JANUARY 201 2 

# Appendix A: Toxicological Information in Support of Hazard Identification and Dose

- Response Analysis for 

# 1,2,4

- and 1,3,5

- TrimethylbenzeneDRAFT

- DO NOT CITE OR QUOTE 2 

# 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.DRAFT

- DO NOT CITE OR QUOTE 3 

# 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. Mo del 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

................................ ................................ ................................ ................................ ........ 121DRAFT

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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

Tabl e 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 me asured 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 expos ed 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. Mod el 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

................................ ................................ ................................ ............................... 31DRAFT

- DO NOT CITE OR QUOTE 5 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 huma n 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 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)

................. 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

Ta ble A.2

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

................................ .......................... 5 0

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 Dat a 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)

................................ ................... 92DRAFT

- DO NOT CITE OR QUOTE 6 Table A.2

- 20. Study Summary and D ata 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 a nd 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)

........................... 120DRAFT

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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 pr evious 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 saturab le Michaelis

Menten 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. T issue: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 concentra tions 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 val ues 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 increase d 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/m 3 ) shows that overall metabolism is
saturable, and that t he metabolic pathway yielding 3,4

- dimethylbenzene becomes more important as exposure concentrations increase.DRAFT

- DO NOT CITE OR QUOTE 8 C: concentration of 1,2,4

- TMB; Cair: concentration in ambient air; Cart: concentration in arterial blood; Cven: concentration in venous blood; Q alv: 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 con stant 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 Values a 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 Q alv C air Q alv C alv Q
co C art

Q rap

Q f

Q
w

Q
r

Q h Q
co C
ven

V
max / K
m CL i

k
e Lungs and arterial blood

Rapidly perfused tissues

Resting muscles Adipose tissue

Working muscles

Liver

3,4

- dimethyl hippuric acid other metabolites

urine urine Q alv C air Q alv C alv Q
co C art

Q rap

Q f

Q
w

Q
r

Q h Q
co C
ven

V
max / K
m CL i

k
eDRAFT

- DO NOT CITE OR QUOTE 9 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, wer e 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 Both a 50 W Body height (m) 1.78 Body weight (kg) 75.5

V
max (μmol/min) 3.49

K
m (μM) 4.35

CL i (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 ofDRAFT

- DO NOT CITE OR QUOTE 10 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/m 3 ) 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 mode l 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 teste d 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. Cleara nce 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 ma thematical 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 b lood, 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.DRAFT

- DO NOT CITE OR QUOTE 11 Physiological parameters appear to be wi thin 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 compart ments) 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 f ollowing 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

m odel 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 w ere 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.DRAFT

- DO NOT CITE OR QUOTE 12 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 i n 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 r ats 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).DRAFT

- DO NOT CITE OR QUOTE 13 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

b rain 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 K m and V max 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 V max data was scaled as described above and K m values were used unchanged.DRAFT

- DO NOT CITE OR QUOTE 14 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 o f

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 expe rimental 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 asses sment.
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 (V max ) during exposure in a manner that was not d ocumented 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 V max 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,DRAFT

- DO NOT CITE OR QUOTE 15 however, did not appear to be entirely consistent with the inclusion of these V max adjustments, raising que stions 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 V max value is described below in the regarding verification of the Hissink et al. ( 2007 )
model ( page 26 )

. Other minor issues were identified by ex amining 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 balan ce 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 tissu es, 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 equiv alent 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, rathe r 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/m 3 WS (7.8% 1,2,4

- TMB) and mean analytical concentrations ran ging from 4,440 to
4,769 mg/m 3

— 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, a nd the text states that the fitted V max and K m were obtained for a 70 kg male ( Hissi nk 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 wer e compared to “CMIX,” the average of arterial and venous blood concentrations, while the protocol states that blood was taken from the cubitalDRAFT

- DO NOT CITE OR QUOTE 16 vein, so a more appropriate measure may have been CVS (venous blood exiting the slowly perfused tissues compartme nt). 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 a nd 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 e t 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 th e 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 tissue s 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/kg 0.7 ) 20 12

- 54 b Yes

Total cardiac output (L/hr/kg 0.7 ) 20 9.6

- 15 No Blood flow (% Cardiac output) Liver (total) 25 13.1

- 22.1 No
Fat 9 7 Acceptable c
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 Acceptable c
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 Hissin k 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 t hose 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?

Alv eolar ventilation rate (L/hr/kg 0.7 ) 20 15 Acceptable Total cardiac output (L/hr/kg 0.7 ) 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 liter ature data

Parameter Hissink et al. Literature Values in agreement?
Value Technique Value Technique
Partition Coefficients
Saline:Air 3 In vitro 1.47

- 1.75 a In vitro Acceptable

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

- 10,400 a In vitro Acceptable

Blood:Air

- human 85 In vitro 59.6

- 61.3 a 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 63 b In vivo Yes
Brain:Blood 2.53 Calculated 2 b In vivo Acceptable
Liver:Blood 2.53 Calculated

