Reaction mass of 1,1-bis(phenethyloxy)ethane and [2-(1-ethoxyethoxy)ethyl]benzene

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:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1,1-bis(phenethyloxy)ethane and [2-(1-ethoxyethoxy)ethyl]benzene

EC Number:

907-481-4

Molecular formula:

C18H22O2 + C12H18O2

IUPAC Name:

Reaction mass of 1,1-bis(phenethyloxy)ethane and [2-(1-ethoxyethoxy)ethyl]benzene

Test material form:

liquid

Specific details on test material used for the study:

- Physical Appearance: Colourless liquid
- Date of Expiry: 12/07/2018
- Storage Condition: Ambient (21 to 29°C)

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Sodium phenobarbitone and β-naphthoflavone induced rat liver S9 homogenate

Test concentrations with justification for top dose:

Pre-test: 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 μL/plate.
Mutation assay: 0.02, 0.06, 0.20, 0.63 and 2 μL/plate (Based on the results of the initial cytotoxicity test)

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 30 minutes (preincubation)
- Exposure duration: 48 hours and 3 minutes (plate incorporation); 48 hours and 2 minutes (preincubation)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn

Evaluation criteria:

EVALUATION AND INTERPRETATION OF RESULTS
The conditions necessary for determining a positive result are: there should be a dose related increase over the range tested and/or a reproducible increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test item, either in the presence or absence of the metabolic activation system. The test will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and TA102 or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537. An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non dose responsive increase that is equal to or greater than the respective threshold cited. A response is evaluated as negative, as it is neither positive nor equivocal.

CRITERIA FOR ACCEPTABILITY OF THE TEST
The mutation test is considered acceptable as it meets the following criteria: All tester strains confirmed to their genetic characteristics. The positive controls showed increase in revertant colony numbers of at least twice or thrice the concurrent vehicle control levels with the appropriate bacterial strain.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Solubility Test: The test item was miscible in dimethyl sulphoxide at a concentration of 50 μL/mL.
- Precipitation Test: The test item resulted in mild precipitation at 5 and 4 μL/plate and minimal precipitation at 3 μL/plate. No precipitation was observed at 0.5, 0.6, 0.7, 0.8, 0.9, 1 and 2 μL/plate.
- Initial Cytotoxicity Test: Based on the results of the solubility and precipitation tests, the concentrations of 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 μL/plate were selected for the initial cytotoxicity test. The tester strain exposed to test item in the presence and absence of metabolic activation system resulted in cytotoxicity and was graded as 1+ (extremely reduced lawn) for 4 and 5 μL/plate, 2+ (moderately reduced lawn) for 3 μL/plate and 3+ (slightly reduced lawn) for 2 μL/plate when compared to vehicle control. On the basis of the cytotoxicity results, 2 μL/plate was considered as the highest test concentration for the mutation assay.
- Plate Incorporation Method: Trial I: All the tester strains treated with the test item, at the concentration of 2 μL/plate showed moderately reduced lawn (grade 3+) when compared to vehicle control. At concentrations of 0.006, 0.02, 0.06, 0.20 and 0.63 μL/plate, all the tester strains showed very close resemblance in the number of revertant colonies and bacterial lawn compared to the vehicle control when tested with and without metabolic activation. There was no appreciable increase in the number of revertant colonies and no change in bacterial background lawn when compared to that of the vehicle control, among the tester strains. The mean number of revertant colonies/plate and bacterial background lawn in the treatment groups for the tested strains were comparable to that of the vehicle control. The specific positive controls tested simultaneously produced approximately a 2.0 to 18.1 fold increase in mean number of revertants as compared to the vehicle control.
- Preincubation Method: Trial II: All the tester strains treated with test item, at the concentration of 2 μL/plate showed moderately reduced lawn (grade 3+) when compared to the vehicle control. At concentrations of 0.006, 0.02, 0.06, 0.20 and 0.63 μL/plate all the tester strains showed very close resemblance in the number of revertant colonies and bacterial lawn compared to the vehicle control when tested with and without metabolic activation. There was no appreciable increase in the number of revertant colonies and no change in bacterial background lawn when compared to that of the vehicle control, among the tester strains. The mean number of revertant colonies/plate and bacterial background lawn in the treatment groups for the tested strains were comparable to that of the vehicle control. The specific positive controls tested simultaneously produced approximately 2.1 to 18.2 fold increase in mean number of revertants as compared to the vehicle control.
- Viable Count: Each tester strain was serially diluted to 10-7 and plated on nutrient agar. After 43 hours and 33 minutes of incubation for the plate incorporation method and pre-incubation method, the numbers of colonies were counted manually and results were expressed as Colony Forming Units (CFU). Each tester strains resulted in an acceptable range of 1 to 2E9 CFU/mL.
- Quality Control Plates: Control plates with S9 mix, phosphate buffer saline, soft agar, 2 μL/plate of test item, dimethyl sulphoxide and minimal glucose agar plates incubated at 37±1°C for 48 hours and 2 minutes for the plate incorporation method and pre-incubation method were checked for contamination. No microbial contamination was observed.