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:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well performed GLP and OECD guidline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HOECHST AG breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation: males mean: 27.7 g (25-31g), females mean: 24.0 g (23-26 g)
- Housing: fully air-conditioned, in Macrolon (TN) cages, in groups of 5 animals
- Diet (e.g. ad libitum): rat/mice diet Altromin 1324 (TN), ad libitum
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 10 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
sesame oil
Duration of treatment / exposure:
animals were treated once
animals were killed 24, 48 or 72 hours after administration
Frequency of treatment:
once
Post exposure period:
24, 48 or 72 hours after administration
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1800
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Endoxan (TN) (50 mg/kg body weight p.o.)

Examinations

Tissues and cell types examined:
bone marrow, polychromatic erythrocytes
Details of tissue and slide preparation:
Extraction of the bone marrow:
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 muscl e tissue. The proxi ma 1 ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was centri fuged for 5 minutes at 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a slide and air-dried for about 24 hours.

Staining procedure:
- 5 minutes in methanol
- 3 minutes in May-Grünwalds solution
- 2 minutes in May-Grünwalds solution diluted 1:1 with distilled water
- rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan
Evaluation criteria:
1000 polychromatic erythrocytes were counted for each animal and the number of cells with micronuclei was recorded. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically. Comparison of dose groups with the simultaneous control group was performed according to Wilcoxon.

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. 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, paired, two sided. All statistical results are based on a 95 % level of significance.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
3000 m/kg body weight: lethality, 2000 mg/kg body weight: severe clinical signs
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Oral administration of 1800 mg Nitroanissaure TTR per kg body weight resulted in death of one femal e' out of 30 animal s treated. Thi s animal was repl aced and survived after treatment. The following signs of toxicity were observed: reduced spontaneous activity, narrowed palpebral fissures, uncoordinated gait, ataxic gait, piloerection and stilted gait. 24 hours after application all animals were free of clinical signs of toxicity.

The incidence of micronucleated polychromatic and normochromatic erythrocytes in the dose groups of Nitroanissäure TTR was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes has been observed. The number of normochromatic erythrocytes with micronuclei did not differ from the values of the simultaneous control animals for each of the three killing times investigated. The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound.

Cyclophosphamid induced a marked and statistically significant increase of the number of polychromatic erythrocytes with micronuclei in both males and females indicating the sensitivity of the test system.

Applicant's summary and conclusion

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

Nitroanissäure TTR was tested in the micronucleus test. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0 and 1800 mg Nitroanissaure TTR per kg body weight. The 1800 mg/kg body weight dose level was chosen since a preliminary study had shown it to be the maximum tolerated dose. The animals were treated once with the test compound. The animals were killed 24, 48 or 72 hours after administration of the test compound. Endoxan (TM) was used as positive control substance and was administered orally at a dose of 50 mg per kg body weight. The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with Nitroanissaure TTR and was statistically not different from the control values. Endoxan (TM) 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 extent.