1,2,4-trimethylbenzene

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

publication

Title:

Inhalation toxicity of high flash aromatic naphtha

Author:

Clark DG, Butterworth ST, Martin JG, Roderick HR and Bird MG

Year:

1989

Bibliographic source:

Toxicol. Ind. Health Vol 5, No. 3, pp415-428

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

yes

Remarks:

No ophthalmology, no detailed clinical observations, bodyweights weekly only for initial 4 weeks, no food consumption and efficiency, haematology only for controls and high dose groups, no clinical chemistry at 3 months, limited number of organs weighed.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

High flash aromatic naphtha (ASTMD-3734)

IUPAC Name:

High flash aromatic naphtha (ASTMD-3734)

Constituent 2

Reference substance name:

50/50 blended mixture of SHELLSOL A and SOLVESSO 100

IUPAC Name:

50/50 blended mixture of SHELLSOL A and SOLVESSO 100

Details on test material:

- Name of test material (as cited in study report): High flash aromatic naphtha.
- Components: The test substance was a 50:50 blend of SHELLSOL A and SOLVESSO 100. Analysis by capillary gas chromatography showed the following major components:
0.46 % non-aromatics
2.27 % o-Xylene
4.05 % n-Propylbenzene
7.14 % 1-Methyl-3-ethylbenzene
16.60% 1-Methyl-4-ethylbenzene
9.35 % 1,3,5-Trimethylbenzene
7.22 % 1-Methyl-2-ethylbenzene
32.7 % 1,2,4-Trimethylbenzene
2.76 % 1,2,3-Trimethylbenzene
6.54% 1-Methyl-3-n-propylbenzene + 1,2-Diethylbenzene
1.77 % 1-Ethyl-3,5-dimethylbenzene

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shell Toxicology Laboratory (Tunstall) Breeding Unit
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 150-300 g
- Fasting period before study: No
- Housing: Singly housed in the exposure chambers in hanging aluminium cages with stainless steel mesh bases during and between exposures. Recovery animals (after 12 months exposure) transferred to an adjacent animal room.
- Diet: ad libitum except during exposure
- Water: ad libitum

ENVIRONMENTAL CONDITIONS: Temperature and relative humidity constantly monitored (no results reported); 6 hours light / 18 hour dark cycle, air flow 3-6 m3/min.

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel with doors fitted with glass windows and a volume of at least 8 m3
- Method of holding animals in test chamber: individually housed in a hanging aluminium cage within the chamber.
- Source and rate of air: Laboratory air at a rate of 3-6 m3/min.
- Method of conditioning air: Filtered.
- Temperature, humidity, pressure in air chamber: Not reported.
- Atmosphere generation: Test atmospheres were generated by completely evaporating the solvent into part of the ventilating air entering each chamber using micrometering pumps and vaporizers.

TEST ATMOSPHERE
- Brief description of analytical method used: Total hydrocarbon analyzer for 10 minutes at intervals of 40 minutes throughout the exposure periods. Each test atmosphere was also analyzed consecutively for a period of two hours during each exposure period using a gas chromatograph fitted with a flame ionization detector.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No significant change in test substance composition over the period of the study. Atmospheric generation did not significantly change the composition of the solvent blend. The actual atmospheric concentrations expressed as the overall means of the daily atmosphere analyses were close to the
nominal concentrations Actual concentrations were 0, 470±29, 970±70 and 1830±130 mg/m3 for target concentrations of 0, 450, 900 and 1800 m3 respectively.

Duration of treatment / exposure:

12 months exposure

Frequency of treatment:

6h/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 450, 900, 1800 mg/m3
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 470±29, 970±70, 1830±130 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

50. 10/sex/group killed after 6 months exposure, 25/sex/group killed after 12 months exposure, 15/sex/group (recovery) killed 4 months after cessation of exposure.

Control animals:

other: yes, exposed to air only

Details on study design:

- Dose selection rationale: Based on results from an earlier 13 week inhalation study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice/day

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: prior to exposure, weekly for initial 4 weeks and monthly thereafter.

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Schedule / numbers: weeks 1, 2, 3, 4, 6, 8, 12, 20, 24, 28 and 32 (tail vein blood) from 10/sex controls and high dose groups; 6 months, 12 months and end of recovery period (cardiac blood) from 10/sex/group.
- Parameters examined: Erythrocyte count (RBC), mean cell volume (MCV), haemoglobin concentration (Hb), leucocyte count (WBC), mean corpuscular haemoglobin (MCHC), haematocrit (Hct). Reticulocytes, differential leukocytes, prothrombin time, kaolin cephalin time and erythrocyte osmotic fragility (Fragilograph) were only measured using the cardiac blood samples.

