481.66
1469.46
3161.1
2841.13
2841.13
BMDL1SD
(mg/m3)
1827.72
798.275
1069.57
1614.44
1969.76
1969.76
Basis for Model
Selection
No model selected as
Test 3 p-value was <
0.10
aModeled variance case presented (Test 3 p-value = 0.01301), this p-value indicates that the modeled variance
does not adequately describe the observed variances. Therefore, this endpoint cannot be modeled in BMDS
and the NOAEL/LOAEL approach is recommended.
Table C-21. Model predictions (modeled variance) for decreased fetal
weight in female Sprague-Dawley rats, 1,3,5-TMB (Saillenfaitetal..
2005)
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5
Hill
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p-value
0.01931
0.05097
0.5334
0.4769
0.0148
0.01552
AIC
-67.53742
-69.49883
-73.06401
-59.505126
-67.061071
-67.061071
BMD5%
(mg/m3)
2,244.13
1,447.04
1,472.61
2,009.89
2,346.47
2,346.47
BMDL5%
(mg/m3)
1,633.96
850.802
1,125.04
928.261
1,739.45
1,739.45
Basis for Model
Selection
No model selected as
Test 3 p-value was <
0.10
aModeled variance case presented (Test 3 p-value = 0.01301), this p-value indicates that the modeled variance
does not adequately describe the observed variances. Therefore, this endpoint cannot be modeled in BMDS
and the NOAEL/LOAEL approach is recommended.
This document is a draft for review purposes only and does not constitute Agency policy.
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Table C-22. Model predictions (constant variance) for decreased
maternal weight gain in female Sprague-Dawley rats, 1,3,5-TMB
(Saillenfait et al.. 2005)
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5
Hill
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p- value
< 0.0001
< 0.0001
< 0.0001
0.00262
0.5141
0.6919
0.4835
AIC
805.8321
807.8353
701.8275
649.4267
639.963339
636.99599
638.991033
BMD1SD
(mg/m3)
3.36 x 10"51
6.29281
Not_Computed
2,057.15
2,035.36
1,982.21
2,014.88
BMDL1SD
(mg/m3)
Bad_Completion
Bad_Completion
0
1,396.23
1,353.4
1,655.52
1,655.77
Basis for Model
Selection
No model selected as
Test 2 p-value was <
0.10
Constant variance case presented (Test 2 p-value =
model does not adequately describe the observed
variance model.
= 0.003114), this p-value indicates that a constant variance
variances. BMDS recommends using a non-homogenous
Table C-23. Model predictions (modeled variance) for decreased
maternal weight gain in female Sprague-Dawley rats, 1,3,5-TMB
(Saillenfait et al.. 2005)
Model"
Exponential 2b
Exponential 3B
Exponential 4
Exponential 5
Hill
Linear
Polynomial 2°
Polynomial 3°
Power
Goodness-of-fit
p-value
< 0.0001
< 0.0001
< 0.0001
< 0.0001
<.0001
0.0003338
<.0001
0.2014
0.1981
AIC
921.089
923.089
698.0766
650.9354
728.727708
645.262934
710.199993
631.886974
631.236865
BMD1SD
(mg/m3)
Not_Computed
Not_Computed
3.76 x 10"46
1,476.12
29.7037
2,749.72
-9,999
1,797.1
1,826.86
BMDL1SD
(mg/m3)
0
0
3.76 x 10"46
601.777
11.8372
2,330.78
2,491.63
Not calculated
1,302.02
Basis for Model
Selection
Only the power model
provided an adequate
fit and calculated a
BMDandBMDL, and
therefore was
selected.