-- Metabolism

VmaxC

– rat (mg/hr/kg 0.7 ) 3.5 Visual optimization --

V
max C

– human (mg/hr/kg 0.7 ) 3.5 Assumed equal to rat 1.2

- 21 c Optimizatio

n Yes

K
m – rat (mg/L) 0.25 Visual optimization --

K
m – human (mg/L) 0.25 Assumed equal to rat 0.42

- 4.0 c Optimizatio

n No

V
max C/K
m – human (L/hr/kg 0.7 ) 14 Assumed equal to rat 2.6

- 15 c Optimizatio

n 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 K m is less than the lower 95 percent confidence interval value for the human K m , the human

V max C/K m 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 num erical estimates. The model code provided (adapted for acslX), with a variable value for V max , 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 )

sim ulations, 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 V max 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/m 3 WS (45 mg/m 3 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 V max , (c) constant V max . 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 hrsDRAFT

- 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 Co llins

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 Sweene y 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 us ed 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. E PA, 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

PARAMET ER RAT HUMAN (AT REST) Body weight (kg) 0.230

- 0.390 a 70 Alveolar ventilation rate (L/hr/kg 0.74 ) 14 15 Total cardiac output (L/hr/kg 0.74 ) 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

V max C (mg/h/kg 0.70 ) 4.17

K m (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 V max C and K m 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 V max C was only modestly different from the value determined by Hissink

et al. ( 2007 ) (initial: 3.5 vs. optimized: 3.08 mg/hr/kg 0.7 ) from visual optimization (with slightly different physiological parameters), but the K m 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 v isualDRAFT

- 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 cour se 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 ) Sprague

Dawley 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. ( 199 2 ) Sprague

Dawley 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 ) Sprague

Dawley 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 TextDRAFT

- 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 V max C and K m 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 Swie rcz 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 V max C = 4.17 mg/hr/kg 0.7 and K m = 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 exp lained 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 hig h concentration alone yields V max C and K m estimates of 7.91 mg/hr/kg 0.7 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 o ther 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 concentrati ons 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 accur ate, 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 Recovery b 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 Recovery b 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 Recovery b 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 Recovery b 0.46 0.24 1.9

Fat 1 491 210 2.3
2 503 165 3.1
3 504 128 3.9 Recovery b 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 ex posure 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 ex posed 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 pred icted 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 o f

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 V max C and K m 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 subseque nt 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 per cent variation explained was 69.74%. When K m was held constant

and V max C was optimized (final value: 3.39 mg/hr/kg 0.7 ), the improvement in fit was minimal (72.14% of variation explained), and not statistically significant, so the rat

- derived values were

co nsidered 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 i n 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 “nati ve 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 ve ntilation (0.56 to 1 m3/h), average of high and low body weights,
BW 0.74 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); alcoh ol 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, BW 0.74 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. TheDRAFT

- 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 1.0 4 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 geomet ric 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

V max C, but smaller K m 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 co

exposed 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 V max C and K m 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

conce ntrations (25 and 100 ppm) was apparent from careful visual inspection and wasDRAFT

- 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 ra ts, 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. Th e 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 mad e 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 venou s 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 V max C and K m 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 V max C and K m (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 co ncentration, 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 i nput, 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 co nsidered relevant. Parameters are grouped ( Table A.2

- 13 ) as relatively insensitiveDRAFT

- 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).

V max C/K m was identifiable from the data (as o pposed to V max C and K m 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 K m and V max C are distinctly identifiable using the Swiercz et al. ( 2003 ; 2002 ) data.

While the focus of this sensitivity analysis was to evaluate the identifiability of chemical

specific parameters from the available data, additional insights can be obtained by considering the other “sensitive” parameters. Predict ed 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 min utes) 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 venou s 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
V
max C L, H
K
m 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 wer e 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 con tribution 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 e ase 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, equiv alent 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 dai ly 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 V max C and K m values were estimated through numerical optimization; and (3) A n 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 V max of 4.17 mg/hr/kg 0.7 and K m 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 m odel 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), n

butanol, 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% co nfidence 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, alcoho l consumption, and social conformity.

• Log

- regression model used for case

control 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 pa inting 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 neuropsychologi cal 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 parti cipants 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,

ex posure varied from undetectable to 111.7 μg/m 3 , with median concentration 4.0 μg/m 3

.

• 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 smokin g 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 d ata 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 Stat istical 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

- s um 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 Estimates a

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 m 3 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 experimen t, 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 sep arate 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.DRAFT

- DO NOT CITE OR QUOTE 52 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 1 st minute of exposure was taken for consideration. The regression line was determine d 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 h ot 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 m 3 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.

• Rot arod 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

te sted 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.DRAFT

- DO NOT CITE OR QUOTE 54 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 pain

sensing 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 behavi ors were not measured.

