Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1982-1983
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report which meets basic scientific principles, basic data given, and comparable to guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: SCHMID, W.; The Micronucleus Test. Mutation Res. 31: 9-15, 1975
Deviations:
not specified
GLP compliance:
yes
Remarks:
The investigation was performed in compliance with; -Non-Clinical Laboratory Studies: Good Laboratory Practice Regulations.- Federal Register. Vol. 431 No. 247. dated December 22, 1978 1 pp. 60013-60025, U.S.A.
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
BATCH-NUMBER: 2/04 lot-Nr. 11-08-10
DESCRIPTION: Powder
STABILITY: Stable at room temperature
STORAGE: The test material and a reserve sample were stored in the dark at room temperature.
SAFETY PRECAUTIONS: Routine hygenic procedures used in handling chemicals were sufficient to assure personnel health and safety.

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Kleintierfarm Madoerin AG 4414 Fuellinsdorf / Switzerland
- Age at study initiation: Five Weeks
- Weight at study initiation: 20 - 36 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: The experiment was performed under standard laboratory conditions. The animals of the positive control and the treatment groups were housed in groups of 6. The animals of the negative control group were housed in groups of 6 and 4. All animals had Macrolan Type 3 cages with wire mesh tops and standardized granulated soft wood bedding (Source:HLignocel N , Schill AG, 4321 Muttenz / SWitzerland).
- Diet (e.g. ad libitum): The diet consisted of pelleted standard Kliba 343-A mouse maintenance diet, ad libitum (Source: Klingentalmuehle AG, 4403 Kaiseraugst I Switzerland).
- Water (e.g. ad libitum): Tap water was available ad libitum, with water quality according to the requirements of the Schweizerische Lebensmittelbuch.
- Acclimation period: Under test conditions, 7 days, with veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10%
- Air changes (per hr): NA
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
For the treated animals, a suspension was prepared by adding the test material to distilled water containing Tween 80 (1%). The suspension was homogenized with a Potter Elvejem Homogenizer (Bender Holbein, 8042 Zuerich, Switzerland). During treatment, homogeneity was maintained by a magnetic stirrer (Auer Bittmann, Switzerland).
Details on exposure:
All mice received an oral gavage, once daily, for two consecutive days, thereby exposing the cell population to the test material during two successive S-phases. For the treated animals, a suspension was prepared by adding the test material to distilled water containing Tween 80 (1%). The suspension was homogenized with a Potter Elvejem Homogenizer (Bender Holbein, 8042 Zuerichl Switzerland). During treatment, homogeneity was maintained by a magnetic stirrer (Auer Bittmann, Switzerland). The positive control material was Cyclophosphamide (Source: Serva Chern, Brunschwig, Basel I Switzerland) dissolved in 0.9% NaCl at a concentration of 50 mg.kg-1 body weight, prepared on the day of the experiment. Negative Control: Distilled water with Tween 80 (1%).
The test material and the control materials were administered in a standard volume of 20 ml.kg-1 body weight.
Duration of treatment / exposure:
Two Days
Frequency of treatment:
Once Daily
Post exposure period:
Twenty four hours after the second treatment (48 hours after first treatment) the mice were sacrificed. The bone marrow was removed from the femora and slides were prepared for examination.
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
In the assay, 6 animals of each sex were included in each treatment group (5 animals were evaluated) and 6 animals of each sex in the positive control group (5 animals were evaluated). The negative control group contained 10 animals of each sex (8 animals were evaluated).
Control animals:
yes
Positive control(s):
The positive control material was Cyclophosphamide (Source: Serva Chern, Brunschwig, Basel / Switzerland) dissolved in 0.9/. NaCl at a concentration of 50 mg.kg-1 body weight, prepared on the day of the experiment.

Examinations

Tissues and cell types examined:
BONE MARROW; Polychromatic Erythrocyte (PCE) and Normochromatic Erythrocytes (NCE)
Details of tissue and slide preparation:
Both femora were removed and freed of adherent tissue. The proximal end of the femur was cut with scissors and the distal end was left intact. The needle of a plastic syringe) containing 0.2 ml of calf serum* was inserted into the proximal marrow canal. The femur was submerged in 1.5 ml of calf serum in a labeled centrifuge tube. Gentle aspiration and flushing dispersed the bone marrow cells into the serum as a fine homogeneous suspension. The tube containing the bone marrow cells of both femora was centrifuged at 1000 r.p.m. for 5 min. (Centra 7; Kontron AG, Zuerich). The supernatant was removed, leaving a thin layer of serum. The cells of the sediment were carefully mixed by aspiration in a siliconized Pasteur pipette. A small drop of the suspension was smeared onto
the slide and air-dried overnight. Two slides were made per animal. Each slide was identified with: project code, date, dose and the animal number. The following day, the preparations were stained with the panoptic staining method, according to Pappenheim (3).
Evaluation criteria:
From each animal, one thousand polychromatic erythrocyte (PCE) and one thousand normochromatic erythrocytes (NCE) were screened under the microscope (magnification 1000x) (Wild and Leitz AG, 8002 Zurich/Switzerland), for the incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was based on 1500 cells (PCE + NCE) per slide. The ratio PCE/NeE was determined to measure the toxicity of the test material. The animal number of the slide was obscured to ensure objective evaluation. The numbers of micronucleated erythrocytes at every dose level of the test material were compared with those of the negative control. A significant increase beyond the negative control value would indicate mutag~nic activity through chromosome-breakage induced by the test material.
Statistics:
Regression Model: Maximum Likelihood Method (Theoretical Statistics, Cox and Hinkley); Poisson heterogenicity test (Armitage, Statistical Methods in Medical Research, P. 214)

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The mice were clinically examined by a veterinarian during the acclimation period. No disease symptoms were observed. The non-toxicity of the test material was confirmed in the mice species used in the test (LD 50 greater than 5000 mg.kg-1 B.W.) The mouse number 2B died spontaneously after the second application of the test material. After two oral applications of the test material 24 hours apart, no significant difference in the numbers of micronucleated polychromatic or normochromatic erythrocytes was found between the treatment groups and the negative control group (appendix B). No evidence of toxic effects of the test material was observed. As expected, the positive control group showed a significant increase in the number of micronucleated erythrocytes in comparison with the negative control group. Most micronuclei were found in polychromatic erythrocytes. The dose used for the positive control also exhibited toxic effects, as shown by the reduced PCE/NeE ratio (appendix AS).

Applicant's summary and conclusion

Conclusions:
None of the three dose levels tested (the highest level was 5000 mg/kg bw) showed any substance-related increase in the numbers of micronucleated polychromatic and normochromatic erythrocytes. Therefore, it can be stated that during the mouse micronucleus assay performed under the conditions described, no chromosome-breakage was detected, and no evidence of potential chromosomal mugenicity could be attributed to CYASORB UV-2908.
Executive summary:

It can be stated that during the mouse micronucleus assay performed under the conditions described, no chromosome-breakage was detected, and no evidence of potential chromosomal mugenicity could be attributed to CYASORB UV-2908. GLP and SCHMID, W.; The Micronucleus Test. Mutation Res. 31: 9-15, 1975 were followed as guidlines. Test substance doses used in the study were as follows: low dose: 200 mg/kg bw; medium dose: 1000 mg/kg bw; high dose: 5000 mg/kg bw.