JANUARY 2012

Appendix A: Toxicological 

Information in Support of Hazard 

Identification and Dose-Response 

Analysis for 

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

Trimethylbenzene
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DISCLAIMER

This document is an Interagency Science Consultation 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 the 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 or recommendation for use.
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CONTENTS

DISCLAIMER.............................................................................................................................................2

CONTENTS.................................................................................................................................................3

LIST OF TABLES AND FIGURES..........................................................................................................4

A.1. Physiologically-Based Pharmakokinetic Models..............................................................................7

A.1.1. Summary of Available PBPK Models for 1,2,4-TMB.......................................................... 7

A.1.2. Model Selection ................................................................................................................. 14

A.1.3. Details of Hissink et al. (2007) Model Analysis................................................................. 14

A.1.4. Modification of the Hissink et al. (2007) model to include oral route of exposure ............ 41

A.1.5. Conclusions......................................................................................................................... 42

A.2. Study Summary Tables for 1,2,4-TMB and 1,3,5-TMB ................................................................43

Study Summary Tables for Human Studies of 1,2,4-TMB and 1,3,5-TMB.................................. 43

Study Summary Tables for Animal Toxicological Studies of 1,2,4-TMB and 1,3,5-TMB........... 50

Study Summary Tables for Toxicokinetic Studies of 1,2,4-TMB and 1,3,5-TMB........................ 92

A.3. References........................................................................................................................................121
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LIST OF TABLES AND FIGURES

Figure A.1-1. Physiological based toxicokinetic model for 1,2,4-TMB in humans...............................8

Table A.1-1. Measured and calculated partition coefficients for TMB isomers at 37○

C.....................8

Table A.1-2. PBPK model parameters for 1,2,4-TMB toxicokinetics in humans using the Jarnberg 

and Johanson model structure...................................................................................................................9

Figure A.1-2. Schematic of human model structure for 1,2,4-TMB using the NLE-based model 

approach. ...................................................................................................................................................11

Figure A.1-3. Schematic of rat and human PBPK model structure.....................................................13

Table A.1-3. Comparison of rat anatomical and physiological parameters in Hissink et al. (2007) to 

those of Brown et al. (1997)......................................................................................................................17

Table A.1-4. Comparison of human anatomical and physiological parameters in Hissink et al. to 

those of Williams and Leggett (1989) as reported by Brown et al.(1997). ...........................................18

Table A.1-5. Comparison of chemical-specific parameters in Hissink et al. (2007) to literature data

....................................................................................................................................................................19

Figure A.1-4. Simulated and measured blood concentrations of 1,2,4,-TMB in rats exposed to 2,400, 

or 4,800 mg/m3 WS for 8 hrs. ..................................................................................................................21

Figure A.1-5. 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 hrs........................................................................................................22

Figure A.1-6. Simulated and measured exhaled air concentrations of 1,2,4-TMB in three volunteers 

exposed to 600 mg/m3 WS for 4 hrs. .......................................................................................................23

Table A.1-6. Parameter values for the rat and human PBPK models for 1,2,4-TMB used by EPA 25

Table A.1-7. Rat 1,2,4-TMB kinetic studies used in model development and testing.........................26

Figure A.1-7. 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. .........27

Figure A.1-8. 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.................................................................................................................................27

Figure A.1-9. 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...............................................................................................................................................28

Figure A.1-10. Comparisons of model predictions to measured rat venous blood concentrations by 

Swiercz et al. (2002) in acutely exposed rats during (a) and after (b) exposure..................................29

Table A.1-8. Model simulated and experimental measured concentrations of 1,2,4-TMB in male 

Wistar rats exposed to 1,2,4-TMB...........................................................................................................30

Table A.1-9. Model simulated and experimental measured concentrations of 1,2,4-TMB in male 

Sprague-Dawley rats exposed to 100 ppm 1,2,4-TMB (1 h/d, for 3 d) at the end of exposure or 12 hr 

after the last exposure...............................................................................................................................31
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Table A.1-10. 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 h exposure............................................32

Table A.1-11. Model simulated and experimental measured concentrations of 1,2,4-TMB in male 

Sprague-Dawley rats exposed to 1,000 ppm 1,2,4-TMB (12 h/d, for 14 d) at the end of exposure....33

Table A.1-12. Human kinetic studies of 1,2,4-TMB used in model validation ...................................34

Figure A.1-11. Comparisons of model predictions to measured human venous blood concentrations 

of Kostrzewki et al. (1997) in human volunteers exposed to 150 mg 1,24-TMB/m3 for 8 hours. ......34

Figure A.1-12. 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 1,2,4-TMB for 2 hours 

while riding a bicycle (50 W)....................................................................................................................35

Figure A.1-13. Comparisons of model predictions to measured human venous blood (a) and end of 

exposure exhaled air (b) 1,2,4-TMB in human volunteers exposed to 100 ppm WS with 7.8% 1,2,4-

TMB (Hissink et al., 2007)........................................................................................................................36

Table A.1-13. Parameter sensitivity for venous blood 1,2,4-TMB concentration in rats exposed to 

1,2,4-TMB via inhalation..........................................................................................................................39

Figure A.1-14. Time course of normalized sensitivity coefficients of moderately sensitive chemicalspecific parameters (response: venous blood concentration) in rats exposed to (a) 25 ppm or (b) 250 

ppm of 1,2,4-TMB via inhalation for 6 hours (Swiercz et al., 2003; Swiercz et al., 2002).................40

Table A.2-1. Study Summary and Data for Chen et al. (1999).............................................................43

Table A.2-2. Study Summary and Data for Billionnet et al. (2011)......................................................46

Table A.2-3. Study Summary and Data for Norseth et al. (1991).........................................................48

Table A.2-4. Study Summary and Data for Korsak et al. (1995)..........................................................50

Table A.2-5. Study Summary and Data for Korsak and Rydzyński (1996) ........................................53

Table A.2-6. Study Summary and Data for Korsak et al. (1997).........................................................55

Table A.2-7. Study Summary and Data for Korsak et al. (2000).........................................................56

Table A.2-8. Study Summary and Data for Saillenfait et al.(2005).....................................................61

Table A.2-9. Study Summary and Data for Gralewicz et al. (1997a)..................................................66

Table A.2-10. Study Summary and Data for Gralewicz et al. (1997b)................................................69

Table A.2-11. Study Summary and Data for Gralewicz and Wiaderna (2001)..................................71

Table A.2-12. Study Summary and Data for Wiaderna et al. (2002)..................................................74

Table A.2-13. Study Summary and Data for McKee et al. (2010).......................................................76

Table A.2-14. Study Summary and Data for Tomas et al. (1999a)......................................................79

Table A.2-15. Study Summary and Data for Tomas et al. (1999b) .....................................................81

Table A.2-16. Study Summary and Data for Tomas et al. (1999c)......................................................82

Table A.2-17. Study Summary and Data for Lammers et al. (2007)...................................................85

Table A.2-18. Study Summary and Data for Maltoni et al. (1997)......................................................90

Table A.2-19. Study Summary and Data for Jarnberg et al. (1996) ...................................................92
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Table A.2-20. Study Summary and Data for Jarnberg et al.(1997a) ..................................................95

Table A.2-21. Study Summary and Data Jarnberg et al. (1997b) .......................................................97

Table A.2-22. Study Summary and Data for Jarnberg et al. (1998) ..................................................98

Table A.2-23. Study Summary and Data for Kostrzewski et al. (1997)..............................................99

Table A.2-24. Study Summary and Data for Jones et al. (2006)........................................................101

Table A.2-25. Study Summary and Data for Swiercz et al. (2002)....................................................103

Table A.2-26. Study Summary and Data for Swiercz et al. (2003)....................................................105

Table A.2-27. Study Summary and Data for Swiercz et al. (2006)....................................................107

Table A.2-28. Study Summary and Data for Zahlsen et al. (1990)....................................................110

Table A.2-29. Study Summary and Data for Eide and Zahlsen et al. (1996)....................................112

Table A.2-30. Study Summary and Data for Huo et al. (1989)..........................................................113

Table A.2-31. Study Summary and Data for Tsujino et al. (2002)....................................................115

Table A.2-32. Study Summary and Data for Tsujimoto et al. (2000)................................................117

Table A.2-33. Study Summary and Data for Tsujimoto et al. (2005)................................................118

Table A.2-34. Study Summary and Data for Mikulski and Wiglusz (1975).....................................119

Table A.2-35. Study Summary and Data for Meulenberg and Vijverberg (2000)...........................120
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A.1. Physiologically-Based Pharmakokinetic 

Models

A.1.1. Summary of Available PBPK Models for 1,2,4-TMB

Jarnberg and Johanson (1999)

Jarnberg and Johanson (1999) describe a PBPK model for inhalation of 1,2,4-TMB in 

humans. The model is composed of six compartments (lungs, adipose, working muscles, resting 

muscles, liver, and rapidly perfused tissues) for the parent compound and one (volume of 

distribution) for the metabolite, 3,4-dimethylhippuric acid (see Figure C-1). The lung 

compartment includes lung tissue and arterial blood. Excretion of parent compound is assumed 

to occur solely by ventilation. As 1,2,4-TMB has a pronounced affinity to adipose tissue, a 

separate compartment for fat is incorporated into the model. Remaining non-metabolizing 

compartments are rapidly perfused tissues, comprising the brain, kidneys, muscles, and skin. 

Because previous experimental data was gathered during exercise (Järnberg et al., 1997a; 

Järnberg et al., 1996), the muscle compartment was divided into two equally large 

compartments, resting and working muscles. Two elimination pathways (a saturable MichaelisMenten pathway for all metabolites other than 2,4-DMHA (pathway I) and a first order pathway 

(pathway II) for formation of 3,4-DMHA) from the hepatic compartment were included. 

Metabolism was assumed to occur only in the liver compartment. Tissue:blood partition 

coefficients of 1,2,4-TMB were calculated from experimentally determined blood:air, water:air, 

and olive oil:air partition coefficients (Järnberg and Johanson, 1995) (Table A.2-1). The model 

was used to investigate how various factors (work load, exposure level, fluctuating exposure) 

influence potential biomarkers of exposure (end-of-shift and prior-to-shift concentrations of 

parent compound in blood and 3,4-DMHA in urine). Biomarker levels estimated at end-of-shift 

remained fairly constant during the week, whereas biomarker levels prior-to-shift gradually 

increase throughout the week. This indicates end-of-shift values represent the same day’s 

exposures, whereas prior-to-shift values reflect cumulative exposure during the entire work 

week. Increased work load increased uptake of 1,2,4-TMB. For example, a work load of 150W 

over an exposure period of 8 hours increased the level of 1,2,4-TMB in the blood more than 2-

fold, compared to levels of 1,2,4-TMB in the blood after an 8 hour exposure at rest. Simulated 

8-hour exposures at air levels 0 to 100 ppm (492 mg/m3

) shows that overall metabolism is 

saturable, and that the metabolic pathway yielding 3,4-dimethylbenzene becomes more important 

as exposure concentrations increase.
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C: concentration of 1,2,4-TMB; Cair: concentration in ambient air; Cart: concentration in arterial blood; Cven: 

concentration in venous blood; Qalv: alveolar ventilation; QCO: cardiac output; Qi: blood flow to compartment 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; CLI: intrinsic hepatic 

clearance of metabolic pathway II; ke: excretion rate constant of 3,4-dimethylhippuric acid. Adapted from Jarnberg 

and Johanson (1999)

Figure A.1-1. Physiological based toxicokinetic model for 1,2,4-TMB in humans. 

Table A.1-1. Measured and calculated partition coefficients for TMB isomers at 37○

C

Substance

Measured Valuesa Calculated 

Values

P Saline:Air

n=42

P Oil:Air

n=25

Human P Blood:Air

n=39

Human P

Blood:Air

b

1,3,5-TMB 1.23 (1.11-1.35) 9880 (9620-10140) 43.0 (40.8-45.2) 60.3

1,2,4-TMB 1.61 (1.47-1.75) 10200 (9900-10400) 59.1 (56.9-61.3) 62.2

a Mean values and 95% confidence intervals

b Calculated as (0.79 * P Saline:Air) + (0.006 * P Oil:Air); 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 (1995)

Lungs and arterial 

blood 

Rapidly perfused

tissues

Resting muscles

Adipose tissue

Working muscles

Liver

3,4-dimethyl 

hippuric acid

other 

metabolites

urine urine

Qalv

Cair

Qalv

Calv

Qco

Cart

Qrap

Qf

Qw

Qr

Qh

Qco

Cven

Vmax / Km CLi

ke

Lungs and arterial 

blood 

Rapidly perfused

tissues

Resting muscles

Adipose tissue

Working muscles

Liver

3,4-dimethyl 

hippuric acid

other 

metabolites

urine urine

Qalv

Cair

Qalv

Calv

Qco

Cart

Qrap

Qf

Qw

Qr

Qh

Qco

Cven

Vmax / Km CLi

ke
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Previously performed experimental human exposures to 1,2,4-TMB were used to estimate 

the metabolic parameters and alveolar ventilation (Järnberg et al., 1997a; Järnberg et al., 1996). 

Individual simulated arterial blood concentrations and exhalation rates of 1,2,4-TMB, as well as 

the urinary excretion rate of 3,4-DMHA, were simultaneously adjusted to the experimentally 

obtained values by varying the alveolar ventilation at rest, the DMHA acid excretion. One 

individual’s compound-specific and physiological parameters were then used for subsequent 

model predictions (Table A.2-2). 

Table A.1-2. PBPK model parameters for 1,2,4-TMB toxicokinetics in humans using the 

Jarnberg and Johanson model structure

Parameters Rest Botha 50 W

Body height (m) 1.78

Body weight (kg) 75.5

Vmax (μmol/min) 3.49

Km (μM) 4.35

CLi(liter/min) 0.149

Elimination rate constant (min -1) 0.0079

Alveolar ventilation 9.05 20.2

Compartment volumes (liter) 1.37

Lungs and arterial blood 1.51

Liver 25.0

Fat 1.49

Brain and kidneys 16.6

Working muscles 16.6

Resting muscles

Blood flows (liter/min)

Cardiac output 5.17 9.16

Liver 1.67

Fat 0.55

Brain and kidneys 1.86

Working muscles 0.55

Resting muscles 0.55

Partition coefficients

Blood:air 59

Fat:blood 125

Liver:blood 5

Rapidly perfused tissues:blood 5

Muscle:blood 5 a Parameters used for both working and resting conditions.

Adapted from Jarnberg and Johanson (1999)

The model was used to investigate how various factors (e.g., work load, exposure level) 

influence potential biomarkers of exposure (end-of-shift and prior-to-shift concentrations of 
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parent compound in blood and 3,4-DMHA in urine). Biomarker levels estimated at end-of-shift 

remained fairly constant during the week, whereas biomarker levels prior-to-shift gradually 

increase throughout the week. This indicates end-of-shift values represent the same day’s 

exposures, whereas prior-to-shift values reflect cumulative exposure during the entire work 

week. Increased work load increased uptake of 1,2,4-TMB. For example, a work load of 150W 

over an exposure period of 8 hours increased the level of 1,2,4-TMB in the blood more than 2-

fold, compared to levels of 1,2,4-TMB in the blood after an 8 hour exposure at rest. Simulated 

8-hour exposures at air levels 0 to 100 ppm (492 mg/m3) shows that overall metabolism is 

saturable, and that the metabolic pathway yielding 3,4-dimethylbenzene becomes more important 

as exposure concentrations increase. 

Emond and Krishnan (2006)

The Emond and Krishnan (2006) model was not developed specifically for 1,2,4-TMB, but 

rather to test a modeling concept. The physiological pharmacokinetic model developed was to 

test the hypothesis that a model could be developed for highly lipophilic volatile organic 

chemicals (HLVOCs) using the neutral lipid-equivalent (NLE) content of tissues and blood as the 

basis. This NLE-based modeling approach was tested by simulating uptake and distribution 

kinetics in humans for several chemicals including α-pinene, d-limonene, and 1,2,4-TMB. For 

the focus of this model review is the use of the model for prediction of 1,2,4-TMB kinetics and 

distribution. 

This model consisted of 5 compartments (see Figure A.2-2) with systemic circulation, where 

the tissue volumes corresponded to the volumes of the neutral lipids (a.k.a. their neutral lipid 

equivalents), rather than actual tissue volume as more commonly found. NLE is the sum of the 

neutral (nonpolar) lipids and 30% of the tissue phospholipid (fraction of phospholipids with 

solubility similar to neutral lipids) content. The model describes inhalation of 1,2,4-TMB using 

a lumped lung/arterial blood compartment. Clearance of 1,2,4-TMB is described in the model 

with exhalation, but more significantly through first order hepatic metabolism. First-order 

metabolism is appropriate in the low dose region (< 100 ppm), where metabolism is not expected 

to be saturated. 

The mathematical descriptions presented by the authors in the manuscript appear to be 

correct. The mixed lung/arterial blood compartment is not a standard structure for the 

lung/blood/air interface. The concentration in lung tissue is assumed equal to alveolar blood, and 

the exhaled air concentration is equal to the lung/blood concentration divided by the blood air 

partition coefficient. This approach is appropriate, and appears to be accurately represented 

mathematically.
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Physiological parameters appear to be within ranges normally reported. The calculation of 

the NLE fraction is clearly explained and values used in the calculations are clear and 

transparent. Other model parameters (e.g., alveolar ventilation, cardiac output, blood flows, and 

volumes of compartments) were taken from Jarnberg and Johanson (1999) and converted to NLE 

equivalents. Hepatic clearance rates were taken from literature on in vivo human clearance 

calculations and then expressed in terms of NLE. The NLE-based model was able to adequately 

predict human blood concentrations of 1,2,4-TMB following inhalation of 2 or 25 ppm for 2hrs 

without alteration to model parameters obtained from literature.

Arrows represent blood flows, gas exchange, and metabolism as indicated. Source: Emond and Krishnan (2006)

Figure A.1-2. Schematic of human model structure for 1,2,4-TMB using the NLE-based 

model approach. 

The PBPK model developed by Emond and Krishnan (2006) is used to test the hypothesis 

that a model could be developed for HLVOCs using the NLE content of tissues and blood as the 

basis. To test this NLE-based approach, the uptake and distribution kinetics in humans for 

several chemicals including 1,2,4-TMB were simulated. The model appeared correct; however, a 

rodent model is needed for this assessment for animal to human extrapolation and no known 

rodent NLE model for 1,2,4-TMB is available. 
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Hissink et al. (2007)

This model was developed to characterize internal exposure following white spirit (WS) 

inhalation. Since WS is a complex mixture of hydrocarbons, including straight and branched 

parrafins, two marker compounds were used including 1,2,4-TMB and n-decane. The rat models 

were developed to predict the levels of 1,2,4-TMB and n-decane in blood and brain, then the rat 

model was scaled allometrically to obtain estimates for human blood following inhalation. 

Toxicokinetic data on blood and brain concentrations in rats of two marker compounds, 1,2,4-

TMB and n-decane, together with in vitro partition coefficients were used to develop the model. 

The models were used to estimate an air concentration that would produce human brain 

concentrations similar to those in rats at the no-observed-effect-level (NOEL) for CNS effects.

This is a conventional 5 compartment PBPK model for 1,2,4-TMB similar to previously 

published models for inhaled solvents. The five compartments were: liver, fat, slowly perfused 

tissues, rapidly perfused tissues, and brain (see Figure A.2-3). 
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Boxes represent tissue compartments, while solid arrows represent blood flows, gas exchange, and metabolism as 

indicated.

Figure A.1-3. Schematic of rat and human PBPK model structure. 

All compartments are described as well mixed/perfusion limited. A lung compartment is 

used to describe for gas exchange. The liver was the primary metabolizing organ where 1,2,4-

TMB metabolism was described as saturable using Michaelis-Menten (M-M) kinetics. Since the 

brain is the target organ for CNS effects due to exposure to hydrocarbon solvents, it was included 

as a separate compartment. For the rat, the authors reported that Km and Vmax values were 

obtained by fitting predicted elimination time courses to observed blood concentration profiles at 

3 different exposure levels (obtained from the rat exposure portion of the study). For the human 

model, rat Vmax data was scaled as described above and Km values were used unchanged.
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The model appears to effectively predict blood concentrations in rats and humans and in the 

brains of rats following inhalation of WS. Changes to the rat model parameters to fit the human 

data were as expected. The model is simple and includes tissues of interest for potential dose 

metrics.

In rats, the model-predicted blood and brain concentrations of 1,2,4-TMB were in 

concordance with the experimentally derived concentrations. In humans, experimental blood 

concentrations of 1,2,4-TMB were well predicted by the model, but the predicted rate of 

decrease in air concentration between 4-12 hours was lower compared to measured values. The 

authors did not provide information on how model predictions compared to data from animals or 

humans exposed to pure 1,2,4-TMB. Based on good model fits of experimental data, the model 

was valid for the purpose of interspecies extrapolation of blood and brain concentrations of 

1,2,4-TMB as a component of WS.

A.1.2. Model Selection

All available 1,2,4-TMB PBPK models were evaluated for potential use in this assessment. 

Of the three deterministic PBPK models available for 1,2,4-TMB (Hissink et al., 2007; Emond 

and Krishnan, 2006; Järnberg and Johanson, 1999), the Hissink et al. (2007) model was chosen 

to utilize in this assessment because it was the only published 1,2,4-TMB model that included 

parameterization for both rats and humans, the model code was available, and the model 

adequately predicted experimental data in the dose range of concern. The Hissink et al. (2007)

model was thoroughly evaluated, including a detailed computer code analysis (details follow in 

Section A.2.3). 

A.1.3. Details of Hissink et al. (2007) Model Analysis

Review and Verification of the Hissink et al. (2007) 1,2,4-TMB PBPK Model

 Verification of accuracy of the model code

In general, the model code and the description of the model in Hissink et al. (2007) were in 

agreement. The one significant discrepancy was that the model code contained an element that 

changed the metabolism rate (Vmax) during exposure in a manner that was not documented in the 

paper. This additional piece of model code, when used in 8 hr rat simulations with a body weight 

of 0.2095 kg, resulted in Vmax remaining at 1.17 from the beginning of exposure to t=1 hr, then 

increasing linearly to 1.87 by the end of the exposure and to 2.67 by the end of the post exposure 

monitoring period (t = 16 hrs, 8 hrs after the end of exposure). The published rat simulations, 
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however, did not appear to be entirely consistent with the inclusion of these Vmax adjustments, 

raising questions as to whether the code that was verified was the code that was actually used in 

the final analyses done for the published simulations. The impact of this deviation from the 

published Vmax value is described below in the regarding verification of the Hissink et al. (2007)

model (page 26). 

Other minor issues were identified by examining the code and comparing it to the model 

documentation in Hissink et al. (2007). The code contained some elements that were not 

necessary (e.g., iv dosing, repeated exposure, interruptions in daily exposure), but since these do 

not hinder proper functioning of the model, these elements were not removed or modified. The 

mass balance equation omitted one term, the amount of 1,2,4-TMB in the brain (ABR); this term 

has been added. The coding for the blood flow was not set up so as to ensure flow/mass balance. 