*, ** statistically significant from controls at p ≤ 0.05 and p≤0.01, respectivelyDRAFT

- 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/m 3 90 days

Additional Study details

• Animals were exposed to 1,2,4

- TMB in a dynamic inhalation chamber (1.3 m 3 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 w ere 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 report ed

Observation Exposure Concentration (mg/m 3 )
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 (10 6 /cm 3 ) a 1.93 ± 0.79 5.82 ± 1.32*** 5.96 ± 2.80** 4.45 ± 1.58* Macrophages (10 6 /cm 3 ) 1.83 ± 0.03 3.78 ± 0.8 4.95 ± 0.2** 3.96 ± 0.3** Polymorphonuclear leucocytes (10 6 /cm 3 ) 0.04 ± 0.02 1.54 ± 0.7 0.52 ± 0.6 0.21 ± 0.3

Lymphocytes (10 6 /cm 3 ) 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/m 3

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 coheren t 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 = 0.0164DRAFT

- 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/m 3 90 days

Additional Study details

• Animals were exposed to 1,2,4

- TMB in a dynamic inhalation chamber (1.3 m 3 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 gro ups.

• 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/m 3 )
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 ± 1.34DRAFT

- 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/m 3 )

0 123 492 1230 1230 a Trend
test b
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 (× 10 3 /mm 3 ) c 9.98 ± 1.68 9.84 ± 1.82 8.50 ±
1.11 7.70 ± 1.38** 7.61 ± 1.6 0.0004

WBCs (× 10 3 /mm 3 ) 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 (× 10 3 /mm 3 ) 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 (× 10 3 /mm 3 ) c 8.22 ± 1.16 7.93 ± 2.04 8.51 ± 1.13 7.71 ± 1.58 6.99 ± 1.8 0.1891

WBCs (× 10 3 /mm 3 ) 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 0.3270DRAFT

- 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 (× 10 3 /mm 3 ) 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/m 3 )

0 123 492 1230 Trend
test b
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 0.4036DRAFT

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- 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/m 3 )

0 123 492 1230 Comparison to Controls Trend
test b

Males
Proliferation of peribronchial lymphatic tissue (0

- 4) j 16.0 k 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/m 3 492 mg/m 3DRAFT

- 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 coheren t 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/m 3 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 d etermining a POD

a effects measured in rats exposed to 1230 mg/m 3 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, respectivelyDRAFT

- 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 Sprague

Dawley F &
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 ± 17 g 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 gain a 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 litters b 21 22 21 17 18 No. of corpora lutea per dam 15.3 ± 1.5 g 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 ± 3.7DRAFT

- DO NOT CITE OR QUOTE 62 Table A.2

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

Mean % post

- implantation loss per litter c 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 litters d 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 malformations e 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 unossified f 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

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- 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 ± 18 g 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 gain a 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 litters b 24 22 22 22 24 No. of corpora lutea per dam 15.4 ± 2.1 g 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 litter c 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 litters d 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

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- 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 malformations e 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 unossified f 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: 6 00 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 fetalDRAFT

- 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.

Li mitations: 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, respectivelyDRAFT

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- 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 m 3 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

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- 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 p assive 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 step

down 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 the rmal 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

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- 9 . Study Summary and Data for Gralewicz et al. ( 1997a )

Figure 4. A comparison of the active avoidance performance increment during a singl e 30

- trial training session in consecutive groups of rats. The testing was performed on day 54 after 4

- week exposure to 1,2,4

- TMB. Bars represent the percentage (group mean and SE, n=15 for each group) of avoidance response in successive five

trial block s. 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 e xposure, 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

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- 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 m 3 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 me ans and
SE.DRAFT

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- 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 repr esent 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 huma n health reference values.DRAFT

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- 11 . Study Summary and Data for Gralewicz and Wiaderna ( 2001 )

Study Design Species Sex N Exposure r oute 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 m 3 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 trial s.

Denotation: Control

- sham exposed group (n=10), XYL

- m

- xylene exposed group (n=11),
PS

- 1,2,4

- TMB exposed group (n=11), MES

1,2,3

- TMB exposed group (n=11), HM

hemimellitene exposed group (n=11). Bars represent group means and SE.DRAFT

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- 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 m

xylene 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

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- 11 . Study Summary and Data for Gralewicz and Wiaderna ( 2001 )

Figure 4. A comparison of sham

- exposed and solvent

exposed rats with respect to the latency of the paw

- lick response to heat (54.5°C) before (L1), several seconds after (L2), and 24 h after a two minute intermittent fo otshock. 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 ex posure to m

xylene or a TMB isomer at a concentration of 100 ppm. In on massed

- trial session (inter

trial 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 perfor med 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 expo sure 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

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- 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 m 3 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, includin g 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 avoid ance 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

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- 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 perform ed 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 e xposure, 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 valuesDRAFT

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- 1 3 . 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/m 3 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.

Observ ation Exposure to 1,2,4

- TMB (mg/m 3 /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/m 3 /8 h/day)

0 125 1250 5000 Visual Discrimination Performance Testing (means±SD)

Trials a

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 obtained b

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±0.0DRAFT

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- 1 3 . Study Summary and Data for McKee et al. ( 2010 )

Discrimination ratio c

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 to d

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 errors e

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 latency g

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 seconds h

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 seconds i

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±0.35DRAFT

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- 1 3 . Study Summary and Data for McKee et al. ( 2010 )

Drink response latency j

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.

Limit ations: 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 highe st exposure group on day 3. Acute d uration 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 int er

- 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 r esponse

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 < 0.05DRAFT

- DO NOT CITE OR QUOTE 79 Table A.2

- 1 4 . 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 chromatograph y.

