Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
The Ames test (in vitro) showed mutagenic effects in TA98 and TA1538 tester strains in the presence of metabolic activation, whereas the micronucleus test (in vivo) showed no mutagenic effects.
Link to relevant study records
Reference
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
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well performed GLP and OECD guideline study following a previous guideline version
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Principles of method if other than guideline:
Study was performed according to a previous guideline version. Following this protocol only a single dose was tested in the main study. This test dose was accurately determined in a dose range finding study in order to find the maximum dose producing distinct toxicity but no lethality.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain specifics: NMRK f (SPF71)
- Source: Hoechst AG, breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation: males: 25 - 33 g, mean 29.8 g, females: 22 - 27 g, mean 24.4 g
- Housing: in groups of five in Macrolon type 3 cages in fully air-conditioned room, softwood granulate
- Diet (e.g. ad libitum): rat/mice diet (Altromin 1324), 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 ± 3 °C
- Humidity (%): 55 ± 10 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: starch mucilage
- Concentration of test material in vehicle: 22 % (w/v)
- Amount of vehicle (if gavage or dermal): 10 ml/kg body weight
Details on exposure:
The test compound was suspended in starch mucilage and dosed once orally at 2200 mg per kg bw to male and female mice, upon the results of the previously conducted dose range finding assay
Duration of treatment / exposure:
treatment: once orally at 2200 mg per kg bw
exposure: animals were killed 24, 48 or 72 h after administration
positive control: 24 h
Frequency of treatment:
once
Remarks:
Doses / Concentrations:
2200
Basis:
nominal conc.
No. of animals per sex per dose:
5 males and 5 females per dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
50 mg/kg body weight cyclophosphamide (Endoxan (R), 5 males and 5 females); dissolved in distilled water 0.5 % (w/v)
Tissues and cell types examined:
polychromatic and normochromatic erythrocytes from the femoral bone marrow of each animal
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 micronuelei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. The number of polychromatic erythrocytes with micronucIei 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 (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) and Wilcoxon (paired, two sided).
All statistical results are based on a 95 % level of significance.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
All animals survived after application of 2200 mg per kg bw. The following signs of toxicity were observed: reduced spontaneous activity, uncoordinated gait, urine orange coloured.

48 h after application most animals were free of clinical signs of toxicity.

The dissection of the animals revealed the following macroscopic findings: content of the stomach orange-yellow coloured, muscle and hypodermis yellow discoloured.
Conclusions:
Interpretation of results (migrated information): negative
Oral administration of the test item did not lead to a substantial increase of micronucleated polychromatic and normochromatic erythocytes and was not mutagenic in the in vivo micronucleus test
Executive summary:

The test item was tested in the micronucleus test according to OECD 474. The substance was suspended in starch mucilage and dosed once orally at 2200 mg per kg bodyweight to male and female rats, upon the results of the previously conducted dose range finding assay. The animals were killed 24, 48 and 72 h after administration.

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 the test item and was statistically not different from the control values.

EndoxanRwas used as positive control substance and was administered orally at a dose of 50 mg per kg bodyweight.

EndoxanR induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity af the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extent.

The results indicate that, under the conditions of the present study, the test item is not mutagenic in the micronucleus test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Justification for selection of genetic toxicity endpoint
micronucleus test in vivo

Justification for classification or non-classification

Available genetic toxicity reports (Ames test, Micronucleus in vivo) presented in this IUCLID are not sufficient to classify the substance 4-chlor-2 -nitroaniline as mutagenic according to 67/548/EEC, (EC) No 1272/2008 respectively.

Results of a Micronucleus test in vivo do not exhibit mutagenic effects for this substance.