That is, values of QRTOTC (sum of fractional flows to rapidly perfused tissues, liver, and brain) 

and QSTOTC (sum of fractional flows to slowly perfused tissues) were selected such that their 

sum equals one, but if one value were to be changed, the model code would not automatically 

compensate by changing the other. Therefore, the code was modified so that QSTOTC = 1-

QRTOTC, to facilitate future sensitivity analyses. Human exhaled breath concentrations were 

compared to CXEQ (= CV/PB based on the model code and consistent with the description of 

the experiment), which would be equivalent to the end-exhaled alveolar air after breath holding, 

but the method used to calculate CXEQ was not noted in Hissink et al. (2007). This is important 

because there can be different definitions of exhaled breath depending on the measurement 

technique. For example, mixed exhaled breath is typically calculated as 70 percent alveolar air 

and 30 percent “inhaled” concentration, due to dead space.

Comparisons between the computer .m files and published descriptions (Hissink et al., 2007)

indicated minor discrepancies and uncertainties in exposure concentrations and body weight. 

Exposure concentrations in the simulations were set at the nominal exposure levels, rather than 

analytically determined levels. The maximum deviation between the nominal level and 

analytically determined levels was for the rat high exposure group, with a nominal exposure of 

4,800 mg/m3 WS (7.8% 1,2,4-TMB) and mean analytical concentrations ranging from 4,440 to 

4,769 mg/m3—as much as 9.2 percent lower. Rat body weights at time of exposure were reported 

as 242 to 296 g (Hissink et al., 2007), but the .m files use values of 210.01, 204.88, and 209.88 g 

in the low-, mid-, and high-exposure groups, respectively. Human volunteer body weights 

reportedly ranged from 69 to 82 kg, and the text states that the fitted Vmax and Km were obtained 

for a 70 kg male (Hissink et al., 2007), but a body weight of 74.9 kg was used in the .m file. No 

changes to these parameters were made in the model code, based on the assumption that 

additional data were available to the model authors.

Measured human blood concentrations were compared to “CMIX,” the average of arterial 

and venous blood concentrations, while the protocol states that blood was taken from the cubital 
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vein, so a more appropriate measure may have been CVS (venous blood exiting the slowly 

perfused tissues compartment). This choice of dose metric is unlikely to have contributed 

significantly to any errors in parameterizing the model (i.e., estimating best-fit metabolism 

parameters) because the difference between the two values is generally small. Revised model 

code and modeling results are provided on EPA’s Health Effects Research Online (HERO) 

database (U.S. EPA, 2011).

 Verification of model parameter plausibility 

 Anatomical and physiological parameters

The anatomical physiological parameters used by Hissink et al. (2007) were taken from Arms 

and Travis (1988), but more current convention is to use the parameters in Brown et al.(1997). 

Comparisons of the rat anatomical and physiological parameters in these sources are found in 

Table A.2-3 below. 

Many disagreements in values were identified, particularly with respect to the blood flows. In 

interpreting the blood flow percentages, it should be noted that the percentages enumerated by 

Brown et al. (1997) do not sum to 100%, which is of course a physiological requirement. 

Perfusion rates of various depots of fat may differ, so the single value or fractional blood flow to 

fat given by Brown et al. (1997) of 7%, may be deemed sufficiently uncertain that the Hissink et 

al. value of 9% is considered acceptable. Brown et al. (1997) report substantially higher blood 

flow percentages to slowly perfused tissues (skin: 5.8% and muscle: 27.8%, for a total of 33.6%) 

than the value of 15% used by Hissink et al. (2007). The difference cannot be due to a smaller set 

of tissues being “lumped” into this compartment, because Hissink et al. (2007) assign a larger 

volume fraction of tissue to this compartment. Hissink et al. (2007) also assign a higher 

percentage of blood flow to the liver than indicated by Brown et al. (1997). Because no 

sensitivity analyses were conducted by the authors, it is unclear what impact these discrepancies 

may have had on the predicted 1,2,4-TMB kinetics and visual optimization of metabolism 

parameters.

Comparisons of the human anatomical and physiological parameters in Hissink et al. 

(2007)and Brown et al. (1997) are found in Table A.2-4 below. 
DRAFT - DO NOT CITE OR QUOTE 17

Table A.1-3. Comparison of rat anatomical and physiological parameters in Hissink et al. 

(2007) to those of Brown et al. (1997).

Parameter Hissink et al. 

(2007)

a

Range from Brown 

et al. (1997)

Values in 

agreement?

Alveolar ventilation rate 

(L/hr/kg0.7) 20 12-54b Yes

Total cardiac output (L/hr/kg0.7) 20 9.6-15 No

Blood flow (% Cardiac output)

Liver (total) 25 13.1-22.1 No

Fat 9 7 Acceptablec

Brain 1.2 1.5-2.6 No

Rapidly Perfused (total) 49.8 15.3-27.4 No

 Adrenals 0.2-0.3

 Heart 4.5-5.1

 Kidneys 9.5-19

 Lung 1.1-3

Slowly Perfused (total) 15 33.6 No

 Muscle 27.8

 Skin 5.8

Total 100 70.5-92.7

Tissue Volume (% Body weight)

Liver 4 2.14-5.16 Yes

Fat 7 3.3-20.4 Yes

Brain 0.72 0.38-0.83 Yes

Rapidly Perfused 4.28 3.702-6.11 Yes

 Adrenals 0.01-0.31

 Stomach 0.4-0.6

 Small intestine 0.99-1.93

 Large intestine 0.8-0.89

 Heart 0.27-0.4

 Kidneys 0.49-0.91

 Lungs 0.37-0.61

 Pancreas 0.24-0.39

 Spleen 0.13-0.34

 Thyroid 0.002-0.009

Slowly Perfused 75 51.16-69.1 Acceptablec

 Muscle 35.36-45.5

 Skin 15.8-23.6

Total 91 60.682-101.6

a

Values from Arms and Travis (1988) b

Assuming a standard 250g rat.

c

Hissink et al. (2007) value outside of literature range, but acceptable (see discussion in text).
DRAFT - DO NOT CITE OR QUOTE 18

Table A.1-4. Comparison of human anatomical and physiological parameters in Hissink et 

al. to those of Williams and Leggett (1989) as reported by Brown et al.(1997).

Parameter Hissink et al. 

(2007)

a

Range from 

Brown et al. 

(1997)

Values in 

agreement?

Alveolar ventilation rate (L/hr/kg0.7) 20 15 Acceptable

Total cardiac output (L/hr/kg0.7) 20 16 Acceptable

Blood flow (% Cardiac output)

Liver (total) 26 11-34.2 Yes

Fat 5 3.7-11.8 Yes

Brain 14 8.6-20.4 Yes

Rapidly Perfused (total) 30 19.9-35.9 Yes

 Adrenals 0.3

 Heart 3-8

 Kidneys 12.2-22.9

 Lung 2.5

 Thyroid 1.9-2.2

Slowly Perfused (total) 25 9-50.8 Yes

 Muscle 5.7-42.2

 Skin 3.3-8.6

Total 100 52.2-153.1

Tissue Volume (% Body weight)

Liver 2.6 2.57 Yes

Fat 14.6 21.42 Acceptable 

(measured)a

Brain 2 2 Yes

Rapidly Perfused 3 3.77 Acceptable

 Adrenals 0.02

 Stomach 0.21

 Small intestine 0.91

 Large intestine 0.53

 Heart 0.47

 Kidneys 0.44

 Lungs 0.76

 Pancreas 0.14

 Spleen 0.26

 Thyroid 0.03

Slowly Perfused 66.4 43.71 Acceptable

 Muscle 40

 Skin 3.71

Total 88.6 73.47

a The Hissink et al. (2007) value differs from Brown et al. , but is acceptable (see discussion in text).

In general, the agreement was better for humans than it was for rats. Brown et al. (1997)

propose a higher default body fat percentage than was used by Hissink et al. (2007), but Hissink 

et al. (2007) used values derived from measurements of the volunteers participating in the study. 
DRAFT - DO NOT CITE OR QUOTE 19

Because these volunteers had relatively low percentages of body fat, it is appropriate that the 

volume of slowly perfused tissue (including muscle) should be increased to compensate. 

 Chemical-specific parameters

The chemical-specific model parameters, the partition coefficients and metabolic parameters are 

summarized in Table A.2-5.

Table A.1-5. Comparison of chemical-specific parameters in Hissink et al. (2007) to 

literature data

Parameter Hissink et al. Literature Values in 

agreement?

Value Technique Value Technique

Partition Coefficients

Saline:Air 3 In vitro 1.47-1.75a In vitro Acceptable

Olive oil:Air 13,200 In vitro 9,900-

10,400a In vitro Acceptable

Blood:Air - human 85 In vitro 59.6-61.3a In vitro Acceptable

Blood:Air - rat 148 In vitro --

Rapidly perfused:Blood 2.53 Calculated --

Slowly perfused:Blood 1.21 Calculated --

Fat:Blood 62.7 Calculated 63b In vivo Yes

Brain:Blood 2.53 Calculated 2b In vivo Acceptable

Liver:Blood 2.53 Calculated --

Metabolism

VmaxC – rat (mg/hr/kg0.7) 3.5 Visual 

optimization --

VmaxC – human (mg/hr/kg0.7) 3.5 Assumed 

equal to rat 1.2-21c Optimization

Yes

Km – rat (mg/L) 0.25 Visual 

optimization --

Km – human (mg/L) 0.25 Assumed 

equal to rat 0.42-4.0c Optimization

No

VmaxC/Km – human (L/hr/kg0.7) 14 Assumed 

equal to rat 2.6-15c Optimization

Yes

a Jarnberg and Johanson (1995)

b Zahlsen et al. (1990) c Jarnberg and Johanson (1999)

Where data were available, the agreement is generally acceptable. While the rat-derived Km

is less than the lower 95 percent confidence interval value for the human Km, the human 

VmaxC/Km ratio is in acceptable agreement. When considering sufficiently low exposure 

concentrations, the performance of the Hissink et al. (2007) human model metabolism 

parameters would be consistent with the Jarnberg and Johanson (1999) value.
DRAFT - DO NOT CITE OR QUOTE 20

 Verification that the model can reproduce all figures and tables in the publication 

by Hissink et al. (2007)

The experimental data in Hissink et al. (2007) were estimated by use of Plot Digitizer 

(version 2.4.1) to convert the symbols on the relevant figures into numerical estimates. The 

model code provided (adapted for acslX), with a variable value for Vmax, does not appear to 

perfectly reproduce the rat simulations in Hissink et al. (2007) (Figures A.2-4 a and b and A.2-5 

a and b) (please note that the Hissink et al. (2007) figures have been “stretched” to produce 

approximately the same x-axis scale found in the acslX figures). It appears to yield end-of 

exposure blood and brain concentrations that are about the same as in the Hissink et al. (2007) 

simulations, but the post-exposure clearance appears faster in our hands (see, for example, the 16 

hr time points for the high exposures). When the simulations were run with Vmax constant 

(Figures A.2-4c and A.2-5c), as documented in Hissink et al. (2007), the rat simulations yield 

higher blood and tissue concentrations than depicted in Hissink et al. (2007), most notably at the 

high exposure concentration. Similar results were obtained for the rat brain concentrations 

(Figure A.2-4). The human simulations of blood and exhaled air appear to be faithfully 

reproduced by the model (Figure A.2-6). The predicted brain concentration for humans exposed 

to 600 mg/m3 WS (45 mg/m3 1,2,4-TMB) for 4 hrs was reported as 721 ng/g (0.721 mg/L) in 

Hissink et al. (2007), whereas the current simulation predicts a concentration of 0.818 mg/L.
DRAFT - DO NOT CITE OR QUOTE 21

(a)

(b)

(c)

(a) Hissink et al., (2007), Figure 2, lower panel (b) variable Vmax, (c) constant Vmax.

Figure A.1-4. Simulated and measured blood concentrations of 1,2,4,-TMB in rats exposed 

to 2,400, or 4,800 mg/m3 WS for 8 hrs. 
DRAFT - DO NOT CITE OR QUOTE 22

(a)

(b)

(c)

(a) Hissink et al., (2007), Figure 3, lower panel. (b) variable Vmax (c) constant Vmax.

Figure A.1-5. 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 hrs 
DRAFT - DO NOT CITE OR QUOTE 23

(a)

(b)

(c)

(a) Hissink et al., (2007), Figure 3 (b) model simulation during exposure, and (c) model simulation after 

exposure.

Figure A.1-6. Simulated and measured exhaled air concentrations of 1,2,4-TMB in three 

volunteers exposed to 600 mg/m3 WS for 4 hrs. 
DRAFT - DO NOT CITE OR QUOTE 24

PBPK Model Optimization and Validation

 Methods and Background

For all optimizations, the Nelder-Mead algorithm was used to minimize the log-likelihood 

function (LLF). A constant heteroscedasticity value of 2 (i.e., relative error model) was assumed. 

Statistical significance of an increase in the LLF was evaluated for 95 % confidence per Collins 

et al. (1999). All kinetic studies were conducted with adult animals or adult human volunteers. 

In many cases, blood and tissue concentration data in a numerical form were available from the 

literature (Swiercz et al., 2003; Swiercz et al., 2002; Kostrzewski et al., 1997; Eide and Zahlsen, 

1996; Zahlsen et al., 1992; Dahl et al., 1988). The 1,2,4-TMB blood, brain, and exhaled breath 

concentration data in Hissink et al. (2007) were published in graphical format and a colleague of 

Dr. Hissink also provided these in numerical form to Dr. Lisa Sweeney for use in this analysis 

(Sweeney personal communication).

Average estimates of the blood concentrations of 1,2,4-TMB (average and standard 

deviation) in humans exposed only to 1,2,4-TMB as presented in graphs in Jarnberg et al. (1998, 

1997a; 1996) were used in this evaluation. Estimates of the blood and tissue 1,2,4-TMB 

concentrations in rats presented in graphs in Zahlsen et al. (1990) were used in this evaluation. 

Prior to model optimization, physiological parameters were modified from those in Hissink et al. 

(2007) to better reflect a more recent literature compilation (Brown et al., 1997) than the 

references cited by Hissink et al. (2007) (Table A.2-6). Where possible, study specific body 

weights and measured concentrations (rather than nominal concentrations) have been used, as 

detailed in the .m files (U.S. EPA, 2011). For the Zahlsen et al. (1990) 14-day study, body 

weights for exposures after the first exposure were estimated based on European growth curves 

for male Sprague-Dawley rats (linear regression of weights for weeks 6-9) (Harlan Laboratories, 

2011). 
DRAFT - DO NOT CITE OR QUOTE 25

Table A.1-6. Parameter values for the rat and human PBPK models for 1,2,4-TMB used 

by EPA

PARAMETER RAT HUMAN (AT REST)

Body weight (kg) 0.230 - 0.390a 70

Alveolar ventilation rate (L/hr/kg0.74) 14 15

Total cardiac output (L/hr/kg0.74) 14 16

Blood flow (% of total cardiac output)

Liver 17.6 17.5

Fat 9 8.5

Brain 2.0 11.4

Rapidly perfused 37.8 37.7

Slowly perfused 33.6 24.9

Volume (% of body weight)

Liver 4 2.6

Fat 7 21.42

Brain 0.57 2

Rapidly perfused 4.43 3

Slowly perfused 75 59.58

Partition coefficients (dimensionless)

Blood: air 148 85

Rapidly perfused: blood 2.53 4.4

Slowly perfused: blood 1.21 2.11

Fat: blood 62.7 109

Brain: blood 2.53 4.4

Liver: blood 2.53 4.4

Liver metabolism

VmaxC(mg/h/kg0.70) 4.17

Km (mg/L) 0.322 a Study specific

 Rat Model Optimization

The rat studies considered in model optimization and model testing (validation) are 

summarized in Table A.2-7. Values for VmaxC and Km were numerically optimized based on the 

fit of the model predictions to the measured blood concentrations of 1,2,4-TMB of Hissink et al. 

(2007) for rats exposed once to one of three concentrations of 1,2,4-TMB as a component of WS. 

The optimized value of VmaxC was only modestly different from the value determined by Hissink 

et al. (2007) (initial: 3.5 vs. optimized: 3.08 mg/hr/kg0.7) from visual optimization (with slightly 

different physiological parameters), but the Km value differed by 5-fold (initial: 0.25 vs. 

optimized: 0.050 mg/L). The increase in the LLF from 42.6 to 58.2, with two adjustable 

parameters, indicates that the improvement in fit (Figure A.2-7) is statistically significant. The 

percentage of variation explained increased from 82.3 to 90.4 percent, and the fit by visual 
DRAFT - DO NOT CITE OR QUOTE 26

inspection appears to be very good during exposure (modestly overpredicting) and excellent in 

the post exposure period. Using the optimized kinetic parameters, the rat brain concentrations of 

1,2,4-TMB were also well-predicted (Figure A.2-8).

Table A.1-7. Rat 1,2,4-TMB kinetic studies used in model development and testing

Reference Strain Gender

Nominal 

Concentration 

(ppm)

Exposure 

Regimen

1,2,4-TMB 

Measurement

Use in 

Model 

Evaluation

Form of 

Comparison

Hissink et 

al. (2007)

WAG/Ri

jCR/BR 

(Wistar 

derived)

Male

102, 410, 820 

(WS with 7.8% 

1,2,4-TMB)

8 h

Mixed blood 

time course

Optimization 

(1,2,4-TMB 

in mixture)

Figure A.2-6

Brain time 

course Testing Figure A.2-7

Swiercz et 

al. (2003) Wistar Male 25, 100, 250

6 h/d, 

5 d/wk 

4 wks

Venous blood 

time course

Optimization 

(1,2,4-TMB 

only)

Figure A.2-8

Arterial blood, 

liver, brain Testing Table A.2-8

6 h Arterial blood, 

liver, brain Testing Table A.2-8

Swiercz et 

al. (2002) Wistar Male 25, 100, 250 6 h Venous blood time course Testing Figure A.2-8

Zahlsen et 

al. (1990)

SpragueDawley Male 1,000 12 h/d

14 d

Blood, brain, 

perirenal fat

on days 1,3, 7, 

10 and 14

Testing Table A.2-9

Zahlsen et 

al. (1992)

SpragueDawley Male 100

12 h/d

3 d

Blood, brain, 

liver, kidney, 

and perirenal 

fat at end of 

exposures and 

after 12 hr 

recovery

Testing Table A.2-10

Eide and 

Zahlsen 

(1996)

SpragueDawley Male 75, 150, 300, 

450 12 h

Blood, brain, 

liver, kidney, 

and perirenal 

fat

Testing Table A.2-11

Dahl et al. 

(1988) F344/N Male 100 80 min Inhalation 

uptake Testing Text
DRAFT - DO NOT CITE OR QUOTE 27

(a)

(b)

Figure A.1-7. 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.

Figure A.1-8. 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.
DRAFT - DO NOT CITE OR QUOTE 28

(a)

(b)

Figure A.1-9. 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 

The VmaxC and Km values derived from optimization to the Hissink et al. (2007) rat data were 

used as the starting values for optimizing fit to the venous blood data of Swiercz et al. (2003), in 

which exposure was to 1,2,4-TMB only, repeatedly for 4 weeks. Venous blood samples were 

collected from the tail vein. The best fit parameters of VmaxC = 4.17 mg/hr/kg0.7 and Km= 0.322 

mg/L produced an increase in the LLF from -28.1 to -15.6, a statistically significant 

improvement, which increased the variation explained from 47.9 to 68.1 percent (Figure A.2-9). 

The deviation between the model and experimental data is primarily in the high concentration 

data set. When this set is not considered, the percent variation explained in the remaining two 

sets is 94.5 percent. Optimization to the low and middle concentrations alone (omitting the high 

concentration) does not substantially change the parameters or increase the LLF (simulations not 

shown). Optimization using the high concentration alone yields VmaxC and Km estimates of 7.91 

mg/hr/kg0.7 and 0.11 mg/L, respectively, with 96.7 percent of variation explained (simulations 

not shown).
DRAFT - DO NOT CITE OR QUOTE 29

 Rat Model Validation

The parameters derived from the Swiercz et al. (2003) venous blood optimizations were used 

to simulate other studies in which rats and humans (see below) were exposed to 1,2,4-TMB 

alone (without co-exposures). The fit to the Swiercz et al. (2002) venous blood data was very 

good (Figure A.2-10). In fact, the fit to the acute, high-exposure blood concentrations was 

superior to the fit to the repeated, high-exposure data (Figure A.2-9b). This may reflect 

adaptation (induction of metabolism) resulting from repeated, high concentration exposures. 

(a)

(b)

Figure A.1-10. Comparisons of model predictions to measured rat venous blood 

concentrations by Swiercz et al. (2002) in acutely exposed rats during (a) and after (b) 

exposure.

The model predictions of arterial blood and tissues in the repeated-exposure Swiercz et al. 

(2003) study were not very accurate, considering that the venous blood data from the same study 

were used for optimization (Table A.2-8). The discrepancies between seemingly 

contemporaneous venous and arterial blood measurements were noted by the authors of the 

original study and may be due to collection delays (i.e., tail vein for venous blood, decapitation 

for arterial samples). The geometric mean error ratio (greater of model/experiment or 

experiment/model) for these data was 2.8.
DRAFT - DO NOT CITE OR QUOTE 30

Table A.1-8. Model simulated and experimental measured concentrations of 1,2,4-TMB in 

male Wistar rats exposed to 1,2,4-TMB

Exopsure 

Concentration 

(ppm)

Model

(mg/L)

Experiment

(mg/L)a

Model: 

Experiment 

Ratio

Repeated exposure (Model t = 606 h)

Arterial blood

25 0.61 0.33 1.8

100 5.0 1.54 3.2

250 22.8 7.52 3.0

Brain

25 1.91 0.45 4.2

100 14.6 2.82 5.2

250 59.0 18.6 3.2

Liver

25 0.41 0.45 0.91

100 10.5 3.00 3.5

250 54.6 22.5 2.4

Acute Exposure (Model t= 6 h)

Arterial blood

25 0.53 0.31 1.7

100 7.10 1.24 5.7

250 18.6 7.76 2.4

Brain

25 2.19 0.49 4.5

100 20.6 2.92 7.0

250 62.1 18.3 3.4

Liver

25 0.49 0.44 1.1

100 16.3 7.13 2.3

250 57.7 28.2 2.0 a

Data from Swiercz et al. (2003).

Zahlsen and co-workers (Eide and Zahlsen, 1996; Zahlsen et al., 1992; Zahlsen et al., 1990)

conducted studies in which male Sprague-Dawley rats were exposed to 1,2,4-TMB by inhalation 

for 12 h/day. For the studies conducted at concentrations similar to those in the Swiercz studies 

(Tables A.2-9 and A.2-10), the model error was similar to that of the arterial blood and tissue 

measurements in the Swiercz studies (geometric mean error of 3.3 for Zahlsen et al.(1990), and 

2.9 for Eide and Zahlsen (1996). 
DRAFT - DO NOT CITE OR QUOTE 31

Table A.1-9. Model simulated and experimental measured concentrations of 1,2,4-TMB in 

male Sprague-Dawley rats exposed to 100 ppm 1,2,4-TMB (1 h/d, for 3 d) at the end of 

exposure or 12 hr after the last exposure

Day Model

(mg/L)

Experiment

(mg/L)a

Model: 

Experiment Ratio

Venous Blood

1 8.52 1.70 5.0

2 8.71 1.51 5.8

3 8.72 2.05 4.2

Recoveryb 1.08 0.024 7.6

Brain

1 22.6 4.57 4.9

2 23.1 4.19 5.5

3 23.1 4.38 5.3

Recoveryb 0.46 Nondetect Not calculated

Liver

1 18.2 4.92 3.7

2 18.7 3.66 5.1

3 18.7 4.25 4.4

Recoveryb 0.077 0.072 1.1

Kidney (Compared to 

rapidly perfused)

1 22.6 13.7 1.7

2 23.1 17.0 1.4

3 23.1 12.4 1.9

Recoveryb 0.46 0.24 1.9

Fat

1 491 210 2.3

2 503 165 3.1

3 504 128 3.9

Recoveryb 29.1 14.4 2.0

a

Data from Zahlsen et al. (1992).

b

Recovery period is designated as 12 h after the last exposure.

