Mesitylene

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Dates not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP, near guideline study, published in peer reviewed literature, minor limitations in design and/or reporting but otherwise adequate for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2000

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

95-65-6

IUPAC Name:

95-65-6

Constituent 2

Reference substance name:

Reference substance 001

Cas Number:

95-63-6

Molecular formula:

C9-H12

Constituent 3

Reference substance name:

pseudocumene

IUPAC Name:

pseudocumene

Details on test material:

1,2,4-trimethylbenzene (pseudocumene) (>97% purity) was supplied by Fluka.

Test animals

Species:

rat

Strain:

other: outbred Imp.WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Nofer Institute of Occupational Medicine, Lodz, Poland
- Age at study initiation: 2-2.5 months old
- Weight at study initiation: 213 ± 20g (males), 160 ± 11g (females)
- Fasting period before study: not applicable
- Housing: polypropylene cages with wire mesh covers, 5 per cage
- Diet : Murigan pelleted rodent chow (Fodder Plant, Motycz, Poland) ad libitum except during exposures.
- Water : ad libitum except during exposures. no other details.
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5-22.5
- Humidity (%): 50-65
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: To: not reported

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: Air

Remarks on MMAD:

MMAD / GSD: not applicable (vapour)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: dynamic inhalation chamber (1.3 m³ volume)
- Method of holding animals in test chamber: not reported
- Source and rate of air: not reported
- Method of conditioning air: not reported
- System of generating vapours: liquid solvent was heated in washers and diluted in air to obtain desired concentrations.
- Temperature, humidity, pressure in air chamber: not reported
- Air flow rate: not reported
- Air change rate: 16 changes per hour
- Method of particle size determination: not applicable
- Treatment of exhaust air: not reported


TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatograph
- Samples taken from breathing zone: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of solvent vapours in the exposure chamber were measured every 30 mins by a Hewlett-Packard gas chromatograph with a flame ionisation detector, using 40 m capillary HP-5 column at 100°C column temperature.

Duration of treatment / exposure:

6 hours/day.

Frequency of treatment:

5 days/week for 3 months.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 25, 100 and 250 ppm
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 123, 492 & 1230 mg/m3
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0, 129±18 , 492±62 & 1207±76 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

10 males and 10 females per group, plus an additional 10 males and 10 females at the high dose group which were maintained untreated for 2 weeks after the final exposure.

Details on study design:

- Dose selection rationale: The authors report that the Polish MAC of 100mg/m3 for trimethylbenzene isomers has been established using incomplete, unreliable and obsolete data. A pseudocumene concentration of 2844 mg/m3 causes respiratory depression in mice (RD50) and may indicate that a lower MAC is needed. There is limited toxicity data on pseudocumeme, which is used extensively, and this study was undertaken to assess the sub-chronic toxicity effects of pseudocumene in rats.
- Rationale for animal assignment (if not random): random assignment
- Rationale for selecting satellite groups: not reported
- Post-exposure recovery period in satellite groups:2 weeks
- Section schedule rationale (if not random): random assignment

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: prior to first exposure and then weekly

FOOD CONSUMPTION:
- Food consumption determined : Yes
- Time scheduleor examinations: weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: Not examined

OPHTHALMOSCOPIC EXAMINATION: Not examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: pre-study and 1 week prior to termination
- Anaesthetic used for blood collection: No data (tail vein)
- Animals fasted: No data
- How many animals: not reported
- Parameters examined: erythrocyte count, haemoglobin concentration, haematocrit, leucocyte count, platelet count and clotting times..

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 18 hours after final exposure
- Anaesthetic used for blood collection: Yes (light ether)
- Animals fasted: Yes
- How many animals: not reported
- Parameters examined: aspartate aminotransferase, alanine aminotransferase, alkaline amnotransferase, sorbitol dehydrogenase, gamma gutamyltransferase, bilirubin, total cholesterol, glucose, total protein, albumin, creatinine, urea, calcium, phophorous, sodium, potassium, chloride.


URINALYSIS: Not examined

NEUROBEHAVIOURAL EXAMINATION: Not examined

OTHER: No data

Sacrifice and pathology:

ORGAN WEIGHTS: Yes
lungs, liver, spleen, kidneys, adrenals, heart and gonads (ovaries, testes).

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
brain, nose, larynx, trachea, thymus, lungs, heart, liver, spleen, kidney, adrenals, thyroid gland, pancreas, gonads, urinary bladder, stomach, duodenum, small and large intestines, and salivary glands were preserved in neutral formalin. The lungs, removed in toto, were perfused with 10 neutral
buffered formalin via trachea at a pressure of 20 cm H2O. The tissue were embedded in paraffin-wax, sectioned at 5um, and stained with hematoxylin and eosin.
The changes in lungs reflecting the degree of proliferation of peribronchial lymphatic tissue, lymphoepitelium in bronchi mucosa, interstitial lymphocytic infiltration, macrophage infiltration and inflammatory processes,were graded using an arbitrary scale of 0-3 or 0-4 (0 = normal status,
1 = minimal, 2 = mild, 3 = moderate, and 4 = marked).

