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Ames-Test:

The test substance LUGALVAN BPC dry was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The study was conducted according to OECD 471 guideline and GLP (BASF SE, 2013).

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5 200 μg/plate (SPT); 33 μg - 5 200 μg/plate (SPT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: No precipitation of the test substance was observed either with or without S9 mix.

TOXICITY: No bacteriotoxic effect was observed under all test conditions.

MUTAGENICITY:

A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

CONCLUSION:

Thus, under the experimental conditions of this study, the test substance LUGALVAN BPC dry is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

HPRT-Test

The substance LUGALVAN BPC dry was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. The study was conducted according to OECD 476 guideline and GLP (BASF SE, 2013).

Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following doses were tested. Test groups printed in bold type were evaluated in this study:

1st Experiment without S9 mix 0; 181.3; 362.5; 725.0; 1 450.0; 2 900.0 μg/mL with S9 mix 0; 181.3; 362.5; 725.0; 1 450.0; 2 900.0 μg/mL 2nd Experiment without S9 mix 0; 500.0; 1 000.0; 2 000.0; 2 900.0 μg/mL with S9 mix 0; 500.0; 1 000.0; 2 000.0; 2 900.0 μg/mL.

Following attachment of the cells for 20-24 hours, cells were treated with the test substance for 4 hours in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. Subsequently, cells were cultured for 6-8 days and then selected in 6- thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line.

Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations.

In this study in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest required concentration evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance LUGALVAN BPC dry is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

In vitro Micronucleus Assay:

The substancec LUGALVAN BPC dry was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) in a study according to OECD 487 guideline and GLP (BASF SE, 2013). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation).

Based on the observations and the toxicity data of a previously performed pretest for a HPRT study the following concentrations were tested. The test groups printed in bold type were evaluated.

1st Experiment

4 hours exposure; 24 hours harvest time; without S9 mix:        
0
; 90.63; 181.25; 362.50;725.00;1450.00;2900.00 µg/mL

4 hours exposure, 24 hours harvest time, with S9 mix:   
0
; 90.63; 181.25; 362.50;725.00;1450.00;2900.00 µg/mL

2nd Experiment

24 hours exposure, 24 hours harvest time, without S9 mix        
0
; 181.25; 362.50;725.00;1450.00;2900.00 µg/mL

4 hours exposure, 24 hours harvest time, with S9 mix     
0
; 362.50; 725.00;1 450.00;2175.00;2900.00 µg/mL

A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group.

The negative controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances,and cyclophos­phamide, led to the expected increase in the number of cells containing micronuclei.

Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC) was observed at the highest applied test substance concentration after 24 hours continuous treatment without metabolic activation only.

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Thus, under the experimental conditions described, LUGALVAN BPC dry is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.


Short description of key information:
Ames-Test (BASF, 2013): negative
HPRT-Test (BASF, 2013): negative
in vitro Micronucleus Assay (BASF, 2013): negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, no classification and labeling is required (according to Directive 67/548/EEC and according to CLP) for genetic toxicity.

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