There was essentially no difference in the measured venous blood concentration of 

1,2,4-TMB in the 1992 study at 100 ppm (1.70 mg/L) and at 75 ppm in the 1996 study (1.69 

mg/L), so there is evidently some inter-study variability or subtle differences in how the studies 

were conducted, perhaps in the rapidity of sample collection. The Zahlsen et al. (1990) study, 

which used a higher nominal concentration of 1,000 ppm, exhibited greater deviation between 

predicted and measured blood and tissue 1,2,4-TMB concentrations (Table A.2-11), which 

generally increased with a greater number of exposure days and then plateaued (geometric mean 

errors of 2.7, 8.4, 12.6, 13.9, and 12.1 on exposure days 1, 3, 7, 10, and 14, respectively).
DRAFT - DO NOT CITE OR QUOTE 32

Table A.1-10. 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 h exposure

Exposure 

Concentration 

(ppm)

Model

(mg/L)

Experiment

(mg/L)a

Model: 

Experiment Ratio

Venous Blood

75 74.21 1.69 2.5

150 17.8 6.9 2.6

300 48.3 13.9 3.5

450 78.6 26.6 3.0

Brain

75 11.5 2.83 4.1

150 46.6 11.7 4.0

300 125 26.5 4.7

450 203 48.0 4.2

Liver

75 7.39 6.41 1.2

150 42.2 14.8 2.9

300 120 30.8 3.9

450 198 56.2 3.5

Kidney (Compared to 

Rapidly perfused)

75 11.5 6.41 1.8

150 46.6 20.2 2.3

300 125 33.9 3.7

450 203 59.1 3.4

Fat

75 255 61.9 4.1

150 987 457 2.2

300 2,636 1,552 1.7

450 4,276 2,312 1.8

a

Data from Eide and Zahlsen (1996).

Dahl et al. (1988) exposed male F344 rats to 1,2,4-TMB at 100 ppm for 80 minutes and 

monitored the total uptake. Under the conditions of the experiment, it was determined that 

average rat took up 3.28 (trial 1) or 3.89 (trial 2) mg 1,2,4-TMB. In a model simulation, the 

predicted uptake was 3.61 mg. Geometric mean model error for the two trials was 1.2.
DRAFT - DO NOT CITE OR QUOTE 33

Table A.1-11. Model simulated and experimental measured concentrations of 1,2,4-TMB in 

male Sprague-Dawley rats exposed to 1,000 ppm 1,2,4-TMB (12 h/d, for 14 d) at the end of 

exposure

Day Model

(mg/L)

Experiment

(mg/L)a

Model: 

Experiment Ratio

Venous Blood

1 181 63.5 2.8

3 293 43.1 6.8

7 372 33.4 11.1

10 395 34.0 11.6

14 399 35.2 11.3

Brain

1 465 120 3.9

3 747 64.9 11.5

7 946 63.5 14.9

10 1,005 62.1 16.2

14 1,014 71.5 14.2

Fat

1 9,919 5,860 1.7

3 17,328 2,282 7.6

7 22,323 1,835 12.2

10 23,763 1,677 14.2

14 23,961 2,169 11.0

a

Data from Zahlsen et al. (1990).

 Human Model Validation

Kinetic parameters derived from optimal fit for rat venous blood data (described above) were 

tested for the applicability to human kinetics by comparison to studies in which humans were 

exposed to 1,2,4-TMB alone or 1,2,4-TMB in co-exposures with WS (Table A.2-12). The key 

data set for validation in humans was deemed to be Kostrzewski et al. (1997) because these 

volunteers were exposed to 1,2,4-TMB alone (no co-exposure, as in Hissink et al.,(2007)) under 

sedentary conditions (i.e., level of effort was not elevated, as in Jarnberg et al., (1998, 1997a; 

1996)).

Using the VmaxC and Km derived from the Swiercz et al. (2003) rat repeated exposure data, 

the simulated blood concentration modestly underestimated those measured during exposure of 

human volunteers by Kostrzewski et al. (1997), then overpredicted blood concentrations up to 7 

hrs post exposure, and underpredicted subsequent measured blood concentrations (Figure A.2-

11). Of 21 blood measurements, only two differed from the simulated value by more than a 

factor of 2 (maximum: 2.6), with a geometric mean deviation of 1.5-fold between the simulated 

and measured values. The percent variation explained was 69.74%. When Km was held constant 

and VmaxC was optimized (final value: 3.39 mg/hr/kg0.7), the improvement in fit was minimal 

(72.14% of variation explained), and not statistically significant, so the rat-derived values were 

considered acceptable (see the section regarding rat model optimization, page 31).
DRAFT - DO NOT CITE OR QUOTE 34

Table A.1-12. Human kinetic studies of 1,2,4-TMB used in model validation

Reference Ethnicity Gender

Nominal 

Concentration 

(ppm)

Exposure 

Regimen

1,2,4-TMB 

Measurements

Use in 

Model 

Evaluation

Form of 

Comparison

Kostrzewski 

et al. 

(1997)

a

Not stated; 

conducted 

in Poland

Sex not 

stated. 

Assumed 

male.

30 8 h Venous blood 

time course

Testing Figure A.2-

11

Jarnberg et 

al. (1999; 

1998, 

1997a; 

1996)

c

Caucasian; 

conducted 

in Sweden

Male 2 and 25 2 h at 50 

W 

(bicycle)

Venous blood 

and exhaled 

air time course

Testing 

(blood 

data only)

Figure A.2-

12

Hissink et 

al. (2007)

b

Not stated; 

spoke 

Dutch as 

“native 

language”

Male 100 ppm WS 

with 7.8% 

1,2,4-TMB

6 h Venous blood 

and end 

exhaled air 

time course

Testing Figure A.2-

13

a

Five volunteers, ages 24-37, with no known occupational exposure to 1,2,4-TMB. Height of 1.70 to 1.86 m and 

weight 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 body fat (VFC), per Deurenberg et al. (1991). QPC 

estimated from the midpoint of the range for total ventilation (0.56 to 1 m3/h), average of high and low body weights, 

BW0.74 scaling, and an assumption that alveolar ventilation was 2/3 of total ventilation.

b

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).

c

Ten 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 

two days before exposure. Average body weight 76.5 kg. QPC estimated from the mean value for total ventilation 

rate during exposure, average body weights, BW0.74 scaling, and an assumption that alveolar ventilation was 2/3 of 

total ventilation. Digitized blood data (group averages) extracted from figures.

Figure A.1-11. Comparisons of model predictions to measured human venous blood 

concentrations of Kostrzewki et al. (1997) in human volunteers exposed to 150 mg 1,24-

TMB/m3 for 8 hours.

For comparisons between the Jarnberg et al. data and the model, simulations were conducted 

with QPC (calculated as described in footnote to Table A.2-12) at the elevated (working) level 

throughout the simulation, but with no other adjustments made for exercise conditions. The 
DRAFT - DO NOT CITE OR QUOTE 35

model consistently underpredicted the measured venous blood concentrations of 1,2,4-TMB 

(Figure A.2-12). At 25 ppm, blood concentrations were underpredicted by a factor of 2.1 to 3.5 

during exposure and by a factor of 1.04 to 1.5-fold in the post-exposure period, for a geometric 

mean discrepancy of 1.7 for this concentration. At 2 ppm, blood concentrations were 

underpredicted by factors of 1.7 to 2.7 during exposure and 1.01 to 1.2 in the post-exposure 

period, for a geometric mean discrepancy of 1.6 for this concentration.

Figure A.1-12. 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 

1,2,4-TMB for 2 hours while riding a bicycle (50 W).

Comparisons of model predictions and experimental data were also made for the human 

study described in Hissink et al. (2007) in which volunteers inhaled 100 ppm WS with 7.8% 

1,2,4-TMB for four hours (Figure A.2-13). The agreement between simulated and measured 

concentrations of 1,2,4-TMB in blood during exposure was excellent. The agreement between 

the modeled and measured 1,2,4-TMB in end-exhaled air during the post-exposure period was 

very good.
DRAFT - DO NOT CITE OR QUOTE 36

(a)

(b)

Figure A.1-13. Comparisons of model predictions to measured human venous blood (a) and 

end of exposure exhaled air (b) 1,2,4-TMB in human volunteers exposed to 100 ppm WS 

with 7.8% 1,2,4-TMB (Hissink et al., 2007).

 Summary of Optimization and Validation

Numerical optimization of the fit to the rat data in Hissink et al. (2007) produced a similar 

VmaxC, but smaller Km than the values determined by Hissink et al. (2007) from visual 

optimization. Changes made to values of physiological parameters may have contributed to the 

differences in optimized values. Because the rats in the Hissink et al. (2007) study were coexposed to other components of WS, the potential for these other components to alter the kinetics 

of 1,2,4-TMB was noted as a possible concern for predicting the kinetics of 1,2,4-TMB in test 

animals with no co-exposures. Another concern was the potential for kinetic changes with 

repeated exposure. As the Swiercz et al. (2003) rat kinetic study involved repeated exposure to 

1,2,4-TMB without potentially confounding co-exposures, and provides post-exposure venous 

blood time course data, it appears to be the most suitable for describing kinetics relevant to 

chronic RfC and RfD development. The VmaxC and Km values from the numerical optimization 

to the Hissink et al. (2007) rat data were used as starting values for optimization of the fit to the 

Swiercz et al. (2003) venous blood data. The improvement in fit for the low and middle 

concentrations (25 and 100 ppm) was apparent from careful visual inspection and was 
DRAFT - DO NOT CITE OR QUOTE 37

statistically significant, and these values were used in subsequent validation simulations. In 

general, the model simulations of venous blood concentrations in exposed Wistar rats, uptake by 

F344 rats, and venous blood and exhaled breath of human volunteers were acceptable. The 

measured Wistar rat arterial blood and tissue concentrations were consistently overpredicted by 

the model, suggesting collection delays in the studies. The model also consistently overpredicted 

the measured Sprague-Dawley rat tissue and blood concentrations, including the “recovery” (12 

hr post-exposure) samples, which should not be subject to collection delays. Many of the 

“validation” comparisons were made at exposure concentrations (250 ppm or greater) for which 

the optimized model did not provide accurate venous blood concentrations. It cannot be 

determined with the available data whether the 2-3-fold differences between the model and 

Sprague-Dawley rat blood concentrations at lower concentrations (75 and 150 ppm) are due to 

methodological differences (e.g. in sample collections and analysis) or true strain differences. 

Overall, we conclude that the optimized model produces acceptable simulations of venous blood 

1,2,4-TMB for chronic exposure to 100 ppm (rats) or 30 ppm (humans) or less 1,2,4-TMB by 

inhalation. If rat exposures of interest exceed 100 ppm, consideration should be given to 

reassessing model validation at high concentrations using VmaxC and Km parameters optimized 

for repeated, high concentration exposures [e.g., 250 ppm from Swiercz et al.(2003)].

Sensitivity Analysis

The primary objective of the sensitivity analysis was to evaluate the ability of the available 

data to unambiguously determine the values of both VmaxC and Km (i.e., parameter 

identifiability). Toward this end, sensitivity analyses were conducted using acslX. Because the 

selected key data set was the venous blood concentrations in the Swiercz et al. (2003) study, 

simulations were conducted to see how small changes in parameters changed the estimated 

venous blood concentrations under the conditions of this study, simulating the first 12 hrs (6 hrs 

exposure, 6 hrs post exposure), conditions that are essentially identical to those in Swiercz et al. 

(2002). The evaluations were limited to the lowest (25 ppm) and highest (250 ppm) exposure 

concentrations. It should be noted that after the optimization (Figure A.2-8b), the agreement 

between the model and the experimental data at the lower exposure concentration was superior to 

the agreement at the high concentration, so the low concentration sensitivity analysis results are 

somewhat more meaningful than the high concentration results. The results are calculated as 

normalized sensitivity coefficients (NSC) (i.e., percent change in output/percent change in input, 

calculated using the central difference method).

The interpretation of the sensitivity analysis outputs focused on the times during which blood 

concentrations were measured, so the sensitivity analyses for the first 15 minutes of exposure 

were not considered relevant. Parameters are grouped (Table A.2-13) as relatively insensitive 
DRAFT - DO NOT CITE OR QUOTE 38

(maximum|NSC| <0.2 for 0.25 hr <t <12 hr), moderately sensitive (0.2<maximum|NSC|<1.0), or 

highly sensitive (maximum|NSC|>1.0).

VmaxC/Km was identifiable from the data (as opposed to VmaxC and Km each being 

identifiable), one would expect that the NSC for these parameters would always be opposite in 

sign, and equal in magnitude, which is not the case. We conclude that Km and VmaxC are 

distinctly identifiable using the Swiercz et al. (2003; 2002) data. 

While the focus of this sensitivity analysis was to evaluate the identifiability of chemicalspecific parameters from the available data, additional insights can be obtained by considering 

the other “sensitive” parameters. Predicted blood concentrations were sensitive to the value of 

QPC (ventilation rate). If high concentrations produce a sedative effect, decreases in ventilation 

could contribute to the model’s greater over-prediction of the experimentally measured values at 

high concentrations [e.g., as high as 1000 ppm, in Zahlen et al. (1990)]. The accuracy of the 

predicted net uptake in the Dahl et al. (1988) study indicates that, at 100 ppm, the model value of 

QPC is likely appropriate, since net uptake in this relatively short experiment (80 minutes) is 

highly sensitive to the breathing rate (simulations not shown). The fractional volumes of the fat 

and slowly perfused tissues compartments are also moderately important parameters (with time 

courses similar to those of the corresponding partition coefficients shown in Figure A.2-14). The 

volume of the fat compartment in particular is known to vary with age and strain (Brown et al., 

1997), so using the same value for all studies might have an impact on the predicted kinetics. 
DRAFT - DO NOT CITE OR QUOTE 39

Table A.1-13. Parameter sensitivity for venous blood 1,2,4-TMB concentration in rats 

exposed to 1,2,4-TMB via inhalation

Parameter Insensitive

(maximum |NSC|<0.2)

Moderately Sensitive

(0.2<maximum 

|NSC|<1.0)

Highly Sensitive 

(maximum |NSC|>1.0)

BW L, H

CONC L, H

QPC L, H

VmaxC L, H

Km H L

PB L H

PF L, H

PS L, H

PR L, H

PL L, H

PBR L, H

VFC L, H

VSTOTC L, H

VRTOTC L, H

VLC L, H

VBRC L, H

QCC H L

QFC L, H

QRTOTC L, H

QLC H L

QBRC L, H

L=low exposure concentration (25 ppm), H=high exposure concentration (250 ppm)
DRAFT - DO NOT CITE OR QUOTE 40

(a)

(b)

Figure A.1-14. Time course of normalized sensitivity coefficients of moderately sensitive 

chemical-specific parameters (response: venous blood concentration) in rats exposed to (a) 

25 ppm or (b) 250 ppm of 1,2,4-TMB via inhalation for 6 hours (Swiercz et al., 2003; 

Swiercz et al., 2002)
DRAFT - DO NOT CITE OR QUOTE 41

A.1.4. Modification of the Hissink et al. (2007) model to include oral route of 

exposure

For derivation of an oral RfD, the updated 1,2,4-TMB PBPK model based on Hissink et al. 

(2007) was further modified by adding code for continuous oral ingestion. It was assumed that 

100% of the ingested 1,2,4-TMB is absorbed by constant infusion of the oral dose into the liver 

compartment. There were no oral data available to calibrate the model for oral absorption and no 

data were available evaluate the model predictions following oral ingestion either. Thus, the 

assumption that 100% of the dose would enter the liver is a common assumption.

The contribution of the first-pass metabolism in the liver for oral dosing was evaluated by 

simulating steady state venous blood levels (at the end of 50 days continuous exposure) for a 

standard human at rest (70 kg) for a range of concentrations and doses. For ease of visual 

comparison (Figure A.2-15), concentrations were converted to daily doses based on the amount 

of 1,2,4-TMB inhaled, as computed by the model. (An inhaled concentration of 0.001 mg/L 

(0.20 ppm) is equivalent to an inhaled dose of 0.12 mg/kg/day.) At both very low and very high 

daily doses by inhalation or oral dosing, steady state CV is essentially linear with respect to the 

daily dose, but with different CV/dose ratios and a transition zone between 1 and 100 mg/kg/day. 

At low daily doses, equivalent inhalation doses result in steady state blood concentrations 4-fold 

higher than an equivalent oral dose due to the hepatic first-pass effect. The first-pass effect 

becomes insignificant with respect to steady-state venous blood concentrations for daily doses in 

excess of ~50 mg/kg/day.
DRAFT - DO NOT CITE OR QUOTE 42

Figure A.2-15. 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.

A.1.5. Conclusions

Several changes were made to the model for use in this assessment: (1) Updated 

physiological parameters were implemented (Brown et al., 1997); (2) Hepatic metabolism was 

revised to omit variation over time and new VmaxC and Km values were estimated through 

numerical optimization; and (3) An oral dosing component was added to the model as constant 

infusion into the liver compartment. The values were optimized to Hissink et al. (2007) data and 

resulted in a Vmax of 4.17 mg/hr/kg0.7 and Km of 0.322 mg/L. In addition, the model was tested 

for its ability to predict published rat data resulting from exposure to 1,2,4-TMB alone (Swiercz 

et al., 2003; Swiercz et al., 2002; Eide and Zahlsen, 1996; Zahlsen et al., 1992; Zahlsen et al., 

1990; Dahl et al., 1988). Using the optimized values, the model adequately predicted the data 

and lower concentrations. Human data (Hissink et al., 2007; Järnberg and Johanson, 1999; 

Järnberg et al., 1998, 1997a; Kostrzewski et al., 1997; Järnberg et al., 1996) were also utilized to 

validate model predictions. 
DRAFT - DO NOT CITE OR QUOTE 43

A.2. Study Summary Tables for 1,2,4-TMB and 

1,3,5-TMB 

Study Summary Tables for Human Studies of 1,2,4-TMB and 1,3,5-TMB

Table A.2-1. Study Summary and Data for Chen et al. (1999)

Study (Location) Outcome Assessment

• Dockyard in Scotland, United Kingdom

• 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) or cases (case-control) Referent or control description

• 1292 TMB-exposed males who worked 

as painters in a dockyard for at least 

one year between 1950 and 1992.

• Follow up period extended from 1960 

through 1994

• 953 individuals matched by age and 

selected from lists of patients of local 

primary care physicians.

Exposure Assessment Statistical Analysis

• Exposure level: Specific concentrations 

not discussed

• Exposure duration: at least one year; 

range 1-41 years

• Compounds to which study participants 

were exposed: white spirit (1,2,4-

TMB), xylene, TMB (unspecified), nbutanol, trichlorethylene, naptha, and 

cumene.

• Intra-cohort proportional mortality 

ratios (PMRs) were calculated, as were 

standardized mortality ratios (SMRs) 

for comparison with all Scottish males. 

95% confidence intervals calculated 

assuming a Poisson distribution.

• χ

2 test 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 casecontrol study.
DRAFT - DO NOT CITE OR QUOTE 44

Table A.2-1. Study Summary and Data for Chen et al. (1999)

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% Confidence Interval)

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.
DRAFT - DO NOT CITE OR QUOTE 45

Table A.2-1. Study Summary and Data for Chen et al. (1999)

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.

Figure 3. The effect of exposure duration on odds ratio for neuropsychological symptoms. 

With increasing years of exposure, odds ratios were found to increase. 
DRAFT - DO NOT CITE OR QUOTE 46

Table A.2-2. Study Summary and Data for Billionnet et al. (2011)

Study (Location) Outcome Assessment

• Random selection of dwellings 

throughout France

• 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 (cohort) or cases (case-control) Referent or control description

• 1612 individuals living in 567 

dwellings, aged 15 or older.

• Surveys were conducted and air 

samples were collected over a period of 

one week.

• 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.

Exposure Assessment Statistical Analysis

• Exposure level: For 1,2,4-TMB, 

exposure varied from undetectable to 

111.7 μg/m3, with median 

concentration 4.0 μg/m3.

• Exposure duration: Not reported; 

reported measurements represent the 

means of one week of monitoring. 

• Pollutant correlations tested by 

Spearman’s rank correlation 

coefficient.

• Generalized estimating equation (GEE) 

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.

RESULTS

Exposure Subgroup

• Statistically significant increase in odds ratios for asthma and rhinitis following 1,2,4-

TMB exposure.

• No statistically significant increase in odds ratio for rhinitis and 1,2,4-TMB.
DRAFT - DO NOT CITE OR QUOTE 47

Table A.2-2. Study Summary and Data for Billionnet et al. (2011)

Effect Estimate ( 95% Confidence Interval)

Figure 1. Odds ratios for asthma and asthma/rhinitis and exposure to 1,2,4-TMB. For all 

models, data was adjusted for confounders. 
DRAFT - DO NOT CITE OR QUOTE 48

Table A.2-3. Study Summary and Data for Norseth et al. (1991)

Study (Location) Outcome Assessment

• Norway

• 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 (cohort) or cases (case-control) Referent or control description

• 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.

• A group of 247 maintenance workers 

who were not exposed to asphalt. The 

group was given a questionnaire similar 

to the exposed group. 

Exposure Assessment Statistical Analysis

• Mean concentration of 1,2,4-TMB was 

0.015 ppm, with range between 0 and 

0.122 ppm.

• Mean concentration of 1,3,5-TMB was 

0.0014 ppm, with range between 0 and 

0.011 ppm.

• Exposure duration: Not reported; 

measurements represent the means of 

five days of monitoring. 

• 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.

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.
DRAFT - DO NOT CITE OR QUOTE 49

Table A.2-3. Study Summary and Data for Norseth et al. (1991)

Effect Estimatesa

Observation

Symptoms Associated with Asphalt Exposure in Exposed and 

Non-Exposed Groups of Workers* 

Days With 

Symptom

Asphalt 

Workers 

(n=79)

Asphalt 

Workers 

(n=254)

Non-Asphalt 

Workers 

(n=247)

Symptoms of Asphalt Exposure

Abnormal fatigue None 64.6 75.2 84.6

1-2 21.5 14.6 9.7

3-5 13.9 10.2 5.7

Reduced appetite None 86.1 89.8 95.1

1-2 12.7 7.5 4.1

3-5 1.3 2.8 0.8

Laryngeal/pharyngeal 

irritation None 63.3 74.0 83.0

1-2 21.5 15.4 11.7

3-5 15.2 10.6 5.3

Eye irritation None 54.4 68.9 85.4

1-2 22.8 22.4 10.5

3-5 22.1 8.7 4.1

Other, unspecified 

symptom None 91.1 85.4 92.3

1-5 8.9 14.6 7.7 a

 For correlation between symptom sum and 1,2,4-TMB exposure, r=0.31, p<0.01

* All differences between asphalt workers (n=254) and non-asphalt workers (n=247) were 

statistically significant (p<0.05).
DRAFT - DO NOT CITE OR QUOTE 50

Study Summary Tables for Animal Toxicological Studies of 1,2,4-TMB and 

1,3,5-TMB 

Table A.2-4. Study Summary and Data for Korsak et al. (1995)

Study Design

Species Sex N Exposure route Dose range Exposure duration

IMP:DAK 

Wistar and 

Balb/C mice

M 8-

10/dose

Inhalation 250-2000 ppm 4 hours

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/hour

• Mean initial body weights were 250-300g for rats and 23-30g 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 two 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 pseudocumene. Rats were exposed to vapours of 

solvent for 4 hours. Rotarod performance was tested immediately after termination of exposure. 

