O,O,O',O'-tetraoctyl-(branched and linear) S,S'-{methylenebis[imino(3-oxopropane-3,1-diyl)]} bis(phosphorothioate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June 2001 and 16 August 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study done according to OECD 471 guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Reaction product of methylenebis(acrylamide) and (O,O)-diisooctyl dithiophosphoric acid

EC Number:

700-945-5

Molecular formula:

Complex UVCB substance

IUPAC Name:

Reaction product of methylenebis(acrylamide) and (O,O)-diisooctyl dithiophosphoric acid

Test material form:

liquid: viscous

Details on test material:

- Physical state: pale yellow viscous liquid
- Analytical purity: 92%
- Storage condition of test material: room temprature in the dark

Method

Target gene:

The mutant strains of Salmonella with a deletion through the excision repair gene (uvrB- Salmonella strains) which renders the organism incapable of DNA excision repair and deep rough mutation (rfa), are incapable of synthesising histidine, and may undergo a reverse mutation to histidine independent forms.
A mutant strain of Escherichia coli (WP2uvrA-, a deletion in an excision repair gene uvrA) requires tryptophan and which can be reverse mutated by base substitution to tryptophan independence.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta-naphthoflavone induced S9

Test concentrations with justification for top dose:

0, 50, 150, 500, 1500 and 5000 ug/plate

Vehicle / solvent:

dimethylsulphoxide (DMSO)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

2 ug/plate for WP2uvrA-, 3 ug/plate for TA100, 5 ug/plate for TA1535

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 ug/plate for TA1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2 ug/plate for TA98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: 5 ug/plate for TA98

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

1ug/plate for TA100, 2 ug/plate for TA1535 and TA1537, and 10 ug/plate for WP2uvrA-

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate plates per dose level

DETERMINATION OF CYTOTOXICITY
- Method: numbers of revertant colonies

OTHER:
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgment about the test material activity. Results of this type will be reported as equivocal.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative 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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

1. Preliminary Toxicity Test:

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

 The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9­mix	Strain	Dose (mg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	157	156	157	173	181	170	173	181	173	167	189P
+	TA100	164	150	195	153	138	139	155	177	159	177	187P
-	WP2uvrA"	28	24	21	22	34	25	29	23	18	21	26P
+	WP2uvrA"	37	29	18	25	23	24	29	26	25	24	31P

2. Mutation Test

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The

S9-mix used in both experiments of the main study was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Study.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5 with the results also expressed graphically in Figure 1 to Figure4.

Information regarding the equipment and methods used in these experiments as required by the

Japanese Ministry of Labour, Japanese Ministry of International Trade and Industry and Japanese

Ministry of Health and Welfare are presented in Appendix 1.

Ahistory profile of vehicle and positive control values is presented in Appendix2.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 pg/plate.Anoily precipitate with associated opaque film was observed at and above 1500 pg/plate; this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of

revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction. The method was designed to conform to the guidelines for bacterial mutagenicity

testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and

MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471

“Reverse Mutation Study”, Method B14 of Commission Directive 92/69/EEC and the USA, EPA

(TSCA) OPPTS harmonised guidelines.

Methods. Salmonella typhimuriumstrains TAl535, TA1537, TA98 and TAl 00 andEscherichia

colistrain WP2uvrA- were treated with the test material using the Ames plate incorporation

method at five dose levels, in triplicate, both with and without the addition of a rat liver

homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was

determined in a preliminary toxicity assay and was 50 to 5000 ug/plate in the range-finding study.

The experiment was repeated on a separate day using the same dose range as the range-finding

study, fresh cultures of the bacterial strains and fresh test material formulations.

Results.T he vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within

the normal range. All of the positive control chemicals used in the test induced marked increases

in the frequency of revertant colonies, both with and without metabolic activation. Thus, the

sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at

any dose level. The test material was, therefore, tested up to the maximum recommended dose

level of 5000 ug/plate. An oily precipitate with associated opaque film was observed at and above

1500 ug/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the

bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion. The test material was considered to be non-mutagenic under the conditions of this

test.