Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

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:
other: Mammalian Bone Marrow Chromosome Aberration Test

Test material

Specific details on test material used for the study:
Sodium cocoyl glycinate (clear liquid, Lot No. 970925, purity: 28.0% w/w) provided by Ajinomoto Co., Inc.

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
For this test 7-week-old male ICR (Crj: CD-1) SPF mice were purchased from Japan Biomaterial Center. The mice were produced by Charles River Japan, Inc.. The animals were kept in the laboratory for eight days for quarantine and acclimatization. Following physical examination, the mice were used in the tests with an age of 8 weeks. The range of body weights at the first administration was 34.5 to 39.1 g in the dose-finding test and 33.2 to 40.6 g in the main test. The animals were stratified with the body weight on the final day of the acclimatization period, and then randomly assigned to one of the test groups using random numbers produced by a computer. Six mice were assigned to each group in both the dose-finding test and the main test. A total of 36 male mice were used in each test. Six mice (the whole group) were kept in one cage.
The animals were kept in aluminum box-type cages. The temperature was set at 20 to 26°C, and relative humidity was set at 40 to 70%. The lighting period was set to 12 hours and the ventilation cycle was set at 10 to 15 cycles per hour.
Autoclaved floor chips were placed on the floor of the cage.
The animals had free access to commercially available radiation-sterilized solid food and tap water from a water bottle.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
physiol. saline
Details on exposure:
The required amount of the test item was weighed out and diluted with physiological saline to prepare 400, 200, 100 and 50 mg/kg solutions (4, 2, 1 and 0.5 % [w/v]) for use in the main test.
Duration of treatment / exposure:
The animals received intraperitoneal administration twice (24-hour interval)
Frequency of treatment:
see above
Post exposure period:
see below
Doses / concentrations
Remarks:
50-400 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6
Control animals:
yes
Positive control(s):
The animals in the positive control group received a single intraperitoneal administration of mitomycin C at 2 mg/kg bw and were put down 24 hours following administration. The dose and time at which they were put down were based on the historical control data.

Examinations

Tissues and cell types examined:
micronuclei / bone-marrow smears
Details of tissue and slide preparation:
Preparation of bone-marrow smears
The mice that were alive at the completion of the treatment period were put down humanely by dislocating the neck bone. The right femur was removed and bone marrow cells were washed out using a small volume (0.5 to 1.0 mL/femur) of fetal bovine serum. The bone marrow cells were centrifuged at 1000 rpm for five minutes. The supernatant was removed and the bone marrow cells in the test tube were mixed well using a Pasteur pipette to prepare a suspension of even consistency. One drop of the suspension was placed on a slide, and a cover glass was used to prepare a bone-marrow smear. The smear was dried at room temperature, fixed using methanol for five minutes, and stained using Giemsa’s staining solution for 30 minutes. Next, the smear was washed with Sörensen phosphate buffer and placed in a 0.004% citric acid solution for several seconds. The smear was washed with distilled water and dried naturally. Two smears were prepared per mouse, and the code number was written on each smear.

Observation of micronuclei
The bone-marrow smear were observed using a 100× oil immersion objective lens and a 10× ocular lens (total: 1000×), and in the order of the code. Information on the test solution for each bone-marrow smear was concealed (blind observation). Erythrocytes were classified as either polychromatic erythrocytes (PCE) or normochromatic erythrocytes (NCE). One thousand PCE were observed per bone-marrow smear and the percentage of micronucleated PCE (MNPCE) was calculated. One thousand erythrocytes (PCE and NCE [total erythrocytes]) were observed per animal (smear) and the proportion of the PCE to total erythrocytes was calculated to examine the cytostatic effect of the test solution on erythroblasts.
Evaluation criteria:
The percentages of MNPCE in the negative control group and the positive control group were compared with historical control data for the negative control. If the following criteria were met, this study was regarded as valid:
The number of MNPCE in the negative control group in this study does not exceed the number of MNPCE estimated on the basis of the historical control data.
The number of MNPCE in the positive control group in this study exceeds the number of MNPCE estimated on the basis of the historical control data.
Statistics:
The test results were assessed using the Kastenbaum and Bowman assessment table. In addition, in order to examine the cytostatic effect of the test item on bone marrow cells, the proportions of PCE to total erythrocytes in the test item and positive control groups were each compared to that in the negative control group using Statistic Library 1, Statistical Analysis Version 5. When three or more groups were compared, the comparisons were carried out in the following manner. Bartlett’s test for homogeneity was conducted for distribution among the groups. If the distribution in the groups was equivalent, one-way analysis of variance (ANOVA) was carried out. If ANOVA revealed a significant difference among the groups, a Dunnett’s test was performed to compare the mean values of the negative control and the test article (or positive control) groups. If the distribution was not equivalent, a Kruskal-Wallis rank-test was performed. If the rank-test revealed a significant difference, a Dunnett’s test (a comparison of the mean values of the negative control and the test article or positive control groups) was performed.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
The test item was shown to inhibit the growth of erythroblasts at dose levels of >= 100 mg/kg bw, but does not have the potency of eliciting micronuclei in vivo.
Executive summary:

An in vivo micronucleus study using the bone marrow of male CD-1 mice was conducted, where groups of 6 mice received doses of 400, 200, 100 or 50 mg/kg bw via intraperitoneal injections (twice with 24 -hour interval). The bone-marrow smears were prepared 24 hours following the final administration. Four deaths were observed in the 400 mg/kg bw group and one death in the 200 mg/kg bw group. A decrease in locomotor activity and bradypnea were observed at >= 50 mg/kg bw, piloerection was observed at >= 100 mg/kg bw and hypothermia, lacrimation and prone position at >= 200 mg/kg bw. A decrease in body weight was observed at >= 100 mg/kg bw one day following the first administration and onwards. The percentage of MNPCE in the MMC (positive control) group 24 hours following single intraperitoneal administration was 4.77%, which is clearly higher than that in the negative control group, 0.07%. The number of MNPCE was 286, which was assessed as positive and exceeded the number of MNPCE estimated on the basis of the historical control data. The number of MNPCE in the negative control group was within the range estimated on the basis of the historical control data. Thus, it was judged that this study was valid.

The percentage of MNPCE in the 400, 200, 100 and 50 mg/kg bw groups 24 hours following the final administration of the test item was 0.10, 0.08, 0.07 and 0.18%, respectively. These values were within the range estimated on the basis of the historical control data for the negative control. The result of the Kastenbaum and Bowman assessment was negative. The proportion of PCE to total erythrocytes was significantly lower in the 100 and 200 mg/kg bw groups than that in the negative control group.

Based on these results it can be concluded that the test item inhibits the growth of erythroblasts at >= 100 mg/kg bw, but does not have the potency of eliciting micronuclei in vivo.