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Endpoint:
basic toxicokinetics in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-guideline experimental study with clearly reported methods and results. Adequate for assessment.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline available
Principles of method if other than guideline:
Male Wistar rats were exposed by inhalation to 1,2,4-trimethylbenzene for 2, 4, or 8 hr; or 8-hr/day for three consecutive days. The concentration of 1,2,4-trimethylbenzene was measured in the blood and brain tissue at these timepoints.
GLP compliance:
not specified
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: approximately 14 weeks of age
Route of administration:
inhalation: vapour
Vehicle:
other: air
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMPER DESCRIPTION
- Exposure apparatus: modified H1000 inhalation chambers
- System of generating vapours: Test atmospheres were created by pumping liquid substance through heated water baths to create vapours. The vapours were transported with an air stream and added to the main airflow systems for the inhalation chambers.

Duration and frequency of treatment / exposure:
2, 4, and 8 hr; and 8 hr/day for three consecutive days
Remarks:
Doses / Concentrations:
The mean average concentrations were 128, 1,255, and 4,980 mg/m3 for the 125, 1,250, and 5,000 mg/m3 exposure groups.
No. of animals per sex per dose / concentration:
3-4
Control animals:
no
Details on dosing and sampling:
Samples of blood and brain tissue was taken immediately from animals exposed to 1,2,4-trimethylbenzene. Tissues were taken and analysed by methods described in Hissink et al., Neurotoxicol. 28: 736-750, 2007. The detection levels were 20 ng/mL in blood and 150 ng/g in brain.
Details on distribution in tissues:
There was rapid uptake of 1,2,4-trimethylbenzene into the blood and brain, with the concentrations in the brain being about 2-3 times higher than the corresponding blood concentrations. Equilibrium appeared to have been reached within 2 hrs In the 125 and 1,250 mg/m3 exposed groups, since the brain concentrations did not substantially increase after that point. In the 5,000 mg/m3 exposed group, the blood and brain concentrations continued to increase over the entire single 8-hr exposure period. The blood and brain concentrations in the 5,000 mg/m3 exposed group after three consecutive days of exposure were approximately half the concentrations recorded in animals after a single 8-hr exposure period.
Conclusions:
Interpretation of results (migrated information): other: Repeated exposures to 1,2,4-trimethylbenzene appeared to induce its own metabolism in the 5,000 mg/m3 exposed animals, but not at lower exposures (125 and 1,250 mg/m3).
Repeated exposures to 1,2,4-trimethylbenzene appeared to induce its own metabolism in the 5,000 mg/m3 exposed animals, but not at lower exposures (125 and 1,250 mg/m3).
Executive summary:

Male Wistar rats were exposed by inhalation to 0, 125, 1,250 or 5,000 mg/m3 1,2,4 -trimethylbenzene for 2, 4, or 8 hours; and to 8 hours/day for three consecutive days. Immediately following exposure, animals were sacrificed and samples of blood and brain were taken. There was rapid uptake of 1,2,4 -trimethylbenzene into the blood and brain, with brain concentrations being about two to three times higher than the corresponding blood concentrations. Equilibrium appeared to have been reached after 2 hours at 125 and 1,250 mg/m3, but blood and brain concentrations continued to increase over the entire 8 -hour period in the 5,000 mg/m3 exposed animals. The blood and brain concentrations in the 5,000 mg/m3 group after three consecutive days of exposure were approximately half the concentrations found in animals after a single 8 -hour exposure. These data suggest that 1,2,4 -trimethylbenzene induced its own metabolism in the 5,000 mg/m3 exposed animals. Metabolism was apparently unaffected at lower concentrations.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-guideline experimental study with clearly reported methods and results. Adequate for assessment.
Objective of study:
excretion
Qualifier:
no guideline available
Principles of method if other than guideline:
The elimination kinetics of unchanged 1,3,5-trimethylbenzene in human urine was examined in six male volunteers exposed by inhalation to 1,3,5-trimethylbenzene. In addition, the relationship between air concentration of 1,3,5-trimethylbenzene and its concentration in urine and blood and the urinary concentration of its metabolite 3,5-dimethylbenzoic acid, was determined.
GLP compliance:
not specified
Species:
human
Sex:
male
Details on test animals or test system and environmental conditions:
Age of volunteers was between 20 and 28 years.
Route of administration:
inhalation: vapour
Vehicle:
other: air
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMPER DESCRIPTION
- Exposure apparatus: 117 cubic meter exposure chamber
- Air turnover rate: 350 m3/hr
- System of generating vapours: Vapour generation was based on continuous injection of liquid substance into the pressed air stream, with controlled speed.


