Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well performed GLP and EPA guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
other: EPA guideline: In vivo mammalian bone marrow cytogenetic tests: micronucleus assay. HG-Chromo-Micronuc, August 1982
Deviations:
not specified
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
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): Acetoacetamid

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG, SPF breeding colony
- Age at study initiation: about 7 weeks
- Weight at study initiation: males: 27 - 35 g; females: 21 - 29 g
- Housing: in fully air-conditioned rooms in Macrolon cages (Type 3), on softwood granulate in groups of 5 animals
- Diet: rat/mice diet Altromin 1324 (Altromin-GmbH, Lage/Lippe), ad libitum
- Water: tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days
- Animal identification: fur-marking with KMn04 and cage numbering

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +-2
- Humidity (%): 55 +-10
- lighting time: 12 hours dally

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
deionized water
Duration of treatment / exposure:
Killing time: 24, 48 or 72 hours after administration of test compound and negative control
Killing time: 24 h after administration of positive control
Frequency of treatment:
single treatment
Post exposure period:
Killing time: 24, 48 or 72 hours after administration of test compound and negative control
Killing time: 24 h after administration of positive control
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
500 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2750 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5 males and 5 females in each dose group at each killing time
Control animals:
yes
Positive control(s):
Endoxan(R) (50 mg/kg body weight p.o.)

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
The animals were killed by carbon dioxide asphyxiation 24, 48 or 72 hours after application. For each animal, about 3 ml foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 24 hours.
Evaluation criteria:
1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. The number of polychroma tic erythrocytes with micronucl i occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated stastistically; comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one·sided, increase).

The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired. two sided). All statistical results are based on a 95 % level of significance. Actual data were also compared with historical controls.
Statistics:
Wilcoxon (paired, one·sided, increase). All statistical results are based on a 95 % level of significance.

Results and discussion

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

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The results indicate that, under the conditions of the present study, Acetoacetamid is not mutagenic in the micronucleus test.
Executive summary:

Acetoacetamid was tested in the micronucleus test. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 500, 2750 and 5000 mg Acetocetamid per kg body weight.

5000 mg per kg body weight was chosen since a preliminary study had shown it to be the maximum applicable dose. The animals were treated once with the test compound and were killed 24, 48 or 72 hours after administration.

Endoxan(R) was used as positiv control and was administered orally at a dose of 50 mg per kg body weight.

The incidence of micronucleated polychromatic erythrocytes of the animals treated with Acetoacetamid was within the normal range of the negative control. The number of normochromatic erythrocytes containing micronucuei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with Acetoacetamid and was statistically not different from the control values.

Endoxan(R) induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extend.