Modeled variance case presented (Test 3 p-value = 0.2221). Selected model in bold; scaled residuals for
selected model for concentrations 0, 497,1,471, 2,974, 5,874 mg/m3 were -0.442, 0.983, -0.47, -0.776, 0.0673,
respectively.
bThe Exponential 2 and 3 models did not return BMD and/or BMDL values and were excluded from further
consideration.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Power Model with 0.95 Confidence Level
40
30
20
10
-10
-20
Power
1000
2000
11:14 04/19 2012
3000
dose
4000
5000
6000
Figure C-12. Plot of mean response by dose (mg/m31,3,5-TMB) for
decreased maternal weight gain in female Sprague-Dawley rats, with
fitted curve for Power model (BMR = 1 SD, modeled variance).
(Saillenfait et al.. 2005)
This document is a draft for review purposes only and does not constitute Agency policy.
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C.2. BENCHMARK DOSE MODELING SUMMARY - ALTERNATIVE ANALYSIS
WITH HIGH DOSES INCLUDED
1 The modeling summaries included in this section are for comparison purposes only.
2 After calculation of internal blood dose metrics using the animal PBPK model, the high
3 doses were not dropped in these modeling analyses, even though the PBPK demonstrates
4 poor model fit at high doses. These modeling results were not used in any RfC derivations
5 in Volume 1 of the Toxicological Review.
This document is a draft for review purposes only and does not constitute Agency policy.
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Table C-24. Model predictions (constant variance) for increased latency
to paw-lick in male Wistar rats, 1,2,4-TMB (Korsak and Rydzynski.
19961
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5b
Hill
Linear
Polynomial 2°
Polynomial 3°
Power
Good ness-of -fit
p-value
0.00061
0.8239
n/a
n/a
0.0009125
AIC
190.1611
177.4066
179.3571
179.357065
189.355645
BMD1SD
(mg/L)
3.62226
0.242222
0.268238
0.237108
3.15451
BMDL1SD
(mg/L)
2.73586
0.104385
0.105201
0.0889465
2.22737
Basis for Model
Selection
No model selected as
Test 2 p-value was <
0.10
Constant variance case presented (Test 2 p-value = 0.07651). BMDS recommends using a non-homogenous
variance model.
bp-value not reported due to estimated model parameters = dose groups
Table C-25. Model predictions (modeled variance) for increased latency
to paw-lick in male Wistar rats, 1,2,4-TMB (Korsak and Rydzynski.
19961
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5b
Hillb
Linear
Polynomial 2°
Polynomial 3°
Power
Good ness-of -fit
p-value
0.000633
0.8604
n/a
n/a
0.001014
AIC
191.8156
179.1164
181.0855
181.982905
190.872265
BMD1SD
(mg/L)
3.38239
0.231414
0.252014
0.292816
2.8175
BMDL1SD
(mg/L)
2.34048
0.09854
0.0990336
Not calculated
1.72529
Basis for Model
Selection
No model selected as
Test 2 p-value was <
0.10
Modeled variance case presented (Test 3 p-value = 0.0371). This p-value indicates that a modeled variance
model does not adequately describe the observed variances. Therefore, this endpoint cannot be modeled in
BMDS and the NOAEL/LOAEL approach is recommended.
b bA goodness-of-fit p-value was not calculated for the Exponential 5 or Hill models, inspection of scaled
residuals and visual fit indicated appropriate model fit. However, the Hill model failed to calculate a BMDL and
was excluded from consideration.
This document is a draft for review purposes only and does not constitute Agency policy.
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Table C-26. Model predictions (constant variance) for decreased red
blood cells in male Wistar rats, 1,2,4-TMB (Korsak et al.. 2000a)
Model3
Exponential 2
Exponential 3
Exponential 4
Exponential 5b
Hillb
Linear
Polynomial 2°
Polynomial 3°
Power
Good ness-of -fit
p-value
0.1671
0.7345
n/a
n/a
0.1498
AIC
78.98918
77.52579
79.41075
79.410749
79.207001
BMD1SD
(mg/L)
3.68518
0.795033
0.842867
0.835638
3.91553
BMDL1SD
(mg/L)
2.30432
0.241565
0.249166
0.212686
2.5963
Basis for Model
Selection
Of the models that
provided an adequate
fit and a valid BMDL
estimate, the Hill
model was selected
(BMDLs differed by
greater than 3-fold)
Constant variance case presented (Test 2 p-value = 0.4329). Selected model in bold; scaled residuals for
selected model for concentrations 0, 0.1339, 0.8671, 5.248 mg/L were -1.93 xlO"08,1.75x 10"08, 4.83 x 10"08and
-6.99 x 10"08, respectively.
bAlthough the Exponential 5 and Hill model returned no goodness-of-fit p-value, inspection of scaled residuals
and visual fit indicated appropriate model fit.