• 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
0.03 2 mol/kg.DRAFT

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- 1 4 . Study Summary and Data for Tomas et al. ( 1999a )

Figure 2. 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.

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 d uration of exposure (single oral gavage) . Generally, acute exposure studies have limited utility in quantitation of human health reference values.DRAFT

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- 1 5 . 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

m mol/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 d ose group.

Limitations: Acute d uration of exposure ( single oral gavage ) . Generally, acute exposure studies have limited utility in quantitation of human health reference values.DRAFT

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- 1 6 . 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.DRAFT

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- 1 6 . 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

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- 1 6 . 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 d uration of exposure (single i.p. injection) . Generally, acute exposure studies have limited utility in quantitation of human health reference values.DRAFT

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- 1 7 . 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/m 3 (rats); 57 or 570 mg/m 3 (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/m 3 for 3 days. Human volunteers were exposed to 57 or 570 mg/m 3 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/m 3 )
0 600 2400 4800 Functional Observation Battery (Mean±SD)
Gait score a

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 response b

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±0.21DRAFT

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- 1 7 . 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/m 3 ) 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

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- 1 7 . 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/m 3 White Spirit for 4 Hours 57 mg/m 3 570 mg/m 3

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 coordinationDRAFT

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- 1 7 . 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.DRAFT

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- 1 7 . 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, whic h 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 g roup comparisons. *,**,*** Statistically significant from controls at p < 0.05, p<0.01, and p<0.001 respectivelyDRAFT

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- 18. Study Summary and Data for Maltoni et al. ( 1997 )

Study Design Species Sex N Exposure route Dose range Exposure duration Sprague

Dawley 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 Neuroesthesioepi

theliomas -- 2.0

Oral cavity

cancers -- 2.0

Total head 2.0 10.0DRAFT

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- 18. Study Summary and Data for Maltoni et al. ( 1997 )

cancers Females Zymbal gland

cancer 2.0 2.0

Ear duct cancer 2.0

-- Neuroesthesioepi

theliomas -- 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 Neuroesthesioepi

theliomas -- 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

- 1 9 . 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 ± 0.29DRAFT

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- 1 9 . Study Summary and Data for Jarnberg et al. ( 1996 )

x hr - 1 x kg - 1 ) f

1 st Phase Half

- Life (minutes) 1.7 ± 0.8 1.3 ± 0.8 1.4 ± 1.8
2 nd Phase Half

- Life (minutes) 27 ± 5 21 ± 5 28 ± 14
3 rd Phase Half

- Life (minutes) 4.9 ± 1.4 3.6 ± 1.1 5.9 ± 2.5
4 th 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)DRAFT

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- 1 9 . 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 r espiratory uptake

f Calculated from respiratory uptake

g Calculated from net respiratory uptakeDRAFT

- DO NOT CITE OR QUOTE 95 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/m 3 2 hours

Additional Study details

• Nine Caucasian males were exposed to 11 mg/m 3 1,2,4

- trimethylbenze alone or 11 mg/m 3
1,2,4

- TMB as a component of 300 mg/m 3 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 level s. 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 0.5 b AUC (μM x min), 0

- 3 h 53 ± 4 86 ± 9 <0.0001 b
Half life of 3,4

- DMHA (h) 3.7 ± 0.4 3.0 ± 0.7 0.2 cDRAFT

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- 20 . Study Summary and Data for Jarnberg et al. ( 1997a )

Excretion of 3,4

- DMHA (% a ), 0

- 6 h 11 ± 2 18 ± 3 0.007 c 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/m 3 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

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- 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 foun d 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 3.8 0 ± 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.DRAFT

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- 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 ch romatography.

• 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 in terfere 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)
AUC blood (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, 1 st Phase (min) 3.9(1.4

- 6.4) 5.9(3.1

- 8.7) 6.1(5.3

- 6.9) Idem, TMB, 2 nd 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) ND c 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<0.01DRAFT

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- 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/m 3 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/m 3

.

• 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/dm 3 ) Std. Deviation Blood Concentration (μg/dm 3 ) 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. DevDRAFT

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- 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, althoug h one volunteer was treated with a 2cm 2 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

- TMBDRAFT

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- 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

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Table A.2

- 2 5 . 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

- TM B 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 ± 4.03DRAFT

- DO NOT CITE OR QUOTE 104 Table A.2

- 2 5 . 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

- 2 6 . 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 ± 0.22DRAFT

- DO NOT CITE OR QUOTE 106 Table A.2

- 2 6 . 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

- 2 7 . 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 ± 28DRAFT

- DO NOT CITE OR QUOTE 108 Table A.2

- 2 7 . 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 ± 0.06DRAFT

- DO NOT CITE OR QUOTE 109 Table A.2

- 2 7 . 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

- 2 8 . Study Summary and Data for Zahlsen et al. ( 1990 )

Study Design

Species Sex n Exposure
route Dose range Exposure duration

Sprague

Dawley 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 concentratio n 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, possibl y 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 14DRAFT

- DO NOT CITE OR QUOTE 111 Table A.2

- 2 8 . 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

Sprague

Dawley 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 ur inary 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 14 C 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 ra dio

- 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 14 C

- 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 ± 6DRAFT

- 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

- TMP a 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

- DMBOH b 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 acidDRAFT

- DO NOT CITE OR QUOTE 115 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.