Duration and frequency of treatment / exposure:
A single 4 or 8 hr exposure.
Remarks:
Doses / Concentrations:
200 mg/m3 (25 ppm) for the 4-hr study; 20, 60 or 100 mg/m3 (5, 15, or 25 ppm) for the 8-hr study.
No. of animals per sex per dose / concentration:
6 male volunteers
Control animals:
no
Details on dosing and sampling:
4-HR EXPOSURE STUDY
Urine samples were collected at the following time points: before exposure, every 2 hr of exposure, and up to 24 hr from the onset of exposure.

8-HR EXPOSURE STUDY
Capillary blood samples obtained from the finger tips were collected before the start of exposure and at 30 min after termination of exposure. Urine samples were collected before exposure and for the last two hours of exposure.
Statistics:
Statistical analysis was carried out using StatGraphics Plus for Windows 4.1. Linear regression analysis was applied to examine the correlation between exposure parameters.
Details on excretion:
The average percentage of the dose excreted as unchanged 1,3,5-trimethylbenzene in urine during the 4-hr exposure period and up to 24 hr post-exposure was 0.0012% (range: 0.00095% to 0.0047%). The elimination kinetics followed an open two-compartment model with half-life values of 0.45 hr and 6.68 hr for the first and second phases, respectively. When 1,3,5-trimethylbenzene was expressed quantitatively in different units, such as urine concentration (uncorrected and corrected for specific gravity or creatinine) and the excretion rate, uncorrected concentration provided the best results.

There was a linear relationship between the air concentration of 1,3,5-trimethylbenzene and the concentration of unchanged substance and its metabolite 3,5-dimethylbenzoic acid in urine samples collected during the last 2 hr of the 8-hr exposure period as well as the concentration of unchanged substance in blood samples collected 30 min after the termination of exposure. Unchanged 1,3,5-trimethylbenzene in urine was found to be a more precise method than those based on the unchanged substance in blood or its metabolite in urine.


Conclusions:
Interpretation of results (migrated information): other: Linear relationships were found between the air concentrations of 1,3,5-trimethylbenzene and the concentration of unchanged 1,3,5-trimethylbenzene and its metabolite 3,5-dimethylbenzoic acid in urine as well as the unchanged substance in blood.
Linear relationships were found between the air concentrations of 1,3,5-trimethylbenzene and the concentration of unchanged 1,3,5-trimethylbenzene and its metabolite 3,5-dimethylbenzoic acid in urine as well as the unchanged substance in blood.
Executive summary:

Six male volunteers were exposed to 200 mg/m3 1,3,5 -trimethylbenzene vapours for 4 hours to determine the kinetics of elimination of unchanged 1,3,5 -trimethylbenzene in urine. Urine samples were collected before exposure, every 2 hours of exposure, and up to 24 hours from the start of exposure. In the subsequent study, six male volunteers were exposed to 20, 60, and 100 mg/m3 1,3,5 -trimethylbenzene vapours for 8 hours. Blood samples were collected before exposure and 30 minutes after the end of the exposure. Urine samples were collected before exposure and for the final two hours of the exposure period. The elimination kinetics of 1,3,5 -trimethylbenzene in urine followed an open two-compartment model with the half-life values of 0.45 and 6.68 hours for the first and second phases, respectively. There was a linear relationship between the air concentration of 1,3,5-trimethylbenzene and the concentration of unchanged substance and its metabolite 3,5-dimethylbenzoic acid in urine as well as the unchanged substance in blood. Unchanged 1,3,5-trimethylbenzene in urine was found to be a more precise method than those based on the unchanged substance in blood or its metabolite in urine.

Endpoint:
basic toxicokinetics in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, non-guideline, animal experimental study, published in peer reviewed literature, no restrictions, fully adequate for assessment.
Objective of study:
toxicokinetics
Principles of method if other than guideline:
Dynamic inhalation chamber study in rats. Time course of 1,2,4-trimethylbenzene in blood and concentrations of dimethylbenzoic acid in urine determined.
GLP compliance:
not specified
Radiolabelling:
no
Species:
rat
Strain:
other: Wistar Imp:DAK
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: no data
- Weight at study initiation: 180-370 g
- no further details

ENVIRONMENTAL CONDITIONS
- Temperature: 20-23°C
- Humidity: 45-60%
- no further details

IN-LIFE DATES:
- no data
Route of administration:
inhalation: vapour
Vehicle:
unchanged (no vehicle)
Details on exposure:
TYPE OF INHALATION EXPOSURE: no data

TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: dynamic inhalation chambers (volume 250 dm3)
- Vapours of pseudocumene were generated by heating liquid solvents in a washer.
- The desired concentrations of vapours were obtained by diluting them with the air.
- Concentrations of solvent vapours in the exposure chamber were measured every 30 min by means of GC-FID.
Duration and frequency of treatment / exposure:
Single 6 hr
Remarks:
Doses / Concentrations:
25, 100 or 250 ppm (nominal concentration)
No. of animals per sex per dose / concentration:
4
Control animals:
no
Details on dosing and sampling:
Blood was collected from the tail vein during the 1st, 2nd, 3rd, 4th, 5th and 6th h of exposure, as well as 3, 15, 30, 45 min and 1, 2, 3, 4, 5, 6 h after its termination
Urine samples were collected 18 h after the termination of exposure in metabolic cages.
Statistics:
The kinetic analysis of pseudocumene in blood was calculated on an open two-compartment model using Sigma Plot 4.0 (Jandel Corporation) for Windows. The Michaelis-Menten parameters (Km and Vmax) for pseudocumene metabolism were estimated by analyzing Lineweaver-Burk plots using Microsoft Excel 5.0.
Details on absorption:
The absorption half life of pseudocumene in blood increased with increasing exposure concentration (38, 68 & 101 minutes at 25, 100 & 250ppm respectively)
Details on excretion:
The terminal half life for elimination of pseudocumene from blood increased with increasing exposure concentration (3.8, 5.3 & 17.3h at 25, 100 & 250ppm respectively)
Metabolites identified:
yes
Details on metabolites:
3,4-dimethylbenzoic acid (3,4-DMBA), 2,4-dimethylbenzoic acid (2,4-DMBA) and 2,5-dimethylbenzoic acid (2,5-DMBA) were measured in urine by GC after hydrolysis. 3,4-DMBA was present at the highest concentration.
Conclusions:
Interpretation of results (migrated information): other:
The rates of uptake and elimination of pseudocumene from blood were dependant on the exposure concentration. All 3 isomers of DMBA were detected in urine, 3,4-DMBA was present at the highest concentration. A significant linear correlation was found between the level of exposure and the concentration of dimethylbenzoic acids. The enzyme kinetic parameters [Km (mg/l) & Vmax (mg/h/kg)] for pseudocumene biotransformation in rats were estimated (3,4-DMBA, Km = 28, Vmax = 96; 2,4-DMBA, Km = 7, Vmax = 25; 2,5-DMBA, Km = 7, Vmax = 23).
Executive summary:

Both the absorption half life and terminal elimination half life of pseudocumene from blood increased with increasing exposure concentration. The three isomers of dimethylbenzoic acid (3,4 -, 2,4- & 2,5 -) were all detected in urine following hydrolysis; the 3,4 - isomer was present at the highest concentration.

Endpoint:
basic toxicokinetics in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, non-guideline animal experimental study, published in peer reviewed literature, no restrictions, fully adequate for assessment.
Objective of study:
toxicokinetics
Qualifier:
no guideline followed
Principles of method if other than guideline:
Dynamic inhalation chamber study in rats. Tissue concentrations of pseudocumene were determined.
GLP compliance:
no
Radiolabelling:
no
Species:
rat
Strain:
other: Wistar IMP:DAK
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
- Weight at study initiation: 152-421 g
No further details reported
ENVIRONMENTAL CONDITIONS
- Temperature: 20-23°C
- Humidity: 45-60%
Route of administration:
inhalation: vapour
Vehicle:
unchanged (no vehicle)
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body (assumed)
GENERATION OF TEST ATMOSPHERE / CHAMPER DESCRIPTION
- Exposure apparatus: Dynamic inhalation chambers (volume, 250 dm3)
- System of generation: Pseudocumene vapours were generated by heating liquid solvents in a washer. The desired concentrations of vapours were obtained by diluting them with the air.
ANALYSIS: Concentrations of solvent vapours in the exposure chamber were measured every 30 min by GC-FID.
Duration and frequency of treatment / exposure:
Single 6h or repeat 6h/day, 5 days/week for 4 weeks
Remarks:
Doses / Concentrations:
25, 100 and 250 ppm (nominal)
No. of animals per sex per dose / concentration:
4
Control animals:
no
Details on dosing and sampling:
Arterial blood, brain, liver and lung samples were obtained after decapitation. Venous blood samples (tail vein) were collected 3, 15, 30 and 45 min and 1, 2, 3, 4, 5 and 6 h after termination of exposure to pseudocumene vapours. The brain was homogenized or divided into anatomical structures: brainstem, hippocampus, temporal cortex and cerebellum. The liver and lung were also homogenized. Pseudocumene was quantitatively assessed In about 100 mg of each brain structure or organ homogenate. In the blood and tissues, pseudocueme concentrations were estimated by gas chromatography combined with the headspace technique.
Statistics:
The kinetic analysis of pseudocumene in blood was calculated on an open two-compartment model using SigmaPlot 4.0 (Jandel Corporation) for Windows. The differences in pseudocumene concentrations between brain structures and tissues were estimated by employing one-way analysis of variance (ANOVA).
Details on distribution in tissues:
Pseudocumene concentrations in brain and liver after both single and repeated exposures were similar to the maximum values in venous blood. Concentrations in the liver were significantly lower after repeated than after a single exposure indicating possible induction of metabolising enzymes. After repeated exposure, concentrations in liver, lungs and brain were similar. Higher concentrations in brainstem than in other regions of the brain may be associated with higher fat levels.
Details on excretion:
The terminal half-lives of pseudocumene in blood after termination of repeated exposure at 25, 100 and 250 ppm were 2 h 53 min, 5 h 47 min and 9 h 54 min respectively.