Statistics:

Except for bodyweight data, other parameters from the treated groups were compared statistically with the control group using the following statistical tests: Bartlett's test of homogeneity of variance was used to assess whether the groups had equivalent variances at the p<0.001 level. if the variances were not significantly different, the groups were analysed by a standard one-way Analysis of Variance (ANOVA). If significant differences among the means were indicated, Dunnett's test was used to determine which treatment groups differed from controls. If the groups did not have equivalent varainces at the p<0.001 level, then the Kruskal-Wallis test was used to assess differences in group means. If means were different, Dunn's summed rank test was used to determine which treatment group differed significantly from controls.
Body weight data for each sex were analysed by one-way analysis of co-variance (ANCOVA) using pre-exposure (day 0) weights as the co-variate.
Histopathological data was compared using the Fisher's exact test. Graduated morphological changes in lungs were analysed by the Kruskall-Wallis test in dunn's modification. The trend analysis was used to evaluate the relation between the extent of changes in haematological and clinical chemistry parameters, morphological changes in lungs and exposure level.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no deaths during the course of the study and no toxicologically significant clinical observations.

BODY WEIGHT AND WEIGHT GAIN
No effects

FOOD CONSUMPTION
No effects

FOOD EFFICIENCY
Not measured

WATER CONSUMPTION
Not measured

OPHTHALMOSCOPIC EXAMINATION
Not examined

HAEMATOLOGY
At the end of the exposure period, statistically significant differences from control included: Decreased red blood cells and increased white blood cells in males at 1230 mg/m³, decreased reticulocyte counts in females at 1230 mg/m³, decreased clotting times in females exposed to 492 and 1230 mg/m³. Two weeks after the end of exposure, white blood cell count had decreased , reticulocyte count and clotting times showed increases. However, red blood cells remained at similar levels to those at the end of exposure.

CLINICAL CHEMISTRY
At the end of the exposure period, statistically significant differences from control included: increased sorbitol dehydrogenase in males in all exposed groups. The slight increases in sorbitol dehydrogenase activity (males only) and blood chemistry changes were considered to be related to respiratory irritation

ORGAN WEIGHTS
No effects

GROSS PATHOLOGY
No effects reported

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no significant histopathological changes in the upper respiratory tract. However, in rats exposed to 492 and 1230 mg/m³, there was an increased number of rats with peribronchial, lung parenchymal and perivascular lymphocytic infiltrations. There were no other significant histopathological changes detected.
In the other examined organs and tissues of all male and female rats exposed to pseudocumene, there were no significant changes which could be related to pseudocumene

Effect levels

Key result

Dose descriptor:

NOAEC

Remarks:

Systemic

Effect level:

1 230 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: No systemic toxicity. Slight increase in sorbitol dehydrogenase activity (males only), blood chemistry changes and lung pathology considered to be related to respiratory irritation

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. Effect of a 3-month exposure to pseudocumene on selected haematological and clinical chemistry parameters

	Pseudocumene (mg/m3)					Jonckheere'strend test
Parameters	Control	123	492	1230	1230°
Red blood cells (x106/mm3)
Male	9.98±1.689.	9.84±1.82	8.50±1.11	7.70**±1.38	7.61±1.6	p=0.0004
Female	8.22±1.16		8.51±1.13	7.71±1.58	6.99±1.8	p = 0.1891
White blood cells (x106/mm3)
Male	8.68±2.89	8.92±3.44	8.30±1.84	15.89**±5.74	7.11±2.1	p = 0.0019
Female	7.50±1.31	6.76±2.95	9.55±4.48	9.83±3.74	7.11±2.4	p = 0.0307
Reticulocyte (%)
Male	3.1±2.3	2.3±1.4	2.8±2.1	3.1±2.5	6.4±3.2	p = 0.4900
Female	3.5±2.6	1.7±2.0	1.8 ±0.9	1.0* ±0.6	5.8±3.6	p=0.0137
Clotting time (s)
Male	43±19	41±17	37±13	33±7	56±21	p = 0.1457
Female	30±10	23±4	19**±5	22*±7	48±19	p = 0.0034
Sorbitol dehydrogenase
Male	6.6±1.4	8.1**±0.8	7.8•±1.0	8.0**±1.1	-	p = 0.0083
Female	5.9±1.5	7.3±1.7	7.1±1.8	7.0±1.6	-	p = 0.0637

º Parallel group,14days after termination of exposure. Mean values ± SD for 10 rats

Statistically significant differenceas compared to the control  *p <0.05, ** p <0.01

Applicant's summary and conclusion

Conclusions:

Following 3 months exposure to vapours of 1,2,4-trimethylbenzene, 1230 mg/m3 was established as a NOAEL for systemic toxicity in rats. Other changes, were considered to represent inflammatory changes as a result of respiratory irritation.

Executive summary:

Inhalation exposure of rats to vapours of pseudocumene (1,2,4-trimethylbenzene) at concentrations of 123 mg/m3, 492 mg/m3and 1230 mg/m3 for 3 months resulted in low systemic toxicity. There were no changes in body weight gain, food consumption or organ weights. There were a number of effects considered to be related to inflammation arising from the respiratory irritative effects of pseudocumene exposure: an increase in peribronchial lung, parenchymnal and perivascular lymphocytic infiltration, at 492 and 1230 mg/m3. Some disturbances in haematological parameters, characterised by a decrease in red blood cells, decreased reticulocytes and slight increase in white blood cells at 1230 mg/m3 occurred and there was an increase in sorbitol dehydrogenase activity in males.