Hill Model with 0.95 Confidence Level
11
10
Hill
: BMDL
BMD
dose
09:24 04/19 2012
Figure C-13. Plot of mean response by dose (mg/L 1,2,4-TMB) for
decreased red blood cells in male Wistar rats, with fitted curve for Hill
model (BMR = 1 SD, constant variance). (Korsak etal.. 2000a)
This document is a draft for review purposes only and does not constitute Agency policy.
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Table C-27. Model predictions (constant variance) for decreased
clotting time in female Wistar rats (Korsaketal.. 2000a)
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5b
Hillb
Linear
Polynomial 2°
Polynomial 3°
Power
Good ness-of -fit
p-value
0.00311
0.3078
n/a
n/a
0.003013
AIC
207.7609
199.2547
201.2538
201.25379
207.824506
BMD1SD
(mg/L)
13.2329
0.119261
0.12336
0.129946
12.5899
BMDL1SD
(mg/L)
4.78502
0.000258705
0.000534297
1.20 x 10"10
5.12676
Basis for Model
Selection
No model selected as
Test 2 p-value was <
0.10
aConstant variance case presented (Test 2 p-value = 0.02286). This p-value indicates that a constant variance
model does not adequately describe the observed variances. BMDS recommends using a non-homogenous
variance model.
bp-value not reported due to estimated model parameters = dose groups
Table C-28. Model predictions (modeled variance) for decreased
clotting time in female Wistar rats (Korsaketal.. 2000a)
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5b
Hillb
Linear
Polynomial 2°
Polynomial 3°
Power
Good ness-of -fit
p-value
0.0001725
0.09227
n/a
n/a
0.0001675
AIC
209.2185
196.7223
198.7223
204.758516
209.276823
BMD1SD
(mg/L)
16.2811
0.297031
0.235929
0.138361
15.0257
BMDL1SD
(mg/L)
5.15229
0.000698259
7.68 x 10"05
Not calculated
5.46511
Basis for Model
Selection
No model selected as
the only appropriate
fitting models
(Exponential 5 and
Hill) either calculated
no BMDL, or
calculated an
implausibly low BMDL
aModeled variance case presented (Test 3 p-value = 0.2001, except Hill model for which Test 3 p-value = <
0.0001).
bAlthough the Exponential 5 and Hill model returned no goodness-of-fit p-value, inspection of scaled residuals
and visual fit indicated appropriate model fit. However, these models either failed to calculate a BMDL or
calculated a BMDL that is biologically unreasonably low. Therefore, this endpoint cannot be modeled in BMDS
and the NOAEL/LOAEL approach is recommended.
This document is a draft for review purposes only and does not constitute Agency policy.
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Table C-29. Model predictions (constant variance) for decreased
reticulocytes in female Wistar rats, 1,2,4-TMB (Korsaketal.. 2000a)
Model"
Exponential 2
Exponential 3
Exponential 4
Exponential 5
Hill
Linear
Polynomial 2°
Polynomial 3°
Power
Good ness-of -fit
p-value
0.05738
0.2784
n/a
0.3149
0.04654
AIC
91.21206
88.67076
90.67077
88.506257
91.631076
BMD1SD
(mg/L)
5.67056
0.107641
0.111117
0.11386
6.34191
BMDL1SD
(mg/L)
0.775822
0.000190582
0.000273446
6.85 x 10"15
3.62271
Basis for Model
Selection
No model selected as
Test 2 p-value was <
0.10
Constant variance case presented (Test 2 p-value = < 0.0001). This p-value indicates that a constant variance
model does not adequately describe the observed variances. BMDS recommends using a non-homogenous
variance model.