Each point represents probit of failures on rotarod in a group of 10 rats.
DRAFT - DO NOT CITE OR QUOTE 51

Table A.2-4. Study Summary and Data for Korsak et al. (1995)

Figure 2. Hot-plate behavior in rats exposed to pseudocumene. Rats were exposed to vapors of 

solvent for 4 hours. 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.

Figure 3. Time-response relationship for the effect of 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.
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Table A.2-4. Study Summary and Data for Korsak et al. (1995)

Figure 4. Respiratory rate of mice exposed to 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.

Health Effect at 

LOAEL NOAEL LOAEL

Decreased respiration 

rate, impaired rotarod test 

performance, decreased 

pain-response time

n/a n/a

Strengths: This study used an appropriate number of animals per dose group and observed 

alterations in rotarod performance, decreases in pain sensitivity, and decreases in respiratory rates. 

Limitations: Paw-licking was used to measure response to being placed on hot plate, though other 

possible resulting behaviors were not measured. No values are provided for dose-specific responses, 

and NOAEL and LOAEL cannot be determined. 
DRAFT - DO NOT CITE OR QUOTE 53

Table A.2-5. Study Summary and Data for Korsak and Rydzyński (1996)

Study Design

Species Sex N Exposure route Dose range Exposure duration

IMP: 

Wistar

M 10/dose Inhalation (4 hrs 

or 6h/day, 5 

days/week, for 

three months) 

250-2000 ppm 4 hours or 3 months

Additional Study details

• Animals were exposed to either 1,2,4-TMB, 1,3,5-TMB or 1,2,3-TMB in a dynamic 

inhalation chamber (1.3 m3 volume) with 16 air changes/hour

• Mean initial body weights were 250-300 grams; 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 activity and 

pain sensitivity respectively.

Figure 1. Rotarod performance of rats exposed to trimethylbenzene isomers. Rats were exposed to 

solvent vapors for 4 hours. Rotarod performance was tested immediately after termination of 

exposure. Each point represents probit of failures on rotarod in a group of 10 rats.

Figure 2. Hot-plate behaviors in rats exposed to trimethylbenzene isomers. 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. 
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Table A.2-5. Study Summary and Data for Korsak and Rydzyński (1996)

Figure 3. Rotarod 

performance of rats exposed to 

1,2,4-TMB at concentrations of 

25, 100, and 250 ppm. Rats 

were exposed to vapours of 

solvents for 6h/day, 

5days/week, 3 months. 

Statistical significance marked 

by asterisks p<0.005 

Observation Latency of the Paw-Lick Response, sec

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

Control 15.4±5.8 9.7±2.1

25 ppm 18.2±5.7 11.8±3.8*

100 ppm 27.6±3.2** 16.3±6.3

250 ppm 30.1±7.9** 17.3±3.4**

250 ppm two weeks after 

termination of exposure 17.3±3.9 11.0±2.4

Health Effect at 

LOAEL NOAEL LOAEL

Decreased pain 

sensitivtiy 25 ppm 100 ppm

Strengths: This study used an appropriate number of animals per dose group and observed 

alterations in rotarod test performance and pain-response time. Additionally, the authors 

investigated the effects of exposure to 1,2,4- and 1,2,3- TMB on rotarod test performance and painsensing response two weeks after the termination of exposure.

Limitations: Paw-licking was used to measure response to being placed on hot plate, though other 

possible resulting behaviors were not measured.

*, ** statistically significant from controls at p ≤ 0.05 and p≤0.01, respectively
DRAFT - DO NOT CITE OR QUOTE 55

Table A.2-6. Study Summary and Data for Korsak et al. (1997)

Study Design

Species Sex N Exposure route Dose range Exposure duration

IMP:DA

K Wistar

M 10/dose Inhalation (6 

h/day, 5 d/week)

0, 123, 492, 1230 

mg/m3

90 days

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/hour

• 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 hours 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 1230

Body weight (mean ± SD)

Body weight (g) 411 ± 28 383 ± 25 409 ± 56 416 ± 27

BAL cell counts (mean ± SD)

Total cells (106/cm3)

a 1.93 ± 0.79 5.82 ± 1.32*** 5.96 ± 2.80** 4.45 ± 1.58*

Macrophages (106/cm3) 1.83 ± 0.03 3.78 ± 0.8 4.95 ± 0.2** 3.96 ± 0.3**

Polymorphonuclear 

leucocytes (106/cm3) 0.04 ± 0.02 1.54 ± 0.7 0.52 ± 0.6 0.21 ± 0.3

Lymphocytes (106/cm3) 0.06 ± 0.01 0.5 ± 0.2 0.5 ± 0.4 0.2 ± 0.1

Cell viability (%) 98.0 ± 1.7 95.5 ± 1.6 95.3 ± 3.5 95.3 ± 3.1

BAL protein levels and enzyme activities (mean ±SD)

Total protein (mg/ml)b 0.19 ± 0.04 0.26 ± 0.07* 0.26 ± 0.06* 0.24 ± 0.08

Mucoproteins (mg/ml)b 0.16 ± 0.03 0.14 ± 0.02* 0.13 ± 0.02 0.12 ± 0.02

Lactate dehydrogenase 

(mU/ml)b 34.2 ± 8.52 92.5 ± 37.2*** 61.3 ± 22.9* 53.8 ± 28.6 

Acid phosphatase mU/ml)b 0.87 ± 0.20 1.28 ± 0.37* 1.52 ± 0.42* 1.26 ± 0.22*

Health Effect at LOAEL NOAEL LOAEL

Increased Total BAL cells n/a 123 mg/m3

Strengths: This study used an appropriate number of animals per dose group and observed 

increases in markers of pulmonary inflammation (although attenuation of effect was observed at the 

high exposure concentration). 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 minutes)

Limitations: The authors did not report at which dose groups the numbers of polymorphonuclear 

leucocytes and lymphocytes were significantly elevated relative to control

a *, **, *** statistically significant from control at p < 0.05, 0.01, and 0.001, respectively

b Jonckheere’s test for trend: total protein, p = 0.0577; mucroprotein, p = 0.3949; lactate dehydrogenase, p = 0.2805; 

acid phosphatase, p = 0.0164
DRAFT - DO NOT CITE OR QUOTE 56

Table A.2-7. Study Summary and Data for Korsak et al. (2000)

Study Design

Species Sex N Exposure route Dose range Exposure duration

IMP: 

Wistar

M & 

F

10/dose Inhalation (6 

h/day, 5 d/week)

0, 123, 492, 1230 

mg/m3

90 days

Additional Study details

• Animals were exposed to 1,2,4-TMB in a dynamic inhalation chamber (1.3 m3 volume) with 

16 air changes/hour

• 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 week prior to termination 

of exposure; blood clinical chemistry parameters were evaluated 18 hours after termination 

of exposure (animals were deprived of food for 24 hours)

• Necropsy was performed on all animals. Pulmonary lesions were graded using an arbitrary 

scale: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked

Observation Exposure Duration (mg/m3

)

0 123 492 1230

Body and Organ weights (mean ± SD)

Males

Terminal Body weight (g) 368 ± 22 390 ± 26 399 ± 22 389 ± 29

Absolute organ weight (g)

Lungs 1.78 ± 0.28 1.83 ± 0.25 2.93 ± 0.26* 1.78 ± 0.36

Liver 10.27 ± 1.82 11.43 ± 1.05 10.78 ± 1.33 10.86 ± 2.04

Spleen 0.68 ± 0.08 0.85 ± 0.19* 0.79 ± 0.09 0.72 ± 0.08

Kidney 2.06 ± 0.13 2.24 ± 0.15 2.14 ± 0.15 2.18 ± 0.16

Adrenals 0.048 ± 0.007 0.046 ± 0.0050. 054 ± 0.011 0.047 ± 0.005

Testes 3.72 ± 0.35 3.90 ± 0.38 4.03 ± 0.27 3.87 ± 0.24

Heart 0.90 ± 0.04 0.94 ± 0.06 0.94 ± 0.08 0.96 ± 0.07

Relative organ weight (g)

Lungs 0.496 ± 0.056 0.475 ± 0.056 0.586 ± 0.115 0.477 ± 0.080

Liver 2.896 ± 0.456 2.894 ± 0.427 2.990 ± 0.465 2.901 ± 0.479

Spleen 0.189 ± 0.011 0.220 ± 0.041 0.210 ± 0.018 0.200 ± 0.018

Kidney 0.588 ± 0.029 0.585 ± 0.022 0.587 ± 0.065 0.586 ± 0.040

Adrenals 0.011 ± 0.003 0.010 ± 0.000 0.022 ± 0.024 0.011 ± 0.003

Testes 1.041 ± 0.076 1.020 ± 0.079 1.067 ± 0.102 1.039 ± 0.077

Heart 0.252 ± 0.013 0.239 ± 0.020 0.249 ± 0.014 0.258 ± 0.020

Females

Terminal Body weight (g) 243 ± 16 243 ± 19 230 ± 14 229 ± 21

Absolute organ weight (g)

Lungs 1.29 ± 0.18 1.32 ± 0.12 1.25 ± 0.13 1.23 ± 0.11

Liver 6.48 ± 1.02 6.54 ± 0.69 5.81 ± 0.83 6.72 ± 1.34
DRAFT - DO NOT CITE OR QUOTE 57

Table A.2-7. Study Summary and Data for Korsak et al. (2000)

Spleen 0.59 ± 0.08 0.61 ± 0.11 0.49 ± 0.06* 0.52 ± 0.08

Kidney 1.55 ± 0.12 1.50 ± 0.14 1.38 ± 0.11* 1.44 ± 0.19

Adrenals 0.065 ± 0.007 0.070 ± 0.008 0.066 ± 0.010 0.061 ± 0.013

Ovaries 0.09 ± 0.02 0.09 ± 0.01 0.09 ± 0.27 0.09 ± 0.02

Heart 0.66 ± 0.07 0.64 ± 0.05 0.61 ± 0.07 0.63 ± 0.06

Relative organ weight (g)

Lungs 0.555 ± 0.058 0.581 ± 0.040 0.596 ± 0.051 0.569 ± 0.053

Liver 2.770 ± 0.222 2.881 ± 0.309 2.758 ± 0.223 3.078 ± 0.434

Spleen 0.255 ± 0.025 0.266 ± 0.031 0.237 ± 0.036 0.24 ± 0.033

Kidney 0.667 ± 0.030 0.661 ± 0.047 0.660 ± 0.042 0.662 ± 0.036

Adrenals 0.0028 ± 0.006 0.031 ± 0.006 0.032 ± 0.006 0.029 ± 0.006

Ovaries 0.043 ± 0.008 0.041 ± 0.006 0.045 ± 0.013 0.047 ± 0.009

Heart 0.284 ± 0.023 0.283 ± 0.025 0.291 ± 0.025 0.289 ± 0.015

Observation

Exposure Duration (mg/m3)

0 123 492 1230 1230a Trend 

testb

Hematological parameters (mean ± SD)

Males

Hematocrit (%) 49.9 ± 1.9 50.4 ± 2.0 50.0 ± 1.9 50.6 ± 1.5 50.1 ± 1.1 0.2993

Hemoglobin (g/dL) 15.1 ± 1.1 15.6 ± 0.9 15.4 ± 0.9 15.4 ± 0.6 16.0 ± 1.0 0.2112

RBCs (× 103/mm3)

c 9.98 ± 1.68 9.84 ± 

1.82

8.50 ± 

1.11

7.70 ± 

1.38** 7.61 ± 1.6 0.0004

WBCs (× 103/mm3)

d 8.68 ± 2.89 8.92 ± 

3.44

8.30 ± 

1.84

15.89 ± 

5.74** 7.11 ± 2.1 0.0019

Rod neutrophil (%) 0.0 ± 0.0 0.4 ± 0.5 0.2 ± 0.4 0.9 ± 1.5 0.7 ± 0.8 0.0589

Segmented neutrophil (%) 24.1 ± 9.2 19.7 ± 6.5 20.7 ± 7.7 18.9 ± 10.8 29.4 ± 6.4 0.0730

Eosinophil (%) 1.2 ± 1.7 1.2 ± 1.0 0.4 ± 0.6 1.7 ± 1.4 1.5 ± 1.5 0.2950

Lymphocyte (%) 73.5 ± 10.3 76.2 ± 7.1 76.8 ± 8.5 75.8 ± 16.0 65.4 ± 8.9 0.1297

Monocyte (%) 1.1 ± 1.3 2.5 ± 2.1 2.3 ± 2.2 1.8 ± 2.5 2.7 ± 2.5 0.3818

Lymphoblast (%) 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.8 ± 1.3 0.3 ± 0.9 0.1387

Myelocyte (%) 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.4 0.0 ± 0.0 0.0 ± 0.0 0.4046

Erythroblase (%) 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.5000

Reticulocyte (%) 3.1 ± 2.3 2.3 ± 1.4 2.8 ± 2.1 3.1 ± 2.5 6.4 ± 3.2 0.4900

Platelet (× 103/mm3) 294 ± 46 293 ± 73 359 ± 46 335 ± 80 386 ± 70 0.0741

Clotting time (sec) 43 ± 19 41 ± 17 37 ± 13 33 ± 7 56 ± 21 0.1457

Females

Hematocrit (%) 46.0 ± 1.6 46.6 ± 2.7 47.0 ± 2.7 46.5 ± 4.1 45.8 ± 1.3 0.2336

Hemoglobin (g/dL) 14.5 ± 0.9 13.8 ± 1.3 14.4 ± 0.9 14.2 ± 0.9 14.9 ± 0.9 0.3461

RBCs (× 103/mm3)

c 8.22 ± 1.16 7.93 ± 

2.04

8.51 ± 

1.13 7.71 ± 1.58 6.99 ± 1.8 0.1891

WBCs (× 103/mm3)

d 7.50 ± 1.31 6.76 ± 

2.95

9.55 ± 

4.48 9.83 ± 3.74 7.11 ± 2.4 0.0307

Rod neutrophil (%) 1.4 ± 1.6 0.5 ± 0.7 0.4 ± 0.5 0.4 ± 0.9 0.5 ± 0.7 0.3270
DRAFT - DO NOT CITE OR QUOTE 58

Table A.2-7. Study Summary and Data for Korsak et al. (2000)

Segmented neutrophil (%) 22.8 ± 6.5 15.5 ± 7.9 20.7 ± 7.5 17.4 ± 9.3 20.5 ± 9.5 0.1868

Eosinophil (%) 1.2 ± 0.6 16 ± 1.6 1.1 ± 1.7 1.2 ± 2.1 2.0 ± 1.7 0.1051

Lymphocyte (%) 73.2 ± 7.9 79.4 ± 8.4 75.5 ± 7.4 78.8 ± 11.6 74.1 ± 9.5 0.2140

Monocyte (%) 1.2 ± 1.3 2.6 ± 2.8 1.3 ± 1.7 1.5 ± 0.8 1.5 ± 1.4 0.4156

Lymphoblast (%) 0.0 ± 0.0 0.1 ± 0.3 0.5 ± 1.5 0.7 ± 1.1 0.8 ± 1.3 0.1361

Myelocyte (%) 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 1.5 0.1 ± 0.3 0.1 ± 0.3 0.3189

Erythroblase (%) 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.5000

Reticulocyte (%) 3.5 ± 2.6 1.7 ± 2.0 1.8 ± 0.9 1.0 ± 0.6* 5.8 ± 3.6 0.0137

Platelet (× 103/mm3) 306 ± 34 234 ± 50* 303 ± 48 325 ± 57 349 ± 77 0.1542

Clotting time (sec) 30 ± 10 23 ± 4 19 ± 5** 22 ± 7* 48 ± 19 0.0034

Observation

Exposure Duration (mg/m3)

0 123 492 1230 Trend 

testb

Clinical Chemistry Parameters (mean ± SD)

Males

AST (U/dL)e 138.7 ± 20.6 141.3 ± 21.0 134.5 ± 27.0 138 ± 35.0 0.2223

ALT (U/dL)f 51.7 ± 5.9 48.3 ± 7.8 49.7 ± 9.1 46.8 ± 5.1 0.0637

ALP (U/dL)g 80.4 ± 12.0 86.2 ± 22.0 84.9 ± 21.0 90.5 ± 19.0 0.1518

SDH (U/dL)h 6.6 ± 1.4 8.1 ± 0.8** 7.8 ± 1.0* 8.0 ± 1.1** 0.0083

GGT (μU/ml)i 0.22 ± 0.44 0.20 ± 0.42 0.20 ± 0.42 0.20 ± 0.42 0.4700

Bilirubin (mg/dL) 1.027 ± 0.193 0.974 ± 0.338 1.106 ± 0.289 0.932 ± 0.175 0.2594

Total cholesterol (mg/dL) 63.6 ± 13.0 69.1 ± 12.0 72.4 ± 14.9 70.6 ± 19.5 0.0920

Glucose (mg/dL) 141.9 ± 23.9 163.8 ± 29.7 157.9 ± 23.2 162.2 ± 28.9 0.0876

Total protein (g) 5.43 ± 1.00 5.47 ± 1.39 5.34 ± 1.29 5.82 ± 1.49 0.3242

Albumin (g) 3.25 ± 0.60 3.45 ± 0.56 3.41 ± 0.83 3.53 ± 0.66 0.2279

Creatinine (mg/dL) 0.506 ± 0.099 0.437 ± 0.138 0.510 ± 0.150 0.490 ± 0.178 0.3982

Urea (mg/dL) 54.2 ± 8.6 48.8 ± 8.3 47.6 ± 3.4 49.0 ± 8.7 0.1145

Calcium (mg/dL) 10.4 ± 0.5 10.8 ± 0.5 10.7 ± 0.8 10.8 ± 0.7 0.2449

Phosphorus (mg/dL) 6.27 ± 0.49 6.50 ± 0.57 6.49 ± 0.61 6.46 ± 0.78 0.1580

Sodium (mmol/L) 139.0 ± 1.4 1393 ± 1.3 139.6 ± 1.4 139.0 ± 1.4 0.4950

Potassium (mmol/L) 4.87 ± 0.36 4.97 ± 0.34 4.97 ± 0.25 4.83 ± 0.40 0.2907

Chloride (mmol/L) 106.6 ± 1.2 106.1 ± 1.7 106.3 ± 1.5 106.7 ± 1.2 0.4353

Females

AST (U/dL)e 139.4 ± 16.6 136.7 ± 27.1 145.5 ± 22.7 141.4 ± 15.6 0.2118

ALT (U/dL)f 49.8 ± 6.3 51.4 ± 8.2 50.4 ± 9.0 55.1 ± 9.5 0.1844

ALP (U/dL)g 41.2 ± 7.8 37.2 ± 6.8 39.8 ± 11.0 49.8 ± 15.5 0.1740

SDH (U/dL)h 5.9 ± 1.5 7.3 ± 1.7 7.1 ± 1.8 7.0 ± 1.6 0.0637

GGT (μU/ml)i 0.20 ± 0.42 0.30 ± 0.48 0.10 ± 0.32 0.44 ± 0.53 0.2821

Bilirubin (mg/dL) 0.745 ± 0.342 0.690 ± 0.396 0.743 ± 0.248 0.642 ± 0.257 0.3092

Total cholesterol (mg/dL) 64.5 ± 11.9 65.7 ± 12.8 64.1 ± 10.8 62.5 ± 7.6 0.4775

Glucose (mg/dL) 118.2 ± 28.8 138.8 ± 38.5 104.5 ± 23.8 129.9 ± 39.7 0.4838

Total protein (g) 6.91 ± 0.53 7.44 ± 0.89 7.08 ± 0.35 6.94 ± 0.64 0.4036
DRAFT - DO NOT CITE OR QUOTE 59

Table A.2-7. Study Summary and Data for Korsak et al. (2000)

Albumin (g) 3.42 ± 0.24 3.46 ± 0.27 3.61 ± 0.26 3.42 ± 0.15 0.2408

Creatinine (mg/dL) 0.655 ± 0.135 0.553 ± 0.104 0.629 ± 0.153 0.577 ± 0.133 0.1641

Urea (mg/dL) 52.7 ± 7.8 49.6 ± 6.7 52.8 ± 10.5 52.2 ± 11.8 0.4718

Calcium (mg/dL) 10.5 ± 0.6 10.8 ± 0.8 10.6 ± 0.5 10.8 ± 0.6 0.3011

Phosphorus (mg/dL) 4.75 ± 0.54 5.05 ± 0.70 5.34 ± 0.74 4.90 ± 1.01 0.4050

Sodium (mmol/L) 137.9 ± 1.7 138.0 ± 1.8 137.8 ± 2.5 138.2 ± 2.2 0.3628

Potassium (mmol/L) 4.54 ± 0.22 4.39 ± 0.61 4.51 ± 0.26 4.46 ± 0.25 0.4108

Chloride (mmol/L) 104.9 ± 2.0 105.5 ± 1.3 105.9 ± 1.6 106.4 ± 1.8 0.0601

Observation

Exposure Duration (mg/m3)

0 123 492 1230 Comparison 

to Controls

Trend 

testb

Males

Proliferation of 

peribronchial lymphatic 

tissue (0-4)j

16.0k 15.6 30.6 17.4 1-3* 0.13

Formation of 

lymphoepithelium in 

bronchii (0-4)

18.1 15.6 27.9 18.2 ns 22

Bronchitis and 

bronchopneumonia (0-4) 19.0 18.3 26.1 16.5 ns 0.49

Interstitial lymphocytic 

infiltration (0-3) 14.8 18.4 26.9 19.4 1-3* 0.12

Alveolar macrophages 

(0-3) 14.1 14.8 24.1 26.4 1-4* 0.002

Cumulative score of all 

individuals 13.9 15.1 29.1 21.3 1-3* 0.02

Females

Proliferation of 

peribronchial lymphatic 

tissue (0-4)j

19.4 21.7 21.2 17.5 ns 0.36

Formation of 

lymphoepithelium in 

bronchii (0-4)

18.3 20.1 25.1 16.1 ns 0.48

Bronchitis and 

bronchopneumonia (0-4) 19.0 22.9 19.0 19.0 ns 0.48

Interstitial lymphocytic 

infiltration (0-3) 15.8 14.5 21.5 29.2 1-4* 0.0017

Alveolar macrophages 

(0-3) 19.7 14.9 16.6 29.8 ns 0.03

Cumulative score of all 

individuals 16.8 15.3 21.3 27.3 ns 0.01

Health Effect at 

LOAEL NOAEL LOAEL

Increased pulmonary 123 mg/m3 492 mg/m3
DRAFT - DO NOT CITE OR QUOTE 60

Table A.2-7. Study Summary and Data for Korsak et al. (2000)

lesions, decreased RBCs, 

and increased WBCs in 

males

Strengths: This study used an appropriate number of animals per dose group and observed 

alterations in a number of hematological parameters and increases in pulmonary inflammatory 

lesions in both female and male animals. The observed inflammatory lesions are coherent with 

observations of increased inflammatory cell populations in bronchoalveolar lavage fluid in Korsak et 

al. (1997). Additionally, the authors investigated the effects of exposure to 1230 mg/m3 1,2,4-TMB 

on hematological effects two weeks after the termination of exposure.