• Trac e 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 d 10 ) (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*DRAFT

- DO NOT CITE OR QUOTE 116 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)DRAFT

- DO NOT CITE OR QUOTE 117 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 speci fic speciation data for 2,4

- , 2,5

- , or 3,4

- dimethylbenzyl mercapturic acid.DRAFT

- DO NOT CITE OR QUOTE 118 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

ra ts (high standard deviation) within exposure groups.DRAFT

- DO NOT CITE OR QUOTE 119 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 wi th 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.DRAFT

- DO NOT CITE OR QUOTE 120  Toxicokinetic Studies in Humans and Animals

Table A.2

- 3 5 . 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

P oil:air 10200 a 9880 a

P saline:air 1.61 a 1.23 a Reported and Predicted P tissue:air values for Various Human Tissues
Blood 59.1 a 43 a

Fat 4566 b 4423
Brain 206 199
Liver 286 277
Muscle 144 140 Kidney 114 110 Reported and Predicted P tissue: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 J ärnberg and Johanson ( 1995 )

b All other values predicted by Meulenberg and Vijverberg ( 2000 )DRAFT

- DO NOT CITE OR QUOTE 121 

# A. **3 . References** 

Arms, AD; Travis, CC. (1988). Reference physiological parameters in pharmacokinetic modeling. (EPA/600/6

- 88/004). Washington, D.C.: U.S. Environmental Proctection Agency. http://www.ntis.gov/search/product.aspx?ABBR=PB88196019.

Bakke, OM; Scheline, RR. (1970). Hydroxylation of aromatic hydrocarbons in the rat. Toxicol Appl Pharmacol
16: 691

- 700.

Billionnet, C; Gay, E; Kirchner, S; Leynaert, B; Annesi

- Maesano, I. (2011). Quantitative assessments of indoor air pollution and respiratory health in a population

- based sample of French dwellings. Environ Res 111: 425

- 434. http://dx.doi.org/10.1016/j.envres.2011.02.008.

Brown, RP; Delp, MD; Lindstedt, SL; Rhomberg, LR; Beliles, RP. (1997). Physiological parameter values for physiologically based pharmacokinetic models. Toxicol Ind Health 13: 407

- 484. http://dx.doi.org/10 .1177/074823379701300401.

Cerf, J; Potvin, M; Laham, S. (1980). Acidic metabolites of pseudocumene in rabbit urine. Arch Toxicol 45: 93

- 100.

Chen, R; Dick, F; Seaton, A. (1999). Health effects of solvent exposure among dockyard painters: Mortality and neuropsychological symptoms. Occup Environ Med 56: 383

- 387. htt p://dx.doi.org/10.1136/oem.56.6.383.

Collins, AS; Sumner, SCJ; Borghoff, SJ; Medinsky, MA. (1999). A physiological model for tert

- amyl methyl ether and tert

- am yl alcohol: Hypothesis testing of model structures. Toxicol Sci 49: 15

- 28.

Cooper, SP; Burau, K; Sweeney, A; Robison, T; Smith, MA; Symanski, E; Colt, JS; Laset er, J; Zahm, SH. (2001). Prenatal exposure to pesticides: a feasibility study among migrant and seasonal farmworkers. Am J Ind Med 40: 578

- 585.

Dahl, AR; Dam on, EG; Mauderly, JL; Rothenberg, SJ; Seiler, FA; McClellan, RO. (1988). Uptake of 19 hydrocarbon vapors inhaled by F344 rats. Fundam Appl Toxicol 10: 262

- 269. http://dx.doi.org/10.1016/0272

- 0590(88)90310

- 7.

Dowty, BJ; Laseter, JL; Storer, J. (1976). The transplacental migration and accumulation in blood of volatile organic constituents. Pediatr Res 10: 696

- 701.

Eide, I; Zahlsen, K. (1996). Inhalation experiments with mixtures of hydrocarbons. Experimental design, statistics and interpretation of kinetics and possible interactions. Arch Toxicol 70: 397

- 404. http ://dx.doi.org/10.1007/s002040050291.

Emond, C; Krishnan, K. (2006). A physiological pharmacokinetic model based on tissue lipid content for simulating inhalat ion pharmacokinetics of highly lipophilic volatile organic chemicals. Toxicol Mech Meth 16: 395

- 403. http://dx.doi.org/10.1080/15376510600860474.

Fukaya, Y; Sait o, I; Matsumoto, T; Takeuchi, Y; Tokudome, S. (1994). Determination of 3,4

- dimethylhippuric acid as a biological monitoring index for trimethylbenzene exposure in transfer printing workers. Int Arch Occup Environ Health 65: 295

- 297. http://dx.doi.org/10 .1007/BF00405692.

Gralewicz, S; Wiaderna, D; Tomas, T; Rydzyński, K. (1997a). Behavioral changes following 4

- week inhalation exposure to pseudocumene (1,2,4

- trimethylbenzene) in the rat. Neurotoxicol Teratol 19: 327

- 333. http://dx.doi.org/10.1016/S0892

- 0362(97)00001

- 9.

