bis[C11-14-(branched and linear)-alkyl]aminium nonadecaoxohexatungstate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

20.1.2011 - 4.4.2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted according to OECD Guideline under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

reaction mass of bis(C11-14-alkyl, branched and linear)amine nonadecaoxo hexatungstate

IUPAC Name:

reaction mass of bis(C11-14-alkyl, branched and linear)amine nonadecaoxo hexatungstate

Test material form:

liquid: viscous

Details on test material:

- Physical state: Pale straw colored liquid
- Analytical purity: 100%
- Lot/batch No.: PB-39-131 (oil free)
- Expiration date of the lot/batch: 9 December 2013
- Storage condition of test material: Room temperature in the dark

Method

Target gene:

Not specified

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:

Rat liver homogenate (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Rangefinding test: 1.5 to 5000 micro g/plate -S9; 5 to 5000 micro g/plate +S9
Main test: 0.5 to 5000 micro g/plate -S9; 5 to 5000 micro g/plate +S9

Vehicle / solvent:

Dimethyl sulphoxide

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Details on test system and experimental conditions:

Tester Strains

The four strains of Salmonella used in the test were obtained from the University of California, Berkeley, on culture discs, on 4 August 1995 and Syngenta CTL, Alderly Edge, as frozen vials, on 20 March 2007. Escherichia coli strain WP2uvrA was obtained from the British Industrial Biological Research Association on 17 August 1987. All of the strains were stored at approximately -196 degrees Centigrade in a Statebourne liquid nitrogen freezer, model SXR 34.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37 degrees Centigrade for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Preparation of Test and Reference Items

The test item was immiscible in sterile distilled water and acetone at 50 mg/ml but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

The test item was accurately weighed and approximately half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at 40 degreesCentigrade on the day of each experiment. All formulations were used within four hours of preparation and were assumed to be stable for this period.

Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pettets with a nominal ore diameter of 4 x 10-4 microns.

Microsomal Enzyme Fraction

The S9 Microsomal fraction was prepared in-house (24 October 2010) from rats induced with Phenobarbitone/beta-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenising the liver in a 0.15 KCl solution (1 g liver to 3 ml KCl) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/ml. Aliquots of the supernatant were frozen and stored at approximately -196 degrees Centigrade. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.

S9-Mix and Agar

The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test.

S9

1.65 M KCl/0.4 M MgCl2 5.0 ml
0.1 M Glucose-6-phosphate 1.0 ml
0.1 M NADP 2.5 ml
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 ml
Sterile distilled water 14.5 ml

A 0.5 ml aliquot of S9- mix and 2 ml of molten, trace histidine or tryptophan supplement, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, intriplicate, on the day of each experiment.

Top agar was prepared using 0.6% Bacto agar and 0.5% sodium chloride with 5 ml of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 ml of top agar. Vogel-Bonner Minimal agar plates were purchased from ILS Ltd.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the 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 of data as determined by UKEMS.
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 test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

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 Test

The test item induced toxicity to TA100, initially from 500 micro g/plate and to WP2uvrAfrom 1500 micro g/plate. The test item formulation and S9-mix used in this experiment were both shown to be sterile.

Mutation Test

In the first experiment (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains (except TA98 dosed with S9-mix), initially from 150 micro g/plate (absence of S9-mix) and 1500 micro g/plate (presence of S9-mix). In the second experiment (pre-incubation method) the test item induced a stronger toxic response to the bacterial background
lawns of all of the Salmonella strains, with toxicity initially observed from 50 micro g/plate (absence of S9-mix) and 500 micro g/plate (presence of S9-mix). Weakened bacterial background lawns were not observed for Escherichia coli strain WP2uvrA at any test item dose level in either experiment. The sensitivity of the tester strains to the toxicity of the test item varied between strain type, exposures with or without S9-mix and experiment number. The test item was, therefore, tested up to either the maximum recommended dose level of 5000 micro g/plate or the toxic limit, depending on bacterial strain type and Experiment number. A test item precipitate (oily in appearance) was observed at 5000 micro g/plate, this observation 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 item, either with or without metabolic activation or exposure method.

All of the reference items (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

In an OECD 471 study ("Bacterial Reverse Mutation Test"), conducted according to GLP, reaction mass of bis(C11-14-alkyl, branched and linear)amine nonadecaoxo hexatungstate (the surrogate substance) is non-mutagenic (Harlan Laboratories Ltd., 2011).