Limitations: 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

a effects measured in rats exposed to 1230 mg/m3 two weeks after termination of exposure

b Jonckheere’s test for trend, p < 0.05 indicates statistical significance

c red blood cells, d white blood cells, e aspartate aminotransferase, f alanine aminotransferase, g

alkaline phosphatase, h sorbitol dehydrogenase, i

γ-glutamyltransferase, j grading system (0-4, 0-3; 

see Additional Study details above)

*, ** statistically significant from controls at p < 0.05 and 0.01, respectively
DRAFT - DO NOT CITE OR QUOTE 61

Table A.2-8. Study Summary and Data for Saillenfait et al.(2005)

Study Design

Species Sex N Exposure route Dose range Exposure duration

SpragueDawleyF & 

M

24 dams 

per dose

Inhalation (6 

h/day GD 6-20)

0, 100, 300, 600, 900 

ppm 1,2,4-TMB; 0, 

100, 300, 600, 1200 

ppm 1,3,5-TMB

Gestational days 6-20 

Additional Study details

• Animals were exposed to 1,2,4- or 1,3,5 TMB in 200 liter glass/steel inhalation chambers for 

6 hours per day starting on gestation day 6 and ending on gestation day 20.

• Animals were randomized and assigned to the experimental groups.

• After gestational day 20, dams were sacrificed and weighed, as were their uteri and any 

fetuses.

• Decreases in maternal body weight and fetal toxicity were observed.

Observation Exposure to 1,3,5-TMB (ppm/6 h/day)

0 100 300 600 1200

Maternal Parameters 

No. treated 24 24 24 24 24

No. (%) pregnant at 

euthanization 21 (87.5) 22 (91.7) 21 (87.5) 17 (70.8) 18 (75.0)

No. deaths 0 0 0 0 0

Body weight (g) on day 6 274 ± 17g 273 ± 16 274 ± 21 270 ± 17 275 ± 14

Body weight change (g)

Days 0-6 31 ± 11 31 ± 8 31 ± 7 29 ± 8 28 ± 8

Days 6-13 25 ± 12 29 ± 4 23 ± 6 16 ± 8** 10 ± 7

Days 13-21 110 ± 14 109 ± 10 95 ± 21* 80 ± 20** 63 ± 26**

Days 6-21 135 ± 15 138 ± 11 118 ± 24* 95 ± 24** 73 ± 28**

Corrected weight gaina 29 ± 14 30 ± 9 20 ± 12 7 ± 20** -12 ± 19**

Food consumption (g/day)

Days 0-6 22 ± 2 22 ± 3 22 ± 2 22 ± 2 23 ± 2

Days 6-13 22 ± 2 22 ± 2 20 ± 1* 18 ± 2** 17 ± 2**

Days 13-21 26 ± 2 25 ± 2 24 ± 2* 21 ± 3** 19 ± 3**

Days 6-21 24 ± 2 24 ± 2 22 ± 2* 20 ± 2** 18 ± 2**

Observation Exposure to 1,3,5-TMB (ppm/6 h/day)

0 100 300 600 1200

Gestational parameters 

All littersb 21 22 21 17 18

No. of corpora lutea per 

dam 15.3 ± 1.5g 15.4 ± 1.7 15.5 ± 1.7 14.9 ± 2.1 15.2 ± 1.5

Mean no. of implantation 

sites per litter 14.9 ± 1.5 14.9 ± 1.8 14.5 ± 3.4 13.0 ± 5.1 13.6 ± 3.7
DRAFT - DO NOT CITE OR QUOTE 62

Table A.2-8. Study Summary and Data for Saillenfait et al.(2005)

Mean % post-implantation 

loss per litterc 4.8 ± 4.2 3.9 ± 4.3 6.8 ± 8.5 1.6 ± 3.7 4.4 ± 6.9

Mean % dead fetuses per 

litter 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

Mean % resorption sites 

per litter 4.8 ± 4.2 3.9 ± 4.3 6.3 ± 6.5 1.6 ± 3.7 4.4 ± 6.9

Live littersd 21 22 21 17 18

Mean no. of live fetuses 

per litter 14.1 ± 1.6 14.3 ± 1.7 13.4 ± 3.4 12.8 ± 5.0 13.1 ± 3.7

Mean % male fetuses per 

litter 49.3 ± 13.5 48.2 ± 16.3 52.1 ± 18.1 51.1 ± 20.9 48.5 ± 18.2

Fetal body weight (g)

All fetuses 5.64 ± 0.35 5.61 ± 0.24 5.43 ± 0.45 5.36 ± 0.68 4.98 ± 

0.56**

Male fetuses 5.80 ± 0.41 5.76 ± 0.27 5.50 ± 0.31 5.39 ± 0.55* 5.10 ± 

0.57**

Female Fetuses 5.50 ± 0.32 5.47 ± 0.21 5.27 ± 0.47 5.18 ± 0.68 4.81 ± 

0.45**

Observation Exposure to 1,3,5-TMB (ppm/6 h/day)

0 100 300 600 1200

Fetal Variations and Malformations

Total no. fetuses 

examined (litters)

External 297 (21) 314 (22) 282 (21) 217 (17) 236 (18)

Visceral 149 (21) 157 (22) 141 (20) 109 (15) 118 (18)

Skeletal 148 (21) 157 (22) 141 (21) 108 (17) 118 (18)

Malformations

Diaphragmatic hernia 0 1 (1) 0 1 (1) 0

Multiple skeletal 

malformationse 1 (1) 0 0 0 0

External variations 0 0 0 0 0

Club foot (bilateral) 0 1 (1) 0 0 0

Visceral variations

Dilated renal pelvis 2 (2) 0 5 (4) 0 2 (2)

Distended ureter 12 (9) 14 (8) 18 (8) 5 (3) 11 (6)

Skeletal variations

Fifth sternebrae incomplete 

ossification or unossifiedf 2 (2) 2 (2) 7 (4) 7 (5) 12 (7)

Fourth sternebrae, split 0 0 0 0 1 (1)

Cervical rib, rudimentary 2 (2) 0 5 (5) 5 (3) 2 (2)

Fourteenth rib, 

supernumerary 11 (8) 9 (6) 11 (6) 15 (8) 17 (8)
DRAFT - DO NOT CITE OR QUOTE 63

Table A.2-8. Study Summary and Data for Saillenfait et al.(2005)

Thoracic vertebra centra, 

incomplete ossification 10 (5) 8 (6) 10 (7) 9 (7) 9 (7)

Observation Exposure to 1,2,4-TMB (ppm/6 h/day)

0 100 300 600 900

Maternal Parameters

No. treated 25 24 24 24 24

No. (%) pregnant at 

euthanization 24 (96.0) 22 (91.7) 22 (91.7) 22 (91.7) 24 (100)

No. deaths 0 0 0 0 0

Body weight (g) on day 6 271 ± 18g 272 ± 21 272 ± 22 275 ± 19 269 ± 18

Body weight change (g)

Days 0-6 27 ± 8 28 ± 6 28 ± 7 28 ± 12 24 ± 8

Days 6-13 27 ± 8 27 ± 6 26 ± 6 19 ± 8** 14 ± 12**

Days 13-21 105 ± 28 98 ± 16 100 ± 20 97 ± 17 82 ± 14**

Days 6-21 131 ± 33 124 ± 18 126 ± 24 116 ± 23 95 ± 19**

Corrected weight gaina 29 ± 12 31 ± 14 27 ± 12 15 ± 17** 0 ± 14**

Food consumption 

(g/day)

Days 0-6 23 ± 2 23 ± 2 23 ± 2 23 ± 3 23 ± 3

Days 6-13 21 ± 3 20 ± 2 20 ± 2 18 ± 2** 17 ± 2**

Days 13-21 26 ± 3 25 ± 2 24 ± 2 23 ± 3** 22 ± 3**

Days 6-21 24 ± 3 23 ± 2 22 ± 2 21 ± 3** 20 ± 2**

Observation Exposure to 1,2,4-TMB (ppm/6 h/day)

0 100 300 600 900

Gestational Parameters

All littersb 24 22 22 22 24

No. of corpora lutea per 

dam 15.4 ± 2.1g 15.2 ± 1.3 15.2 ± 2.1 15.8 ± 1.7 15.7 ± 2.5

Mean no. of implantation 

sites per litter 14.2 ± 3.3 13.7 ± 2.9 14.1 ± 3.2 14.9=-2.4 15.0 ± 2.4

Mean % post-implantation 

loss per litterc 10.0 ± 22.1 8.6 ± 8.9 5.8 ± 6.8 5.0 ± 5.7 5.4 ± 6.7

Mean % dead fetuses per 

litter 0.0 ± 0.0 0.3 ± 1.5 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

Mean % resorption sites 

per litter 10.0 ± 22.1 8.3 ± 9.1 5.8 ± 6.8 5.0 ± 5.7 6.4 ± 6.7

Live littersd 23 22 22 22 24

Mean no. of live fetuses 

per litter 13.9 ± 2.5 12.5 ± 3.0 13.3 ± 3.2 14.1 ± 2.3 14.3 ± 2.6

Mean % male fetuses per 

litter 46.6 ± 17.1 46.0 ± 14.1 49.9 ± 13.4 46.2 ± 15.4 50.4 ± 16.2

Fetal body weight (g)

All fetuses 5.71 ± 0.34 5.64 ± 0.31 5.56 ± 0.47 5.40 ± 0.39* 5.60 ± 0.40**

Male fetuses 5.86 ± 0.34 5.79 ± 0.30 5.72 ± 0.49 5.55 ± 0.48* 5.20 ± 0.42**
DRAFT - DO NOT CITE OR QUOTE 64

Table A.2-8. Study Summary and Data for Saillenfait et al.(2005)

Female Fetuses 5.57 ± 0.33 5.51 ± 0.31 5.40 ± 0.45 5.28 ± 0.40* 4.92 ± 0.40**

Observation Exposure to 1,2,4-TMB (ppm/6 h/day)

0 100 300 600 900

Fetal Variations and Malformations

Total no. fetuses 

examined (litters)

External 319 (23) 275 (22) 293 (22) 310 (22) 342 (24)

Visceral 160 (23) 137 (22) 147 (22) 155 (22) 171 (24)

Skeletal 159 (23) 138 (22) 146 (22) 155 (22) 171 (24)

Malformations

Diaphragmatic hernia 0 0 1 (1) 0 1 (1)

Multiple skeletal 

malformationse 0 0 0 1 (1) 0

External variations

Club foot (bilateral) 3 (3) 0 0 0 0

Visceral variations

Dilated renal pelvis 3 (3) 3 (3) 3 (3) 3 (3) 3 (2)

Distended ureter 7 (4) 5 (3) 8 (5) 8 (5) 2 (2)

Skeletal variations

Third sternebrae, 

incomplete ossification 0 1 (1) 0 0 0

Fifth sternebrae 

incomplete ossification 

or unossifiedf

1 (1) 0 4 (4) 5 (4) 6 (6)

Extra ossification site 0 1 (1) 0 0 0

Cervical rib, rudimentary 1 (1) 2 (2) 0 3 (2) 2 (2)

Fourteenth rib, 

supernumerary 25 (10) 13 (8) 18 (12) 21 (10) 34 (16)

Thirteenth rib, short 

(unilateral) 1 (1) 0 0 0 0

Thoracic vertebral centra, 

incomplete ossification 8 (6) 4 (4) 7 (4) 6 (6) 7 (5)

Health Effect at 

LOAEL NOAEL LOAEL

Maternal toxicity: 

decrease in maternal 

body weight and food 

consumption

Developmental toxicity: 

significant reduction in 

fetal body weight

Maternal toxicity: 300 ppm for 

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

Fetal toxicity: 300 ppm for 1,2,4-

and 1,3,5-TMB 

Maternal toxicity: 600 ppm for 

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

Fetal toxicity: 600 ppm for 1,2,4-

and 1,3,5-TMB

Strengths: This study used an appropriate number of animals per dose group and observed 

alterations in a number of maternal and fetal parameters, including decreased maternal and fetal 
DRAFT - DO NOT CITE OR QUOTE 65

Table A.2-8. Study Summary and Data for Saillenfait et al.(2005)

weight. Values reported by authors can be used to determine NOAEL and LOAEL. 

Limitations: No investigation of preimplantation developmental toxicity due to TMB isomer 

exposure 

a Body weight gain during GD 6-21 minus gravid uterine weight b Includes all animals pregnant at euthanization

c Resorptions plus dead fetuses

d Includes all animals with live fetuses at euthanization

e Runt showing skeletal alterations including missing ribs, missing thoracic vertebrae, incomplete ossification of 

sternebrae and skull bones

f Unossified=alizarine red S negative

g Mean ± standard deviation

*, ** statistically significant from controls at p < 0.05 and 0.01, respectively
DRAFT - DO NOT CITE OR QUOTE 66

Table A.2-9. Study Summary and Data for Gralewicz et al. (1997a)

Study Design

Species Sex N Exposure route Dose range Exposure duration

Wistar M 15 rats 

per dose

Inhalation (6 

h/day, 5 days/wk)

0, 25, 100, or 250 ppm 

1,2,4-TMB

4 weeks 

Additional Study details

• Animals were exposed to 1,2,4-TMB in 1.3 m3 dynamic inhalation exposure chambers for 6 

hours per day, 5 days per week for 4 weeks. 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 exposure 

levels.

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 

TMB. The bars represent group means and SE 

(n=15 for each group). *p<0.05 compared with 

TMB0 group (0 ppm control group)
DRAFT - DO NOT CITE OR QUOTE 67

Table A.2-9. Study Summary and Data for Gralewicz et al. (1997a)

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-h intervals. The stepdown response was punished by a 10-s 

foot shock only in trial 3. Trials 4, 5, 

and 6 were performed 24 h, 3 days, and 

7 days after trial 3, respectively. The 

maximum step-down latency was 180s. 

The bars represent group means and SE 

(n=15 for each group). ***p<0.001 

compared with respective data from 

group TMB0 (0 ppm control group). 

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. L1: 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 L1 in the 

same group. 

Lower diagram: A comparison of the change 

in the paw-lick latency noted 24 hours after 

foot shock (trial 3). ***p<0.001, **p<0.01 

when compared to TMB0 (0 ppm control 

group).
DRAFT - DO NOT CITE OR QUOTE 68

Table A.2-9. Study Summary and Data for Gralewicz et al. (1997a)

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 fivetrial 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. 

Health Effect at 

LOAEL NOAEL LOAEL

Open field grooming 

significantly increased, 

lower than expected step 

down latency 

25 ppm 100 ppm

Strengths: This study used an appropriate number of animals per dose group and observed 

alterations in a number of behavioral tests. Values reported by authors can be used to determine 

NOAEL and LOAEL. CNS disturbances observed up to 2 months after termination of exposure, 

indicating the persistence of effects.

Limitations: Duration of exposure only 4 weeks. Generally, short-term exposure studies have 

limited utility in quantitation of human health reference values. 
DRAFT - DO NOT CITE OR QUOTE 69

Table A.2-10. Study Summary and Data for Gralewicz et al. (1997b)

Study Design

Species Sex N Exposure route Dose range Exposure duration

Wistar M 9 rats 

per dose

Inhalation (6 

h/day, 5 days/wk)

0, 25, 100, or 250 ppm

1,2,4-TMB

4 weeks 

Additional Study details

• Animals were exposed to 1,2,4-TMB in 1.3 m3 dynamic inhalation exposure chambers for 6 

hours per day, 5 days per week for 4 weeks. 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 did not show an increase in SWD activity. 

Rats exposed to 0 or 25 ppm 1,2,4-TMB showed progressively decreasing levels of SWD 

activity. 

Figure 1. Diagrams showing the 

effect of a four-week inhalation 

exposure to TMB on the 

contribution of TRANS (upper 

diagram, HA (middle diagram), 

and SWS (lower diagram)) states 

in the rat EEG during successive 

on-hour recording periods. The 

bars represent group means and 

SE. 
DRAFT - DO NOT CITE OR QUOTE 70

Table A.2-10. Study Summary and Data for Gralewicz et al. (1997b)

Figure 2. Diagram 

showing the effect of a 

four-week inhalation 

exposure to TMS on the 

SWD burst occurrence 

(upper diagram) and on 

the percent contribution 

of SWD activity within 

TRANS state (lower 

diagram) during 

successive one-hour 

recording periods. The 

bars represent group 

means and SE. * denotes 

p<0.05 in comparison to 

the preexposure value in 

the same group. 

Health Effect at LOAEL NOAEL LOAEL

Decreased spike-wave 

discharges 25 ppm 100 ppm

Strengths: This study used an appropriate number of animals per dose group. CNS disturbances 

observed up to 4 months after termination of exposure, indicating the persistence of effects.

Limitations: Duration of exposure only 4 weeks. Generally, short-term exposure studies have 

limited utility in quantitation of human health reference values. 
DRAFT - DO NOT CITE OR QUOTE 71

Table A.2-11. Study Summary and Data for Gralewicz and Wiaderna (2001)

Study Design

Species Sex N Exposure route Dose range Exposure duration

Wistar M 10 or 11 

rats per 

dose

Inhalation (6 

h/day, 5 days/wk)

0 or 100 ppm 1,2,4- or 

1,3,5-TMB

4 weeks 

Additional Study details

• Animals were exposed to 1,2,4- or 1,3,5-TMB in 1.3 m3 dynamic inhalation exposure 

chambers for 6 hours per day, 5 days per week for 4 weeks. 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 two weeks post-exposure.

• 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.

Figure 1. Radial maze performace of rats 

exposed for 4 weeks to m-xylene or a TMB 

isomer at a concentration of 100 ppm. The test 

(one trial a day) was performed on days 14-18 

after exposure. The diagrams illustrate the 

number of perseveration (upper diagram) and 

omission (lower diagram) errors in successive 

daily trials. 

Denotation: Control- sham exposed group 

(n=10), XYL- m-xylene exposed group (n=11), 

PS- 1,2,4-TMB exposed group (n=11), MES1,2,3-TMB exposed group (n=11), HMhemimellitene exposed group (n=11). Bars 

represent group means and SE. 
DRAFT - DO NOT CITE OR QUOTE 72

Table A.2-11. Study Summary and Data for Gralewicz and Wiaderna (2001)

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. 

Bars represent group 

means and SE. 

Figure 3. Diagram illustrating 

the effect of a four-week 

inhalation exposure to mxylene or a TMB isomer at 

concentration of 100 ppm 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. 

Trials 4, 5, and 6 were 

performed 24 h, 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. 
DRAFT - DO NOT CITE OR QUOTE 73

Table A.2-11. Study Summary and Data for Gralewicz and Wiaderna (2001)

Figure 4. A comparison of 

sham-exposed and solventexposed rats with respect to 

the latency of the paw-lick 

response to heat (54.5°C) 

before (L1), several seconds 

after (L2), and 24 h after a 

two minute 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. 

Bars represent group means 

and SE. 

Figure 5. Active avoidance 

learning in rats after a 4-week 

inhalation exposure to mxylene or a TMB isomer at a 

concentration of 100 ppm. In 

on massed-trial session (intertrial interval 20-40s;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. 

Health Effect at LOAEL NOAEL LOAEL

Deleterious effects on 

locomotor activity, passive 

avoidance learning, and 

paw-lick latencies 

n/a 100 ppm

Strengths: This study used an appropriate number of animals per dose group and observed 

alterations in a number of behavioral tests. CNS disturbances observed up to 2 months after 

termination of exposure, indicating the persistence of effects.

Limitations: Only one exposure group, unable to determine NOAEL for this study. Duration of 

exposure only 4 weeks. Generally, short-term exposure studies have limited utility in quantitation of 

human health reference values.
DRAFT - DO NOT CITE OR QUOTE 74

Table A.2-12. Study Summary and Data for Wiaderna et al. (2002)

Study Design

Species Sex N Exposure route Dose range Exposure duration

LOD: 

Wistar

M 12 rats 

per dose

Inhalation (6 

h/day, 5 days/wk)

0, 25, 100, or 250 ppm 

1,3,5-TMB

4 weeks 

Additional Study details

• Animals were exposed to 1,3,5-TMB in 1.3 m3 dynamic inhalation exposure chambers for 6 

hours per day, 5 days per week for 4 weeks. 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.

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 mesitylene. 

Leaving the platform in trial 3 

was punished by an electric 

shock. Trials 1, 2, 3 and 4 

were performed at 24-h 

intervals, while trials 5 and 6 

were effected 3 and 7 days 

after trial 3, respectively.
DRAFT - DO NOT CITE OR QUOTE 75

Table A.2-12. Study Summary and Data for Wiaderna et al. (2002)

Figure 2. Hot plate. The 

comparison of latency of the 

reaction (paw lick) to the 

thermal stimulus before (L1), 

immediately after (L2) and 24 

h after (L3) intermittent 2 min 

electric shock in rats exposed 

to mesitylene. The test was 

performed on days 50 and 51 

after the exposure.

Figure 3. Active avoidance. 

The comparison of the rat 

groups exposed to mesitylene 

for the number of trials 

(attempts) 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. 

Health Effect at LOAEL NOAEL LOAEL

Shorter retention of 

passive avoidance reaction n/a 25 ppm

Strengths: This study used an appropriate number of animals per dose group and 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.

