Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

GENE MUTATION

The Ames test was performed according to the OECD guideline using 5 bacteria strains (S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A). Following concentrations of the test substance were used: 1st Experiment (standard plate test): 0, 20, 100, 500, 2500 and 5000 μg/plate; 2nd experiment (preincubation test): 0, 312.5, 625, 1250, 2500 and 5 000 μg/plate; 3rd experiment (standard plate test):0, 23, 115, 575, 2875 and 5750 μg/plate; 4th experiment (preincubation test): 0, 23, 115, 575, 2875 and 5750 μg/plate. Water was used as vehicle.

Positive controls used, all dissolved in DMSO were

- with S9 mix: 2-aminoanthracene (2-AA; 2.5 μg/plate, for TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate for E. coli WP2 uvrA);

- Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; 5 μg/plate for TA 1535, TA 100); 4-nitro-o-phenylenediamine (NOPD; 10 μg/plate for TA 98), 9-aminoacridine (AAC; 100 μg/plate for TA 1537 and 4-nitroquinoline-N-oxide (4-NQO; 5 μg/plate for E. coli WP2 uvrA).

The preincubation time was 20 min in the preincubation test followed by 48-72 hours expression duration. In the plate incorporation test the bacteria strains were exposed to the test substance 48-72 hours during the expression time.

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in four experiments carried out independently of each other (standard plate test and preincubation assay).

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above. Thus, under the experimental conditions chosen here, it is concluded that Gelb LD 6259 is not a mutagenic substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

 

CHROMOSOME ABERRATION

Chinese hamster lung fibroblasts (V79) in culture were exposed to the test substance for 4 hours in the presence or absence of S-9 mix. Thereafter, the exposure medium was replaced by MEM supplemented with 10% [v/v] FCS after being rinsed twice with HBSS and subsequently, the Quadriperm dishes were incubated again for the respective recovery time (14 hours) until the cells were harvested (sampling time: 18 hours).

 

2-3 hours prior to cell harvest, 100 μl colcemid (stock: 10 μg/ml colcemid dissolved in PBS [Phosphate Buffered Saline]) was added to each chamber in order to arrest mitosis in the metaphase. Subsequently to spindle inhibition, the culture medium was completely removed. The cells were then fixed with 5 ml of fixative (methanol:glacial acetic acid, ratio 3:1; +4°C), mounted on slides, dried, and the slides were stained with 7.5% (v/v) Giemsa/Titrisol solution pH 7.2 for 10 minutes. After being rinsed twice in purified water and clarified in xylene, the slides were mounted in Corbit-Balsam.

A sample of 100 metaphases for each culture were analyzed for chromosomal aberrations, except for the positive control cultures and the two highest scored concentrations in the presence of S9 mix were only 50 metaphases were scored due to clearly increased aberration rates.

 

The test substance led to a statistically significant and biologically relevant increase in the number of structural chromosomal aberrations incl. and excl. gaps after the addition of a metabolizing system. At all concentrations (156.25, 312.5 and 625 μg/ml) scored for cytogenetic damage aberration rates (6.5%, 21.0% and 26.0% aberrant metaphases, exclusive gaps, respectively) exceeding our historical negative control data range (0.0% - 5.5% aberrant metaphases, exclusive gaps) were observed. Due to this clear dose-dependent increase in the number of structurally aberrant metaphases, a 2nd Experiment for further substantiation of the data was not carried out.

The structural chromosome aberration rates of the vehicle control groups were within the historical negative control data range and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of structural chromosome aberrations induced by the positive control substances and CPP clearly demonstrated the sensitivity of the test system and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. Thus, under the experimental conditions chosen here, the conclusion is drawn that Gelb LD 6259 is a chromosome-damaging (clastogenic) substance under in vitro conditions using V79 cells in the presence of metabolic activation.

Short description of key information:
Two in vitro studies are available, including an Ames test (BASF AG, 2009; report No. 40M0646/084225) performed according to the OECD Guideline 471 (Bacterial Reverse Mutation Assay) and one in vitro mammalian chromosome aberration test (BASF AG, 2009; report No. 32M0646/084227). While Gelb LD 6259 is not a mutagenic substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation, it is a chromosome-damaging (clastogenic) substance under in vitro conditions using V79 cells in the presence of metabolic activation.

Endpoint Conclusion:

Justification for classification or non-classification