CLINICAL CHEMISTRY: Yes
- Schedule / numbers: 6 months, 12 months and end of recovery period (cardiac and tail vein blood) from 10/sex/group.
- Parameters examined: Total protein, urea nitrogen, alkaline phosphatase (AP), chloride, bilirubin, calcium, inorganic phosphate, uric acid, sodium, potassium, alanine amino transferase (ALT), aspartate amino transferase (AST), glucose and protein electrophoresis. (The six-month bloods were limited to measurement of protein, urea, AP, ALT, AST, chloride, sodium, potassium and protein electrophoresis).

URINALYSIS: Yes
- Schedule / numbers: prior to exposure, after 3, 6, 9 and 12 months' exposure and three months after exposure ended. Individual samples collected over 4 hours from 12/sex/group.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: Glucose, protein, ketones, bilirubin, blood pigments, pH, nitrite and urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes - all animals.

ORGAN WEIGHTS: Yes - all animals surviving to the scheduled necropsies (liver, kidneys, spleen, brain, heart and testes).

HISTOPATHOLOGY: Yes - all animals except recovery groups were examined (salivary gland, stomach, heart, brain, spinal cord, pituitary gland, lungs, prostate, seminal vesicles, testes, ovaries, uterus, skeletal muscle, adrenal glands, thyroids with oesophagus and trachea, caecum, spleen, thymus, lymph node, mammary gland, small and large intestine, pancreas, liver, kidneys, urinary bladder, eye and lachrymal glands, nasal cavity, spinal cord, tongue, knee joint and femur, sciatic and posterior tibial nerves and any gross lesion).

Statistics:

Body weights, organ weights: covariance analysis using initial body weight as the covariate. Means adjusted for initial body weight if a significant covariance relationship found, otherwise unadjusted means reported. To adjust for differences in terminal body weight, organ weights were also considered with terminal body weight as a covariate.
Haematology and clinical chemistry: analysis of variance using Williams’ ‘t’ test. If monotonic dose response could not be assumed, Dunnett's test used.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY: 2 male and 1 female control and 2 males at 450 mg/m3 died during study. 7 animals were removed from the study during the 12-month exposure period, and a total of 30 rats was removed during the recovery period but none were related to exposure. A range of minor clinical signs was observed. None of the signs were treatment-related except for a possible increase in male "aggression" at the high exposure, when the animals were slightly more difficult to handle. Some "aggression" was also noted in three or four rats during the recovery phase.

BODY WEIGHT AND WEIGHT GAIN: Initial reduction in body weight gain occurred in both male and female rats at the higher exposures (1800 mg/m3 males, weeks 1-4 -2% compared to controls; 900 mg/m3 females, weeks 1-4 -2% and 1800 mg/m3 females, weeks 1-12 -3%). Thereafter, there were no differences between control and experimental animals for the duration of the study.

HAEMATOLOGY: Various statistically significant changes were transiently seen in males up to six months, but were considered not to be biologically significant.

CLINICAL CHEMISTRY: Various statistically significant changes were probably not of biological significance, since they were within the range of values obtained in normal rats of this strain and age and they were not supported by histopathological or any other evidence of organ damage.

ORGAN WEIGHTS: 1800 mg/m3 exposure male liver and kidney weights were increased at 6 and 12 months but, in the absence of histopathological changes, were considered to be physiological adaptive responses.

HISTOPATHOLOGY: NON-NEOPLASTIC: Histopathological examination of the animals revealed a variety of changes, none of which could be attributed to exposure.

HISTOPATHOLOGY: NEOPLASTIC (if applicable): Only one tumor was found in the six-month exposed animals: a malignant mammary adenocarcinoma in a high-exposure female. After 12 months' exposure, several tumours were found. Pituitary adenomas in females were most common, several being found in each group. Of the remaining tumours, one high-exposure female had a leiomyoma on the left uterine horn, one low-exposure male had a malignant glioblastoma under the left lobe of the cerebellum, and one high-exposure male had a malignant lymphoma of the spleen. The tumours were mostly of a type frequently found in this strain and age of rat and were considered not to be treatment-related.

Effect levels

Dose descriptor:

NOAEC

Effect level:

1 800 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: no significant systemic toxicity following 12 months exposure in this study

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Chronic exposure to this high aromatic naphtha (a 50:50 blend of SHELLSOL A and SOLVESSO 100, which contains 9.35% 1,3,5-trimethylbenzene) was without systemic toxicity in rats under the conditions of this study.

Executive summary:

Rats were exposed to a 50:50 blend of SHELLSOL A and SOLVESSO 100 at exposure concentrations of 0, 450, 900 and 1800 mg/m3 for 12 months. Initial reduction in body weight gain occurred in both male and female rats at the higher exposures. Various statistically significant haematological changes were transiently seen in males up to six months, but were not considered biologically significant. High exposure male liver and kidney weights were increased at 6 and 12 months but, in the absence of histopathological changes, were considered to be physiological adaptive responses. No treatment-related histopathological abnormalities were found. It is concluded that chronic exposure to this high aromatic naphtha is without systemic toxicity in rats under the conditions of this study with a NOAEC of 1800 mg/m³.