bp-value not reported due to estimated model parameters = dose groups
Table C-30. Model predictions (modeled variance) for decreased
reticulocytes in female Wistar rats, 1,2,4-TMB (Korsak et al.. 2000a)
Model"
Exponential 2
Exponential 3
Exponential 4b
Exponential 5b
Hill1
Linear
Polynomial 2°
Polynomial 3°
Power
Good ness-of -fit
p-value
0.01667
0.3582
n/a
0.009093
AIC
75.37239
70.02825
89.127269
76.584735
BMD1SD
(mg/L)
12.0859
Not_Computed
Not_Computed
8.44761
BMDL1SD
(mg/L)
4.65557
0
Not_Computed
5.29336
Basis for Model
Selection
No model selected as
the only appropriate
fitting model
(Exponential, 5, and
Hill) calculated no
BMDL
aModeled variance case presented (Test 3 p-value = 0.253).
bAlthough the Exponential 4 and 5 models display appropriate goodness-of-fit p-values, these models do not
calculate BMD or BMDL values. As these are the only appropriately fitting models, this endpoint cannot be
modeled in BMDS and the NOAEL/LOAEL approach is recommended.
c p-value not reported due to estimated model parameters = dose groups
This document is a draft for review purposes only and does not constitute Agency policy.
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APPENDIX D. DOCUMENTATION OF IMPLEMENTATION OF THE 2011
NATIONAL RESEARCH COUNCIL RECOMMENDATIONS
1 Documentation of the IRIS Program's Implementation of the 2011 NRC Recommendations in the
2 External Peer Review Draft Toxicological Review of Trimethylbenzenes (June 2012)
3
4 Background: On December 23, 2011, The Consolidated Appropriations Act, 2012, was signed into law1. The report language included direction
5 to EPA for the IRIS Program related to recommendations provided by the National Research Council (NRC) in their review of EPA's draft IRIS
6 assessment of formaldehyde2. The report language included the following:
7
8 "The Agency shall incorporate, as appropriate, based on chemical-specific datasets and biological effects, the recommendations of
9 Chapter 7 of the National Research Council's Review of the Environmental Protection Agency's Draft IRIS Assessment of Formaldehyde
10 into the IRIS process...For draft assessments released in fiscal year 2012, the Agency shall include documentation describing how the
11 Chapter 7 recommendations of the National Academy of Sciences (NAS) have been implemented or addressed, including an explanation
12 for why certain recommendations were not incorporated."
13
14 The NRC's recommendations, provided in Chapter 7 of their review report, offered suggestions to EPA for improving the development of IRIS
15 assessments. Consistent with the direction provided by Congress, documentation of how the recommendations from Chapter 7 of the NRC
16 report have been implemented in this assessment is provided in the table below. Where necessary, the documentation includes an explanation
17 for why certain recommendations were not incorporated.
18
19 The IRIS Program's implementation of the NRC recommendations is following a phased approach that is consistent with the NRC's "Roadmapfor
20 Revision" as described in Chapter 7 of the formaldehyde review report. The NRC stated that "the committee recognizes that the changes
21 suggested would involve a multi-year process and extensive effort by the staff at the National Center for Environmental Assessment and input
22 and review by the EPA Science Advisory Board and others."
23
24 Phase 1 of implementation has focused on a subset of the short-term recommendations, such as editing and streamlining documents, increasing
25 transparency and clarity, and using more tables, figures, and appendices to present information and data in assessments. Phase 1 also focused
Vub. L No. 112-74, Consolidated Appropriations Act, 2012.
