Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
December 1992
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
mouse
Strain:
Swiss
Remarks:
OF-1, SPF-quality
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Switzerland
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation:
30-39 g males
23-29 g females
- Assigned to test groups randomly: yes, allocated to treatment groups as they came to hand from delivery boxes.
- Housing: In groups Of 5 per Sex in polycarbonate cages. Bedding: Purified Sawdust (Woody SPF, from BMI, Helmond, The Netherlands).
- Diet (e.g. ad libitum): Standard laboratory animal diet (Kliba 343 from Klingentalmühle Ag, Kaiseraugst, Switzerland). Feed was available ad libitum. The feed was analysed for contaminants by the manufacturer, and the results were archived.
- Water (e.g. ad libitum): Tap Water, ad libitum. Results of chemical and contaminant analyses were archived.
- Acclimation period: At least 6 days under laboratory conditions.

ENVIRONMENTAL CONDITIONS
Standard laboratory conditions. Air-conditioned room, 15 air changes per hour, temperature 213 °C, relative humidity around 40-70%. Fluctuations from these optimal conditions were noted, but Were considered not to have affected integrity. Lighting was 12 hours artificial fluorescent light and 12 hours dark.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Milli-RO water (Millipore Corp. Bedford, Mass., USA)
Duration of treatment / exposure:
Single injection
Frequency of treatment:
Single dosing
Doses / concentrations
Dose / conc.:
5 mg/kg bw (total dose)
Remarks:
Dosing volume = 10 mg/kg body weight
No. of animals per sex per dose:
At least 5 male and 5 female mice per sampling time in each treatment group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CP; CAS no. 50-18-0; Endoxan, Asta - Werke, F. R. G. ) at 50 mg/kg body weight dissolved in 0.9% NaCl (Merck) in Milli-RO water .

The route and frequency of administration and the volume administered were Consistent With those of the test article.

Examinations

Tissues and cell types examined:
Analysis of the bone marrow smears for micronuclei
All slides were randomly coded before examination. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of Suitable technical quality, i. e. where the cells were well spread, undamaged and Well stained. Slides were scored at a magnification of 1000 X. The number of micronuclei was counted in 1000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.
Details of tissue and slide preparation:
The animals were sacrificed by cervical dislocation at 24 and 48 h after dosing of the test substance and the vehicle and at 48 h after dosing of the positive control. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min.
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 h immersed in a 1:1 mixture of 96% ethanol/ether and cleaned with a tissue) and marked (with the NOTOX study identification number and the animal number). The drop was spread by moving a clean slide With round-whetted sides at an angle of approximately 45° Over the slide with the drop of bone marrow suspension.

The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 h immersed in a 1:1 mixture of 96% ethanol/ether and cleaned with a tissue) and marked (with the NOTOX study identification number and the animal number). The drop was spread by moving a clean slide With round-whetted sides at an angle of approximately 45° Over the slide with the drop of bone marrow suspension.
The preparations were then air-dried and thereafter fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal,

Staining of the bone marrow smears
The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B. V.). The dry slides were dipped in xylene before they were embedded in DePex and mounted With a cover slip.
Evaluation criteria:
A micronucleus test is considered acceptable if it meets the following criteria :
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronuclei.
b) The incidence of micronuclei in the control animals should reasonably fall within the laboratory historical control data range.

Statistics:
A test substance is considered positive in the micronucleus test if:
It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P K 0.05) increase in the frequency of micronuclei (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.

A test substance is considered negative in the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronuclei neither in the combined data for both sexes nor in the data for male Or female groups a one.

The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Pilot study / Dose selection
In a preliminary study 36 animals (3 males and 3 females per group) were dosed intraperitoneally with 200, 100, 50, 25, 10 and 5 mg/kg body weight (groups A, B, C, D, E and F, respectively). Higher concentrations were not dosed because of expected toxicity of the test substance. All animals of group A, B, C and D died within 2 to 24 hours after dosing. The male animals of group E died within 24 hours and the female animals within 48 to 72 hours. Two male and one female animal of group F died within 72 to 96 hours. The other animals were lethargic, showed pilo-erection and ataxia. The male animals showed also bloody eye encrustation. One female animal showed bradypnoea and only one female recovered after 96 hours. Based on the results of this pilot study, 5 mg/kg body weight was selected as an appropriate dose for the Micronucleus Test.

Micronucleus test
The mean number of micronuclei scored in the test substance treated groups was compared with the corresponding control groups. No increase in the frequency of micronuclei was observed in the polychromatic erythrocytes of the bone marrow of test substance treated animals.
The polychromatic erythrocytes in the bone marrow of the 48 h sampling time of the groups treated with V190110 showed a basophilic stippling effect, this effect is rare and could be caused by anemia.
The incidence of micronuclei in the control animals was found to be in the range of historical data (0.59 + 0.89; mean + standard deviation, N = 1120). Both the male and female groups that were treated with cyclophosphamide and the female groups treated with the test substance showed a decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a toxic effect of this compound on the erythropoiesis. The positive control substance induced in both sexes a statistically significant increase in the number of micronuclei (see Appendix 2).

Applicant's summary and conclusion

Conclusions:
It is concluded that this test is valid and that V1901.10 is not mutagenic in the micronucleus test under the experimental conditions described in this report.