Limitations: 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
DRAFT - DO NOT CITE OR QUOTE 76

Table A.2-13. Study Summary and Data for McKee et al. (2010)

Study Design

Species Sex N Exposure route Dose range Exposure duration

Wistar M 8 rats 

per 

group

Inhalation 0, 125, 1250, or 5000 

mg/m3

8 hours/day for 3 

consecutive days

Additional Study details

• Animals were exposed to 1,2,4-TMB for 8 hours per day for three days 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 Exposure to 1,2,4-TMB (mg/m3

/8 h/day)

0 125 1250 5000

Results of Functional and Motor Activity Observations 

Forelimb grip strength (g)

One-day pre-exposure 1107±41.2 1065±52.3 1223±25.9 1090±47.0

First 8 hour exposure 1064±39.9 814±91.7* 1059±59.8 1023±55.7

Third 8 hour exposure 908±56.1 847±64.3 956±67.7 1156±68.7*

Total distance traveled (cm)

One-day pre-exposure 3773±120 3598±301 3543±167 3575±119

First 8 hour exposure 2479±110 3048±257 2125±171 1897±200

Third 8 hour exposure 2459±118 2740±226 1967±316 1172±226*

Number of movements

One-day pre-exposure 1054±31 999±80 990±44 998±32

First 8 hour exposure 697±29 848±66 600±48 529±53

Third 8 hour exposure 687±31 744±56 541±82 329±61*

Observation Exposure to 1,2,4-TMB (mg/m3

/8 h/day)

0 125 1250 5000

Visual Discrimination Performance Testing (means±SD)

Trialsa

One-day pre-exposure 100±0.0 100±0.0 100±0.0 100±0.0

First 8-hour exposure 100±0.0 100±0.0 100±0.0 99.13±0.88

Third 8-hour exposure 100±0.0 100±0.0 100±0.0 100±0.0

One-day post-exposure 100±0.0 100±0.0 100±0.0 100±0.0

Percentage reinforcements 

obtainedb

One-day pre-exposure 99.88±0.13 99.88±0.13 99.88±0.13 100±0.0

First 8-hour exposure 100±0.0 100±0.0 99.38±0.63 99.74±0.17

Third 8-hour exposure 99.63±0.26 99.63±0.26 99.63±0.38 100±0.0

One-day post-exposure 99.63±0.26 99.88±0.13 99.88±0.13 100±0.0
DRAFT - DO NOT CITE OR QUOTE 77

Table A.2-13. Study Summary and Data for McKee et al. (2010)

Discrimination ratioc

One-day pre-exposure 0.81±0.84 0.84±0.03 0.83±0.02 0.83±0.03

First 8-hour exposure 0.86±0.02 0.91±0.03 0.91±0.01 0.95±0.01*

Third 8-hour exposure 0.89±0.02 0.88±0.03 0.94±0.01 0.95±0.02

One-day post-exposure 0.87±0.03 0.89±0.03 0.92±0.02 0.88±0.03

Percentage inter-trial 

intervals responded tod

One-day pre-exposure 12.88±2.00 10.13±1.56 10.75±1.94 10.38±1.84

First 8-hour exposure 12.50±2.12 8.88±2.03 11.50±2.60 10.19±1.28

Third 8-hour exposure 12.00±1.65 8.88±2.24 8.25±1.71 5.75±1.39

One-day post-exposure 10.88±1.39 10.63±1.81 11.25±0.92 8.50±1.40

Repetitive errorse

One-day pre-exposure 8.25±3.71 7.63±1.70 10.75±2.73 7.25±1.75

First 8-hour exposure 2.00±0.50 3.25±1.47 4.63±1.58 1.88±0.67

Third 8-hour exposure 2.63±1.70 4.75±1.81 3.00±0.78 1.25±0.73

One-day post-exposure 4.75±2.81 2.75±1.35 4.63±3.09 4.13±1.38

Repetitive inter-trial 

responses

f

One-day pre-exposure 3.63±1.02 5.88±1.33 7.25±1.93 3.25±1.35

First 8-hour exposure 6.13±1.73 3.88±1.22 5.63±1.97 8.38±2.50

Third 8-hour exposure 7.25±1.24 3.25±0.88 2.25±1.52* 1.63±0.98*

One-day post-exposure 6.63±1.94 2.88±0.83 5.13±1.54 2.63±0.68

Trial response latencyg

One-day pre-exposure 1.83±0.18 2.25±0.55 2.06±0.40 2.28±0.43

First 8-hour exposure 1.70±0.18 2.38±0.43 2.52±0.40 3.91±0.73*

Third 8-hour exposure 1.91±0.23 2.69±0.69 2.75±0.94 1.82±0.13

One-day post-exposure 1.68±0.16 2.70±0.60 2.18±0.73 1.45±0.06

Standard deviation of 

response latency

One-day pre-exposure 2.16±0.38 3.82±1.57 3.33±1.42 4.65±2.23

First 8-hour exposure 2.06±0.38 3.64±1.32 4.19±1.65 7.33±3.43

Third 8-hour exposure 2.74±0.71 4.03±1.50 5.25±3.04 2.34±0.40

One-day post-exposure 1.84±0.38 5.95±2.40 5.88±4.21 1.81±0.38

Latency<2 secondsh

One-day pre-exposure 61.75±4.55 70.13±2.23 67.75±66.88 66.88±3.22

First 8-hour exposure 68.50±3.84 69.75±3.75 65.76±3.13 52.13±3.96

Third 8-hour exposure 70.38±4.34 64.13±4.35 74.88±1.75 79.00±2.32

One-day post-exposure 69.38±2.98 67.63±3.20 78.13±3.05 78.00±2.34

Latency>6 secondsi

One-day pre-exposure 3.38±0.71 5.38±1.48 4.63±1.15 4.00±1.05

First 8-hour exposure 3.88±0.58 5.00±1.69 6.00±1.34 10.63±1.80*

Third 8-hour exposure 4.25±0.98 5.63±2.44 5.63±1.92 3.13±0.61

One-day post-exposure 2.13±0.67 6.00±1.68 3.38±1.40 1.88±0.35
DRAFT - DO NOT CITE OR QUOTE 78

Table A.2-13. Study Summary and Data for McKee et al. (2010)

Drink response latencyj

One-day pre-exposure 0.29±0.01 0.32±0.02 0.38±0.03* 0.33±0.02

First 8-hour exposure 0.26±0.01 0.30±0.02 0.43±0.03* 0.49±0.03*

Third 8-hour exposure 0.30±0.02 0.32±0.03 0.37±0.02 0.34±0.03

One-day post-exposure 0.27±0.01 0.34±0.03 0.36±0.03 0.30±0.02

Health Effect at LOAEL NOAEL LOAEL

n/a n/a n/a

Strengths: This study used an appropriate number of animals per dose group and observed 

alterations in a number of parameters, including forelimb grip strength, total distance traveled, 

number of movements, and several visual discrimination performance tests. 

Limitations: 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.

a Total number of trials completed during each session, maximum=100 

b Number of reinforcements obtained divided by the number of reinforcements delivered (x100)

c Number of correct trial responses divided by the number of trial responses

d The number of inter-trial intervals (ITI) in which at least 1 response was made divided by the total number of ITI 

(x100)

e The total number of incorrect trial responses following an initial incorrect response.

f The total number of ITI responses following an initial ITI response g The latency (seconds) to make a correct trial response

h The number of responses within 2 seconds

i The number of responses taking more than 6 seconds

j

The mean latency (seconds) to obtain reinforcement

* statistically significant from controls at p < 0.05
DRAFT - DO NOT CITE OR QUOTE 79

Table A.2-14. Study Summary and Data for Tomas et al. (1999a)

Study Design

Species Sex N Exposure route Dose range Exposure duration

WAG/Rij 

Rats

M 6 rats 

per dose

Oral (gavage, in 

olive oil) 

0,2, 8, or 32 mol/kg 

b.w.

Acute

Additional Study details

• 1,2,4- and 1,3,5-TMB were tested for their effects on electrocortical arousal by an 

electrocardiogram before and after oral administration (in olive oil) of 0, 0.002, 0.008, or 

0.032 mol/kg body weight of either isomer.

• Solvent concentration in peripheral blood was determined via head space gas 

chromatography.

• TMB was found to cause a slight increase in locomotor activity.

Figure 1. Changes 

in total duration of 

high-voltage 

spindle episodes 

following acute 

exposure to toluene 

and TMB isomers, 

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

1,3,5-TMB at doses 

of 0.002, 0.008, and 

0.032 mol/kg.
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Table A.2-14. Study Summary and Data for Tomas et al. (1999a)

Figure 2. Changes in 

total duration of highvoltage spindle episodes 

following acute 

exposure to toluene and 

TMB isomers, 1,2,3-, 

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

doses of 0.002, 0.008, 

and 0.032 mol/kg.

Health Effect at LOAEL NOAEL LOAEL

Abnormal electrocortical 

stimulation n/a 2 mmol/kg 1,2,4-TMB and 1,3,5-TMB 

Strengths: This study used an appropriate number of animals per dose group. 

Limitations: Acute duration of exposure (single oral gavage). Generally, acute exposure studies 

have limited utility in quantitation of human health reference values.
DRAFT - DO NOT CITE OR QUOTE 81

Table A.2-15. Study Summary and Data for Tomas et al. (1999b)

Study Design

Species Sex N Exposure route Dose range Exposure duration

WAG/Rij 

Rats

M 10 rats 

per dose

Oral (in olive oil) 0, 8, 16, or 32 

mmol/kg b.w.

Acute

Additional Study details

• 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, 0.002, 0.008, or 0.032 mol/kg body 

weight of either isomer.

• TMB was found to cause a slight increase in locomotor activity.

Figure 1. 

Locomotor 

activity following 

acute exposure to 

toluene and TMB 

isomers at doses 

of 0.008 mol/kg, 

0.016 mol/kg, and 

0.032 mol/kg. 

Health Effect at LOAEL NOAEL LOAEL

Increased locomotor activity 16 mmol/kg 1,2,4-TMB 

8 mmol/kg 1,3,5-TMB

32 mmol/kg 1,2,4-TMB

16 mmol/kg 1,3,5-TMB 

Strengths: This study used an appropriate number of animals per dose group. 

Limitations: Acute duration of exposure (single oral gavage). Generally, acute exposure studies 

have limited utility in quantitation of human health reference values. 
DRAFT - DO NOT CITE OR QUOTE 82

Table A.2-16. Study Summary and Data for Tomas et al. (1999c)

Study Design

Species Sex N Exposure route Dose range Exposure duration

Wistar M 4 rats 

per dose

Intraperitoneal 

injection 

6.6 mmol/kg body 

weight

Acute

Additional Study details

• 1,2,4- and 1,3,5-TMB were tested for their effects on the CNS by monitoring evoked 

hippocampal and cortical activity following ip injection of 6.6mmol/kg body weight of either 

isomer.

• Solvent concentration in peripheral blood was determined via head space gas 

chromatography.

• Significant differences in hippocampal and cortical activity occurred following injection.

Figure 1. 

Amplitude 

abnormalities of 

the cortical N1 

wave 30 and 60 

min after i.p. 

solvent injection. 

Figure 2. 

Amplitude 

abnormalities 

of the cortical 

P1-N1 wave 30 

and 60 minutes 

after i.p. 

solvent 

injection.
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Table A.2-16. Study Summary and Data for Tomas et al. (1999c)

Figure 3. Amplitude 

abnormalities of the 

hippocampal N1 

wave 30 and 60 min 

after i.p. solvent 

injection 

Figure 4. The 

effect of i.p. solvent 

injection on the 

cortical EEG in the 

13-20.75 Hz 

frequency band.
DRAFT - DO NOT CITE OR QUOTE 84

Table A.2-16. Study Summary and Data for Tomas et al. (1999c)

Figure 5. The 

effect of i.p. solvent 

injection on the

hippocampal EEG 

in the 1-3.75 Hz 

frequency band.

Figure 6. The 

effect of i.p. solvent 

injection on the 

hippocampal EEG 

in the 7-9.75 Hz 

frequency band.

Health Effect at LOAEL NOAEL LOAEL

n/a (acute exposure study, one 

dose level) n/a 6.6 mmol/kg 1,2,4-TMB and 1,3,5-TMB 

Strengths: This study used an appropriate number of animals per dose group. A number of effects 

were found to correlate with TMB injection, including differences in hippocampal and cortical 

activity.

Limitations: Unable to quantify dose-response relationship from data because only one dose group 

used. Acute duration of exposure (single i.p. injection). Generally, acute exposure studies have 

limited utility in quantitation of human health reference values. 
DRAFT - DO NOT CITE OR QUOTE 85

Table A.2-17. Study Summary and Data for Lammers et al. (2007)

Study Design

Species Sex N Exposure route Dose range Exposure duration

WAG/RijCR/

BR Wistar 

and humans

M 3, 4, or 8 

/group

Inhalation 0, 600, 2400, or 

4800 mg/m3 (rats); 

57 or 570 mg/m3 

(humans)

3 days (rats), 4 hours 

(humans)

Additional Study details

• Rats were exposed to 1,2,4-TMB as a constituent of white spirit at concentrations of 0, 600, 

2400, or 4800 mg/m3 for 3 days. Human volunteers were exposed to 57 or 570 mg/m3 during 

2 test sessions separated by one week, each lasting 4 hours.

• Several tests were conducted to evaluate impact of white spirit on CNS. These included tests 

of observation, spontaneous motor activity and learned visual discrimination.

• White spirit was found to affect performance and learned behavior in rats.

• In humans, attention deficit was observed following white spirit inhalation.

Observation

Functional Observations and Physiological Parameters in Rats 

Following Exposure to White Spirit (mg/m3) 

0 600 2400 4800

Functional Observation Battery (Mean±SD)

Gait scorea

Before first 8h 

exposure

1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00

After first 8h 

exposure

1.00±0.00 1.00±0.00 1.13±0.13 1.25±0.16

After third 8h 

exposure

1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00

Click responseb

Before first 8h 

exposure

2.13±0.13 2.63±0.18 2.38±0.18 2.50±0.19

After first 8h 

exposure

2.88±0.13 2.50±0.19 2.75±0.37 2.63±0.18

After third 8h 

exposure

2.13±0.13 3.25±0.31* 2.88±0.23 2.75±0.25

Physiological Parameters (Mean±SD)

Body weight (g)

Before first 8h 

exposure

270.0±2.61 269.2±2.48 273.3±3.52 272.8±2.20

After first 8h 

exposure

279.7±2.53 277.7±3.11 278.0±3.21** 273.8±2.51***

After third 8h 

exposure

280.9±2.68 278.4±2.44 275.9=-2.83*** 268.5±2.67***

Body temperature (°C)

Before first 8h 

exposure

37.60±0.34 37.33±0.39 37.49±0.39 37.29±0.37

After first 8h 36.41±0.05 36.25±0.12 36.16±0.11 35.95±0.21
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Table A.2-17. Study Summary and Data for Lammers et al. (2007)

exposure

After third 8h 

exposure

36.60±0.10 36.44±0.17 36.25±0.05 36.11±0..09**

Figure 1. Effects of 

white spirit on total 

distance run during 

motor activity 

assessment in rats. 

Observation

Visual Discrimination Performance in Rats Exposed to White Spirit 

for Three Consecutive Days (mg/m3)

c

0 600 2400 4800

Lever response latency 

(seconds)

Before first 8h 

exposure

1.93±0.34 2.09±0.24 1.70±0.15 2.29±0.31**

After first 8h 

exposure

2.44±0.56 2.66±0.29 3.24±0.21 12.00±2.37**

After second 8h 

exposure

2.17±0.41 2.32±0.29 2.10±0.18 4.88±1.53**

After third 8h

exposure

3.21±1.22 2.68±0.41 3.86±0.65 6.31±1.35**

One day after 

third 8h exposure 2.27±0.52 1.93±0.16 1.88±0.16 2.34±0.31**

Number of lever response 

latencies <2 seconds

Before first 8h 

exposure

68.00±5.46 67.38±2.58 77.12±4.32*** 71.25±4.00**

After first 8h 

exposure

70.38±2.93 61.88±3.92 58.75±2.58*** 45.62±4.87**

After second 8h 

exposure

70.62±3.60 68.00±3.81 69.00±2.98*** 61.50±5.00**

After third 8h 71.50±3.38 66.38±3.34 63.75±5.04*** 55.62±5.12**
DRAFT - DO NOT CITE OR QUOTE 87

Table A.2-17. Study Summary and Data for Lammers et al. (2007)

exposure

One day after 

third 8h exposure 72.50±3.58 69.75±2.90 73.38±2.93*** 64.88±4.23**

Number of lever response 

latencies >6 seconds

Before first 8h 

exposure

3.88±0.90 5.25±0.84 3.25±0.45* 5.62±0.92**

After first 8h 

exposure

5.00±1.10 7.62±1.83 11.12±0.85* 25.75±5.05**

After second 8h 

exposure

4.38±0.96 5.62±0.78 5.00±0.65* 12.25±3.80**

After third 8h 

exposure

7.38±2.07 6.88±1.16 10.88±1.96* 17.50±2.76**

One day after 

third 8h exposure 4.62±1.31 4.38±1.07 3.75±0.70* 6.50±1.86**

Drink response latency 

(seconds)

Before first 8h 

exposure

0.35±0.04 0.29±0.03 0.36±0.03 0.32±0.02

After first 8h 

exposure

0.37±0.04 0.31±0.03 0.39±0.02 0.52±0.04

After second 8h 

exposure

0.36±0.04 0.28±0.03 0.33±0.02 0.39±0.04

After third 8h 

exposure

0.38±0.05 0.32±0.04 0.39±0.02 0.43±0.07

One day after 

third 8h exposure 0.36±0.03 0.31±0.02 0.34±0.02 0.33±0.04

Observation

Test Scores (Mean±SD) at Various Time Points in Humans Exposed 

to 57 or 570 mg/m3 White Spirit for 4 Hours

57 mg/m3 570 mg/m3

Mood and Affect

Fatigue (scale score)

Pre-test 1.11±0.04 1.11±0.05

1 hour 1.06±0.03 1.17±0.09

3 hours 1.21±0.12 1.29±0.13

Post-test 1.38±0.15 1.51±0.23

Vigor (scale score)

Pre-test 3.35±0.20 3.53±0.09

1 hour 3.58±0.16 3.23±0.20

3 hours 3.27±0.20 3.32±0.22

Post-test 2.98±0.23 3.05±0.22

Psychomotor Skills (Hand-Eye Coordination and Finger Tapping)

Hand-eye coordination 
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Table A.2-17. Study Summary and Data for Lammers et al. (2007)

test (pixels in lnMAE)

Pre-test 1.69±0.05 1.67±0.04

1 hour 1.56±0.05 1.64±0.04

3 hours 1.64±0.05 1.63±0.04

Post-test 1.62±0.04 1.55±0.06

Finger tapping test (no. 

of taps in 30 seconds)

Pre-test 201±7 203±6

1 hour 205±5 194±6

3 hours 202±8 196±6

Post-test 198±7 200±6

Attention

Reaction time test 

(latency, ms)

Pre-test 251±9 246±8

0.25 hours 248±10 252±9

1 hours 248±9 254±9

2.25 hours 253±9 266±12

3 hours 253±11 257±10

Post-test 258±11 269±13

Color word vigilance test 

(latency, ms)

Pre-test 579±28 595±22

1 hours 550±20 569±20

3 hours 537±17 561±23

Post-test 532±18 557±22

Figure 2. Performance on 

finger tapping test with the 

dominant hand at different 

time points during and after 

exposure.
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Table A.2-17. Study Summary and Data for Lammers et al. (2007)

Health Effect at 

LOAEL NOAEL LOAEL

n/a n/a n/a

Strengths: This study used an appropriate number of animals per dose group and observed 

alterations in a number of parameters, including performance and learned behavior in mice, and 

attention in humans. 

Limitations: Exposure to 1,2,4-TMB was via white spirit, which is comprised of additional 

substances. LOAEL and NOAEL cannot be extracted from this study because other constituents of 

the white spirit mixture may confound results. 

a Gait score indicates the severity of gait changes and is scored as 1 (normal) to 4 (severely abnormal) b Click response was scored as 0 (no reaction) to 5 (exaggerated reaction)

c 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
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Table A.2-18. Study Summary and Data for Maltoni et al. (1997)

Study Design

Species Sex N Exposure route Dose range Exposure duration

SpragueDawley Rats: 

CRC/BT

M 50 males, 

50 females 

per group

Stomach tube 

(in olive oil)

0 or 800 mg/kg 

body weight

4 days/week for 104 

weeks

Additional Study details

• Rats were exposed to 1,2,4-TMB for two years via stomach tube administration four days per 

week. 

• Animals were 7 weeks 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 54.0 62.0

Malignant tumors 24.0 26.0

No. malignant 

tumors/100 rats 26.0 34.0

Females

Total benign and 

malignant tumors 70.0 66.0

Malignant tumors 22.0 24.0

No. malignant 

tumors/100 rats 22.0 32.0

Both Sexes

Total benign and 

malignant tumors 62.0 64.0

Malignant tumors 23.0 25.0

No. malignant 

tumors/100 rats 24.0 33.0

Head Cancers

Males

Zymbal gland 

cancer

2.0 4.0

Ear duct cancer -- 2.0

Neuroesthesioepitheliomas -- 2.0

Oral cavity 

cancers -- 2.0

Total head 2.0 10.0
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Table A.2-18. Study Summary and Data for Maltoni et al. (1997)

cancers

Females

Zymbal gland 

cancer

2.0 2.0

Ear duct cancer 2.0 --

Neuroesthesioepitheliomas -- 4.0

Oral cavity 

cancers

2.0 --

Total head 

cancers

6.0 6.0

Both Sexes

Zymbal gland 

cancer

2.0 3.0

Ear duct cancer 1.0 1.0

Neuroesthesioepitheliomas -- 3.0

Oral cavity 

cancers

1.0 1.0

Total head 

cancers

4.0 8.0

Health Effect at 

LOAEL NOAEL LOAEL

Various malignant and 

non-malignant cancers n/a 800 mg/kg

Strengths: This study used an appropriate number of animals per dose group and observed increases 

in total number of tumors and number of head cancers in males. Neuroesthesioepithelioma is 

uncommon in Sprague-Dawley rats, although there were increases in the number of 

neuroesthesioepithelioma in both males and females. 

Limitations: Only one dose level was tested (800 mg/kg). Statistical significance of data not 

discussed. 
DRAFT - DO NOT CITE OR QUOTE 92

Study Summary Tables for Toxicokinetic Studies of 1,2,4-TMB and 1,3,5-

TMB 

Toxicokinetic Studies in Humans

Table A.2-19. Study Summary and Data for Jarnberg et al. (1996)

Study Design

Species Sex n Exposure 

route Dose range Exposure duration

Caucasian 

humans M 9 per dose Inhalation 2 ppm and 25 ppm 2 hours exposure, followed by 4 hours observation 

Additional Study details

• Caucasian males were exposed to 2 ppm or 25 ppm 1,2,4-trimethylbenzene (1,2,4-TMB) 

and 25 ppm 1,3,5-TMB in an inhalation chamber for 2 hours.

• Study subjects were asked to perform light cycling to simulate a work environment, with 

participants generating 50W power during 2 hour exposure.

• 1,2,4- and 1,3,5-TMB concentration was determined via gas chromatography. 

• No significant irritation or CNS effects were observed. 

• Results imply extensive deposition in adipose tissue. 

• Exhalation accounted for 20-37% of absorbed amount while urinary excretion of unchanged 

TMBs accounted for ≤0.002%

Respiratory Uptake and Urinary Excretion of TMB Isomers Following 2 Hour Inhalation 

Exposure (Mean±95%CI)

Exposure 25 ppm 1,3,5-TMB 25 ppm 1,2,4-TMB 2 ppm 1,2,4-TMB

Respiratory Uptake (%)a 62 ± 3 64 ± 3 63 ± 2

Net Respiratory Uptake (%)b 55 ± 2 60 ± 3 61 ± 2

Respiratory Uptake (mmol)a 1.6 ± 0.1 1.6 ± 0.1 0.16 ± 0.01

Net Respiratory Uptake (mmol)b 1.4 ± 0.1 1.5 ± 0.1 0.15 ± 0.01

Respiratory Excretion (%)c 25 ± 6 20 ± 3 15 ± 5

Net respiratory excretion (%)d 16 ± 4 14 ± 2 9 ± 4

Urinary Excretion (%)e 0.0016 ± 0.0015 0.0010 ± 0.0004 0.0005 ± 0.0002

Kinetic Values of TMB Isomers Following 2 Hour Inhalation Exposure (Mean±95%CI)

Kinetic Parameter 25 ppm 1,3,5-TMB 25 ppm 1,2,4-TMB 2 ppm 1,2,4-TMB

Total Calculated Blood Clearance 

(liters x hr-1 x kg-1)

f 0.97 ± 0.16 0.68 ± 0.13 0.87 ± 0.37

Total Apparent Calculated Blood 

Clearance (liters x hr-1 x kg-1)

g 0.86 ± 0.12 0.63 ± 0.11 0.82 ± 0.32

Exhalatory Blood Clearance 

(liters x hr-1 x kg-1)

f 0.24 ± 0.10 0.14 ± 0.04 0.14 ± 0.10

Metabolic Blood Clearance (liters 0.72±0.11 0.54 ± 0.10 0.74 ± 0.29
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Table A.2-19. Study Summary and Data for Jarnberg et al. (1996)

x hr-1 x kg-1)

f

1st Phase Half-Life (minutes) 1.7 ± 0.8 1.3 ± 0.8 1.4 ± 1.8

2nd Phase Half-Life (minutes) 27 ± 5 21 ± 5 28 ± 14

3rd Phase Half-Life (minutes) 4.9 ± 1.4 3.6 ± 1.1 5.9 ± 2.5

4th Phase Half-Life (minutes) 120 ± 41 87 ± 27 65 ± 20

AUC (μM x hours) 22 ± 4 35 ± 10 3.6 ± 2.0

Volume of Distribution (liters/kg) 39 ± 8 38 ± 11 28 ± 3

Mean Residence Time (hours) 42 ± 11 69 ± 32 47 ± 22

Figure 1. Concentration of 1,2,4-TMB in capillary blood during and after 2 hour exposure to 25 

ppm 1,2,4-TMB (Mean values ± 95% confidence interval)

Figure 2. Concentration of 1,3,5-TMB in capillary blood during and after 2 hour exposure to 25 

ppm 1,3,5-TMB (Mean values ± 95% confidence interval)
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Table A.2-19. Study Summary and Data for Jarnberg et al. (1996)

Figure 3. Concentration of 1,2,4-TMB in capillary blood from 10 subjects exposed to 2 and 25ppm 

of 1,2,4-TMB (Mean values ± 95% confidence interval)

Strengths: Adequate number of study subjects per dose group. Multiple exposure concentrations 

were tested. . 

Limitations: Exposure duration possibly not sufficient to detect metabolic changes. Metabolites 

not measured. 

a Percent of dose calculated as net uptake + amount cleared by exhalation during exposure b Percentage of dose calculated as net uptake

c During and post-exposure, percentage of the respiratory uptake d Post-exposure, percentage of net respiratory uptake e Post-exposure, percentage of respiratory uptake f Calculated from respiratory uptake

g Calculated from net respiratory uptake
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Table A.2-20. Study Summary and Data for Jarnberg et al.(1997a)

Study Design

Species Sex n Exposure 

route Dose range Exposure duration

Caucasian 

Human M 9 Inhalation 11 mg/m3 2 hours 

Additional Study details

• Nine Caucasian males were exposed to 11 mg/m3 1,2,4-trimethylbenze alone or 11 mg/m3 

1,2,4-TMB as a component of 300 mg/m3 white spirit.

• Exposure lasted two hours, during which study subjects were required to cycle producing 

50W 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.

Figure 1. Mean (±standard deviation) 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 white spirit

Results from 2 Hour Exposure to 1,2,4-TMB Alone or 1,2,4-TMB as a Component of White 

Spirit (mean±SD)

Exposure 1,2,4-TMB 

Alone

1,2,4-TMB in White 

Spirit P-value

Net Respiratory Uptake (mmol) 0.15 ± 0.01 0.14 ± 0.02 0.5b

AUC (μM x min), 0-3 h 53 ± 4 86 ± 9 <0.0001b

Half life of 3,4-DMHA (h) 3.7 ± 0.4 3.0 ± 0.7 0.2c
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Table A.2-20. Study Summary and Data for Jarnberg et al.(1997a)

Excretion of 3,4-DMHA (%a), 0-6 h 11 ± 2 18 ± 3 0.007c

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/m3 of 1,2,4-TMB, either alone or as a component 

of white spirit. (Mean±95% confidence interval)

Strengths: Metabolites (DMBAs) measured in urine. Appropriate number of animals study 

subjects. 

Limitations: Exposure duration possibly not sufficient to detect other metabolic changes. Only one 

exposure group; multiple concentrations not tested. 
DRAFT - DO NOT CITE OR QUOTE 97

Table A.2-21. Study Summary and Data Jarnberg et al. (1997b)

Study Design

Species Sex n Exposure 

route Dose range Exposure duration

Caucausian 

Humans M 10 Inhalation 25 ppm 2 hours

Additional Study details

• Ten males were exposed to 25ppm 1,2,4-TMB or 1,3,5-TMB for two hours.

• Study subjects were asked to perform light cycling to simulate a work environment, with 

participants generating 50W power during 2 hour exposure.

• Isomers of all DMHA metabolites were detected via HPLC.

• Approximately 22% of inhaled 1,2,4-TMB was found to be excreted in urine within 24 

hours following exposure.

Half-Times of Urinary Excretion Rate, Recoveries, and Rates of Urinary DMHA Isomer 

Excretion (Mean±95% Confidence Interval)

Exposure Isomer Half-time (h)

Urinary 

Recovery % (24 

Hours)

Excretion Rate, 

μg/min, 0-24 hrs.

1,2,4-TMB 3,4-DMHA 3.80 ± 0.4 18 ± 3 44 ± 6

1,2,4-TMB 2,4-DMHA 5.8 ± 0.9 3 ± 0.8 8.2 ± 1.4

1,2,4-TMB 2,5-DMHA 5.3 ± 1.5 <1 ± 0.2 1.6 ± 0.5

1,3,5-TMB 3,5-DMHA 16 ± 6 3 ± 2 8.9 ± 2.1

Strengths: Metabolites (DMBAs) measured in urine. Adequate number of study subjects. 

Limitations: Exposure duration possibly not sufficient to detect metabolic changes associated with 

longer time points. Toxicokinetics studied at only one concentration. 
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Table A.2-22. Study Summary and Data for Jarnberg et al. (1998)

Study Design

Species Sex n Exposure 

route Dose range Exposure duration

Caucasian 

humans M 9 subjects Inhalation 2 ppm, 2 ppm in white spirit, 25 ppm 

2 hours exposure, followed 

by 6 hours observation 

Additional Study details

• Caucasian males were exposed to 2 ppm, 2 ppm in white spirit, or 25 ppm 1,2,4-

trimethylbenzene (1,2,4-TMB) in an inhalation chamber for 2 hours.

• 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 high performance liquid chromatography.

• Blood levels of 1,2,4 TMB and its urinary metabolites were found to be higher in the white 

spirit exposure group suggesting that components of white spirit could interfere with TMB 

metabolism.

• No significant irritation or CNS effects were observed. 

Kinetic Results following 2 Hour Inhalation Exposure to 1,2,4-TMB and 1,2,4-TMB in White 

Spirit—Mean Values (95% Confidence Interval)

Kinetic Parameter 2 ppm Group 2 ppm in white 

spirit 25 ppm alone

Actual [TMB] (ppm) 2.22 (2.13-2.31) 2.26 (2.20-2.32) 23.9(22.7-25.1)

Respiratory Uptake (mmol)a 0.16 (0.14-0.18) 0.16(0.14-0.18) 1.73(1.61-1.85)

Net Respiratory Uptake 0.15 (0.14-0.16) 0.14(0.12-0.16) 1.52(1.37-1.67)

AUCblood (μMxmin) 95 (54-137) 157(136-178)* 1286(1131-1441)

Total Blood Clearance (l/min) 2.09(1.52-2.66) 1.06(0.89-1.23)** 1.38(1.23-1.53)* 

Metabolic Blood Clearance (l/min) 1.71(1.15-2.26) 0.79(0.62-0.96)* 1.06(0.87-1.25)* 

Exhalatory Blood Clearance (l/min) 0.39(0.28-0.50) 0.28(0.20-0.36) 0.32(0.24-0.40)

Mean Residence Time (h) 4.6(-1.3-10.5) 4.8(2.1-7.5) 3.8(1.8-5.8)

Volume of Distribution, Steady State (l) 293(69-517) 271(139-403) 294(165-423)

Half-Life in Blood, TMB, 1st Phase (min) 3.9(1.4-6.4) 5.9(3.1-8.7) 6.1(5.3-6.9)

Idem, TMB, 2nd Phase (h) 4.3(-0.5-9.0) 4.8(2.1-7.5) 4.0(2.2-5.8)

Half-Life in Urine, 34DMHA (h) NDc 3.0(2.3-3.7) 3.8(3.4-4.2)

Urinary Recovery, 34DMHA (%)b, 0-6 h 11 (9-13) 18(15-21) † 14(12-16)

Idem (%)b, 0-22 h ND 27(23-31) 18(15-21)

Strengths: Adequate number of study subjects per dose group. Multiple exposure concentrations 

were tested and multiple tissues were analyzed. Study of 1,2,4-TMB as a component of white 

spirit. 

Limitations: Toxicokinetics of 1,2,3- and 1,3,5-TMB not studied. 

a Net respiratory uptake + amount cleared by exhalation during exposure

b % of net respiratory uptake

c Not determined

*p<0.05, ** p<0.01
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Table A.2-23. Study Summary and Data for Kostrzewski et al. (1997)

Study Design

Species Sex n Exposure 

route Dose range Exposure duration

Human M/F 5 Inhalation Between 5 and 150 

mg/m3 4 or 8 hours

Additional Study details

• Five humans were exposed to 1,2,4-trimethylbenzene, 1,3,5-trimethylbenzene, and 1,2,3-

trimethylbenzene at concentrations between 5 and 150 mg/m3

. 

• Exposure durations were either four or eight hours.

• TMBs were measured via gas chromatography.

• DMBA excretion was found to follow an open, two-compartment model.

TMB Concentration in Blood Before, During, and After Exposure TMB Exposure

Sampling 

Time (hours)

1,2,4-TMB 1,3,5-TMB

Blood 

Concentration 

(μg/dm3)

Std. 

Deviation

Blood 

Concentration 

(μg/dm3)

Std. Deviation

0 0 0.00 0 0.00

0.25 194 19.80 181 25.01

.50 460 57.36 308 5.29

1 533 46.61 355 44.80

2 730 128.89 482 201.57

4 810 112.40 603 184.13

8 979 171.12 751 122.87

0.05 580 36.2 434 36.40

.10 496 85.03 388 64.16

.15 447 106.69 309 38.78

.25 387 65.83 298 65.48

.50 246 128.54 247 34.00

1 131 19.87 190 41.13

2 101 14.17 121 24.60

4 85 13.65 94 16.52

6 63 11.03 76 25.81

8 69 7.09 74 20.16

25 54 3.74 45 13.93

32 48 10.24 44 20.19

49 46 9.98 42 7.93

56 31 9.32 42 9.81

73 26 9.49 -- --

Excretion Rate (V, mg/h) of Dimethylbenzoic Acid (DMBA) in Urine During and After 

Exposure to 1,2,4-TMB or 1,3,5-TMB

Sampling 

time (h)

1,2,4-TMB Exposure 1,3,5-TMB Exposure

2,4- and 2,5-DMBA 3,4-DMBA 3,5-DMBA

V(mg/h) Std. Dev V(mg/h) Std. Dev V(mg/h) Std. Dev
DRAFT - DO NOT CITE OR QUOTE 100

Table A.2-23. Study Summary and Data for Kostrzewski et al. (1997)

0 0.000 0.000 0.000 0.000 0.000 0.000

0-2 6.632 3.069 19.949 5.489 3.538 0.833

2-4 12.931 4.315 22.731 4.536 8.854 2.955

4-6 21.148 7.067 26.906 6.525 12.334 3.905

6-8 29.263 9.240 35.346 11.017 19.204 6.092

8-10 16.616 11.451 12.082 10.205 19.413 6.329

10-12 15.619 2.935 6.198 2.325 23.535 7.606

12-14 17.328 2.218 6.029 2.135 22.460 3.254

14-16 13.832 2.176 4.415 1.372 16.941 4.350

16-23 7.023 2.565 2.520 1.043 10.790 3.116

23-27 4.052 0.674 1.870 0.525 6.908 2.691

27-31 2.570 0.760 2.005 0.460 6.558 3.657

31-35 2.209 0.666 1.523 0.610 3.983 2.367

35-39 1.211 1.075 1.247 0.895 3.946 2.073

39-47 1.262 0.256 0.957 0.099 3.110 0.838

47-51 1.174 0.459 0.953 0.623 3.244 1.140

51-55 0.370 0.228 0.659 0.231 2.343 1.355

55-59 0.928 0.327 0.936 0.515 3.669 1.882

59-63 1.591 1.162 1.286 0.391 2.436 1.303

63-71 0.948 0.276 0.869 0.141 1.600 1.305

71-75 1.122 0.049 0.851 0.246 1.025 0.639

75-79 0.748 0.441 0.422 0.231 1.044 0.825

79-83 1.082 0.733 0.744 0.328 0.750 0.645

83-87 -- -- -- -- -- --

87-95 -- -- -- -- -- --

Strengths: Metabolites (DMBAs) measured in urine. Toxicokinetics studied over a range of 

exposures. 

Limitations: Exposure duration possibly not sufficient to detect other metabolic changes. Only one 

study subject per exposure group. 
DRAFT - DO NOT CITE OR QUOTE 101

Table A.2-24. Study Summary and Data for Jones et al. (2006)

Study Design

Species Sex n Exposure 

route Dose range Exposure duration

Human M/F 2 per 

sex

Inhalation 25 ppm 4 hours 

Additional Study details

• Two males and two females were exposed to 25 ppm 1,3,5-trimethylbenzene (1,3,5-TMB) 

in an inhalation chamber for 4 hours.

• 1,3,5-TMB concentration was determined via gas chromatography. 

• No significant irritation or CNS effects were observed during inhalation study, although one 

volunteer was treated with a 2cm2 gauze 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.

Figure 1. Mean ± standard deviation urinary total DMBAs. Black and grey arrows represent 24 

and 48 hours respectively, following a single 4 hour exposure to 25 ppm 1,3,5-TMB
DRAFT - DO NOT CITE OR QUOTE 102

Table A.2-24. Study Summary and Data for Jones et al. (2006)

Figure 2. Mean ± standard deviation blood levels of 1,3,5-TMB during and after 4 hour exposure to 

25 ppm 1,3,5-TMB

Figure 3. Mean ± standard deviation breath levels of 1,3,5-TMB during and after 4 hour exposure 

to 1,3,5-TMB

Strengths: Metabolite (DMBA) concentration measured in urine. Subjects tested included males 

and females. 

Limitations: Small number of study subjects (n=4). Exposure duration possibly not sufficient to 

detect metabolic changes. Other metabolites not measured. 
DRAFT - DO NOT CITE OR QUOTE 103

 Toxicokinetic Studies in Animals

Table A.2-25. Study Summary and Data for Swiercz et al. (2002)

Study Design

Species Sex n Exposure 

route Dose range Exposure duration

Imp:DAK 

Wistar M 4/dose Inhalation 25, 100, or 250 ppm 6 hours 

Additional Study details

• Two males and two females were exposed to 25, 100, or 250 ppm 1,2,4-trimethylbenzene 

(1,2,4-TMB) in an inhalation chamber for 6 hours.

• 1,2,4-TMB concentration was determined via gas chromatography. 

• Blood samples were taken from the tail vein at various timepoints up to 6 hours 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 1,2,4-TMB Nominal 

Concentration (ppm)

1,2,4-TMB Actual 

Concentration (ppm)

Rat Body Weight 

(g)

Blood During 6h Exposure 

25 25 ± 2 200 ± 10

100 109 ± 10 228 ± 10

250 262 ± 21 190 ± 12

Blood After 6h Exposure

25 26 ± 3 349 ± 6

100 101 ± 3 333 ± 18

250 238 ± 9 336 ± 5

Urine After 6h Exposure

25 27 ± 3 355 ± 10

100 98 ± 3 338 ± 10

250 240 ± 7 330 ± 12

Blood 1,2,4-TMB Concentration During 6 Hour Inhalation Exposure (Mean±SD)

1,2,4-TMB Concentration (mg/l)

Time 25 ppm 100 ppm 250 ppm 

15 (minutes) 0.22 ± 0.07 1.12 ± 0.80 4.02 ± 0.85

30 0.33 ± 0.08 1.99 ± 1.09 4.87 ± 1.61

45 0.49 ± 0.16 3.56 ± 0.49 6.97 ± 1.22

1 (hours) 0.53 ± 0.14 4.29 ± 0.60 8.67 ± 0.54

2 0.73 ± 0.16 5.10 ± 0.34 14.5 ± 2.6

3 0.80 ± 0.17 6.22 ± 0.70 17.8 ± 1.6

4 0.72 ± 0.15 7.40 ± 1.05 20.0 ± 0.5

5 0.79 ± 0.22 7.72 ± 1.48 23.3 ± 2.6

6 0.94 ± 0.16 8.32 ± 1.34 23.6 ± 1.8

Blood Concentrations of 1,2,4-TMB Following 6 Hour Exposure (Mean±SD)

1,2,4-TMB Concentration (mg/l)

Time 25 ppm 100 ppm 250 ppm 

3 (minutes) 0.68 ± 0.09 4.44 ± 1.54 20.9 ± 4.03
DRAFT - DO NOT CITE OR QUOTE 104

Table A.2-25. Study Summary and Data for Swiercz et al. (2002)

15 0.47 ± 0.04 3.72 ± 0.96 20.7 ± 5.13

30 0.40 ± 0.05 2.98 ± 0.88 17.1 ± 4.71

45 0.36 ± 0.04 2.89 ± 0.86 15.9 ± 5.74

1 (hours) 0.34 ± 0.03 1.79 ± 0.49 14.9 ± 3.77

2 0.23 ± 0.04 1.25 ± 0.33 10.2 ± 3.04

3 0.17 ± 0.04 0.88 ± 0.29 8.05 ± 2.25

4 0.12 ± 0.02 0.61 ± 0.20 6.13 ± 1.64

5 0.10 ± 0.02 0.41 ± 0.14 3.98 ± 0.43

6 0.08 ± 0.02 0.33 ± 0.06 3.20 ± 0.52

Dimethylbenzoic Acid (DMBA) Urine Concentrations after 6 h Exposure to 1,2,4-TMB 

(Mean±SD)

1,2,4-TMB (ppm) 2,5-DMBA (mg/l) 2,4-DMBA (mg/l) 3,4-DMBA (mg/l)

25 23.6 ± 8.6 37.6 ± 12.9 79.9 ± 33.3

100 54.0 ± 5.4 130.9 ± 22.1 200.8 ± 25.8

250 109.4 ± 71.1 308.8 ± 220.1 571.8 ± 381.6

Strengths: Metabolites (DMBAs) measured in urine. Appropriate number of animals per dose 

group (n=4). 

Limitations: Exposure duration possibly not sufficient to detect other metabolic changes. 
DRAFT - DO NOT CITE OR QUOTE 105

Table A.2-26. Study Summary and Data for Swiercz et al. (2003)

Study Design

Species Sex n Exposure 

route Dose range Exposure duration

Wistar M 4/dose Inhalation 25, 100, or 250 ppm 6 hours or 4 weeks

Additional Study details

• Male Wistar rats were exposed to either 25, 100, or 250 ppm pseudocumene (1,2,4-

trimethylbenzene) in an inhalation chamber for either six hours or four weeks.

• Rats were sacrificed following exposure period and tissues were analyzed 1,2,4-

trimethylbenzene 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

1,2,4-TMB Nominal 

Concentration In 

Inhaled Air (ppm)

1,2,4-TMB Actual 

Concentration In 

Inhaled Air (ppm)

Rat Body Weight (g)

Arterial blood and 

brain structure from 

rats after 6 hours

25 21 ± 2 219 ± 13

100 116 ± 5 180 ± 28

250 215 ± 15 220 ± 24

Arterial blood and 

brain structure from 

rats after 4 weeks

25 24 ± 3 327 ± 21

100 99 ± 7 295 ± 31

250 249 ± 19 268 ± 21

Liver, lung, and brain 

homogenate after 6 

hours

25 28 ± 1 227 ± 15

100 123 ± 9 246 ± 11

250 256 ± 7 228 ± 12

Liver, lung, and brain 

homogenate after 4 

weeks

25 25 ± 2 310 ± 10

100 103 ± 8 328 ± 23

250 249 ± 13 320 ± 20

Venous blood 

collected following 4 

week exposure

25 24 ± 3 321 ± 6

100 99 ± 7 300 ± 22

250 249 ± 19 373 ± 48

Venous Blood 1,2,4-TMB Concentrations After Four Week Inhalation Exposure

1,2,4-TMB Concentration (mg/l), Mean±SD

Time 25 ppm 100 ppm 250 ppm 

3 (minutes) 0.56 ± 0.18 4.06 ± 0.46 13.77 ± 3.34

15 0.43 ± 0.10 3.73 ± 1.21 11.82 ± 3.05

30 0.33 ± 0.03 3.02 ± 1.43 8.28 ± 2.07

45 0.28 ± 0.05 2.86 ± 0.89 7.21 ± 1.84

1 (hour) 0.22 ± 0.02 2.62 ± 0.82 6.27 ± 1.72

2 0.17 ± 0.06 1.83 ± 0.17 4.50 ± 1.04

3 0.11 ± 0.04 0.88 ± 0.24 3.17 ± 0.76

4 0.07 ± 0.04 0.64 ± 0.21 1.73 ± 0.37

5 0.07 ± 0.01 0.39 ± 0.11 1.30 ± 0.22

6 0.06 ± 0.02 0.37 ± 0.14 1.25 ± 0.22
DRAFT - DO NOT CITE OR QUOTE 106

Table A.2-26. Study Summary and Data for Swiercz et al. (2003)

Liver, Lung, and Brain Homogenates and Arterial Blood 1,2,4-TMB Concentrations 

Following Inhalation Exposure (Mean±SD)

Exposure 25 ppm 100 ppm 250 ppm 

Blood 6 hours (mg/l) 0.31 ± 0.12 1.24 ± 0.41 7.76 ± 1.64

Blood 4 weeks (mg/l) 0.33 ± 0.11 1.54 ± 0.32 7.52 ± 2.11

Brain 6 hours (mg/kg) 0.49 ± 0.06 2.92 ± 0.73 18.34 ± 1.92

Brain 4 weeks (mg/kg) 0.45 ± 0.05 2.82 ± 0.40 18.63 ± 4.27

Liver 6 hours (mg/kg) 0.44 ± 0.01 7.13 ± 1.31 28.18 ± 5.34

Liver 4 weeks (mg/kg) 0.45 ± 0.15 3.00 ± 0.49* 22.47 ± 4.10

Lung 6 hours (mg/kg) 0.43 ± 0.11 4.14 ± 0.54 18.90 ± 3.72

Lung 4 weeks (mg/kg) 0.47 ± 0.20 3.74 ± 0.82 22.47 ± 4.10

Psuedocumene in Various Brain Structures Following Pseudocumene Inhalation Exposure

1,2,4-TMB Concentration (mg/kg), Mean±SD

Brain Structure (time) 25 ppm 100 ppm 250 ppm 

Brain Stem (6hrs) 0.54 ± 0.11 3.38 ± 0.84 26.91 ± 5.33

Temporal Cortex (6hrs) 0.31 ± 0.06** 2.30 ± 0.71 13.54 ± 2.33**

Hippocampus (6hrs) 0.28 ± 0.09** 1.89 ± 0.29** 12.99 ± 2.18**

Cerebellum (6hrs) 0.32 ± 0.09** 1.99 ± 0.40** 12.91 ± 2.05**

Brain Stem (4wks) 0.38 ± 0.23 2.33 ± 1.24 21.95 ± 3.81

Temporal Cortex (4wks) 0.25 ± 0.07 2.03 ± 0.66 15.71 ± 3.54

Hippocampus (4wks) 0.41 ± 0.27 3.03 ± 0.48 12.44 ± 2.63**

Cerebellum (4wks) 0.33 ± 0.05 3.20 ± 0.40 10.85 ± 2.47**

Strengths: Adequate number of animals per dose group.

