bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide

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:

1996-03-26 - 1996-04-09 (experimental phase)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well-documented study according to OECD 471 with minor deviations: only four strains of S. typhimurium (TA1535, TA1537, TA98, TA100) were used, data on E.coli WP2 strains or S. typhimurium TA102 are lacking. However, since these strains were mainly included in the recent version of OECD 471 because the four formerly only recommended (OECD 471 version of May 1983) S. typhimurium strains TA1535, TA1537, TA98 and TA100 may not detect certain oxidising mutagens, cross-linking agents and hydrazines, and this mode of action is not likely to occur based on the chemical structure of the test item, this restriction is considered to be negligible.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 471: Genetic Toxicology: Salmonella typhimurium, Reverse Mutation Assay, 26 May 1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Methods for Determination of Toxicity, Annex to Directive 92/69/EEC, (OJ No. L383A, 29.12.92), Part B, Method B.14. Other effects - Mutagenicity: Salmonella typhimurium - Reverse Mutation Assay

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US Environmental Protection Agency, Method: HG-Gene Muta ― S. typhimurium: The Salmonella typhimurium reverse mutation assay, 1984

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide

EC Number:

605-708-9

Cas Number:

174125-93-0

Molecular formula:

n.a.

IUPAC Name:

bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide

Test material form:

liquid

Remarks:

Orange/brown

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor

Method

Target gene:

his-

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The strains were obtained from Professor B.N. Ames, University of California, California, USA.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

Top Dose: 5000 µg/plate, as demanded by the guideline, no toxic effects in the pre-test noted. Ideally the concentrations chosen for the mutation tests should include a minimum of three non-toxic concentrations.
Doses: 0, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: At 50 mg/ml the test item was immiscible with water and dimethyl sulphoxide, and miscible with ethanol.

Controls

Untreated negative controls:

yes

Remarks:

solvent controls

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene

Remarks:

Solvent control for positive controls: DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: none
- Exposure duration: 3 days
- Expression time (cells in growth medium): = exposure duration

SELECTION AGENT (mutation assays): histidine-minimal agar

NUMBER OF REPLICATIONS: Three petri dishes were used for each dose level.

DETERMINATION OF CYTOTOXICITY
Any toxic effects of the test substance can be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn. In the absence of any toxic effects the top concentration used in the main tests is the same as that used in the preliminary toxicity test.

Rationale for test conditions:

As stipulated by the guideline

Evaluation criteria:

ASSESSMENT OF RESULTS
The mean number of revertant colonies for all treatment groups is compared with the solvent control groups. The mutagenic activity is assessed by applying the following criteria:
(a) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
(c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue of the substance's mutagenic activity in this test system.
(i) Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(ii) If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al. (1989) and will usually be analysis of variance followed by Dunnett's test.

Statistics:

see above

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 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

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate (1st experiment), at 1500 & 5000 µg/plate (2nd experiment)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate and at 1500 µg/plate -S9 only (1st experiment), at 1500 & 5000 µg/plate (2nd experiment)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Testing for mutagenicity in bacteria was performed according to OECD TG 471 of 1983. Here, only four strains of S. typhimurium (TA1535, TA1537, TA98, TA100) were used, data on E.coli WP2 strains or S. typhimurium TA102 are lacking. However, since these strains were mainly included in the recent version of OECD 471 because the four formerly only recommended (OECD 471 version of May 1983) S. typhimurium strains TA1535, TA1537, TA98 and TA100 may not detect certain oxidising mutagens, cross-linking agents and hydrazines, and this mode of action is not likely to occur based on the chemical structure of the test item, this restriction is considered to be negligible. Also, the testing was sufficiently documented, positive and negative controls gave the appropriate response. Hence, the results can be considered as sufficiently reliable to assess the mutagenic potential of the test item. In both presence and absence of metabolic activation, the test item did not induce a significant increase in the number of revertant colonies per plate over those of the negative control plates. In consequence, the test item is considered to be non-mutagenic under the conditions of this test.

Executive summary:

In this in vitro assessment of the mutagenic potential of the test item according to OECD TG 471 (1983) under GLP, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) were exposed to the test substance, diluted in ethanol which was also used as a negative control.

Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats.

In the preliminary toxicity test with dose levels of up to 5000 µg/plate no toxicity was observed. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 1500, 500, 150 and 50 µg/plate.

No evidence of mutagenic activity was seen at any dose level of the test item in either mutation test.

The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, when tested in ethanol, the test item shows no evidence of mutagenic activity in this bacterial system.