Executive summary:

Pseudocumene concentrations in brain and liver after both single and repeated exposures were similar to the maximum values in venous blood. Concentrations in the liver were significantly lower after repeated than after a single exposure indicating possible induction of metabolising enzymes. After repeated exposure, concentrations in liver, lungs and brain were similar. Higher concentrations in brainstem than in other regions of the brain may be associated with higher fat levels. The terminal half-lives of pseudocumene in blood after termination of repeated exposure at increased with increasing exposure concentration.

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, non-guideline in-vitro experimental study, published in peer reviewed literature, minor restrictions in reporting but otherwise adequate for assessment.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The permeation of the test substances through human skin was determined using static Franz diffusion cells.
GLP compliance:
not specified
Radiolabelling:
no
Species:
human
Sex:
not specified
Details on test animals or test system and environmental conditions:
Human skin from one donor obtained from a local hospital and stored for approximately 1 week at -20C.
Type of coverage:
occlusive
Vehicle:
other: Neat TMB, 10 or 50% TMB in ethanol
Duration of exposure:
8h
Doses:
128uL/0.64cm3
Absorption in different matrices:
Steady state status had not be attained after 8h exposure.
64ug penetrated untreated skin from neat 1,2,4-TMB, 77ug penetrated from a 50% ethanolic solution of 1,2,4-TMB.
The presence of skin barrier cream enhanced penetration of 1,2,4-TMB from ethanolic solutions.
1,2,4-TMB was detected in the receptor fluid within 30min of application to glove material.
Dose:
128ul/0.64cm2
Parameter:
percentage
Absorption:
ca. 0.1 %
Remarks on result:
other: 8h
Remarks:
Value calculated by reviewer = 0.057% (not reported in original publication).
Executive summary:

Steady state status had not be attained after 8h exposure.

64 ug (0.057 %) penetrated human skin when 1,2,4-TMB was applied at a rate of 128uL/0.64 cm2.

Description of key information

Short description of key information on bioaccumulation potential result:

Available data indicate that the metabolism of the trimethylbenzene isomers is comparable and therefore this summary includes reference to data on 1,2,4 -trimethylbenzene as well as 1,3,5 -trimethylbenzene.

Short description of key information on absorption rate:

Dermal absorption of 1,2,4-TMB is low at 0.057 %

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
0.06
Absorption rate - inhalation (%):
64

Additional information

Available data indicate that the metabolism of the trimethylbenzene isomers is comparable and therefore this summary includes reference to data on 1,2,4 -trimethylbenzene as well as 1,3,5 -trimethylbenzene.

Non-human information

Absorption

When male rats were exposed by inhalation to 25, 100 or 250 ppm 1,2,4-trimethylbenzene for 6 hours, concentrations of 1,2,4-trimethylbenzene in blood increased rapidly reaching a plateau before the end of the exposure period. Absorption half lives increased with dose from 38 minutes at 25ppm to 101 minutes at 250ppm (Swiercz et al., 2002).

