Registration Dossier

Diss Factsheets

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 guidelineopen allclose all
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
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

Constituent 1
Chemical structure
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 resultsopen allclose all
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
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
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
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
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.