2National Research Council, 2011. Review of the Environmental Protection Agency's Draft IRIS Assessment of Formaldehyde.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
1 on assessments near the end of the development process and close to final posting. The IRISTMBs assessment is in Phase 2 of implementation,
2 which addresses all of the short-term recommendations from Table 1. The Program is implementing all of these recommendations but
3 recognizes that achieving full and robust implementation of certain recommendations will be an evolving process with input and feedback from
4 the public, stakeholders, and external peer review committees. Phase 3 of implementation will incorporate the longer-term recommendations
5 made by the NRC as outlined below in Table 2, including the development of a standardized approach to describe the strength of evidence for
6 noncancer effects . On May 16, 2012, EPA announced3 that as a part of a review of the IRIS Program's assessment development process, the NRC
7 will also review current methods for weight-of-evidence analyses and recommend approaches for weighing scientific evidence for chemical
8 hazard identification. This effort is included in Phase 3 of EPA's implementation plan.
9
10
11
3EPA Announces NAS' Review of IRIS Assessment Development Process (www.epa.gov/iris)
This document is a draft for review purposes only and does not constitute Agency policy.
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Table D-l. National Research Council recommendations
that EPA is implementing in the short term
Implementation status
General recommendations for completing the IRIS formaldehyde assessment that EPA will adopt for all IRIS assessments (p. 152 of the NRC report)
1. To enhance the clarity of the document, the draft IRIS assessment needs
rigorous editing to reduce the volume of text substantially and address
redundancies and inconsistencies. Long descriptions of particular studies
should be replaced with informative evidence tables. When study details are
appropriate, they could be provided in appendices.
Implemented. The overall document structure has been revised in consideration
of this NRC recommendation. The new structure includes a concise Executive
Summary and an explanation of the literature review search strategy, study
selection criteria, and methods used to develop the assessment. The main body of
the assessment has been reorganized into two sections, Hazard Identification and
Dose-Response Analysis, to help reduce the volume of text and redundancies that
were a part of the previous document structure. Section 1 provides evidence
tables and a concise synthesis of hazard information organized by health effect,
More detailed summaries of the most pertinent epidemiology and experimental
animal studies are provided in Appendix B. Information on chemical and physical
properties and toxicokinetics is also provided in Appendix B. The main text of the
Toxicological Review is approximately 90 pages, which is a major reduction from
previous IRIS assessments. Technical and scientific edits were performed to
eliminate any redundancies or inconsistencies.
2. Chapter 1 needs to be expanded to describe more fully the methods of the
assessment, including a description of search strategies used to identify
studies with the exclusion and inclusion criteria articulated and a better
description of the outcomes of the searches and clear descriptions of the
weight-of-evidence approaches used for the various noncancer outcomes.
The committee emphasizes that it is not recommending the addition of long
descriptions of EPA guidelines to the introduction, but rather clear concise
statements of criteria used to exclude, include, and advance studies for
derivation of the RfCs and unit risk estimates.
Implemented. Chapter 1 has been replaced with a Preamble that describes the
application of existing EPA guidance and the methods and criteria used in
developing the assessment. The term "Preamble" was chosen to emphasize that
these methods and criteria are being applied consistently across IRIS assessments.
The new Preamble includes information on identifying and selecting pertinent
studies, evaluating the quality of individual studies, weighing the overall evidence
of each effect, selecting studies for derivation of toxicity values, and deriving
toxicity values. These topics correspond directly to the five steps that the NRC
identified in Figure 7-2 of their 2011 report.
A new section, Literature Search Strategy and Study Selection, provides detailed
information on the search strategy used to identify health effect studies, search
outcomes, and selection of studies for hazard identification. This information is
chemical-specific and has been designed to provide enough information that an
This document is a draft for review purposes only and does not constitute Agency policy.
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Table D-l. National Research Council recommendations
that EPA is implementing in the short term
Implementation status
independent literature search would be able to replicate the results. This section
also includes information on how studies were selected to be included in the
document and provides a link to EPA's Health and Environmental Research Online
(HERO) database (www.epa.gov/hero) that contains the references that were
cited in the document, along with those that were considered but not cited.
3. Standardized evidence tables for all health outcomes need to be
developed. If there were appropriates tables, long text descriptions of studies
could be moved to an appendix of deleted.