Distribution

After repeated exposures (6 hour/day, 5 day/week for 4 weeks), concentrations of 1,2,4-trimethylbenzene in liver, lung and brain were similar and increased with atmospheric concentration of 1,2,4-trimethylbenzene. Concentrations of 1,2,4-trimethylbenzene in liver were significantly lower following repeated exposures than after a single exposure; this may indicate induction of metabolising enzymes (Swiercz et al., 2003).

Metabolism

Following exposure by inhalation to 1,2,4-trimethylbenzene, 3,4-, 2,4-, and 2,5-dimethylbenzoic acids (3,4-DMBA, 2,4-DMBA and 2,5-DMBA) were all present in blood; concentrations of 3,4-DMBA were greatest. There was an approximately linear relationship between the concentration of each metabolite in blood and the atmospheric concentration of 1,2,4-trimethylbenzene. The relative amounts of each DMBA isomer present in blood is dependent on the metabolic rate constants; those for 3,4-DMBA (Vmax 96mg/h/kg; Km 28mg/L) being greater than for the other isomers (Vmax 25mg/h/kg; Km 7mg/L) (Swiercz et al., 2003). Repeated inhalation exposure to high vapour concentrations of 1,2,4-trimethylbenzene (5,000 mg/m3, 8 hr/d for 3 days) appeared to result in increased metabolism (and clearance) of 1,2,4-trimethylbenzenefrom blood and brain (McKee et al., 2010).

Excretion

Blood concentrations of 1,2,4-trimethylbenzene showed a biphasic decline following exposure by inhalation to 1,2,4-trimethylbenzene for 6 hours. Terminal half lives increased with dose from 217 minutes at 25ppm to 1140 minutes at 250ppm (Swiercz et al., 2002).  After repeated exposures (6 hour/day, 5 day/week for 4 weeks), terminal elimination half lives again increased with atmospheric concentration of 1,2,4-trimethylbenzene from 173 minutes at 25 ppm to 594 minutes at 250ppm but were shorter than after a single exposure indicating possible induction of metabolising enzymes (Swiercz et al., 2003).

Human information

When human volunteers were exposed to 2 or 25 ppm 1,2,4-trimethylbenzene by inhalation for 2 hours, the respiratory uptake was approximately 64% and was independent of atmospheric concentration of 1,2,4-trimethylbenzene. Concentration of 1,2,4-trimethylbenzene in arterial blood increased rapidly without reaching any plateau within the exposure period. Approximately 20% of the respiratory uptake of 1,2,4-TMB was exhaled unchanged during and after exposure while less than 0.002% was excreted unchanged in urine within 4 hours post-exposure. The kinetics of 1,2,4-trimethylbenzene were characterised by a large volume of distribution and long terminal elimination half life (Jarnberg et al., 1996). 

Following a 4 hour exposure by inhalation (5-150 mg/m3), the retention of 1,2,4-trimethylbenzene in the lungs was 68%. Elimination of 1,2,4-trimethylbenzene from capillary blood following exposure at 150 mg/m3showed a triphasic decline with a terminal life of approximately 44 hours. The elimination of DMBA isomers from urine following exposure at 150 mg/m3was biphasic with a terminal half life of approximately 63 hours for all isomers (Kostrzewski et al., 1997).

Linear relationships were found between the air concentrations of the read-across substance 1,3,5-trimethylbenzene and the concentration of unchanged 1,3,5-trimethylbenzene and its metabolite 3,5-dimethylbenzoic acid as well as the unchanged substance in blood (Janasik et al., 2008).

Comparative metabolism and uptake of trimethylbenzene isomers

Both Jarnberg et al. (1996) and Kostrzewski et al. (1997) studied the toxicokinetics of the three isomers of trimethylbenzene in human volunteers. Respiratory uptake was similar; 64±3, 56±4 and 62±3% for 1.2,4, 1,2,3 and 1,3,5-trimethylbenzene respectively. The percentage of the respiratory uptake exhaled unchanged was similar for 1,2,4 and 1,3,5-trimethylbenzene (20±3 and 25±6% respectively) but was higher for 1,2,3-trimethylbenzene (37±9%); in all cases the amount excreted unchanged in urine was negligible. The three isomers were all metabolised to isomers of dimethylbenzoic acid; the number of isomers formed depending on the number of unique substitution patterns available.

Discussion on absorption rate:

The in-vitro percutaneous absorption of 1,2,4 -trimethylbenzene has been determined after applying a dose of 128 µL 1,2,4 -trimethylbenzene to occluded skin from a human donor (exposed area = 0.64 cm2).  Steady state conditions were not attained within 8 hours; the amount absorbed was 64 µg, equivalent to 0.057% of the applied dose (Korinth et al., 2003).