4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline)

Endpoint summary

Additional information

The in-vitro genetic toxicity of 4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline) (CAS 107934-68-9) was assessed in a bacterial reverse mutation assay (Ames test) (Riccio, 1988). The study was performed equivalent to OECD 471, but without the required TA 102 or E. coli strain and for the characterisation of S9-mix only 2-aminoanthracene was used. The plate incorporation method was applied using S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 at concentrations up to 5000 µg/plate with and without metabolic activation. The test substance did not induce reversions in any of the S. typhimurium strains with or without metabolic activation. Cytotoxicity was observed at 5000 µg/plate without metabolic activation in the first experiment with TA 1537. All the positive controls were valid. Precipitation was seen from 500µg/plate and upwards, for 1000 and 5000 µg/plate it interfered with counting of colonies wherefore plates were handcounted at those concentrations.

In addition to the bacterial reverse mutation assay a test in saccharomyces cerevisae equivalent to OECD 480 was performed with 4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline). Saccharomyces cerevisae D3 was tested with 0.05, 0.1, 0.5, 1.0, 2.5% (w/v) in the presence and absence of metabolic activation. The cells from the overnight culture were preincubated with the test material for 4 h at 30°C. After pre-incubation serial dilutions of the mixture were plated on agar plates for 3 days at 30°C and 1 day at 4°C. Thereafter the plates were scanned with a microscope and the red colonies, indicative of adenine-deficient homozygosity, were counted. The positive controls (-S9: 1,2,3,4 -diepoxybutane; +S9: sterigmatocystin) were valid. No genotoxic and cytotoxic effects were observed in the experiments with the negative control and the test substance.

The potential of 4,4'-(9H-fluoren-9-ylidene)bis(2 -chloroaniline) (CAS 107934-68-9) to induce chromosomal aberrations was assessed using peripheral human lymphocytes, in a study performed according to OECD 473 (Adams, 1991). The lymphocytes were exposed to 3.1, 12.5, and 30 µg/mL (without S9-mix, 24 h treatment) or 12.5, 50, and 100 µg/mL (with S9-mix, 3 h treatment) test substance diluted in DMSO. The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, with or without metabolic activation. Reduction of mitotic index to 23.9% of controls was seen at 30 µg/mL in the absence of S9 mix. Precipitation was observed at concentrations from 100 µg/mL, while no cytotoxicity was noted at any concentration. The vehicle and positive controls were valid.

An in vitro mammalian cell gene mutation assay was performed using 4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline) (CAS 107934-68-9), according to OECD 476 (Adams, 1991). Mouse Lymphoma cells were treated with the test substance at concentrations of up to 125 µg/mL for 3 h both with and without metabolic activation. Duplicate cultures were used in 2-3 independent experiments each, in the absence or presence of S9-mix, respectively. In the first experiment no increase of the induced mutant frequency (IMF) exceeding the Global Evaluation Factor of 90 (GEF) was observed up to 125 µg/mL and a relative survival of 8% in the absence of S9-mix. In a second experiment two concentrations of 50 and 87.5 µg/mL were included for a more narrow distribution at higher concentrations, yielding a IMF of 94 and 133, respectively. As both concentrations led to a relative survival <10% a third test including 40 µg/mL was performed. A IMF of 87 and a relative survival of 9% was observed after treatment with 40 µg/mL. In all 3 experiments 25 µg/mL was the highest concentration tested yielding a relative survival >10%. IMF values of 18, 65, and 49 were observed after treatment with 40 µg/mL with a relative survival of 14%, 22%, and 13%, respectively.

In the presence of S9-mix no increase of the IMF over the GEF was observed up to concentrations of 125 µg/mL and down to a relative survival of 14%.

In summary, no mutagenic effects were observed in the presence or absence of S9-mix. IMF exceeding the GEF were only observed in the presence of cytotoxicity (relative survival <10%) and in the absence of S9 -mix. Testing in the range of 10 -100% relative survival revealed no positive results. Therefore, the evaluation criteria for a positive result were not fulfilled.

In conclusion,4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline) is considered to be not genotoxic based on the negative results in an Ames test and Saccharomyces cerevisae gene mutation test as well as in a chromosome aberration test and mouse lymphoma assay. 

Justification for selection of genetic toxicity endpoint
No study was selected, since all available in vitro genetic toxicity studies were negative.

Short description of key information:
Gene mutation in bacteria (similar to OECD 471, Ames test with S. typhimurium TA 100, TA 1535, TA 98, TA 1537): negative with and without metabolic activation
Gene mutation in yeast (similar to OECD 480, S. cerevisiae): negative with and without metabolic activation
Chromosome aberration in mammalian cells (similar to OECD 473): negative with and without metabolic activation
Gene mutation in mammalian cells (according to OECD 476): negative with and without metabolic activation

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the negative results in in-vitro gene mutation assays in yeast, bacteriae, and mammalian cells and the lack of clastogenic properties in a chromosome aberration assay, 4,4´-(9H-fluoren-9 -ylidene)bis(2 -chloroaniline) does not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and is therefore conclusive but not sufficient for classification.