Implemented. In the new document template, standardized evidence tables that
present key study findings that support how toxicological hazards are identified
for all major health effects are provided in Section 1.1. More detailed summaries
of the most pertinent epidemiology and experimental animal studies are provided
in Appendix B.
4. All critical studies need to be thoroughly evaluated with standardized
approaches that are clearly formulated and based on the type of research, for
example, observational epidemiologic or animal bioassays. The findings of
the reviews might be presented in tables to ensure transparency.
Implemented. Information in Section 4 of the Preamble provides an overview of
the approach used to evaluate the quality of individual studies. Critical evaluation
of the epidemiologic and experimental animal studies and is included in the
evidence tables in Section 1.1. Additional information on study characteristics is
found in Appendix B. The study information for TMBs is presented in table format
that clearly presents detailed study summary information and key study
characteristics.
5. The rationales for the selection of the studies that are advanced for
consideration in calculating the RfCs and unit risks need to be expanded. All
candidate RfCs should be evaluated together with the aid of graphic displays
that incorporate selected information on attributes relevant to the database.
Implemented. The Dose-Response Analysis section of the new document
structure provides a clear explanation of the rationale used to select and advance
studies that were considered for calculating toxicity values. Rationales for the
selection of studies advanced for reference value derivation are informed by the
weight-of-evidence for hazard identification as discussed in Section 1.2. In
support of the RfC derivations for individual TMB isomers, an exposure-response
array was included that compares effect levels for several toxicological effects
(Figures 2-1, 2-3, and 2-5). The exposure-response array provides a visual
representation of points of departure for various effects resulting from exposure
to TMB isomers. The array informs the identification of doses associated with
specific effects, and the choice of principal study and critical effects. In the case of
TMBs, the database supported development of multiple candidate RfC's. Such
This document is a draft for review purposes only and does not constitute Agency policy.
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Table D-l. National Research Council recommendations
that EPA is implementing in the short term
Implementation status
values have been developed previously and will be developed in future
assessments, where the data allow.
6. Strengthened, more integrative and more transparent discussions of
weight-of-evidence are needed. The discussions would benefit from more
rigorous and systematic coverage of the various determinants of weight-of-
evidence, such as consistency.
Partially implemented. A new section, Hazard Identification (Section 1), provides
a more strengthened, integrated and transparent discussion of the weight of the
available evidence. This section includes standardized evidence tables to present
the key study findings that support how potential toxicological hazards are
identified and exposure-response arrays for each potential toxicological effect.
Weight-of-evidence discussions are provided for each major effect (Section
1.1.1—neurotoxic effects, Section 1.1.2—respiratory effects, Section 1.1.3—
reproductive/developmental effects, and Section 1.1.4—hematological and
clinical chemistry effects). A more rigorous and formalized approach for
characterizing the weight-of-evidence will be developed as a part of Phase 3 of the
implementation process.
Other specific recommendations (p. # in NRC report)
General Guidance for the Overall Process (p. 164)
7. Elaborate an overall, documented, and quality-controlled process for IRIS
assessments.
8. Ensure standardization of review and evaluation approaches among
contributors and teams of contributors; for example, include standard
approaches for reviews of various types of studies to ensure uniformity.
9. Assess disciplinary structure of teams needed to conduct the assessments.
Implemented. EPA has created Chemical Assessment Support Teams to formalize
an internal process to provide additional overall quality control for the
development of IRIS assessments. This initiative uses a team approach to making
timely, consistent decisions about the development of IRIS assessments across the
Program. This team approach has been utilized for the development of theTMBs
assessment. Additional objectives of the teams is to help ensure that the
necessary disciplinary expertise is available for assessment development and
review, to provide a forum for identifying and addressing key issues prior to
external peer review, and to monitor progress in implementing the NRC
recommendations.
This document is a draft for review purposes only and does not constitute Agency policy.
