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GENETIC TOXICITY IN VITRO

1- Gene mutation in bacteria:

The mutagenic activity of sodium hypophosphite was assessed in the Ames test performed in five strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) according to the OECD guideline 471 and the EU Method B13/14. The study was in compliance with the Principles of Good Laboratory Practice. Sodium hypophosphite was tested in two independent experiments, with and without a metabolic activation system, both performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Bacteria were exposed to sodium hypophosphite at five dose-levels (three plates/dose-level) selected from a preliminary toxicity test. In the first experiment, the dose-levels ranged from 312.5 to 5000 μg/plate for all strains, with and without S9 mix. In the second experiment, the dose-levels ranged from 625 to 5000 µg/plate for all strains, with and without S9 mix. After 48 to 72 hours of incubation, the revertant colonies were scored.

The numbers of revertants for the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered valid. Sodium hypophosphite did not induce any significant increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Sodium hypophosphite was considered to be non-mutagenic under the conditions of this test.

2- Chromosomal aberrations in mammalian cells:

The genotoxic activity of sodium hypophosphite was assessed in cultured human lymphocytes according to OECD guideline 473 and EU Method B.10. The study was in compliance with the Principles of Good Laboratory Practice. Sodium hypophosphite was tested in two independent experiments, with and without a metabolic activation system, together with vehicle and positive controls. In the first experiment, lymphocytes cultures were exposed to sodium hypophosphite or control items for 3 hours without or with S9 mix and cells were harvested 20 hours after the beginning of treatment. In the second experiment, treatment in absence of S9 mix was continuous until harvest whilst the cultured cells were exposed for 3 hours to sodium hypophosphite in the presence of S9 mix. Cells were harvested 20 and 44 hours after the beginning of the treatment.

The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Sodium hypophosphite did not exhibit significant cytotoxicity and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range from 19.53 to 2500 µg/ml, the maximal practicable concentration regarding the osmolality of the culture medium.

Sodium hypophosphite was considered to be non-clastogenic to human lymphocytes in vitro.

3- Gene mutation in mammalian cells:

The mutagenic activity of sodium hypophosphite was assessed in L5178Y TK+/- mouse lymphoma cells according to the OECD guideline 476 and the EU Method B17. The study was in compliance with the Principles of Good Laboratory Practice. Sodium hypophosphite was tested in two independent experiments, with and without a metabolic activation system, together with vehicle and positive controls. As positive control methylmethanesulfonate was used in experiments without S9 mix and cyclophosphamide in the experiments with S9 mix. Cells as suspension cultures were exposed to sodium hypophosphite at six dose-levels (two cultures/dose-level) selected from a preliminary toxicity test. For both experiments the dose-levels ranged from 78.13 to 2500 µg/mL for all lymphoma cell cultures, with and without S9-mix. In the first experiment lymphoma cell cultures were exposed to sodium hypophosphite or control items for 3 hours without or with S9 mix. In the second experiment lymphoma cell cultures were exposed to sodium hypophosphite or control items for 3 hours in the presence of S9 mix and for 24 hours in the absence of S9 mix. In both experiments cells were harvested at the end of the treatment period and to enable the expression of the mutant genotype were incubated for 48 hours at 37°C in a humidified atmosphere. Afterwards cells were plated with trifluorothymidine as selection agent.

The cloning efficiency and mutation frequency of the vehicle controls and the increase in the mutation frequency of the positive controls were as specified in the acceptance criteria. The study was therefore considered valid. Sodium hypophosphite did not exhibit significant cytotoxicity and no noteworthy increase in the mutation frequency in either of the experiments, both without and with S9 mix, at any dose-level.

Sodium hypophosphite was considered to be non-mutagenic to mouse lymphoma cells in vitro.

Short description of key information:
The genetic toxicity of sodium hypophosphite was assessed in 3 in vitro studies conducted in compliance with the principles of Good Laboratory Practices:
- a gene mutation assay on Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 (OECD guideline 471)
- an in vitro chromosomal aberration assay in cultured human lymphocytes (OECD guideline 473)
- an in vitro gene mutation assay in mouse lymphoma L5178Y cells (OECD guideline 476)
In all three studies negative results were reported in the presence and absence of metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to the criteria laid down in EU regulation (EC) n° 1272/2008 (CLP) and the EU directive 67/548/EEC, sodium hypophosphite is not classified for genetic toxicity as all in vitro mutagenicity assays are negative.