4-fluoroaniline

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-07-05 to 1999-07-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

EPA 712-C-98-226, August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

3,5-difluoroaniline

EC Number:

206-752-8

EC Name:

3,5-difluoroaniline

Cas Number:

372-39-4

Molecular formula:

C6H5F2N

IUPAC Name:

3,5-difluoroaniline

Test material form:

other: solid

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd. Biotechnology & Animal Breeding Division, CH-4414 Füllinsdorf
- Age at study initiation: 8-12 weeks
- Weight at study initiation: males 40 g, females 32.1 g
- Assigned to test groups randomly: yes
- Housing: Single, animals were kept conventionally; Cage type: Makrolon Type 1, with wire mesh top; Bedding: granulated soft wood bedding
- Diet: Pelleted standard diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: min. 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +/- 3 °C
- Humidity: 30-78 %
- Photoperiod: Artificial light from 6.00 am to 6.00 pm

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle used: DMSO
- Amount of vehicle: 4 mL/kg bw
- Purity: 99.5%

Frequency of treatment:

Once

Post exposure period:

24, 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
62.5, 125 and 250 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

84 (42 males /42 females)
6 males/ 6 females to each test group

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: Intraperitoneally, once
- Doses: 40 mg/kg bw
- Volume: 10 mL/kg bw

Solution prepared on day of administration

Examinations

Tissues and cell types examined:

Bone marrow cells oft he mouse
2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on the pre-experiment of toxicity maximum tolerated dose estimated to be 250 mg/kg bw .

Pre-Experiment for Toxicity

A preliminary study on acute toxicity was performed with two or five animais per sex and group, respectively, under identical conditions as in the mutagenicity study concerning: animal strain; vehicle; route, frequency of administration, and application volume.
The animals were treated intraperitoneally with the test article and examined for acute toxic symptoms 1h, 6h, 24h, and 48h after treatment.
The pre-experiments were not conducted under GLP-regulations. However, the experimental performance was in accordance to the SOP for pre-experiments. The results will be archived together with the data and materials of the present study.

TREATMENT AND SAMPLING TIMES
Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUTKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed as quotient PCEs/NCEs. The analysis was performed with coded slides. Five animals per group were evaluated as described.

Evaluation criteria:

Acceptance Criteria

The study is considered valid as the following criteria are met:
- the vehicle controls are in the range of our historical control data (0.3 - 2.6 0/00) PCEs with micronuclei.
- the positive controls show statistically significant increased values (the upper range of the historical range of 9.5 - 23.6 0/00 PCEs with micronuclei was slightly exceeded [23.9 0/00]).
- more than 80 % of animals are evaluable

Evaluation of Results
A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test artiele producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test.
However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p <0.05) was evaluated by means ofthe non-parametric Mann-Whitney test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

maximum tolerated dose estimated to be 250 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 200-400 mg/kg bw
- Solubility: in DMSO
- Clinical signs of toxicity in test animals (maximum tolerated dose 250 mg/kg bw): Reduction of spontaneous activity, eyelid closure, apathy, abdominal position, death (two female animals at 48 h)
- Evidence of cytotoxicity in tissue analyzed: No

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE: 62.5 mg/kg bw: 1.14; 125 mg/kg bw: 1.17; 250 mg/kg bw: 1.08
- Statistical evaluation: Mann-Whitney test 250 mg test item at 24 h p=0.8264; 40.00 mg CPA/kg bw at 24 h <0.0001
The mean values of micronuclei observed after treatment with the test item were in the same range as compared to the vehicle control groups and within the laboratory's historical negative control range.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative