Reaction products of diphenyl ether and 9-methylene nonadecane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Standard for Investigation by Mutagenicity Studies using Microorganisms of Notification No. 77 of Ministry of Labor, 1988 (partly revised by Notification No. 67 of Ministry of Labor, 1997).

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of diphenyl ether and 9-methylene nonadecane

EC Number:

943-303-1

Molecular formula:

Variable, substance is a UVCB

IUPAC Name:

Reaction products of diphenyl ether and 9-methylene nonadecane

Test material form:

liquid

Method

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:

Negative control, 4.9, 9.8, 20, 39 and 78 µg/plate.
Doses based on findings in dose finding phase.

Vehicle / solvent:

- Vehicle(s)/solvent(s): acetone
- Justification for choice of solvent/vehicle: Low dispensing volume (0.05 mL) of test solutions in acetone applied to account for toxicity of acetone. Acetone was selected based on formulation work which showed the test material was uniformly dispersed at 10% in acetone. The test material was not soluble in DMSO.
- Positive control solutions were dissolved in DMSO or distilled water.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-2(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 6-chloro-9-(3-(2-chloroethylamino)-propylamine)-2-methoxyacridine dihydrochloride and 2-aminoanthracene.

Details on test system and experimental conditions:

S9 Mixture preparation: S9 mix was prepared from the livers of male sprague-dawley rats dosed with Phenobarbitol and 5,6-benzoflavone. Sterility of the mix was assessed prior to use.

Bacterial pre-culture: Stock solutions of bacterial cultures were thawed and pre-cultured under standard conditions. The viable cell number was calculated prior to testing via optical density measurements taken with a spectrophotometer (660 nm). Viable cell numbers ranged from 2.8 to 5.4 x 10^9/mL across the strains used in the main test.

Experimental method: The pre-incubation method was used following standard guideline approaches. In summary the test material (with or without S9 mix) was pre-incubated with bacterial cultures and prepared with agar. The tests were conducted using duplicate plates for each dose of test substance, with triplicates for the vehicle controls. The prepared plates were incubated at 37°C for 48 hours ahead of counting.

Plate observation: Observations of growth inhibition were observed with a stereoscopic microscope, with precipitation observed visually.

Colony counting: Colonies were counted using an automatic colony counter. When colonies on a plate were greater than 1500, colonies were manually counted. Where precipitate was observed manual counting procedures were performed.

Rationale for test conditions:

Following standard guidelines.

Evaluation criteria:

A positive reponse is indicated when the number of revertant colonies is increased by 2-fold or more compared with negative controls with dose dependancy and reproducibility observed. All other results are considered negative.

Statistics:

No statistical analysis is performed.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at and above 20 µg/plate without S9 mix, and at and above 78 µg/plate with S9 mix.

RANGE-FINDING/SCREENING STUDIES: Conducted at the highest dose of 5000 µg/plate. The results of the range-finding phase showed no significant increase in revertant colonies.

VALIDITY OF TESTS: The values in the vehicle control groups were within the background range of historical control data. The positive controls all showed a 2-fold increase in revertant colonies compared with vehicle controls. No contamination was observed in the sterility tests. All these factors indicate that the test was conducted adequately.

Applicant's summary and conclusion

Conclusions:

Based on the results, it was concluded that the substance was negative for genotoxicity under the conditions of the test, and the test was considered valid.