Gralewicz, S; Wiaderna, D; Tomas, T. (1997b). Retardation of the age

- related increase in spontaneous cortical spike

- wave discharges (SWD) in rats after a 28

- day inhalat ion (SWD) in rats after a 28

- day inhalationDRAFT

- DO NOT CITE OR QUOTE 122 exposure to an industrial solvent, pseudocumene (1,2,4

- trimethylbenzene). Int J Occup Med Environ Health
10: 213

- 222.

Gralewicz, S; Wiaderna, D. (2001). Behavioral effects following subacute inhalation exposure to m

- xylene or trimethylbenzene in the rat: A comparative study. Neurotoxicology 22: 79

- 89. http://dx.doi.org/10.1016/S0161

- 813X(00)00003

- 6.

Harlan Laboratories. (2011). Sprague Dawley: Outbred Rat Retrieved June 7, 2011, from http://www.harlan.com/products_and_services/research_models_and_services/research_models _by_produc t_type/outbred_rats/sprague_dawley_sd

Hissink, AM; Krüse, J; Kulig, BM; Verwei, M; Muijser, H; Salmon, F; Leenheers, LH; Owen, DE; Lammers, JH; Freidig , AP; McKee, RH. (2007). Model studies for evaluating the neurobehavioral effects of complex hydrocarbon solvents III. PBPK modeling of white spirit constituents as a tool for integrating animal and human test data. Neurotoxicology 28: 751

- 760. http://d x.doi.org/10.1016/j.neuro.2007.03.005.

HSDB. (Hazardous Substances Data Bank). (2011a). 1,2,4

- Trimethylbenzene [Database]. Bethesda, MD: National Library of M edicine. Retrieved from http://toxnet.nlm.nih.gov

HSDB. (Hazardous Substances Data Bank). (2011b). 1,3,5

- Trimethylbenzene [Database]. Bethesda, MD: National L ibrary of Medicine. Retrieved from http://toxnet.nlm.nih.gov

Huo, JZ; Aldous, S; Campbell, K; Davies, N. (1989). Distribution and metabolism of 1,2,4

- trimethy lbenzene (pseudocumene) in the rat. Xenobiotica 19: 161

- 170. http://dx.doi.org/10.3109/00498258909034688.

Ichiba, M; Hama, H; Yukitake, S; Kubota, M; Kawasaki, S ; Tomokuni, K. (1992). Urinary excretion of 3,4

- dimethylhippuric acid in workers exposed to 1,2,4

- trimethylbenzene. Int Arch Occup Environ Health 64: 325

- 327. http://dx.doi.org/10.1007/BF00379541.

Janasik, B; Jakubowski, M; Jałowiecki, P. (2008). Excretion of unchanged volatile organic compounds (toluene, ethylbenzene, xylene and mesitylene) in urine as result of experimental human volunteer exposure. Int Ar ch Occup Environ Health 81: 443

- 449. http://dx.doi.org/10.1007/s00420

- 007

- 0233

- 9.

Järnberg, J; Johanson, G. (1995). Liquid/air partition coefficients of the t rimethylbenzenes. Toxicol Ind Health
11: 81

- 88. http://dx.doi.org/10.1177/074823379501100107.

Järnberg, J; Johanson, G; Löf, A. (1996). Toxicokinetics of inha led trimethylbenzenes in man. Toxicol Appl Pharmacol 140: 281

- 288. http://dx.doi.org/10.1006/taap.1996.0223.

Järnberg, J; Johanson, G; Löf, A; Stahlbom, B. (1 997a). Inhalation toxicokinetics of 1,2,4

- trimethylbenzene in volunteers: Comparison between exposure to white spirit and 1,2,4

- trimethylbenzene alone. Sci Total Environ 199: 65

- 71. http://dx.doi.org/10.1016/S0048

- 9697(97)05482

- X.

Järnberg, J; Stahlbon, B; Johanson, G; Löf, A. (1997b). Urinary excretion of dimethylhippuric acids in humans after exposure to trimethylbenzenes. Int Arch Occup Environ Health 69: 491

- 497. http://dx.doi.org/10.1007/s004200050179.

Järnberg, J; Johanson, G; Löf, A; Stahlbom, B. (1998). Toxicokinetics of 1,2,4

- trimethylbenzene in humans exposed to vapours of white spirit: Comparison with exposure to 1,2,4

- trimethylbenzene alone. Arch Toxicol
72: 483

- 491. htt p://dx.doi.org/10.1007/s002040050532.

Järnberg, J; Johanson, G. (1999). Physiologically based modeling of 1,2,4

- trimethylbenzene inhalation toxicokinetics. To xicol Appl Pharmacol 155: 203

- 214. http://dx.doi.org/10.1006/taap.1998.8596.

