Brightener 0503E

Endpoint summary

Additional information

1. Genetic toxicity in vitro testing:

The mutagenic effects of the test item were determined in a reverse mutation assay with Salmonella typhimurium. Test systems were the strains TA 97a, TA 98, TA 100., TA 102, and TA 1535 with (+) and without (-) the metabolic activation system S9 (from male Wistar rats). Concentrations tested were 0.003 - 0.01 - 0.1- 0.333 - 1.0 - 2.5 – 5.0 mg/plate. Three replicates per concentration level and control were performed. Based on the results of this study the test item was found to have no mutagenic effects on Salmonella typhimurium strains TA 1535 , TA 1537, TA 98 and TA 100, and the Escherichia strain WP2 uvrA with (+) and without (-) the metabolic activation system S9 from Wistar rats at concentrations up to 5.0 mg/plate.

The potential of the test item to induce chromosome aberrations was investigated in V79 Chinese hamster lung fibroblasts (V79) in vitro. For each experiment duplicate cell cultures were used for each concentration. The test compound was tested at concentrations of 46.9 to 2850 µg/mL including solvent controls with (+) and without (-) metabolic activation system from Spraque Dawley-rat liver. The test substance registered was clastogenic in V79 cells without S9 mix at the 28 hrs preparation interval, whereas in all other parts of the experiment no clastogenicity was observed. The observed increase of the aberration rate under this experimental conditions (28 hrs preparation interval without S9 mix) appeared together with an increase of cytotoxicity at these dose levels. It is striking, that the aberration rate increased with increasing cytotoxicity, indicating that clastogenicity correlated with cytotoxicity. As it has been shown for many chemicals that positive results are often irrelevant when correlated with cytotoxcity and that such chemicals are not genotoxic in vivo, the relevance of the present result with the test substance registered for the in vivo situation is questionable. Furthermore, in two in vivo mutagenicity tests (i.e. the in vivo micronucleus test and the Comet assay in rat liver and intestine cells with the test substance registered) it is proofed that the test substance is not genotoxic in vivo. Thus, the result of the chromosomal aberration study can be regarded as an irrelevant positive finding .

2. Genetic toxicity in vivo testing:

Two in vivo genetic toxicity tests, the in vivo micronucleus test and the Comet assay in rat liver and intestine cells, were investigated with the test substance of interest.

The potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse was assessed in the micronucleus assay (OECD 474). The test substance registered was administered once orally to 12 mice per test group (6 per sex) at dose levels of 500, 1000 and 2000 mg/kg bw for the 24 hour preparation interval. A dose level of 2000 mg/kg bw was investigated for the 48 hour interval. The mean number of polychromatic erythrocytes was not decreased after treatment with the test item which indicates that the test substance did not have any cytotoxic properties in the bone marrow. In comparison to the respective controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of the micronuclei detected were below or near the value of the vehicle control. In parallel the treatment with the positive control (cyclophospahamide) showed a statistically significant increase of induced micronucleus frequency. Based on the experimental conditions reported the oral application of the substance to mice did not induce micronuclei as determined by the micronucleus test in the bone marrow cells (polychromatic erythrocytes).

In a second in vivo test, the test item was assessed in the in vivo alkaline single cell gel electrophoresis analysis (i.e. Comet assay) for its potential to induce primary DNA damage in cells prepared from the liver and small intestine of test substance treated Wistar rats. For the Comet assay no internationally accepted guideline is available. This study was conducted according to the procedure indicated by the recommendations of Tice, R.R. et al. (2000) and Hartmann, A. et al. (2003). Single oral administration of 1000 and 2000 mg test item/ kg b.w. did not induce any DNA-damage in the in vivo Comet assay performed in primary liver cells and cells from the small intestine isolated 4 or 16 hours after oral administration of the test item.For each dose group and at each treatment time 100 cells were evaluated for primary DNAdamage. None of the tested dose levels revealed a biologically relevant or statistically significant increase in DNA damage compared to corresponding controls. In addition, all % tail intensities of the test item treated animals were within the historical control data range for the hepatocytes and even below the historical control range for the small intestine. Based on the experimental conditions reported, the test substance registered is considered to be non-genotoxic in this in vivoalkaline single cell gel electrophoresis assay.

Short description of key information:
Results from different in vitro (2) and in vivo (2) test systems with test substance registered are available:
in vitro tests:
1. Ames test according to OECD 471; result: negative (+/-) metabolic activation system.
2. Chromosome aberration test according to OECD 473; result: negative with metabolic activation system, positive without metabolic system at the 28h interval at concentrations with increasing cytotoxicity.
in vivo tests:
1. Mouse micronucleus test (OECD 474); result: negative
2. In vivo Comet assay in rat liver and small intestine cells (no internat. Guideline available); result negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test item was found to have no mutagenic effects on Salmonella typhimurium strains TA 1535 , TA 1537, TA 98 and TA 100, and the Escherichia strain WP2 uvrA with (+) and without (-) the metabolic activation system S9 from Wistar rats at concentrations up to 5.0 mg/plate. In the chromosome aberration test the test substance registered was clastogenic in V79 cells without S9 mix at the 28 hrs preparation interval, whereas in all other parts of the experiment no clastogenicity was observed. It is striking, that the aberration rate increased with increasing cytotoxicity, indicating that clastogenicity correlated with cytotoxicity. As it has been shown for many chemicals that positive results are often irrelevant when correlated with cytotoxcity and that such chemicals are not genotoxic in vivo, the relevance of the present result for the in vivo situation is questionable. Furthermore, in two in vivo mutagenicity tests (i.e. the in vivo micronucleus test and the Comet assay in rat liver and intestine cells with the test substance registered) it is proofed that the test substance registered is not genotoxic in vivo. Thus, the result of the chromosomal aberration study can be regarded as an irrelevant positive finding. In conclusion, mostly negative results, particularly in in vivo mammalian studies. The compound is not considered a genotoxin of relevance for humans.