Registration Dossier

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no deviations from standard test guidelines and no methodological deficiences, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
Allyl bromide was obtained from Fluka Chemical Corporation (Buchs, Switzerland) in one lot (330638) and from Aldrich Chemical Co. in one lot (03614HN). Lot 330638 was used in the 2-week studies, and lot 03614HN was used in the 40-week studies. Identity and purity analyses were conducted by the analytical chemistry laboratory, Midwest Research Institute (Kansas City, MO) and the study laboratory, BioReliance (Rockville, MD). Reports on analyses performed in support of the allyl bromide studies are on file at the National Institute of Environmental Health Sciences.
Both lots of allyl bromide, a clear, colorless liquid, were identified by the analytical chemistry laboratory using infrared and proton nuclear magnetic resonance (NMR) spectroscopy and by the study laboratory using infrared spectroscopy. All infrared and NMR spectra were consistent with the literature spectra and spectra of a reference standard from the same lot. The purity of each lot was determined by the analytical chemistry and study laboratories using gas chromatography (GC). For lot 330638, GC indicated one major peak and five impurities with a combined peak area of 0.7% relative to the total peak area. GC by a second system indicated one major peak and three impurities with a combined peak area of less than 0.5%. The relative purity was 102% when compared to a reference standard from the same lot. The overall purity of lot 330638 was greater than 99%. For lot 03614HN, GC indicated one major peak and four impurities with a combined peak area of 0.45% relative to the total peak area. GC by a second system indicated one major peak and three impurities with a total combined area less than 0.3% of the total peak area. The relative purity was 102% when compared to a frozen reference from the same lot. The overall purity of lot 03614HN was greater than 99%. During the 40-week studies, additional purity analyses were performed by the study laboratory at 26 weeks and at the end of the study using GC.
To ensure stability, the bulk chemical was stored in a sealed container under a nitrogen headspace, protected from light, at 2° to 8° C. No degradation of the bulk chemical was detected.

Test animals

Species:
mouse
Strain:
other: FVB/N - C57BL/6 - Tg.AC hemizygous- p53 haploinsufficient mice
Sex:
male/female
Details on test animals and environmental conditions:
See paragraphe 7.7

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
At the end of the 40-week studies of paragraphe 7.7, peripheral blood samples were obtained from male and female mice from each strain. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs) in each of up to 15 mice per group. In addition, the percentage of polychromatic erythrocytes (PCEs) in a population of 1,000 erythrocytes was determined as a measure of bone marrow toxicity.
Duration of treatment / exposure:
40-week study
Frequency of treatment:
Groups of 15 male and 15 female FVB/N and C57BL/6 mice received 0 or 8 mg allyl bromide/kg body weight in corn oil by gavage, in a volume of 10 mL/kg body weight, 5 days per week for 40 weeks. Groups of 15 male and 15 female Tg.AC hemizygous and p53 haploinsufficient mice received 0, 0.5, 1, 2, 4, or 8 mg allyl bromide/kg body weight in 10 mL/kg corn oil by gavage, 5 days per week for 40 weeks. Vehicle control mice received corn oil only.
Post exposure period:
None
No. of animals per sex per dose:
15 males and 15 females per group
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control groups of 15 male and 15 female Tg.AC hemizygous mice received dermal applications of 1.25 μg TPA in 100 μL acetone (12.5 μg TPA/L solution), three times per week until removal from study. Positive control mice were removed after the appearance of 20 or more skin papillomas and discarded. The TPA solution was applied to the clipped dorsal skin from the mid-back to the interscapular area.

Examinations

Tissues and cell types examined:
Necropsies and microscopic examinations were performed on all mice except positive controls. The heart, right kidney, liver, lung, right testis, and thymus were weighed. At necropsy, all organs and tissues were examined for grossly visible lesions, and all major tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin for microscopic examination. For all paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined.
Statistics:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Animals found dead of other than natural causes or missing were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Negative controls validity:
valid
Additional information on results:
The frequency of micronucleated erythrocytes was assessed in each of the four mouse strains treated with allyl bromide for 40 weeks. Results in all four strains of mice were concluded to be negative; in addition, no significant, consistent changes in the percentage of polychromatic erythrocytes (reticulocytes) among total erythrocytes were observed in any of the four strains.
Some observations of note in these micronucleus tests include the small increase in micronucleated erythrocytes seen in the single dosed group (8 mg/kg) of female C57BL/6 mice that was evaluated for micronucleus frequency. Although the P value was significant (<0.05), these results were judged to be negative because the small increase represented less than half a micronucleus per 1,000 cells, which is not biologically relevant. In the male p53 haploinsufficient mice, one dosed group (1.0 mg/kg) showed a small but significant increase (P=0.0006) in micronucleated erythrocyte frequency, but none of the three higher doses showed an effect; therefore, this small increase at a single dose concentration in one sex, even though statistically significant (P<0.005), was not considered sufficient evidence of the ability of allyl bromide ti induce effect in this assay.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Not sufficient evidence of the ability of allyl bromide to induce an effect in this assay
Executive summary:

The frequency of micronucleated erythrocytes was assessed in male and female mice for each of the four mouse strains administered allyl bromide by corn oil gavage for 40 weeks. Results in all four micronucleus studies with allyl bromide were concluded to be negative; in addition, no significant changes in the percentage of polychromatic erythrocytes (reticulocytes) among total erythrocytes were observed in any of the four strains of mice.