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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Not GLP, but key cytogenetic parameters measured comparable to guideline study.

Data source

Reference
Reference Type:
publication
Title:
Benzene-induced micronuclei in erythrocytes: an inhalation concentration-response study in B6C3F1 mice
Author:
Farris GM, Wong VA, Wong BA, Janszen DB and Shah RS
Year:
1996
Bibliographic source:
Mutagenesis 11, 455-462

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
- no positive control.
Principles of method if other than guideline:
Mice were exposed to benzene vapour (up to 200 ppm; 640 mg/m3) for up to 8 weeks and blood and bone marrow samples evaluated for the presence of micronuclei within 2 hours of end of exposure
GLP compliance:
no
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): High purity liquid chromatography grade benzene
- Supplier: VWR Scientific, Marietta, GA, USA
- Analytical purity: >99.9%

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Nil, USA
- Age at study initiation: 12 weeks±2 days
- Assigned to test groups randomly: yes, by weight
- Housing: one per cage in 8 m3 stainless steel and glass inhalation chambers
- Diet: NIH-07 rodent chow (Ziegler Bros., Gardener, PA, USA) ad libitum
- Water: Filtered tap water ad libitum
- Acclimation period: 10-17 days

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
- Vehicle(s)/solvent(s) used: air
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION: Benzene exposures conducted in 8 m3 stainless steel and glass Hinners-style whole body inhalation chambers. Air was conditioned to 72°F and 50% humidity and maintained at a flow of 2000 L/min through the chambers. Temperature, relative humidity, airflow and static pressure continuously monitored.
- System of generation: Atmospheres of 10, 100 and 200ppm benzene generated by vaporizing liquid benzene contained in a 10-gallon stainless steel pressure vessel. A controlled flow of nitrogen was bubbled through the liquid benzene and carried vapours out of the vessel into the chamber air inlet. The 1 ppm benzene atmosphere was also generated from liquid benzene, however, the pressure vessel was maintained at 10 p.s.i. gauge pressure with nitrogen. The flow of the benzene vapours and nitrogen into the chambers controlled by mass flow controllers placed on the outlet side of the pressure vessels.

TEST ATMOSPHERE
- Brief description of analytical method used: An infrared (IR) gas analyser used to monitor the 0, 10, 100 and 200 ppm benzene concentrations. The 1 ppm chamber was monitored by gas chromatography.
Duration of treatment / exposure:
1, 2, 4 and 8 weeks of exposure
Frequency of treatment:
6 h/day, 5 days/week
Post exposure period:
None. Blood and bone marrow samples collected within 2 h of end of final exposure.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1, 10, 100 or 200 ppm
Basis:
other: target concentrations
No. of animals per sex per dose:
7
Control animals:
yes, sham-exposed
Positive control(s):
none

Examinations

Tissues and cell types examined:
Micronucleated polychromatic erythrocytes (MPCE) in the bone marrow and blood and micronucleated normochromatic erythrocytes (MNCE) in the blood.
Statistics:
For each timepoint, the data from each benzene exposure group was compared to the controls using Dunnett's analysis of variance (ANOVA) with a 5% significance level. Linear and quadratic regression models were fit to the bone marrow NIPCE data. Evaluation of linear and quadratic curves with the data required a weighted analysis because of nonhomogeneous variability. A P value of <0.05 for lack of fit test meant that the curve was not appropriate for the data set.

Results and discussion

Test results
Sex:
male
Genotoxicity:
positive
Remarks:
at exposure concentrations of 100 and 200 ppm
Toxicity:
yes
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Additional information on results:
Atmosphere analysis: The analytical concentrations of benzene within the individual chambers were stable during each exposure period and over the time course of the studies. The actual concentrations of benzene averaged 0.95±0.09, 9.9±1.2, 98.5±1.5 and 198.5±4.8 ppm.

Any other information on results incl. tables

100 and 200 ppm benzene induced a statistically significant increased frequency of micronucleated erythrocytes in the bone marrow and blood. No such increase was observed at levels of 1 or 10ppm benzene. The increase was seen at weeks 1, 2, 4 and 8 of exposure, and the frequency of MPCE plateaued at week 2 with 43/1000 (100 ppm) and 86/1000 (200 ppm) in the bone marrow as compared with 10/1000 for controls. The frequency of MNCE in the blood progressively increased to 13.4/1000 (100 ppm) and 32.5/1000 (200 ppm) at week 8 as compared with 1.8/1000 for controls. Cytotoxicity was seen at 100 and 200 ppm with decreases in both PCE and total erythrocyte counts.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): positive at exposure levels of 100 ppm and 200 ppm
Benzene produced an increased frequency of micronuclei following inhalation exposure in mice and is considered to be an in vivo clastogen.
Executive summary:

The frequencies of micronucleated polychromatic erythrocytes (MPCE) in the bone marrow and blood and micronucleated normochromatic erythrocytes (MNCE) in the blood of male B6C3F1 mice were measured following inhalation of benzene at 0, 1, 10, 100 or 200 ppm for 1, 2, 4 or 8 weeks of exposure. 100 and 200 ppm benzene induced a statistically significant increased frequency of micronucleated polychromatic erythrocytes and/or micronucleated erythrocytes in the bone marrow and blood of exposed animals at all time points. The frequency of MPCE was not statistically raised in animals exposed to 1ppm or 10ppm benzene, indeed in a manual count the frequency of MPCE was lower than control levels. It is concluded that benzene is an in vivo clastogen in the mouse.