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Gene mutagenic activity was tested in the Ames test. The study followed the OECD guideline 471 (adopted 1997). The assay was performed in two independent experiments both with and without liver microsomal activation system in S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2uvrA. In experiment I, concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate were used and in experiment II, concentrations of 156, 312, 625, 1250, 2500 and 5000 µg/plate (concentrations related to dry mass). The plates incubated showed normal background growth up to 5000 µg/plate with and without metabolic activation. No substantial increase in revertant colonies in any of the five tester strains was oberserved. Appropriate positive controls showed a distinct increase of induced revertant colonies. Due to the results obtained, Biofert Plusz is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

The mutagenic properties were also tested in the mouse lymphoma assay using the cell line L5178Y in the absence and presence of metabolic activation. The study was perfomed according to OECD Guideline 476 (adopted 1997). The assay was performed in two independent experiments, using two parallel cultures each. Concentrations based on total mass in Experiment I (4 hour treatment with and without S9 mix) and in Experiment II (24 hour treatment without S9 mix) were 139, 278, 556, 1112, 2225 and 4450 µg/mL corresponding to 39, 78,156, 312, 625 and 1250 µg/mL related to dry mass. No substantial and reproducable dose-dependent increase in mutant colony numbers was observed in both main experiments. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies indicating that the tests were sensitive and valid.

In a GLP study according to OECD guideline 473 (adopted 1997) clastogenic and aneugenic properties of Biofert Plusz were investigeated in primary cultures of human lymphocytes with and without metabolic activation at concentrations of 32, 56, 99, 174, 304, 533, 932, 1632, 2857, 5000 µg/mL (total mass; corresponding to 9, 16, 28, 49, 86, 150, 262, 459, 803, 1405 µg/mL, respectively, related to dry mass). The exposure duration was 4 (Experiment I) or 22 h (Experiment II); cultures were harvested 22 h after beginning of the treatment (three hours before harvesting, colcemid was added). Two cultures were studied for each concentration in Experiment I and II. For each culture 100 metaphases were evaluated (200 per concentration). The mitotic index was calculated from at least 1000 cells per culture. Solvent controls and positive controls were valid. In Experiment I, visible precipitation of the test item in the culture medium was observed at 174 µg/mL (total mass) and above in the absence of S9 mix and at 533 µg/mL (total mass) and above in the presence of S9 mix. In addition, precipitation occurred in Experiment II, in the absence of S9 mix, at >= 304 µg/mL (total mass) and at >= 174 µg/mL (total mass) in the presence of S9 mix.

In both experiments, in the absence and presence of S9 mix, no biologicaily relevant increase in the number of cells carrying structural chromosome aberrations was detected. No increase in polyploid metaphases was noted. No cytotoxicity was detected except in Experiment II without S9 mix at 5 mg/mL (total mass). In conclusion, at dose levels up to and above precipitating concentrations no clastogenic or aneugenic activity was induced.


Short description of key information:
The test item did not induce gene mutations in S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2uvrA with and without metabolic activation at concentrations up to 5 mg/plate (related to dry mass).
The test item was not mutagenic in the mouse lymphoma assay using the cell line L5178Y in the absence and presence of metabolic activation and concentrations up to and above the precipitation threshold of 2225 µg/mL (total mass corresponding to 625 µg/mL dry mass).
Biofert Plusz did not induce structurai chromosomal aberrations in human Iymphocytes in vitro when tested with and without metabolic activation at dose levels up to and above precipitating concentrations (varying between 174 and 533 µg/mL related to total mass).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification