Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Near guideline, GLP animal experimental study, available as published report, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
Cited as Directive 87/302/EEC, part B, p. 8
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Toluene was obtained in one lot (lot no. H-12-19-80) from Exxon Company, USA (Baytown, TX, USA) as a clear, colourless liquid. Purity was greater than 99% as determined by elemental analysis, Karl Fischer water analysis and gas chromatography (GC).

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI., USA
- Age at study initiation: approx. 6-7 weeks
- Housing: 5 per cage in polycarbonate cages with hardwood bedding
- Diet: IH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA) ad libitum
- Water: ad libitum
- Acclimation period: 18days

ENVIRONMENTAL CONDITIONS:
- Temperature: mean temperature 72.4°F (~22.4°C), range 63-82°F (~17-28°C)
- Humidity: mean humidity 59.8%, range 44-82%
- Air changes (per hr): not reported
- Photoperiod: 12hrs dark / 12hrs light

IN-LIFE DATES: Not reported

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Toluene was dissolved in corn oil. Solutions at 20 mg/kg were stable for at least 2 weeks at room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 31.2, 62.5, 126, 250 and 500 mg/mL
- Amount of vehicle (if gavage): dose volume of 10 mL/kg
- Lot/batch no. (if required): no data
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose mixtures were analysed several times during the 13-week studies and found to be within 10% of the target concentrations
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily, 5 days/ week
Doses / concentrations
Remarks:
Doses / Concentrations:
312, 625, 1250, 2500, 5000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
Post-exposure period: none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (for dead and moribund animals)
- Time schedule: twice/day

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: beginning of the study and weekly

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination
- Anaesthetic used for blood collection: no data
- Animals fasted: yes
- How many animals: all
- Parameters examined: eosinophils, haematocrit, haemoglobin, lymphocytes, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean cell volume, methaemoglobin, monocytes, platelets, erythrocytes, reticulocytes, segmented neutrophils, leukocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination
- Animals fasted: yes
- How many animals: all
- Parameters examined: blood urea nitrogen (BUN), albumin, calcium, lactate dehydrogenase, inorganic phosphorus, total protein.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight prior to termination
- Animals fasted: yes
- How many animals: all
- Metabolism cage used for collection: yes
- Parameters examined: No data
Sacrifice and pathology:
Necropsy was performed on all animals.
Tissues examined in control, 2500 and 5000 mg/kg/day groups: adrenal glands, aorta, brain, caecum, colon, duodenum, oesophagus, heart, ileum, jejunum, kidneys, liver, lungs, and bronchi, mammary gland, mesenteric lymph nodes, nasal cavity and turbinates, pancreas, parathyroid glands, pituitary gland, preputial or clitoral glands, prostate/testes or ovaries/uterus, rectum, salivary glands, spinal cord, spleen, sternebrae, including marrow, stomach, thymus, thyroid gland, tissue masses, trachea, urinary bladder and gross lesions. Tissues examined in other groups were: brain, kidneys, liver, and urinary bladder.

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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
All rats in the 5000 mg/kg group died within the first week. In the 2500 mg/kg group 8 males and one female died before termination of the study. Clinical signs included prostration, hypoactivity, ataxia, piloerection, lachrymation, and excessive salivation in the 5000 and 2500 mg/kg groups.

BODY WEIGHT AND WEIGHT GAIN
The final mean body weight of males that received 2500 mg/kg was 19% lower than that of vehicle controls.

HAEMATOLOGY, CLINICAL CHEMISTRY AND URINALYSIS
A there were no treatment related effects in the haematological and serum chemical analyses or urinalyses.

ORGAN WEIGHTS
Absolute and relative kidney weight was increased in males at 625 mg/kg and higher, and in females at 1,250 mg/kg and higher. The differences in absolute and relative liver weights for female rats that received 2500 or 1250 mg/kg (22-62% relative liver weight increase) and for males that received 1250 or 625 mg/kg (8-78% relative liver weight increase) were statistically significant. Statistically significant increases were also seen in absolute and relative heart weight for male and female rats at 2500 and females at 1250 mg/kg. Absolute, but not relative brain weight was reduced in both sexes at 2500 mg/kg.

HISTOPATHOLOGY
Neuropathological changes in the brain, consisting of neuronal cell necrosis in the dentate gyrus and Ammons horn of the hippocampus, was seen in male and female rats that received 2500 or 1250 mg/kg. In addition to the hippocampal lesions, necrosis and/or mineralisation were present in the granular layer of the cerebellar cortex. Haemorrhage was present in the mucosa, submucosa, or muscularis of the urinary bladder of males and females of the two highest dose groups. A dose of 312 mg/kg/kg did not cause any effects.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
625 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: neuropathology (neuronal cell necrosis in hippocampus)
Dose descriptor:
LOAEL
Effect level:
1 250 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: adverse clinical signs, differences in brain, liver, kidney and heart weight and neuropathology (neuronal cell necrosis in hippocampus).

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL considered to be 625 mg/kg/day. Increased relative weights of liver and kidney are interpreted as toxicologically insignificant differences in liver and kidney weight unaccompanied by histological findings.
Executive summary:

Groups of 10 male and 10 female F344N rats received 0, 312, 625, 1250, 2500, or 5000 mg toluene/kg in corn oil by gavage for 13 weeks. All rats in the 5000 mg/kg group died within the first week. In the 2500 mg/kg group 8 males and one female died before termination of the study. The final mean body weight of males that received 2500 mg/kg was 19% lower than that of vehicle controls. Clinical signs included prostration, hypoactivity, ataxia, piloerection, lachrymation, and excessive salivation in the 5000 and 2500 mg/kg groups. Absolute and relative kidney weight was increased in males at 625 mg/kg and higher, and in females at 1,250 mg/kg and higher.

The differences in absolute and relative liver weights for female rats that received 2500 or 1250 mg/kg (22-62% relative liver weight increase) and for males that received 1250 or 625 mg/kg (8-78% relative liver weight increase) were statistically significant. Statistically significant increases were also seen in absolute and relative heart weight for male and female rats at 2500 and females at 1250 mg/kg. Absolute, but not relative brain weight was reduced in both sexes at 2500 mg/kg. There were no treatment related effects in the haematological and serum chemical analyses or urinalyses. Neuropathological changes in the brain, consisting of neuronal cell necrosis in the dentate gyrus and Ammons horn of the hippocampus, was seen in male and female rats that received 2500 or 1250 mg/kg. In addition to the hippocampal lesions, necrosis and/or mineralisation were present in the granular layer of the cerebellar cortex. Haemorrhage was present in the mucosa, submucosa, or muscularis of the urinary bladder of males and females of the two highest dose groups. A dose of 312 mg/kg/kg did not cause any effects.

The NOAEL for repeat dose oral toxicity is considered to be 625 mg/kg. At this dose increased absolute and relative liver and kidney weight (up to 46% increase in relative kidney weight, up to 78% increase in relative liver weight) were unaccompanied by histopathological findings were present. Therefore, the increased relative weights of liver and kidney are interpreted as toxicologically non-significant signs of metabolic activity related to exposure.