C10-14-alkyl-1-(ethenyloxy)-

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:

24 November 2017 to 08 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ec 2008: Part B: Methods for the Determination of Toxicity and other Health Effects, B.13/14, “Mutagenicity: Reverse Mutation Test Using Bacteria.” Commission Regulation (EEC) No. 440/2008 of 30 May 2008.

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Kyoeisha Chemical Co., Ltd. 7080101
- Expiration date of the lot/batch: 01/08/2019
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, away from heat or sunlight
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Method

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: mutants derived from Salmonella typhimurium LT2
- Suitability of cells:
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:
- Whether whole blood or separated lymphocytes were used if applicable:
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes:
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: [yes/no]
- Periodically checked for Mycoplasma contamination: [yes/no]
- Periodically checked for karyotype stability: [yes/no)
- Periodically 'cleansed' against high spontaneous background: [yes/no]

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 ; The S9 fraction procured from M/s G. P. Meshram, Nagpur was used in the study.
- method of preparation of S9 mix ; The S9 fraction is buffered and supplemented with the essential co-factors β-NADP and Glucose-6-phosphate to form “S9 mix”. This mix is added to the top agar in this activated assay.
- concentration or volume of S9 mix and S9 in the final culture medium ; A volume of 0.1 mL of S9 mix (5% v/v S9 mix for initial toxicity-mutation assay and 10% v/v S9 mix for confirmatory mutation assay
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Not specified

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Used in absence of metabolic activation system

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Used in absence of metabolic activation system

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Used in absence of metabolic activation system

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Used in absence of metabolic activation system

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

Used in presence of metabolic activation system

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration; single
- Number of independent experiments ; 8

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: 48h
- Harvest time after the end of treatment (sampling/recovery times):

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
- Selection time (if incubation with a selective agent):
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER:

Evaluation criteria:

Once criteria for a valid assay had been met, responses observed in the assay were evaluated. The conditions necessary for determining a positive result were: there should be a dose related increase in the mean revertants per plate in at least one tester strain over the range tested and/or at one or more doses of the test item either in the absence or presence of the metabolic activation system.

Statistics:

Simple linear regression analysis was performed for tester strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and Escherichia coli WP2uvrA, separately, to assess the dose dependent nature of any increase in revertant colonies.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Applicant's summary and conclusion

Conclusions:

From the results of this study, C10-C14 alkyl ether is concluded to be non-mutagenic in the Bacterial Reverse Mutation Test in tester strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and Escherichia coli WP2uvrA.