4-fluoroaniline

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In the bacterial reverse mutation tests with different strains, the obtained results were ambiguous. Based on the results of the test substance and the Read-across substance 3,5 -Difluoroaniline (CAS 372 -39 -4) it can be stated the test substance is non-mutagenic.

Genetic toxicity in vivo

Description of key information

In the in vivo Micronucleus assay, the test item(Read-across substance 3,5 -Difluoroaniline (CAS 372 -39 -4)) was determined to be not mutagenic. In the in vitro Mouse lymphoma assay, the test item (Read-across substance 3,5 -Difluoroaniline (CAS 372 -39 -4)) was determined to be not mutagenic. This can be supported by the unscheduled DNA synthesis (UDS) test with hepatocytes, where the test item was determined to be not mutagenic. In the bacterial reverse mutation tests with different strains, the obtained results were ambiguous. Based on the results of the test substance and the Read-across substance 3,5 -Difluoroaniline (CAS 372 -39 -4) it can be stated the test substance is non-mutagenic.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted July 21, 1997

Deviations:

no

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

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

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

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

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.

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.

Conclusions:

Interpretation of results (migrated information): negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

For this endpoint partially a Read-across with the structural analogue substance 3,5 -Difluoroaniline (CAS 372 -39 -4) was used. For further justification see IUCLID section 13.

   

Key study

BASF SE, Study No.: 634102, 2000

Genetic toxicity in vivo - Micronucleus assay

 

Read-across approach to structural analogue substance 3,5 -Difluoroaniline (CAS 372 -39 -4) was used. This study was performed GLP compliant and according to the OECD 474 and EPA 870.5395 to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of mice.

The test item was formulated in Dimethylsulfoxide (DMSO). DMSO was used as vehicle control. The volume administered intraperitoneally was 4 mL/kg bw. 24 and 48 hours after a single intraperitoneal administration of the test article the bone marrow cells were collected for micronuclei analysis.

 

Ten animals per test group (exception was test article group 250 mg/kg bw at preparation interval 48 h in which due to lethalities only 4 females could be scored) were evaluated for the occurrence of micronuclei.

2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes (NCEs) was determined in the same sample.

 

The following dose levels of the test item were investigated:

24 h preparation interval: 62.5,125, and 250 mg/kg bw

48 h preparation interval: 250 mg/kg bw

The highest dose applied (250 mg/kg bw) was estimated by pre-experiments to be close to the maximum tolerated dose. The animals expressed toxic reactions. In the main study 2 out of 12 females treated with 250 mg/kg bw test item died.

 

After treatment with the test item the numbers of NCEs were not substantially increased as compared to the corresponding vehicle controls thus indicating that the test item had no cytotoxic effect in the bone marrow.

In comparison to the corresponding vehicle controls there was no significant or biologically relevant enhancement in the frequency of the detected micronuclei.

40 mg/kg bw Cyclophosphamide administered intraperitoneally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

 

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.

 

 

Key study

BASF SE, Study No.: 634103, 2000

Genetic toxicity in vitro - Mouse lymphoma assay

 

Read-across approach to structural analogue substance 3,5 -Difluoroaniline (CAS 372 -39 -4) was used.

The GLP and guideline compliant study was performed to investigate the potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y (microtiter version).

The assay was performed in two independent experiments, using two parallel cultures. Both main experiments were performed with and without liver microsomal activation.

The first experiment was performed with a treatment period of 4 hours (with and without S9 mix). In the second experiment the treatment duration was 24 hours without and 4 hours with metabolic activation.

The test substance was evaluated at the following concentrations:

Experiment I:

without S9 mix: 40.6, 81.3; 162.5; 325.0 and 650.0 µg/mL

with S9 mix: 20.3; 40.6; 81.3; 162.5 and325.0 µg/mL

 

Experiment II:

without S9 mix: 40 6; 81.3; 162.5; 325.0 and 650.0 µg/mL

with S9 mix: 25.0, 50.0, 100.0; 200.0 and 400.0 µg/mL

 

According to the pre-experiment on toxicity the concentration range was selected. After 4 hour treatment strong toxic effects were observed at 325.0 µg/mL and above in the absence and at 81.3 µg/mL and above in the presence of S9 mix. After 24 hours of treatment relevant toxic effects occurred at 650 µg/mL and above. No precipitation was observed up to the maximum concentration tested.

