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Toxicological information

Repeated dose toxicity: oral

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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: GLP compliant, near guideline study, published as NIH publication, some limitations in design but fully adequate for evaluation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
no clinical chemistry analysis, no ophthalmological examination, no neurobehaviour, no organ weights.
GLP compliance:
yes
Remarks:
assumed - audit of 2 year data documented
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
>99.7% pure obtained from Burdick and Jackson Laboratories (Muskegon, Michigan) lot numbers AB223 and AB490.

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)
- Age at study initiation: 6 weeks
- Weight at study initiation: mean weights per group males 110-137 g ; females 75-101 g
- Housing: 5 per sex per cage in polycarbonate cages
- Diet: Purina Lab Chow 5001 - pellets (Ralston-Purina Co., St. Louis, NJ) ad libitum
- Water: ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22±1°C
- Humidity: 40-65%
- Air changes: 15 per h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: 14 October 1978 To: 13 February 1979

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: A weighed amount of benzene was mixed with the appropriate amount of corn oil and stirred for 5 minutes. Rats were dosed at a rate of 5 mL/kg. Benzene in corn oil was found to be stable at 25º C for at least 7 days. Dose mixtures were used within 2 weeks of preparation.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All benzene/corn oil mixtures analyzed by gas chromatography and were within ±10% of the target concentrations.
Duration of treatment / exposure:
120 days (17 weeks). Sub-group of animals from 0, 200, and 600 mg/kg groups killed after 60 days (8-9 weeks).
Frequency of treatment:
Once per day, 5 days/week.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 25, 50, 100, 200, 400, 600 mg/kg bw/day
Basis:
other: nominal in corn oil
No. of animals per sex per dose:
10/sex/group for 0, 25, 50, 100 and 400 mg/kg; 15/sex/group for 0, 200 and 600 mg/kg
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: none

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: once per week

FOOD CONSUMPTION: Yes
- Time schedule: once per week

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule / number of animals for collection of blood: animals killed at day 0 and day 60 and on 5 animals/group at terminal kill and on any animals killed in a moribund condition.
- Method of collection: from the orbital sinus
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- Parameters examined: haemoglobin, haematocrit, white blood cell count, red blood cell count, mean corpuscular volume, reticulocyte count, coagulation time.

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Examinations on all animals except those with excessive autolysis or cannibalized.

HISTOPATHOLOGY: Yes. The following tissues were examined histologically in the predosing vehicle control, study vehicle control, and interim-kill animals and the 600 mg/kg animals at terminal kill: mandibular lymph node, salivary glands, femur, thyroid gland, parathyroid, small intestine, colon, liver, prostate/testes or ovaries/uterus, lungs and mainstem bronchi, mammary gland, heart, oesophagus, stomach, brain, thymus, trachea, pancreas, spleen, kidneys, adrenal glands, urinary bladder, pituitary gland; in addition, spleens were examined in all dose groups. Special histology studies performed on animals killed at days 0 and 60 and on 5 animals/group at 0, 200, and 600 mg/kg at terminal kill and on any animals killed in a moribund condition.
Statistics:
Tests of significance included pairwise comparisons of high dose and low dose groups with vehicle controls and tests for overall dose-response trends. Haematology data was initially screened for outliers and as the same animals were examined across time, a repeated measures analysis of variance (Winer, 1971) method was used to investigate temporal and dose-related variation.

Results and discussion

Results of examinations

Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
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:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
BODY WEIGHT AND WEIGHT GAIN: Final mean bodyweights, relative to vehicle controls, were depressed: 4, 2, 7, 14, 20 and 22% for males and 6, 9, 6, 16, 15 and 20 % for females at dose levels of 25, 50, 100, 200, 400 and 600 mg/kg respectively.

HAEMATOLOGY: A dose-related leukopenia was observed for both male and female rats. Day 60 mean WBC counts were 6.4, 2.5 and 1.7 (males) and 4.3, 2.3 and 1.7 (females) at 0, 200 and 600 mg/kg respectively. Day 60 mean LYM counts were 5.8, 2.0 and 1.3 (males) and 3.8, 1.9 and 1.5 (females) for 0, 200 and 600 mg/kg respectively. Day 120 mean WBC counts were 5.0, 7.0, 5.7, 6.0, 5.2, 3.5 and 3.1 (males) and 8.1, 6.2, 4.9, 5.0, 4.8, 3.4 and 3.8 (females) for 0, 25, 50, 100, 200, 400 and 600 mg/kg respectively. Day 120 mean LYM counts were 4.1, 5.1, 3.9, 3.9, 3.4, 2.3, and 2.4 (males) and 6.6, 4.7, 3.9, 3.6, 3.7, 2.7 and 2.8 (females) for 0, 25, 50, 100, 200, 400 and 600 mg/kg respectively.

HISTOPATHOLOGY: NON-NEOPLASTIC: Lymphoid depletion in the B-cell of the spleen was observed in 3/5 male and 4/5 female rats at 200 mg/kg and 5/5 male and 5/5 female rats at 600 mg/kg at 60 days and in 1/10 male and 10/10 females at 600 mg/kg at 120 days. Increased extramedullary haematopoiesis was observed in the spleen of 4/5 male and 3/5 female rats that received 600 mg/kg for 120 days.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: decreased final body weight, white blood cell and lymphocyte counts and lymphoid depletion of B-cells in the spleen at 200 mg/kg and above
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: decreased final bodyweight, white blood cell and lymphocyte counts and lymphoid depletion of B-cells in the spleen at 200 mg/kg and above
Dose descriptor:
LOAEL
Effect level:
25 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: reduction in white blood cell and lymphocyte counts at 25 mg/kg (lowest dose tested)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Repeat oral administration of benzene to rats is associated with adverse effects in the haematopoietic system. NOAEL for males was 200 mg/kg. No NOAEL was established for females. LOAEL for females was 25 mg/kg/day (lowest dose tested).
Executive summary:

Groups of 10 or 15 rats/sex were administered 0, 25, 50, 100, 200, 400 or 600 mg/kg benzene in corn oil by gavage, 5 days/ week for 17 weeks. Five rats/sex were killed on days 0 and 60 from the 0, 200, and 600 mg/kg groups, remaining surviving animals were killed on day 120. Clinical observations, bodyweights, food consumption, haematological analyses and histopathological examinations were performed. No compound-related deaths occurred. Final mean body weights (relative to those of the vehicle controls) were depressed 14%-22% for male and female rats that received ≥200 mg/kg benzene. A dose-related leukopenia and lymphocytopenia was observed in males at ≥200 mg/kg and in females at ≥25 mg/kg. In the spleen, lymphoid depletion of B-cells was observed in both sexes at ≥200 mg/kg benzene and increased extramedullary haematopoiesis was observed 600 mg/kg.

NOAEL for males was 100 mg/kg/day. LOAEL for males was 200 mg/kg and for females was 25 mg/kg/day.