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Repeated dose toxicity: inhalation

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Administrative data

sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP status not known, guideline compliant, animal experimental study, published in peer reviewed literature, adequate for assessment

Data source

Reference Type:
Subchronic inhalation toxicity of benzene in rats and mice.
Ward CO, Kuna RA, Snyder NK, Alsaker RD, Coate WB and Craig PH.
Bibliographic source:
American Journal of Industrial Medicine 7:457-473

Materials and methods

Test guidelineopen allclose all
equivalent or similar to
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
equivalent or similar to
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
not specified
Limit test:

Test material

Details on test material:
- Name of test material (as cited in study report): benzene- Source: American Petroleum Institute, Washington, D.C.- Analytical purity: >99.9% (by gas chromatography)- Stability confirmed by GC throughout the study

Test animals

Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River Breeding Laboratories, Kingston, New York- Age at delivery: Approximately 6 weeks- Housing: individually in stainless steel wire mesh cages- Diet: Purina Certified Lab Chow (#5002 Ralston Purina, St. Louis, MO) ad libitum except during exposure- Water: ad libitum except during exposure- Acclimation period: 40 daysENVIRONMENTAL CONDITIONS- Temperature: 71-79°C- Humidity: 35-80%- Photoperiod: 12 h dark / 12 h lightIN-LIFE DATES: no data

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
other: air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION- Exposure apparatus: 6 m3 glass and stainless-steel exposure chambers- Method of holding animals in test chamber: no data- System of generating test atmosphere: the test atmosphere was generated by forced evaporation of warmed liquid benzene under a stream of dried filtered air. - The air flows through the generation system and exposure chamber were monitored. - The airflow through the chambers was maintained at 1,200 L/minute. - All generation flasks were seated in water baths agitated with air and maintained at room temperature (Groups 2-4) or heated at 30±2°C (Group 5) with tank-type heaters. TEST ATMOSPHERE- Brief description of analytical method used: The chamber concentrations of benzene were established using gas chromatography with infrared (IR) analyzer
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
GC samples were obtained and analyzed by peak height at least once a day from each exposure chamber. Overall mean GC analyses (for Groups 2-5) varied from the target concentrations by 20.0, 18.0, 2.0, and 2.4% (respectively)
Duration of treatment / exposure:
10 rats/sex/group sacrificed after 7, 14, 28, 56, and 91 days of treatment
Frequency of treatment:
5 days/week for up to 13 weeks
Doses / concentrations
Doses / Concentrations:1, 10, 30 or 300 ppm (3.2, 32, 96 or 960 mg/m³)Basis:nominal conc.
No. of animals per sex per dose:
Control animals:
yes, sham-exposed


Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (mortality and morbidity)- Time schedule: twice dailyDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: weeklyBODY WEIGHT: Yes- Time schedule for examinations: weeklyFOOD CONSUMPTION: NoWATER CONSUMPTION: NoOPHTHALMOSCOPIC EXAMINATION: NoHAEMATOLOGY: Yes- Time schedule for collection of blood: prior to initiation and on days 7, 14, 28, 56 and 91- Anaesthetic used for blood collection: sodium pentobarbital - Animals fasted: Yes- How many animals: 10/sex/group- Parameters examined: haematocrit, total haemoglobin, erythrocyte count, mean cell volume, mean cell haemoglobin, total and differential leukocyte count, platelet count, reticulocyte count (only on days 28, 56, and 91), myeloid/erythroid (M/E) ratio (from bone marrow smears), leukocyte alkaline phosphatase (from peripheral blood smears), total protein, alkaline phosphatase, total bilirubin, creatinine, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, and erythrocyte glycerol lysis timeCLINICAL CHEMISTRY: NoURINALYSIS: NoNEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: 10 rats/sex/group randomly selected, fasted (overnight), weighed, and killed by exsanguination under sodium pentobarbital anaesthesia on days 0, 7, 14, 28, 56, and 91. Complete necropsies were performed on all these animals and on all animals found dead or sacrificed in a moribund condition during the study. ORGAN WEIGHTS: - Organs were weighed and organ/terminal body weight ratios determined on the following: brain, pituitary, adrenals, thyroids, lungs, heart, liver, spleen, kidneys and testes. HISTOPATHOLOGY: - The following tissues were taken and preserved in 10% neutral buffered formalin: heart, lungs (all lobes), bronchi, trachea, cervical lymph nodes, salivary gland, oesophagus, stomach, small intestine (three sections), caecum, colon, bone marrow (femur), bone marrow section and smear (fixed in alcohol), adrenals, testes or ovaries, prostate or uterus, mesenteric lymph nodes, urinary bladder, mammary gland, brain, pituitary, spinal cord (two sections), sciatic nerve/ muscle, thymus, spleen, liver, pancreas, kidneys, bone, eye (fixed in Bouin's solution), Harderian gland, nasopharynx, and Zymbal's gland. - Sections from these tissues, from control and high-level groups at each sacrifice period were examined plus sections of lung, trachea, cervical lymph nodes, thymus, spleen, bone marrow (femur), mesenteric lymph nodes, and nasal turbinates from all animals at each interval.
Body weight data were analyzed by one-way classification analysis of covariance (ANCOVA) using pre-exposure (day 0) weights as the covariate. Clinical pathology data, organ weight and organ/body weight ratio data of the control group were analysed by Barlett's test for homogeneity of variance. If ANOVA of homogeneous data was significant, Scheffe's multiple pairwise comparison procedure was used to compare the group mean values. If ANOVA of heterogeneous data was significant, Games and Howell's multiple pairwise comparison procedure was used to compare the group mean values. If ANOVA was not significant, no pairwise comparisons were made. The null hypothesis was rejected only if p <0.05 (one-tailed) in all cases (ie, the 5% level of significance was chosen).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
no mortality observed
Body weight and weight changes:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related

Effect levels

open allclose all
Dose descriptor:
Effect level:
30 ppm (nominal)
Basis for effect level:
other: decreased white blood cell count, percentage of lymphocytes and femoral marrow cellularity at 300 ppm
Dose descriptor:
Effect level:
96 mg/m³ air (nominal)
Basis for effect level:
other: decreased white blood cell count, percentage of lymphocytes and femoral marrow cellularity at 960 mg/m3

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

No exposure-related mortality or effects on mean body weight and clinical signs were seen. Female rats of the 30 ppm dose group showed lower thyroid weight on day 14 only. At 300 ppm statistically significant decreases in WBC counts were seen in males on day 14 and females on day 91; statistically significant decreases in percent lymphocytes were noted in both males and females on days 14, 28, 56 and 9 (no further detail provided). At this same dose level decreased femoral marrow cellularity was seen on day 7 only.

Applicant's summary and conclusion

Inhalation exposure of rats to 300 ppm (960 mg/m3) benzene for up to 90 days induced decreased lymphocyte counts, a relative increase in neutrophil percentages and slightly decreased femoral marrow cellularity. The NOAEC was 30 ppm (96 mg/m3) in males and females.
Executive summary:

A subchronic inhalation toxicity study of benzene was conducted in Sprague-Dawley rats. Four groups of animals consisting of 50 rats/sex each were exposed to concentrations of 0, 1, 10, 30, and 300 ppm (0, 3.2, 9.6, 960 mg/m3) benzene vapour, 6 h/day, 5 days/week, for 13 weeks. Ten rats/sex in each group were sacrificed after 7, 14, 28, 56, and 91 days of treatment. Criteria used to evaluate exposure-related effects included behaviour, bodyweights, organ weights, clinical pathology, gross pathology, and histopathology.

No consistent exposure-related trends were seen in the clinical observations and bodyweight data. Exposure-related clinical pathology changes were seen in the high-level (300 ppm) animals. Decreased lymphocyte counts and a relative increase in neutrophil percentages were the only exposure-related clinical pathology alterations. The only exposure-related lesion consisted of slightly decreased femoral marrow cellularity in the animals exposed to 300 ppm.

The NOAEC for toxicity at 28 and 90 days was 30 ppm (96 mg/m3) for both male and female rats.