Dibutyl [[bis[(2-ethylhexyl)oxy]phosphinothioyl]thio]succinate

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:

10 December 1996 to 21 May 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

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:

Dibutyl [[bis[(2-ethylhexyl)oxy]phosphinothioyl]thio]succinate

EC Number:

270-220-1

EC Name:

Dibutyl [[bis[(2-ethylhexyl)oxy]phosphinothioyl]thio]succinate

Cas Number:

68413-48-9

Molecular formula:

C28H55O6PS2

IUPAC Name:

1,4-dibutyl 2-({bis[(2-ethylhexyl)oxy](sulfanylidene)-λ⁵-phosphanyl}sulfanyl)butanedioate

Test material form:

liquid

Specific details on test material used for the study:

Purity: >94%
Description: Extremely pale straw coloured liquid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Obtained from the University of California at Berkeley.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Obtained from the British Industrial Biological Research Association.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 50, 150, 500, 1500 and 5000 µg/plate.
5000 µg is the standard top dose recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.

Vehicle / solvent:

Acetone

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Microsomal Enzyme Fraction
S9 was prepared in-house from the livers of male Sprague-Dawley rats, treated with Aroclor 1254. Prior to use, all batches of S9 were checked for suitability using a recognised mutagenic compound (2AA) and stored at -196 °C in a liquid nitrogen freezer.

S9-Mix and Agar
The S9-mix was prepared at 4 °C as follows:
S9: 5.0 mL
1.65 M KCI/0.4 M MgCI2: 1.0 mL
0.1 M Glucose-6-phosphate: 2.5 mL
0.1 M NADP: 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL
Sterile distilled water: 14.5 mL

A preliminary test using the test material at 0, 50, 150, 500, 1500 and 5000 µg/plate was carried out initially to determine the toxicity of the test material to the tester organisms.

Five concentrations of the test material at 50, 150, 500, 1500 and 500 µg/plate were assayed in triplicate in each tester strain, using the direct plate incorporation method.

All of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

A second experiment was performed using methodology as described for experiment 1, using fresh bacterial cultures, test material and control solutions in triplicate.

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate of at least twice the spontaneous reversion rate in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than two fold at each dose level employed.

Statistics:

A statistical analysis of the data was not required to determine the result of the test.

Results and discussion

Test results

Key result

Species / strain:

other: TA 100, TA 1535, TA 98, TA 1537 and WP2uvrA

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:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

In a study performed to the standardised guideline OECD 471, under GLP conditions, Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA 100 and Escherichia coli strain WP2uvrA were treated with the test material using the plate incorporation method at five dose levels, in triplicate, both with and without the addition of S9.

 

The vehicle (acetone) control plates produced 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 the metabolising system.

 

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation. The test material has been determined to be not-mutagenic.