Phosphorodithioic acid, O,O-bis(2-methylpropyl) ester, compd. with N,N-dimethylmethanamine (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 June - 12 August 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

O,O-bis(2-methylpropyl) sulfanidylphosphonothioate; trimethylazanium

EC Number:

700-910-4

Cas Number:

1354201-99-2

Molecular formula:

C11H28NO2PS2

IUPAC Name:

O,O-bis(2-methylpropyl) sulfanidylphosphonothioate; trimethylazanium

Test material form:

solid

Remarks:

pale yellow glassy solid

Specific details on test material used for the study:

Name of test material (as cited in study report): S-10713
- Physical state: solid
- Colour: brown
- Lot/batch No.: S20227-179
- Storage condition of test material: at room temperature in the dark

Method

Target gene:

Salmonella typhimurium: histidine gene
Escherichia coli: tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : in-house
- method of preparation of S9 mix: rats induced with phenobarbitone/beta-naphthoflavone at 80/100 mg/kg orally for 3 days.
- concentration or volume of S9 mix and S9 in the final culture medium: 10% S9 in S9 mix, 20% S9 mix in culture medium
- quality controls of S9: each batch tested for its capability to activate known mutagens

Test concentrations with justification for top dose:

Preliminary study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1 and 2: 0, 50, 150, 500, 1500 and 5000 µg/plate (with and without S9-mix)

Vehicle / solvent:

dimethylsulphoxide; the substance was insoluble in sterile distilled water at 50 mg/ml, but was fully soluble in dimethylsulphoxide at the same concentration in solubility checks performed in-house.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation for preliminary test and experiment 1; pre-incubation for experiment 2

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 0.9 to 9*10E9 per plate

DETERMINATION OF CYTOTOXICITY
- Method: Effects on the bacterial background lawn, and reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

Any, one, or all of the following are considered to give a positive result to this study:
1. a dose-related increase in mutant frequency over the dose range tested
2. a reproducible increase at one or more concentrations
3. biological relevance against in-house historical control ranges
4. statistical analysis fo data as determined by UKEMS (Mahon et al 1989)
5. fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response)
A substance is considered negative if the above criteria are not met.

Statistics:

Not applicable.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 98, 100, 1535, 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

PRELIMINARY STUDY/EXPERIMENT 1 and 2:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

The substance was not mutagenic under the conditions of this test.

Executive summary:

The test material was tested in the Salmonella typhymurium reverse mutation assay with TA1535, TA1537, TA100 and TA98 and in the Escherichia coli reverse mutation assay with WP2uvrA, in accordance with OECD 471 guideline and GLP principles.

In both mutation assays, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

No increase in the number of revertants was observed upon treatment up to concentrations of 5000 µg/plate. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhymurium reverse mutation assay and the Escherichia coli reverse mutation assay.