Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1987-01-13 to 1987-02-13
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from a study comparable to guideline with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
Mammalian erythrocyte micronucleus test as described by described by W. Schmid - The Micronucleus test, Mutation Research, 31, 9-15 (1975), the later released OECD Guideline 474 (1983) is based to a great extend on this publication.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Test animals

Species:
mouse
Strain:
other: OF1 (I. O. P. S. Caw)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: no data
- Weight at study initiation: no data
- Assigned to test groups randomly: animals were not formally randomized
- Fasting period before study: no data
- Housing: 2 (preliminary study) or 5 (main study) in plastic cages, on dust free bedding
- Diet: ad libitum, commercial pelleted diet UAR - A04C-R
- Water: ad libitum from plastic bottles
- Acclimation period: minimum 24 h


ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C +/- 1.5 °C
- Humidity: relative humidity 55 +/- 15%
- Air changes): minimum of 8 air changes per hour
- Photoperiod: 12 h light / 12 h dark cycle


Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: sterile distilled water. The pure compound and all dilutions were kept at 4°C during the test.
Details on exposure:
Preliminary study:
- No. of animals per dose: 2 males/2 females
- Frequency of treatment: two times; 24 h between injections
- Doses: 100, 200, 500, 1000, 2000 mg/kg bw i.p.
- Time of sacrifice: 6 hours after second injection
- Observation time points: 15 min after first administration, then periodically up to 30 hours.
- Type of observations: clinical signs, mortality
- Tolerated doses: 100 - 500 mg/kg bw

Main study
- No. of animals per dose: 5 males/5 females
- Frequency of treatment: two times; 24 h between injections
- Doses: 20, 200 mg/kg bw i.p., injected volume: each 10 ml/kg bw
- Positive control: cyclophosphamide 100 mg/kg bw i.p., injected volume: each 10 ml/kg bw
- Negative control: sterile distilled water, injected volume 10 ml/kg bw
- Time of sacrifice: 6 hours after second injection by cervical dislocation
- Number of samples: two smears of bone marrow/each animal
- EXAMINATIONS: Number of cells with micronuclei / 1000 polychromatic erythrocytes (PCE/NCE ratio: not given)
Duration of treatment / exposure:
30 hours
Frequency of treatment:
Two times, 24 h between the two injections
Post exposure period:
30 h after first injection
6 h after second injection
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 20, 200 mg/kg bw /injection (two injections at an interval of 24 h)
Basis:
nominal conc.
No. of animals per sex per dose:
Preliminary study: 2 males, 2 females
Main study: 5 males, 5 females
Control animals:
yes
Positive control(s):
Cyclophosphamide, 5 males and 5 females
- Doses / concentrations: 100 mg/kg bw

Examinations

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
preliminary study (details see "any other information on materials and methods")
In this preliminary study the test animals were administered twice (in a 24 hours interval) each 100, 200, 500, 1000 and 2000 mg/kg bw/day by intraperitoneal injection. Clinical signs and mortality were observed up to 30 hours after the first administration. Clinical signs like piloerection and ptosis were seen at doses of >/= 100 mg/kg bw/day. At doses >/= 1000 mg/kg bw/day the mice died within 30 and 4 hours after the first administration. The tolerated doses were in the range of 100 to 500 mg/kg bw/day. Therefore, the dose of 200 mg/kg bw/day was selected as the high dose and 20 mg/kg bw/day (10 % of the high dose) as the low dose. The dose level of 200 mg/kg bw/day (corresponding to 60 mg active substance/kg bw/day) is considered to be sufficiently high based on the effects found in the preliminary study and due to the highly irritating properties of the compound.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): As the test substance was applied twice with a 24 h interval (although only one timepoint was chosen for sacrifice), the result of the sacrifice 6h later may be regarded as a result of a 30h and a 6h treatment.

DETAILS ON SMEAR PREPARATION: The bone marrow was extracted from the femurs using foetal calf serum centrifuged and put into suspension again. For each animal, two smears were prepared, air-dried and stained with freshly filtered May Grunwald and Giemsa staining.
Statistics:
Student´s test
Exact bilateral comparison test

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
in preliminary dose finding study lethal at doses >/= 1000 mg/kg bw
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Preliminary study:

Mortality:1000 mg/kg bw: 4/4 animals died within 30 hours after first administration 2000 mg/kg bw: 4/4 animals died within 4 hours after first administration
Clinical signs:
>= 100 mg/kg bw: piloerection
>= 500 mg/kg bw: ptosis
1000 mg/kg bw: loss of righting reflex

Main study:
Mean numbers of micronucleated erythrocytes/1000 polychromatic erythrocytes:
- 20 mg/kg bw: 0.8, Sd dev 0.7 (males), 1.6 Sd dev 1.5 (females)
- 200 mg/kg bw: 1.2 Sd dev 1.0 (males), 1.0 Sd dev 1.3 (females)
- Negative control (vehicle sterile distilled water): 1.4 Sd dev 0.8 (males), 1.8 Sd dev 2.2 (females)
- Positive control (cyclophosphamide): 67.8 Sd dev 11.9 (males), 44.8 Sd dev 7.7 (females)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
No significant increase in the number of micronucleus-bearing erythrocytes was observed following two intraperitoneal administration of Coco AAPB (a.i.: 30 % ). The results for each treated group were comparable with those obtained for the negative control group. The results obtained with cyclophosphamide positive control (100 mg/kg) are significantly positive. Test article Coco AAPB induced no mutagenic effect in the mouse.
Executive summary:

In a OF1 (I.O.P.S. Caw) mouse bone marrow micronucleus assay, performed as described by W. Schmid - The Micronucleus test, Mutation Research, 31, 9-15 (1975), 5 male and 5 female animals were treated i. p. with Coco AAPB (30 % a.i). The test method by W. Schmidt is almost equivalent to the procedure described by OECD guideline 474. In a preliminary study the test animals were administered twice (in a 24 hours interval) each 100, 200, 500, 1000 and 2000 mg/kg bw/day by intraperitoneal injection. Clinical signs and mortality were observed up to 30 hours after the first administration. Clinical signs like piloerection and ptosis were seen at doses of >/= 100 mg/kg bw/day. At doses >/= 1000 mg/kg bw/day the mice died within 30 and 4 hours after the first administration. The tolerated doses were in the range of 100 to 500 mg/kg bw/day. Therefore, the dose of 200 mg/kg bw/day was selected as the high dose and 20 mg/kg bw/day (10 % of the high dose) as the low dose. As the test substance was applied twice with a 24 h interval (although only one timepoint was chosen for sacrifice), the result of the sacrifice 6h later may be regarded as a result of a 30h and a 6h treatment. The dose level of 200 mg/kg bw/day (corresponding to 60 mg active substance/kg bw/day) is considered to be sufficiently high based on the effects found in the preliminary study and due to the highly irritating properties of the compound.

The mean number of micronucleated erythrocytes/1000 polychromatic erythrocytes in males and female mice at 20 and 200 mg/kg bw/day were unaffected compared to the negative controls. The administration of 100 mg cyclophosphamide/kg bw serving as the positive control led to clearly elevated numbers of micronucleated erythrocytes.

It can be concluded, that Coco AAPB (30 % a.i) induced no clastogenic effect in this in vivo cytogenicity study on mice at dose levels of 20 and 200 mg/kg bw/day.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.