Reaction mass of N-butylphosphorothioic triamide and N-propylphosphorothioic triamide

Endpoint summary

Additional information

In vitro:

Ames test:

The substance LIMUS-Sambaydestillation 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 according to OECD 471 guideline and GLP (BASF, 2008). The following strains were used: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA and tested in the Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S9 mix).

The dose range used was 26 μg - 6 500 μg/plate (SPT) and 406.3 μg - 6 500 μg/plate (PIT). No precipitation of the test substance was found. A bacteriotoxic effect was occasionally observed depending on the strain and the test conditions from 3 250 μg/plate onward. An 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. According to the results of the present study, the test substance LIMUS-Sambaydestillation is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.

Micronucleus Assay:

The substance LIMUS-Sambaydestillation was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) both in the absence and the presence of a metabolizing system based on OECD guideline 487 and GLP (BASF, 2010).

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

1st Experiment

4 hours exposure; 24 hours harvest time; without S9 mix:

0; 362.5; 725.0; 1 450.0; 2 900.0; 4 350.0; 5 800.0 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix:

0; 362.5; 725.0; 1 450.0; 2 900.0; 4 350.0; 5 800.0 μg/mL

2nd Experiment

24 hours exposure, 24 hours harvest time, without S9 mix

0; 250.0; 500.0; 1 000.0; 1 500.0; 2 000 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix

0; 250.0; 500.0; 1 000.0; 2 000.0; 3 000.0 μg/mL

A sample of at least 1 000 cells for each culture were analyzed for micronuclei, i.e. at least 2 000 cells for each test group.

The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to a statistically significant and biologically relevant increase in the number of cells containing micronuclei.

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 in two experiments carried out independently of each other. The single statistically significant outlier value observed in the 2nd Experiment in the presence of metabolic activation after 4 hours treatment with 1 000 μg/mL has to be considered biologically irrelevant due to missing dose-dependency and lacking confirmation in the 1st Experiment at a comparable concentration range.

Thus, under the experimental conditions described, LIMUS-Sambaydestillation 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.

HPRT:

The substance LIMUS-Sambaydestillation 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 according ot OECD 476 guideline and GLP (BASF, 2011). Five independent experiments were carried out 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 an in

vitro micronucleus assay and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested and the doses in bold type were evaluated:

1st Experiment (invalid due to technical error)

without S9 mix (4-hour exposure period)

0; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL

with S9 mix (4-hour exposure period)

0; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL

2nd Experiment

without S9 mix (24-hour exposure period) (failed recommendations of OECD 476)

0; 125.0; 250.0; 500.0; 1 000.0; 2 000.0; 4 000.0; 6 000.0 μg/mL

with S9 mix (4-hour exposure period)

0; 250.0; 500.0; 1 000.0; 2 000.0; 4 000.0; 6 000.0 μg/mL

3rd Experiment

without S9 mix (24-hour exposure period) (failed recommendations of OECD 476)

0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL

4th Experiment

without S9 mix (24-hour exposure period)

0; 7.8; 15.6; 31.3; 62.5; 125.0; 250.0; 500.0; 1 000.0; 2 000.0; 4 000.0 μg/mL

5th Experiment

without S9 mix (4-hour exposure period)

0; 46.9; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL

with S9 mix (4-hour exposure period)

0; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. 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 MCA, led to the expected increase in the frequencies of forward mutations.

Due to a technical error in the 1st Experiment in the absence and presence of S9 mix the

data obtained has to be regarded as invalid and, therefore, they are not reported.

In the 2nd and 3rd Experiment after 24 hours treatment in the absence of metabolic activation the recommendations of the current OECD Guideline 476 were not fulfilled due to excessive cytotoxicity or lacking cytotoxicity, respectively. Therefore, these experimental parts were discontinued.

In all experimental parts assessed as valid regarding the current OECD Guideline 476 and evaluated for gene mutations the highest concentrations applied were clearly cytotoxic.

On the basis from the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in five experiments performed independently of each other.

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

Short description of key information:
Ames test: not mutagenic (BASF, 2008)
in vitro Micronucelus assay: negative (BASF, 2010)
HPRT: negative (BASF, 2011)

Endpoint Conclusion:

Justification for classification or non-classification

Based on the available data, there is no need to classify the substance for genetic toxicity according to Directive 67/548/EEC and CLP.