Jones, K; Meldrum, M; Baird, E; Cottrell, S; Kaur, P; Plant, N; Dyne, D; Cocker, J. (2006). Biological monitoring for trimethylbenzene exposure: A human volunteer study and a practical example in the workplace. Ann Occup Hyg 50: 593

- 598. http://dx.doi.org/10.1093/annhyg/mel016.DRAFT

- DO NOT CITE OR QUOTE 123 Korsak, Z; Swiercz, R; Rydzyński, K. (1995). Toxic effects of acute inhalation exposure to 1,2,4

- trimethylbenzene (pseudocumene) in experimental animals. Int J Occup Med Environ Health 8: 331

- 337.

Korsak, Z; Rydzyński, K. (1996). Neurotoxic effects of acute and subchronic inhalation exposure to trimethylbenzene isomers (pseudocumene, mesitylene, hemimellitene) in rats. I nt J Occup Med Environ Health 9: 341

- 349.

Korsak, Z; Rydzyński, K; Jajte, J. (1997). Respiratory irritative effects of trimethylbenzenes: An experimental
ani mal study. Int J Occup Med Environ Health 10: 303

- 311.

Korsak, Z; Stetkiewicz, J; Majcherek, W; Stetkiewicz, I; Jajte, J; Rydzyński, K. (2000). Sub

- chronic i nhalation toxicity of 1,2,4

- trimethylbenzene (pseudocumene) in rats. Int J Occup Med Environ Health 13: 155

- 164.

Kostrewski, P; Wiaderna

- Brycht, A. (1995). K inetics of elimination of mesitylene and 3,5

- dimethylbenzoic acid after experimental human exposure. Toxicol Lett 77: 259

- 264. http://dx.doi.org/10.1016/0378

- 4274(95)03305

- X.

Kostrzewski, P; Wiaderna

- Brycht, A; Czerski, B. (1997). Biological monitoring of experimental human exposure to trimethylbenzene. Sci Total Environ 199: 73

- 81. http://dx.doi.org/10.1016/S0048

- 9697(97)05504

- 6.

Laham, S; Potvin, M. (1989). Identification and determination of mesitylene acidic metabolites in rabbit urine. Toxicol Environ Chem 24: 57

- 69. http://dx.doi.org/10.1080/02772248909357477.

Lammers, JH; Emmen, HH; Muijser, H; Hoogendijk, EM; McKee, RH; Owen, DE; Kulig, BM. (2007). Model studies for evaluating the neurobehavioral effects of compl ex hydrocarbon solvents II. Neurobehavioral effects of white spirit in rat and human. Neurotoxicology 28: 736

- 750. http://dx.doi.org/10.1016/j.neuro.2007.03.003.

Maltoni, C; Ciliberti, A; Pinto, C; Soffritti, M; Belpoggi, F; Menarini, L. (1997). Results of long

- term experimental carcinogenicity studies of the effects of gasoline, correlated fuels, and major gasoline aromatics on rats. Ann N Y Acad Sci 837: 15

- 52. http://dx.doi.org/10.1111/j.1749

- 6632.1997.tb56863.x.

McKee, RH; Lammers, JH; Muijser, H; Owen, DE; Kulig, BM. (2010). Neurobehavioral effects of acute exposure to aromatic hydrocarbons. Int J Toxicol 29: 277

- 290. http://dx.doi.org/10 .1177/1091581810365089.

Meulenberg, C; Vijverberg, H. (2000). Empirical relations predicting human and rat tissue: Air partition coefficients of volatile organic compounds. Toxicol Appl Pharmacol 165: 206

- 216. http://dx.doi.org/10.1006/taap.2000.8929.

Mikulski, PI; Wiglusz, R. (1975). The comparative metabolism of mesitylene, pseudocumene, and hemimellitene in rats. Toxicol Appl Pharmacol 31: 21

- 31. http://dx.doi.org/10.1016/0041

- 008X(75)90048

- 4.

Norseth, T; Waage, J; Dale, I. (1991). Acute effects and exposure to organic compounds in road maintenance workers exposed to asphalt. Am J Ind Med 20: 737

- 744. http://dx.doi. org/10.1002/ajim.4700200604.

OSHA. (Occupational Safety & Health Administration). (1996). Occupational safety and health guideline for trimethylbenzene Retri eved August 1, 2007, from http://www.osha.gov/SLTC/healthguidelines/trimethylbenzene/recognition.html

Pyykko, K. (1980). Effects of methylbenzenes on microsom al enzymes in rat liver, kidney and lung. Biochim Biophys Acta 633: 1

- 9. http://dx.doi.org/10.1016/0304

- 4165(80)90032

- X.

Saillenfait, AM; Gallissot, F; Sabate, J P; Morel, G. (2005). Developmental toxicity of two trimethylbenzene isomers, mesitylene and pseudocumene, in rats following inhalation exposure. Food Chem Toxicol 43: 1055

- 1063. http://dx.doi.org/10.1016/j.fct.2005.02.008.DRAFT

- DO NOT CITE OR QUOTE 124 Swiercz, R; Rydzyński, K; Wasowicz, W; Majcherek, W; Wesolowski, W. (2002). Toxicokinetics and metabolism of pseudocumene (1,2,4

- trimethylbenzene) after inhalation exposure in rats. Int J Occup Med Environ Health 15: 37

- 42.