Limitations: Adipose tissue was not examined for 1,2,4-TMB content. Metabolite concentration 

was not measured. No control group. 
DRAFT - DO NOT CITE OR QUOTE 107

Table A.2-27. Study Summary and Data for Swiercz et al. (2006)

Study Design

Species Sex n Exposure 

route Dose range Exposure duration

IMP:WIST

Wistar M 5/dose Inhalation 0, 25, 100, or 250 ppm 6 hours or 4 weeks

Additional Study details

• Male Wistar rats were exposed to either 0, 25, 100, or 250 ppm mesitylene (1,3,5-

trimethylbenzene) in an inhalation chamber for either six hours or four weeks.

• 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 and 250 ppm. 

Air Concentrations of 1,3,5-TMB in Inhalation Chamber and Body Weight (Mean±SD) 

Biological Material

1,3,5-TMB Nominal 

Concentration In 

Inhaled Air (ppm)

1,3,5-TMB Actual 

Concentration In 

Inhaled Air (ppm)

Rat Body Weight (g)

Liver, lung, and 

kidney homogenates 

after 6 hour exposure

Control (0) 0 246 ± 9

25 25 ± 2 254 ± 11

100 97 ± 14 242 ± 14

250 254 ± 20 249 ± 7

Liver, lung, and 

kidney homogenates 

after 4 week exposure

Control (0) 0 331 ± 17

25 23 ± 2 311 ± 26

100 101 ± 8 320 ± 38

250 233 ± 16 328 ± 21

Blood collected after 6 

hour exposure

Control (0) 0 251 ± 7

25 24 ± 2 250 ± 5

100 101 ± 7 239 ± 7

250 240 ± 22 249 ± 10

Blood collected after 4 

week exposure

Control (0) 0 310 ± 9

25 23 ± 2 307 ± 15

100 101 ± 8 310 ± 33

250 233 ± 16 309 ± 19

Urine collected after 6 

hour exposure

Control (0) 0 280 ± 9

25 25 ± 2 278 ± 10

100 102 ± 10 335 ± 15

250 238 ± 27 273 ± 18

Urine collected after 4 

week exposure

Control (0) 0 310 ± 10

25 25 ± 2 295 ± 15

100 102 ± 10 331 ± 19

250 238 ± 27 320 ± 28
DRAFT - DO NOT CITE OR QUOTE 108

Table A.2-27. Study Summary and Data for Swiercz et al. (2006)

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 (ppm)

Liver (μg/g 

tissue)

Lung (μg/g 

tissue)

Kidney (μg/g 

tissue)

Blood (μg/g 

tissue)

6 Hours—25 ppm 0.30 ± 0.07 0.31 ± 0.12 4.49 ± 1.93 0.31 ± 0.12

6 Hours—100 ppm 3.09 ± 0.50 2.87 ± 0.57 13.32 ± 2.58 3.06 ± 0.65

6 Hours—250 ppm 17.00 ± 6.08 17.36 ± 5.56 31.80 ± 9.44 13.36 ± 1.54

4 Weeks—25 ppm 0.22 ± 0.01 0.42 ± 0.12 1.73 ± 0.30* 0.31 ± 0.08

4 Weeks—100 ppm 3.01 ± 0.58 1.99 ± 0.75 15.61 ± 2.14 2.30 ± 0.52

4 Weeks—250 ppm 12.98 ± 4.16 11.20 ± 3.61 35.97 ± 8.53 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)

Liver (μg/g 

tissue)

Lung (μg/g 

tissue)

Kidney (μg/g 

tissue)

Urine (mg/18 

hours)

6 Hours—25 ppm 12.62 ± 1.62 2.87 ± 0.55 8.77 ± 0.99 0.52 ± 0.03

6 Hours—100 ppm 26.05 ± 2.77 5.50 ± 0.55 27.01 ± 9.86 3.66 ± 0.57

6 Hours—250 ppm 36.92 ± 1.61 13.39 ± 1.90 60.91 ± 19.78 10.99 ± 3.90

4 Weeks—25 ppm 6.52 ± 0.67** 3.69 ± 1.21 11.06 ± 4.33 0.83 ± 0.15*

4 Weeks—100 ppm 21.67 ± 3.14** 8.90 ± 0.98** 31.03 ± 18.56 4.36 ± 0.86

4 Weeks—250 ppm 53.07 ± 5.41** 19.79 ± 2.70** 82.10 ± 14.48 11.92 ± 3.05

Venous Blood 1,3,5-TMB Concentration Following 6 Hour 1,3,5-TMB Inhalation Exposure

Time 1,3,5-TMB (μg/ml)

25 ppm 100 ppm 250 ppm

3 (minutes) 0.31 ± 0.12 3.06 ± 0.65 13.36 ± 1.54

15 0.26 ± 0.13 2.51 ± 0.17 13.05 ± 1.61

30 0.15 ± 0.04 2.35 ± 0.57 12.06 ± 1.23

45 0.10 ± 0.03 1.41 ± 0.27 10.53 ± 1.71

1 (hours) 0.06 ± 0.02 1.35 ± 0.30 8.85 ± 0.90

2 0.04 ± 0.02 1.34 ± 0.39 6.14 ± 0.53

3 ND*** 0.79 ± 0.30 4.54 ± 0.67

4 ND 0.57 ± 0.14 3.49 ± 1.16

5 ND 0.38 ± 0.14 2.31 ± 0.67

6 ND 0.20 ± 0.04 0.76 ± 0.06
DRAFT - DO NOT CITE OR QUOTE 109

Table A.2-27. Study Summary and Data for Swiercz et al. (2006)

Venous Blood 1,3,5-TMB Concentration Following 4 Week 1,3,5-TMB Inhalation Exposure

Time 1,3,5-TMB (μg/ml)

25 ppm Exposure 100 ppm Exposure 250 ppm Exposure

3 (minutes) 0.31 ± 0.08 2.30 ± 0.52 7.55 ± 1.43

15 0.26 ± 0.03 1.83 ± 0.47 6.51 ± 1.50

30 0.19 ± 0.02 1.57 ± 0.39 4.56 ± 0.98

45 0.17 ± 0.03 1.41 ± 0.13 3.65 ± 0.62

1 (hours) 0.12 ± 0.03 1.33 ± 0.15 3.69 ± 1.25

2 0.05 ± 0.01 0.95 ± 0.22 3.14 ± 0.64

3 ND 0.72 ± 0.17 2.28 ± 0.19

4 ND 0.41 ± 0.11 1.74 ± 0.17

5 ND 0.39 ± 0.05 1.23 ± 0.34

6 ND 0.29 ± 0.13 1.14 ± 0.20

Strengths: Adequate number of animals per dose group (n=5). Kinetics of 1,3,5-TMB elimination 

are reported and discussed in detail. Extensive analysis of 3,5-DMBA.

Limitations: Adipose tissue was not examined for 1,3,5-TMB content. 
DRAFT - DO NOT CITE OR QUOTE 110

Table A.2-28. Study Summary and Data for Zahlsen et al. (1990)

Study Design

Species Sex n Exposure 

route Dose range Exposure duration

SpragueDawley M 24 Inhalation 1000 ppm 12 hour exposures on days 

1, 3, 7, 10, and 14

Additional Study details

• Male Sprague-Dawley rats were exposed to 1000 ppm (1,2,4-trimethylbenzene) in an 

inhalation for 12 hours on days 1, 3, 7, 10, and 14.

• Food and water was give ad libitum except during exposure, and animal weight ranged 

between 150g and 200g prior to exposure on day 1. 

• Hydrocarbon concentration 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 hour exposures on days 1, 3, 7, 10, and 14
DRAFT - DO NOT CITE OR QUOTE 111

Table A.2-28. Study Summary and Data for Zahlsen et al. (1990)

Figure 2. Brain concentrations (+SD) of n-nonane, 1,2,4-TMB, and 1,2,4-trimethylcyclohexane 

following 12 hour exposures on days 1, 3, 7, 10, and 14

Figure 3. Perirenal fat concentrations (+SD) of n-nonane, 1,2,4-TMB, and 1,2,4-

trimethylcyclohexane following 12 hour exposures on days 1, 3, 7, 10, and 14

Brain:Blood and Fat:Blood TMB Distribution After 12 Hour Exposure at End of Day 14

Compound Concentration Ratio

Brain:Blood TMB Ratio 2.0

Fat:Blood TMB Ratio 63

Strengths: Adequate number of animals per dose group. Perirenal fat was analyzed and shown to 

retain higher concentrations of 1,2,4-TMB than blood. 

Limitations: Exposure was not continuous (only occurred on days 1, 3, 7, 10, and 15). Only one 

exposure concentration (1000 ppm) was tested, and there were no control groups.
DRAFT - DO NOT CITE OR QUOTE 112

Table A.2-29. Study Summary and Data for Eide and Zahlsen et al. (1996)

Study Design

Species Sex n Exposure 

route Dose range Exposure duration

SpragueDawley M 4 per 

dose Inhalation 0, 75, 150, 300, 450 ppm

12 hour exposures in 

inhalation chamber 

Additional Study details

• Male Sprague-Dawley rats were exposed to 75, 150, 300, or 450 ppm 1,2,4-

trimethylbenzene (1,2,4-TMB) in an inhalation chamber for 12 hours.

• Food and water was give ad libitum except during exposure, and animal weight ranged 

between 200g and 250g prior to exposure. 

• Hydrocarbon concentration was 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 

(ppm)

Blood 

(μmol/kg)

Brain 

(μmol/kg)

Liver 

(μmol/kg)

Kidneys 

(μmol/kg) Fat (μmol/kg)

75 14.1 23.6 53.4 53.4 516

150 57.5 97.5 123.1 168.5 3806

300 115.5 220.9 256.3 282.4 12930

450 221.3 400.2 468.6 492.5 19270

Strengths: Adequate number of animals per dose group. 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. 

Limitations: No data on urinary elimination. No data on metabolites of 1,2,4-TMB. 
DRAFT - DO NOT CITE OR QUOTE 113

Table A.2-30. Study Summary and Data for Huo et al. (1989)

Study Design

Species Sex n Exposure 

route Dose range Exposure duration

Wistar M 3 rats 

per dose

Oral, in olive 

oil

0.08mmol/kg, 0.8 

mmol/kg, 0.49 

μCi/kg

3, 6, 12, and 24 hours

Additional Study details

• Single doses of 14C labeled 1,2,4-trimethylbenzene administered orally to rats.

• Tissues were analyzed at 3, 6, 12, and 24 hours time points for the tissue distribution study 

and continuously for 24 hours in the metabolism study.

• 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 hours.

• Three most common metabolites were 3,4-dimethylhippuric acid (30.2%), 2,4-

dimethylbenzyl alcohol (12.7%), and 2,5-dimethylbenzyl alcohol (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 3 hours 6 hours 12 hours 24 hours

Liver 2.76 ± 0.39 2.69 ± 0.60 1.54 ± 0.38 0.13 ± 0.04

Kidney 0.56 ± 0.11 0.52 ± 0.12 0.14 ± 0.10 0.06 ± 0.05

Lung 0.10 ± 0.03 0.06 ± 0.03 0.03 ± 0.03 0.01 ± 0.01

Heart 0.03 ± 0.01 0.01 -- --

Testis 0.09 ± 0.04 0.12 ± 0.03 0.04 ± 0.04 --

Spleen 0.03 ± 0.02 0.03 ± 0.01 0.01 ± 0.01 --

Brain 0.08 ± 0.04 0.03 ± 0.02 0.03 ± 0.03 --

Stomach 2.39 ± 1.47 1.33 ± 0.98 0.09 ± 0.06 0.04 ± 0.03

Intestine 2.96 ± 1.82 3.33 ± 1.31 1.39 ± 1.03 0.25 ± 0.35

Serum 0.67 ± 0.14 0.57 ± 0.09 0.26 ± 0.15 0.12 ± 0.21

Muscle 2.38 ± 0.23 1.88 ± 1.63 0.64 ± 0.10 --

Skin 3.99 ± 1.51 2.29 ± 0.98 0.16 ± 0.25 --

Adipose Tissue 28.05 ± 9.28 26.31 ± 18.18 4.97 ± 0.97 0.67 ± 0.15

Urine 15.0 ± 1.1 32.6 ± 7.9 50.7 ± 7.9 99.8 ± 4.1

Concentration (μg/g) radioactive material in tissue (Mean ± SD)

Tissue 3 hours 6 hours 12 hours 24 hours

Liver 72 ± 9 81 ± 20 45 ± 12 5 ± 2

Kidney 68 ± 16 60 ± 13 17 ± 12 7 ± 6

Lung 17 ± 9 12 ± 6 4 ± 4 2 ± 4

Heart 8 ± 2 2 ± 1 -- --

Testis 8 ± 4 11 ± 2 3 ± 4 --

Spleen 11 ± 5 13 ± 5 5 ± 5 --

Brain 11 ± 5 6 ± 2 4 ± 4 --

Stomach 509 ± 313 263 ± 218 18 ± 11 10 ± 7

Intestine 35 ± 22 47 ± 17 21 ± 15 4 ± 6
DRAFT - DO NOT CITE OR QUOTE 114

Table A.2-30. Study Summary and Data for Huo et al. (1989)

Serum 17 ± 3 15 ± 1 6 ± 3 3 ± 6

Muscle 6 ± 1 5 ± 4 1 ± 0 --

Skin 20 ± 7 12 ± 4 1 ± 1 --

Adipose Tissue 200 ± 64 193 ± 125 33 ± 8 5 ± 1

Urinary Metabolites of 1,2,4-TMB 24 Hours After Single Oral Dose In Rats (Values±SD)

Metabolite

%Dose (0.08mmol/kg) in Urine %Dose (0.8mmol/kg) in Urine

Free Conjugate

d Total Free Conjugated Total

All Rats All Rats All Rats Rat 

1

Rat 

2

Rat 

1

Rat 

2

Rat 

1

Rat

2

2,3,5-AND 2,4,5-

TMPa 2.6 ± 1.2 5.1 ± 1.4 7.7 ± 2.2 2.5 1.5 4.3 2.0 6.7 3.5

2,3,6-TMP -- 3.9 ± 0.7 4.0 ± 0.6 0.1 0.4 2.1 1.5 2.1 1.8

Total phenols 2.7 ± 1.1 9.0 ± 2.0 11.8 ± 2.9 2.6 1.9 6.3 3.5 8.8 5.3

2,4-DMBOHb 0.1 ± 0.1 12.5 ± 2.6 12.7 ± 2.6 0.1 0.4 11.5 7.2 11.6 7.6

2,5-DMBOH 0.1 ± 0.0 11.6 ± 2.7 11.7 ± 2.7 0.1 0.2 8.7 8.7 8.8 8.9

3,4-DMBOH -- 1.9 ± 0.9 1.9 ± 0.8 -- 0.1 0.9 0.8 0.9 0.9

Total alcohols 0.2 ± 0.1 26.0 ± 5.5 26.3 ± 5.4 0.1 0.7 21.1 16.8 21.2 17.5

2,4-DMBA 0.8 ± 0.1 5.2 ± 2.0 6.0 ± 2.0 0.8 2.5 6.8 1.5 7.6 4.0

2,5-DMBA 0.5 ± 0.0 3.1 ± 1.3 3.6 ± 1.3 0.3 1.2 3.5 2.1 3.9 2.3

3,4-DMBA 0.2 ± 0.1 0.7 ± 0.2 0.8 ± 0.2 0.1 0.2 0.5 0.2 0.5 0.4

Total benzoic 

acids 1.5 ± 0.1 8.9 ± 3.4 10.4 ± 3.3 1.2 3.9 10.8 3.8 11.9 6.7

2,4-DMHA 5.0 ± 1.9 2.0 ± 1.0 7.0 ± 2.6 3.3 2.7 4.8 1.2 8.1 3.7

2,5-DMAH 0.5 ± 0.2 0.3 ± 0.3 0.8 ± 0.3 0.2 0.1 0.5 0.1 0.7 0.2

3,4-DMHA 27.3 ± 8.4 3.3 ± 1.2 30.2 ± 9.4 23.1 17.9 15.6 7.1 38.7 25.0

Total hippuric 

acids 32.7 ± 10.5 5.6 ± 2.3 37.9 ± 

12.1 26.6 20.8 20.9 8.4 47.5 28.9

Total metabolies 37.1 ± 11.4 49.5 ± 

13.0

86.4 ± 

23.0 30.4 27.2 59.1 32.4 89.5 58.4

Strengths: Many tissues examined for radioactive and metabolite content. Many metabolites 

measured. 

Limitations: Small numbers of rats per dose group, particularly for the 0.8mmol/kg group (n=2). 

Time points only extend to 24 hours. 

a trimethylphenol, b dimethylbenzoic alcohol, c dimethylbenzoic acid, d dimethylyhippuric acid
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Table A.2-31. Study Summary and Data for Tsujino et al. (2002)

Study Design

Species Sex n Exposure route Dose range Exposure 

duration

Wistar M 3 for tests of TMBs:IS, 

36 for data in Figure 3 

Dermal (via 

saturated cotton)

1 mL 

kerosene 0, 1, 3, or 6 hours

Additional Study Details

• In first experiment, rats were dermally exposed to kerosine on a saturated, sealed piece of 

cotton for 1h 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. 

1h Exposure and Ratio of TMBs to Internal Standard (o-xylene d10) (mean ± SD)

Tissue Source Post-mortem samples spiked 

with kerosene (Positive Control)

Post-mortem samples following 

dermal exposure 

Blood 3.6 ± 1.6 0.4 ± 0.4

Brain 3.6 ± 1.6 0.14 ± 0.05*

Lung 1.2 ± 0.5* 0.09 ± 0.03

Liver 1.1 ± 0.5 0.3 ± 0.09*

Spleen 0.7 ± 0.3 0.1 ± 0.04

Kidney 1.0 ± 0.4 0.5 ± 0.1**

Muscle 1.2 ± 0.5* 0.09 ± 0.02

Adipose 0.9 ± 0.3* 0.15 ± 0.07

OVERALL 1.4 ± 0.3** 0.21 ± 0.05*
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Table A.2-31. Study Summary and Data for Tsujino et al. (2002)

Pseudocumene in Various Tissues following 1h Exposure and Ante vs. Post-Mortem Exposure

Strengths: Number of tissues tested and number of animals used in the ante- and post-mortem 1h 

exposure groups (20 and 16 respectively). The authors conclude that their data shows that TMBs 

are dispersed throughout the body by circulation in blood following dermal exposure. 

Limitations: Small number of animals used to determine dermal absorption at 1 hour (n=3). No 

data provided for effects of exposure (if any)
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Table A.2-32. Study Summary and Data for Tsujimoto et al. (2000)

Study Design

Species Sex N Exposure route Dose range Exposure duration

Slc Wistar M 4 per dose Intraperitoneal 

in corn oil

0, 0.3, 1, and 3 

mmol/kg body 

weight

2 days

Additional Study details

• Groups of four male Wistar rats dosed with 0, 0.3, 1, or 3 mmol/kg body weight 1,2,4-

trimethylbenzene.

• Urine samples collected for two days.

• HPLC used to quantify amount of dimethylbenzyl mercapturic acid in urine.

Urinary Excretion of Dimethylbenzyl Mercapturic Acid in 1,2,4-TMB Treated Rats

Dose (mmol/kg)

% of dose ± SD

0-24 h 24-48 h Total

0.3 14.0 ± 1.2 ND 14.0 ± 1.2

1.0 19.4 ± 1.8 ND 19.4 ± 1.8

3.0 16.7 ± 6.2 2.5 ± 1.6 19.2 ± 4.8

Strengths: This study used an appropriate number of animals per dose group and observed a 

marked decrease in dimethylbenzyl mercapturic acid excretion between 24 and 48 hours following 

exposure. 

Limitations: Authors do not report specific speciation data for 2,4-, 2,5-, or 3,4- dimethylbenzyl 

mercapturic acid. 
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Table A.2-33. Study Summary and Data for Tsujimoto et al. (2005)

Study Design

Species Sex N Exposure 

route Dose range Exposure duration

Wistar M 4 per dose Intraperitoneal 

in corn oil

0, 0.3, 1, and 3 

mmol/kg body weight 2 days

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 body weight.

• Urine samples collected for two days, then analyzed for TMPs 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 3,4,5-Trimethylphenol

0-24 h 24-48 h Total 0-24 h 24-48 h 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 h 24-48 h 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

Strengths: This study used an appropriate number of animals per dose group and observed a 

marked decrease in TMP excretion between 24 and 48 hours following exposure. 

Limitations: Does not contain data for 1,2,4 TMB and phenolic metabolites. Variation between 

rats (high standard deviation) within exposure groups. 
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Table A.2-34. Study Summary and Data for Mikulski and Wiglusz (1975)

Study Design

Species Sex N Exposure route Dose range Exposure duration

Wistar M 9 rats/dose Unspecified 1.2 g/kg 48 hours

Additional Study details

• Rats weighing between 210 and 350 grams were with treated with the three TMB isomers at 

1.2g/kg body weight.

• In one experiment, urine was collected every four hours over a period of three days.

• In a second experiment, metabolites were collected from rats were treated with mesitylene, 

pseudocumene, or hemimellitene.

• 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 

% of dose (mean±SD)

Glycine 

Conjugates

Glucuronides Organic 

Sulphates

Total

Mesitylene 59.1 ± 5.2 4.9 ± 1.0 9.2 ± 0.9 73.2

Pseudocumene 23.9 ± 2.3 4.0 ± 0.5 9.0 ± 2.1 36.9

Hemimellitene 10.1 ± 1.2 7.9 ± 1.3 15.0 ± 3.5 33.0

Treated with Phenobarbital

Mesitylene 35.1 ± 3.4 9.8 ± 1.3 8.1 ± 1.4 53.0

Pseudocumene 30.6 ± 2.5 12.2 ± 2.8 17.4 ± 3.6 60.2

Hemimellitene 5.7 ± 1.1 11.3 ± 2.0 22.3 ± 3.0 39.3

Strengths: Kinetic data for all three TMB isomers and their metabolites.

Limitations: Authors do not report method for dosing.
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 Toxicokinetic Studies in Humans and Animals

Table A.2-35. Study Summary and Data for Meulenberg and Vijverberg (2000)

Study Design

Species Sex N Exposure route Dose range Exposure duration

Rat and 

Human

F & 

M

Varies n/a not given not given

Additional Study details

• Authors examined partition coefficients for many volatile organic compounds from many 

studies. 

• 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 Partition Coefficients for 1,3,5- and 1,2,4-TMB

1,2,4-TMB 1,3,5-TMB

Reported and Predicted Partition Coefficients For Oil, Saline, and Air 

Poil:air 10200a 9880a

Psaline:air 1.61a 1.23a

 Reported and Predicted Ptissue:air values for Various Human Tissues

Blood 59.1a 43a

Fat 4566b 4423

Brain 206 199

Liver 286 277

Muscle 144 140

Kidney 114 110

Reported and Predicted Ptissue:air values for Various Rat Tissues

Blood 55.7 55.7

Fat 6068 5878

Brain 552 535

Liver 269 260

Muscle 104 100

Kidney 995 963

Strengths: 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. 

Limitations: Values for 1,2,4- and 1,3,5-TMB based on one study. 

a Averaged values as reported by Järnberg and Johanson (1995) b All other values predicted by Meulenberg and Vijverberg (2000)
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