In experiment I, strong toxic effects occurred at 650 µg/mL in the absence and 81.3 µg/mL and above in both parallel cultures in the presence of metabolic activation (4 h treatment).

Strong toxic effects were observed in the second experiment at 650 µg/mL without metabolic activation (continuous treatment for 24 h) and at 25 µg/mL and above in both parallel cultures with metabolic activation (4 h treatment).

 

No relevant increase in the number of mutant colonies was observed in the first experiment and the threshold of twice the colony count of the corresponding solvent control was not exceeded at any concentration showing acceptable toxicity (relative total growth > 10%). In the second experiment without metabolic activation (24 h treatment) a weak increase in the colony count exceeding the threshold of twice the colony number of the corresponding solvent control occurred in one of the parallel cultures at the maximum concentration of 650 µg/mL showing very strong toxic effects (relative total growth of 14 %). The colony count of the second parallel culture did not exceed this threshold at the same concentration showing less severe toxic effects (relative total growth of 30.8 %). In the presence of metabolic activation exceedingly strong toxic effects reducing the relative total growth down to values below 10 % were already observed at 100 µg/mL. The colony count exceeded the threshold only at those extremely toxic concentrations.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large colonies.

 

In conclusion it can be stated that under the experimental conditions reported the test substance did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the presence and absence of metabolic activation at concentrations leading to acceptable toxic effects.

Therefore, the test item is considered to be non-mutagenic in this mouse lymphoma thymidine kinase locus assay.

 

 

Mutagenicity tests in bacterial systems

 

A weight of evidence approach was conducted for the results of the bacterial reverse mutation tests, UDS and a fluctuation test.

 

Thompson et al. (1983) reported that in a modified Ames gradient plate test with several S. typhimurium and E. coli strains, the test item was determined to be not mutagenic.

 

In Zimmer et al. (1980) a bacterial reverse mutation test with Salmonella typhimurium TA100, TA1537 and TA98 was conducted. The item was mutagenic with metabolic activation in the test strain TA 98 only.

 

In Topham (1980) the test item was determined to be not mutagenic in the test strain TA1535 without metabolic activation. The test item was positive with metabolic activation when Phenobarbitone was used and negative when Aroclor was used as inducing agent.

 

Furthermore, in Pai et al. (1985), a fluctuation test where the score for turbidity is indicating the presence of mutagenicity with E. coli WP2 uvrA was reported. The test item was determined to be not mutagenic without metabolic activation in this test.

 

Unscheduled DNA synthesis (UDS) test

Thompson et al. (1983) reported that the test item is non- mutagenic in the UDS test in hepatocytes.

 

Conclusion:

 

The bacterial reverse mutation tests and fluctuation test revealed different results so that it is inconclusive to make a final decision regarding bacterial mutagenicity.

 

 

Overall conclusion:

 

The bacterial reverse mutation tests and fluctuation test revealed different results In most cases the test system is not completely in line with current requirements, so that it is inconclusive to make a final decision regarding bacterial mutagenicity.

Nevertheless, these studies are quite old literature results and neither guideline no GLP compliant.

 

In contrast, there are new studies available with structure analogue test items which are state of the art (GLP and guideline compliant).

In the in vitro Mouse lymphoma assay and in the in vivo Micronucleus assay in bone morrow cells, the test item was determined to be negative.

This can be also supported by the unscheduled DNA synthesis test (UDS) with hepatocytes reported in Thompson et al. (1983), where the test item is non-mutagenic.

These data in mammalian cells or in vivo show that the inconclusive results obtained in bacterial systems are not biologically relevant.

 

Based on these results it can be stated that the test item is not critical regarding mutagenicity and therefore is not considered to be classified for mutagenicity.

 

Justification for selection of genetic toxicity endpoint
GLP and guideline compliant study. The test substance was a structural analogue substance, please refer to IUCLID section 13 for read across justification.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)
The available study is considered reliable and suitable for classification purposes under Directive 67/548/EEC. As a result the substance is not considered to be classified for mutagenic toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.

 

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for mutagenic toxicity under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation (EC) No 605/2014.