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Table D-l. National Research Council recommendations
that EPA is implementing in the short term
Evidence Identification: Literature Collection and Collation Phase (p. 164)
10. Select outcomes on the basis of available evidence and understanding of
mode of action.
11. Establish standard protocols for evidence identification.
12. Develop a template for description of the search approach.
13. Use a database, such as the Health and Environmental Research Online
(HERO) database, to capture study information and relevant quantitative
data.
Evidence Evaluation: Hazard Identification and Dose-Response Modeling (p.
165)
14. Standardize the presentation of reviewed studies in tabular or graphic
form to capture the key dimensions of study characteristics, weight-of-
evidence, and utility as a basis for deriving reference values and unit risks.
15. Develop templates for evidence tables, forest plots, or other displays.
16. Establish protocols for review of major types of studies, such as
epidemiologicand bioassay.
Implementation status
Implemented. A new section, Literature Search Strategy and Study Selection,
contains detailed information on the search strategy used for the TMBs
assessment, including key words used to identify relevant health effect studies.
Figure LS-1 depicts the study selection strategy and the number of references
obtained at each stage of literature screening. This section also includes
information on how studies were selected to be included in the document and
provides a link to an external database (www.epa.gov/hero) that contains the
references that were cited in the document, along with those that were
considered but not cited. Each citation in the Toxicological Review is linked to
HERO such that the public can access the references and abstracts to the scientific
studies used in the assessment.
Section 3 of the Preamble summarizes the standard protocols for evidence
identification that are provided in EPA guidance. For each potential toxicological
effect identified for ammonia, the available evidence is informed by the mode of
action information as discussed in Section 1.1.
Implemented. Standardized tables have been developed that provide summaries
of key study design information and results by health effect. The inclusion of all
positive and negative findings in each health effect-specific evidence table
supports a weight-of-evidence analysis. In addition, exposure-response arrays are
utilized in the assessment to provide a graphical representation of points of
departure for various effects resulting from exposure to TMB. The exposure-
response arrays inform the identification of doses associated with specific effects
and the weight-of- evidence for those effects.
Implemented. Templates for evidence tables and exposure-response arrays have
been developed and are utilized in Section 1.1.
Implemented. General principles for reviewing epidemiologic and experimental
animal studies are described in Section 4 of the Preamble.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table D-l. National Research Council recommendations
that EPA is implementing in the short term
Selection of Studies for Derivation of Reference Values and Unit Risks (p.
165)
17. Establish clear guidelines for study selection.
a. Balance strengths and weaknesses.
b. Weigh human vs. experimental evidence.
c. Determine whether combining estimates among studies is warranted.
Calculation of Reference Values and Unit Risks (pp. 165-166)
18. Describe and justify assumptions and models used. This step includes
review of dosimetry models and the implications of the models for
uncertainty factors; determination of appropriate points of departure (such as
benchmark dose, no-observed-adverse-effect level, and lowest observed-
adverse-effect level), and assessment of the analyses that underlie the points
of departure.
19. Provide explanation of the risk-estimation modeling processes (for
example, a statistical or biologic model fit to the data) that are used to
develop a unit risk estimate.
20. Provide adequate documentation for conclusions and estimation of
Implementation status
Implemented. EPA guidelines for study selection, including balancing strengths
and weaknesses and weighing human vs. experimental evidence are described in
the Preamble (Sections 3-6). These guidelines have been applied in Section 2 of
theTMBs assessment to inform the evaluation of the weight-of-evidence across
health effects and the strengths and weaknesses of individual studies considered
for reference value derivation.
In the case of TMBs, the database did not support the combination of estimates
across studies. In future assessments, combining estimates across studies will be
routinely considered.
Implemented as applicable.
The rationale for the selection of the point of departure (a 95% lower confidence
limit on the benchmark dose; BMDL) for the derivation of the inhalation reference
value for 1,2,4-TMB and 1,2,3-TMB is transparently described in Section 2. The
determination of sufficient similarity regarding 1,3,5-TMB and 1,2,4-TMB, and the
decision to adopt the RfC for 1,2,4-TMB as the RfC for 1,3,5-TMB, is transparently
described in Section 2.
The rationale for the route-to-route extrapolation in order to use inhalation data
for derivation of an RfD for 1,2,4-TMB is transparently described in Section 2. The
determination of sufficient similarity regarding 1,2,3-, 1,2,4-, and 1,3,5-TMB, and
the decision to adopt the RfD for 1,2,4-TMB as the RfDs for 1,2,3-TMB and 1,3,5-
TMB, is transparently described in Section 2.
A summary of the benchmark dose modeling for the derivation of the reference
values for effects other than cancer, including an alternative analysis with high
doses included, is described in Appendix C.
Not applicable. The TMB assessment concludes that there is inadequate
information to assess the carcinogenic potential. Therefore, a unit risk estimate
for cancer was not derived.
Implemented. The new template structure that has been developed in response
This document is a draft for review purposes only and does not constitute Agency policy.
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Table D-l. National Research Council recommendations
that EPA is implementing in the short term
Implementation status
reference values and unit risks. As noted by the committee throughout the
present report, sufficient support for conclusions in the formaldehyde draft
IRIS assessment is often lacking. Given that the development of specific IRIS
assessments and their conclusions are of interest to many stakeholders, it is
important that they provide sufficient references and supporting
documentation for their conclusions. Detailed appendixes, which might be
made available only electronically, should be provided when appropriate.
to the NRC recommendations provides a clear explanation of the literature search
strategy, study selection criteria, and methods used to develop the TMBs
assessment. It provides for a clear description of the decisions made in developing
the hazard identification and dose-response analysis. Information contained in the
Preamble and throughout the document reflects the guidance that has been
utilized in developing the assessment. As recommended, supplementary
information is provided in the accompanying appendices.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofTrimethylbenzene
Table D-2. National Research Council recommendations
that EPA is implementing in the long-term (p. # in NRC
report)
Implementation status
Weight-of-Evidence Evaluation: Synthesis of Evidence for Hazard
Identification (p. 165)
1. Review use of existing weight-of-evidence guidelines.
2. Standardize approach to using weight-of-evidence guidelines.
3. Conduct agency workshops on approaches to implementing weight-of-
evidence guidelines.
4. Develop uniform language to describe strength of evidence on noncancer
effects.
5. Expand and harmonize the approach for characterizing uncertainty and
variability.
6. To the extent possible, unify consideration of outcomes around common
modes of action rather than considering multiple outcomes separately.
As indicated above, Phase 3 of EPA's implementation plan will incorporate
the longer-term recommendations made by the NRC, including the
development of a standardized approach to describe the strength of
evidence for noncancer effects. On May 16, 2012, EPA announced that as
a part of a review of the IRIS Program's assessment development process,
the NRC will also review current methods for weight-of-evidence analyses
and recommend approaches for weighing scientific evidence for chemical
hazard identification. In addition, EPA may hold additional workshops on
issues related to weight-of-evidence to inform future assessments.
Calculation of Reference Values and Unit Risks (pp. 165-166)
7. Assess the sensitivity of derived estimates to model assumptions and end
points selected. This step should include appropriate tabular and graphic
displays to illustrate the range of the estimates and the effect of uncertainty
factors on the estimates.
As discussed in Section 1.2, although the nervous system is the primary and
most sensitive target of inhaled TMB toxicity, there is evidence of effects in
other organ systems. Candidate RfCs for 1,2,4-TMB and 1,2,3-TMB are
evaluated together in Figures 2-2 and 2-4 (respectively), including the
uncertainty factors applied to individual endpoints.
EPA Announces NAS' Review of IRIS Assessment Development Process (www.epa.gov/iris)
This document is a draft for review purposes only and does not constitute Agency policy.
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APPENDIX E. SUMMARY OF EXTERNAL PEER
REVIEW AND PUBLIC COMMENTS AND EPA'S
DISPOSITION
To be added
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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