1,2,4-trimethylbenzene

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

neurobehavioural assessment not carried out

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Mesitylene

EC Number:

203-604-4

EC Name:

Mesitylene

Cas Number:

108-67-8

Molecular formula:

C9H12

IUPAC Name:

1,3,5-trimethylbenzene

Constituent 2

Reference substance name:

1,3,5-trimethylbenzene

IUPAC Name:

1,3,5-trimethylbenzene

Details on test material:

- Name of test material (as cited in study report): 1,3,5-trimethylbenzene
- Physical state: Clear, colourless liquid
- Analytical purity: 99.2%
-Impurities (identity and concentrations): pseudocumene at 0.7%
- Lot/batch No.: 4388-01
- Storage condition of test material: in original containers at room temperature

Test animals

Species:

rat

Strain:

other: Sprague-Dawley CD (Crl:CD®BR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI, USA
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: random sample of 10/sex, at approximately 6 weeks of age, weighed 133-158g
- Housing: Individually in stainless steel wire mesh cages (24.0 x 17.8 x 17.6 cm)
- Diet: Purina Certified Chow 5002 (PMI Feeds Inc., St. Louis, MO, USA) ad libitum (except for approximately 16-20 hours prior to scheduled blood collection and necropsy)
- Water: Reverse osmosis-purified water ad libitum
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23°C
- Humidity: 21-79%
- Air changes (per hr): Not reported
- Photoperiod: 12hrs dark / 12 hrs light

IN-LIFE DATES: From: 30 August 1994 To: 27 December 1994

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Stock dosing solutions of test substance in corn oil were prepared weekly during the study. The stock solutions were stored at room temperature and dosing aliquots were dispensed daily.

VEHICLE
- Concentration in vehicle: 10, 40 and 120 mg/mL for 50, 200 and 600 mg/kg/day dose groups, respectively.
- Amount of vehicle (if gavage): All animals were dosed at a constant dosing volume of 5 mL/kg of body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing formulations prepared for the first two weeks of dosing were analyzed for homogeneity and concentration at all dose levels. The low and high dose formulations prepared for the following three weeks of dosing were analyzed to confirm their concentration followed by biweekly concentration analysis of the low and high dose formulations for the remainder of the 13 week dosing period. Stability analyses were performed on low and high concentration solutions prepared for the first week of dosing. Stability was determined at 24 hours, I and 2 weeks after formulation.
Similar purity and composition percentages were found upon completion of the treatment phase of the study, indicating the test substance was stable during the period of its use. Low and high concentration solutions stored at room temperature were stable for at least 14 days. All preparations were found to be homogeneous. Overall mean analyzed concentrations were 98.9%, 96.9% and 100.8% of the target concentration for the 10, 40 and 120 mg/mL dosing solutions respectively. All individual sample concentrations were within 10% of the target concentrations. No test substance was detected in any vehicle control sample.

Duration of treatment / exposure:

90-91 days

Frequency of treatment:

once daily, 5 days per week (a total of 65-66 doses)

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 200 & 600 mg/kg bw/day
Basis:
other: nominal in corn oil

No. of animals per sex per dose:

10 (for controls, low and mid-dose groups), 20 for high dose group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Post-exposure recovery period in satellite groups: 29 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once/day for morbidity and mortality during acclimatisation, once or twice daily thereafter

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to treatment and weekly thereafter

BODY WEIGHT: Yes
- Time schedule: Immediately prior to first dose, weekly thereafter and Fasted weight immediately prior to termination.

FOOD CONSUMPTION:
- Time schedule: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to study initiation and during last week of dosing
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 30 days dosing and at termination
- Anaesthetic used for blood collection: Yes :70% CO2:30%O2 (prior to 30 day samples collected from the orbital sinus); sodium pentobarbitol (termination samples collected from abdominal aorta)
- Animals fasted: Yes
- How many animals: All
- Parameters examined: total erythrocyte count (RBC), haemoglobin (HGB), mean corpuscular volume (MCV), total white blood cell count (WBC), differential white blood cell count and platelet count (PLT). The following values were calculated from the data globulin (GLOB), albumin/globulin ratio (A/G Ratio), haematocrit (HCT), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 30 days dosing and at termination
- Anaesthetic used for blood collection: Yes :70% CO2:30%O2 (prior to 30 day samples collected from the orbital sinus); sodium pentobarbitol (termination samples collected from abdominal aorta)
- Animals fasted: Yes
- How many animals: All
- Parameters examined: glucose (GLU), gamma glutamyl transpeptidase (GGT), alanine aminotransferase (ALT), aspartate amino tranasferase (AST), alkaline phosphatase (ALK P), creatine kinase (CK), urea nitrogen (BUN), creatinine (CREA), sodium (Na), potassium (K), calcium (Ca), chloride (Cl), phosphorus (PHOS), total protein (T PRO), albumin (ALB), total bilirubin (MIL) and cholesterol (CHOL).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Full necropsy of all animals

ORGAN WEIGHTS: Yes. adrenal glands, brain, gonads, kidneys, liver and lungs of all rats were weighed at necropsy and the organ weights relative to body weights (fasted) were calculated.

HISTOPATHOLOGY: Yes. Following tissues from all animals collected and fixed: adrenals, brain, eyes, exorbital lachrymal glands, oesophagus, gonads, heart, duodenum, jejunum, ileum, caecum, colon, rectum, kidneys, liver, lungs, mesenteric lymph nodes, pancreas, aorta, pituitary, prostate, epididymides and seminal vesicles, salivary glands, sciatic nerve, skeletal muscle, skin (with mammary gland), spinal cord (cervical, mid-thoracic and lumbar), spleen, sternum (bone and marrow), stomach, thymus, thyroid (with parathyroids), trachea, urinary bladder, uterus, Zymbal glands, nasal turbinate, femur, gross lesions and ear. The complete set of collected tissues (except skin and mammary gland, ear, skeletal muscle, femur, spinal cord, eyes, exorbital lacrimal glands, Zymbal glands, epididymides, prostate and seminal vesicles) from all vehicle control and 600 mg/kg/day dose group animals sacrificed at the end of the 90-day dosing period was processed and examined by light microscopy. In addition, the lungs from the 50 and 200 mg/kg/day dose group animals were also processed and examined microscopically, along with gross lesions observed in all animals. The remaining tissues from the 50 and 200 mg/kg/day dose group animals were collected, but were not processed and examined microscopically. After the 28-day recovery period, a complete set of tissues was collected from all of the recovery 600 mg/kg/day animals; however, no tissues from any of these animals were examined microscopically.

Statistics:

Data collected electronically (i.e., body weights, body weight gains, food consumption, haematology) were analyzed using the statistical capabilities provided by the software, which consisted of analysis of variance (ANOVA) followed by Dunnett's multiple range comparison test. Other data (i.e, clinical chemistry, absolute and relative organ weights) were analyzed by ANOVA followed by Dunnett's multiple-range comparison using SYSTAT software (SYSTAT, SPSS, Inc, Chicago, IL, version 5.0). Analyses were performed on the two sexes independently. Analyses consisted of comparing the treated groups with the vehicle control at the 30-day interim and dose termination time points. Since no vehicle control animals were retained for recovery observation, statistical comparison of recovery animal parameters could not be performed. A minimum significance level of p ≥ 0.05 was used in all comparisons.

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

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: No test substance-related deaths occurred during the study. Clinical signs observed predominantly in the high dose animals consisted of discoloured inguinal fur, wet inguinal fur and salivation. All treatment related effects appeared to be reversible, since these effects were no longer apparent in the recovery animals at the end of the 28-day recovery period.

BODY WEIGHT AND WEIGHT GAIN: No statistically significant effects on body weight, body weight gain (except for a significant decrease in high dose males after 5 weeks of treatment) were observed; however, cumulative body weight gain was decreased approximately 11% in the high dose males. All treatment related effects appeared to be reversible, since these effects were no longer apparent in the recovery animals at the end of the 28-day recovery period.

FOOD CONSUMPTION: No statistically significant effects.

OPHTHALMOSCOPIC EXAMINATION: No treatment-related ophthalmic lesions were observed following the 90-day treatment period.

HAEMATOLOGY: No treatment-related changes.

CLINICAL CHEMISTRY: Increased phosphorus levels in high dose males and females. All treatment related effects appeared to be reversible, since these effects were no longer apparent in the recovery animals at the end of the 28-day recovery period.

ORGAN WEIGHTS: Absolute liver weight was significantly increased in high dose females and relative liver weights were significantly increased in high dose males and females at treatment termination. Relative kidney weight was also increased in high dose males at treatment termination.

GROSS PATHOLOGY: No treatment-related effects.

HISTOPATHOLOGY: No treatment related microscopic lesions were observed in the liver or kidneys of the high dose animals, or in any other tissue or organ of any animal.

Effect levels

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Increases in phosphorus concentrations at 600 mg/kg bw/day not evident after recovery period and not associated with any histopathological findings. Increased liver and kidney weights at 600 mg/kg/day attributed to a-2µ-globulin related nephropathy.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL following 90 days oral gavage of 1,3,5-trimethylbenzene to rats is concluded to be 600 mg/kg bw/day.

Executive summary:

1,3,5-Trimethylbenzene was administered at dose levels of 0, 50, 200 and 600 mg/kg/day to 10 male and 10 female Sprague Dawley CD rats for 90 days. An additional 10 rats/sex were dosed at 600 mg/kg/day group on the same dosing schedule but were subsequently retained without treatment for an additional 28 days to evaluate recovery from any toxic effects.  

In the 600 mg/kg/day group an increase in serum phosphorus levels (males and females), liver weight increase (absolute and relative weight increase in females; relative increase only in males) and kidney weight increase in males were reported. In the recovery group, no apparent effects were reported at the end of the 28-day recovery period.   

Based on the recovery group data, the effects reported at the highest dose were considered reversible and were attributed to adaptive response (enzyme induction) to the test substance. In the liver this could be attributed to microsomal enzymes induction while in the male rat kidney, to a-2µ-globulin related nephropathy.  With respect to the elevated serum phosphorus levels observed, these levels werenot statistically different at the end of the 28 -day recovery period and no associated histopathological effects were reported which could be correlated with the increased serum phosphorus levels.  The NOAEL for this study can therefore be concluded to be 600 mg/kg/day.