Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
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: Acceptable, well-documented study report similar or equivalent to OECD 474. GLP.

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
yes
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): MCP 968

Test animals

Species:
rat
Strain:
not specified
Sex:
male/female

Administration / exposure

Route of administration:
dermal
Vehicle:
neat
Details on exposure:
The test material was applied evenly over the back of the animal begining at the spine and scapula working posteriorly and laterally.
Duration of treatment / exposure:
animals were dosed daily, five days per week for four weeks.
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 125, 500, 2000 mg/kg/day
Basis:
other: dermal application
No. of animals per sex per dose:
20 (10 males and 10 femals per dose)
Control animals:
yes, concurrent no treatment

Examinations

Tissues and cell types examined:
Femurs were taken from five animals per sex per dose group. Bone marrow was removed from the femurs and dissaggregated in heat inactivated fetal bovine serum containing 25 mM EDTA. A nearly pure erythrocyte fraction was then obtained by passing the bone marrow cell suspension through a cellulose column using a procedure based on the method developed by Romagna.
Details of tissue and slide preparation:
The resulting erythrocyte fraction was collected by centrifugation, and the cells were resuspended in a small volume of fetal bovine serum. Two bone marrow slides were made for each animal. Slides were randomized by a computer-generated random numbers table. One thousand PCEs and 1000 NCEs were scored to determine the percentage of micronucleated erythrocytes. One thousand erythrocytes were scored to determine the ratio of PCEs to NCEs.Slides were dried on a slide warmer at 57 °C and fixed in absolute methanol for fifteen minutes. After the slides had dried, the slides were placed in acridine orange (0.125 mg/ml in Giordano's buffer, pH 6.4-6.5) solution for 7 minutes, rinsed in Giordano's buffer, washed in Giordano's buffer for an additional 10 minutes, and finally rinsed in fresh Giordano's buffer. Fluorescent microscopy was used to evaluate the erythrocytes.
Evaluation criteria:
To determine if the test chemical caused cell toxicity in the bone marrow, PCEs and NCEs were counted until 1000 total erythrocytes were tallied for each animal, and the ratios of PCEs to NCEs was calculated. A smaller ratio for the test substance-treated animals compared to the controls would indicate that the test substance had induced toxicity in the bone marrow. Toxicity can reduce the sensitivity of the assay to detect the induction of micronuclei by preventing the formation of new erythrocytes. However, the presence of toxicity in bone marrow would indicate that the test substance had reached the assay tissue.
Statistics:
ANOVA and GLM models were applied to the data. The statistical analyses compared test values to negative control data; a significant increase in micronuclei is an indication of clastogenic activity by the test agent.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not applicable

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The in vivo micronucleus assay of the test material was negative. This finding does warrant the classification of the test material as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.
Executive summary:

The micronucleus assay was used to determine if the test material caused a significant increase in the number of micronucleated red blood cells from bone marrow of rats treated dermally over thirteen weeks, five days a week at doses of 125, 500, and 2000 mg/kg. The test material was not cytotoxic to red blood cell formation nor did it induce any statistically significant increase in the formation of micronucleated PCEs or NCEs in the bone marrow of dermally-treated rats. The test material was not considered to a genotoxin or clastogen under the conditions of the test. This finding does not warrant the classification of the test material as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.