Reaction mass of Octadec-9-en-1-yl ammonium di-n-hexyl phosphorodithioate and Octadec-9-en-1-yl ammonium mono- and di-butylphosphate

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:

The study was conducted between 25 March 2002 and 15 April 2002.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

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 870.5100 - Bacterial Reverse Mutation Test (August 1998)

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:

Octadec-9-en-1-yl ammonium mono and di-butylphosphate

Molecular formula:

C22H48NO4P - C26H56NO4P

IUPAC Name:

Octadec-9-en-1-yl ammonium mono and di-butylphosphate

Constituent 2

Reference substance name:

Octadec-9-en-1-yl ammonium di-n-hexyl phosphorodithioate

Molecular formula:

C30H64NO2PS2

IUPAC Name:

Octadec-9-en-1-yl ammonium di-n-hexyl phosphorodithioate

Test material form:

liquid: viscous

Details on test material:

Material is a yellow viscous liquid

Method

Target gene:

Salmonella: histidine
E. coli: tryptophan

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary Toxicity Study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate

Mutation Study - Experiment 1 (Range-finding Study):
All tester strains (without S9): 0.5, 1.5, 5, 15, 50, 150 ug/plate
All tester strains (with S9): 1.5, 5, 15, 50, 150, 500 ug/plate

Mutation Study -Experiment 2 (Main Study)
All Salmonella tester strains (with S9), TA1535, TA1537 and WP2uvrA- (without S9): 0.5. 1.5, 5, 15, 50, 150 ug/plate
All Salmonella tester strains (without S9): 0.15, 0.5, 1.5, 5, 15, 50 ug/plate
WP2uvrA- (with S9): 1.5, 5, 15, 50, 150, 500 ug/plate

Vehicle / solvent:

Ethanol

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

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

The Salmonella typhimurium strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 whilst Escherichia coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association on 17 August 1987. All of the strains were stored at -196°C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB+ or uvrA- mutation and the spontaneous reversion rate.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37°C for approximately 10 hours.

Preparation of Test and Control Materials
Following information provided by the Sponsor the test material was initially tested and found to be soluble in ethanol which is an acceptable vehicle for use in the Ames test. The test material appearance in solution was clear and colourless and dosed very well onto Vogel- Bonner agar plates. The test material was observed to form an oily precipitate at 5000 ?g/plate. The test material was accurately weighed and approximate half-log dilutions prepared in ethanol by a short mix on an autovortex mixer on the day of each experiment. Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) i.e. 2 mm pellets with a nominal pore diameter of 4E+04 microns.

Microsomal Enzyme Fraction
S9 was prepared in-house on 03 March 2002 from the livers of male Sprague-Dawley rats weighing - 250g. These had each orally received three consecutive daily doses of phenobarbitone/?-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation. Before use, each batch of S9 was assayed for its ability to metabolize the indirect mutagens 2-Aminoanthracene and Benzo(a)pyrene. The S9 was stored at - 196°C.

Test Procedure
Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material. The dose range of the test material was 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ?g/plate. The study was performed by mixing 0.1 mL of bacterial culture (TA 100 or WP2uvrA-), 0.1 mL of test material formulation, 0.5 mL of phosphate buffer or S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar was overlaid onto sterile plates of Vogel-Bonner Minimal agar. Ten concentrations of the test material (single dose) and a vehicle control (ethanol) were tested. In addition, 0.1 mL of the maximum concentration of the test material and 2 mL of molten, trace histidine or tryptophan supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. The plates were incubated for a nominal 48 hours at 37°C after an initial overnight equilibration period and then assessed for revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation Study - Experiment 1 (Range-finding Study)
Six concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method. The dose range was determined from a preliminary toxicity assay and was allocated as follows:

All tester strains (without S9): 0.5, 1.5, 5, 15, 50, 150 ?g/plate
All tester strains (with S9): 1.5, 5, 15, 50, 150, 500 ?g/plate

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. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix. The plates were incubated for a nominal 48 hours at 37°C after an initial overnight equilibration period and the frequency of revertant colonies assessed using a Domino colony counter.

Mutation Study - Experiment 2 (Main Study)
The second experiment was performed using methodology as described for Experiment 1 using fresh bacterial cultures, test material and control solutions. The test material dose range was amended slightly, following information derived from Experiment 1 and was as follows:

All Salmonella tester strains (with S9), TA1535, TA1537 and WP2uvrA- (without S9): 0.5. 1.5, 5, 15, 50, 150 ?g/plate
All Salmonella tester strains (without S9): 0.15, 0.5, 1.5, 5, 15, 50 ?g/plate
WP2uvrA- (with S9): 1.5, 5, 15, 50, 150, 500 ?g/plate

Evaluation criteria:

Evaluation Criteria
The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria. If a greater than two or threefold increase in revertant count (depending on tester strain type) is observed in two experiments then this is taken as evidence of a positive response.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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

Additional information on results:

Preliminary Toxicity Study
The dose range of the test material used in the preliminary toxicity study was 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ?g/plate. The test material exhibited toxicity to Salmonella strain TA100 at 50 and 150 ?g/plate without and with S9 respectively. The test material exhibited toxicity to Escherichia coli strain WP2uvrA- at 150 and 500 ?g/plate without and with S9 respectively. The test material formulation and S9-mix used in this experiment were both shown to be sterile.


Mutation Study
The test material caused a visible reduction in the growth of the bacterial background lawn to all of the Salmonella tester strains, initially at 50 and 150 ?g/plate without and with S9 respectively. Toxicity was also observed to Escherichia coli strain WP2uvrA-, initially at 50 and 500 ?g/plate without and with S9 respectively. The test material was, therefore, tested up to the toxic limit.

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 and the activity of the S9 fraction was shown to be satisfactory.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

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