Swiercz, R; Wiaderna, D; Wasowicz, W; Rydzyński, K. (2003). Pseudocumene in brain, liver, lung and blood of rats after si ngle and repeated inhalation exposure. Int J Occup Med Environ Health 16: 61

- 66.

Swiercz, R; Wasowicz, W; Majcherek, W. (2006). Mesitylene (1,3,5

- trimethylbe nzene) in the liver, lung, kidney, and blood and 3,5

- dimethylbenzoic acid in the liver, lung, kidney and urine of rats after single and repeated inhalation exposure to mesitylene. Pol J Environ Stud 15: 485

- 492.

Tomas, T; Lutz, P; Wiaderna, D. (1999a). Changes in electrocortical arousal following acute trimethylbenzene administration in rats. Int J Occup Med Environ Health 12: 67

- 78.

Tomas, T; Swiercz, R; Wiaderna, D; Gralewicz, S. (1999b). Effects of acute exposure to aromatic hydrocarbons C 9 on locomotor activity in rats. Trimethylbenzene isomers. Int J Occup Med Environ Health 12: 331

- 343.

Tomas, T; Wiaderna, D; Swiercz, R. (1999c). Neurotoxicity assessment of selected organic solvents based on spontaneous and evoked cortical and hippocampal activity in rats. Int J Occup Med Environ Health 12: 73

- 84.

Tsujimoto, Y; Noda, T; Shimizu, M; Moriwaki, H; Tanaka, M. (2000). Identification of the dimethylbenzyl mercapturic acid in urine of rats administered with 1,2,4

- trimethylbenzene. Chemosphe re 40: 893

- 896. http://dx.doi.org/10.1016/S0045

- 6535(99)00467

- 1.

Tsujimoto, Y; Warashina, M; Nam, VD; Noda, T; Shimizu, M; Yamaguchi, Y; Moriwaki, H; Morimoto, T ; Kakiuchi, K; Maeda, Y; Tanaka, M. (2005). Determination of urinary phenolic metabolites from rats treated with 1,2,3

- and 1,3,5

- trimethylbenzenes. J Occup Health 47: 337

- 339.

Tsujino, Y; Hieda, Y; Kimura, K; Eto, H; Yakabe, T; Takayama, K; Dekio, S. (2002). Distribution of kerosene components in rats following dermal exposure. Int J Legal Med 116: 207

- 211. http://dx.doi.org/10.1007/s00414

- 001

- 0282

- 7.

U.S. EPA. (U.S. Environmental Protection Agency). (1987). Health effects assessment for trimethylbenzenes. (EPA/600/8

- 88/060). Cincinnati, OH: U.S. Environmental P rotection Agency, Office of Health and Environmental Assessment. http://nepis.epa.gov/Exe/ZyPURL.cgi?Dockey=2000T8ZG.txt.

U.S. EPA. (U.S. Environmental Protec tion Agency). (1994). Chemical summary for 1,2,4

- trimethylbenzene. (EPA/749/F

- 94/022A). Washington, DC: U.S. Environmental Protection Agency, Office of Pollution Prevention and Toxic Substances. http://www.epa.gov/chemfact/s_trimet.txt.

U.S. EPA. (U.S. Environmental Protection Agency). (2011). PBPK data files for TMB [PBPK]. Research Triangle Park, NC.

Wiaderna, D; Gralewicz, S; Tomas, T. (2002). Assessment of long

- term neurotoxic effects of exposure to mesitylene (1,3,5

- trimethylbenzene) based on the analysis of selected behavioral responses. Int J Occup Med
Envir on Health 15: 385

- 392.

Wiglusz, R. (1979). The effect of 1, 3, 5

- trimethylbenzene inhalation exposure on the glucuronic acid pathway and activity of some xen obiotic

- metabolizing enzymes. Bull Inst Marit Trop Med Gdynia 30: 189

- 196.

Williams, LR; Leggett, RW. (1989). Reference values for resting blood flow to organ s of man. Clinical Physics and Physiological Measurement 10: 187

- 217. http://dx.doi.org/10.1088/0143

- 0815/10/3/001.

Yoshida, T. (2010). Estimation of absorpti on of aromatic hydrocarbons diffusing from interior materials in automobile cabins by inhalation toxicokinetic analysis in rats. J Appl Toxicol 30: 525

- 535. http://dx.doi.org/10.1002/jat.1522.DRAFT

- DO NOT CITE OR QUOTE 125 Zahlsen, K; Nilsen, AM; Eide, I; Nilsen, OG. (1990). Accumulation and distribution of aliphatic (n

- nonane), aromatic (1,2,4

- trimethylbenzene) and naphthenic (1,2,4

- trimethylcyclohexane) hydrocarbons in the rat after repea ted inhalation. Pharmacol Toxicol 67: 436

- 440. http://dx.doi.org/10.1111/j.1600

- 0773.1990.tb00859.x.

Zahlsen, K; Eide, I; Nilsen, AM; Nilsen, OG. (1992). Inhalation kinetics of C6 to C10 aliphatic, aromatic and naphthenic hydrocarbons in rat after repeated exposures. Pharmacol Toxicol 71: 144

- 149. http://dx.doi.org/10.1111/j.1600

- 0773.1992.tb00534.x.