C12-16 alkyl, glycerol ether

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of an AMES test, performed according to OECD guideline 471 and GLP principles, C12-16 alkyletherdiol is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 June 2018 - 16 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium: histidine gene
Escherichia coli: tryptophan gene

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) from rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).

Test concentrations with justification for top dose:

Dose range finding test (TA100 and WP2uvrA, with and without S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (reported as part of the first experiment)
First experiment (TA1535, TA 1537 and TA98): without S9: 0.52, 1.7, 5.4, 17, 52, 164, 512 and 1600 μg/plate
First experiment (TA1535, TA 1537 and TA98): with S9: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate

Second experiment (all strains) without S9: 6.3, 12.5, 25, 50, 100, 500, 1000 and 5000 μg/plate
Second experiment (all strains) with S9: 12.5, 25, 50, 100, 500, 1000, 2500 and 5000 μg/plate

Third experiment (TA1537) with S9: 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Solvent used: ethanol
- Justification for choice of solvent/vehicle: the solvent is according to OECD guideline 471 and the test substance formed a clear colorless solution in ethanol.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene

Remarks:

See table 1 for more details on postive control substances

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 +/- 4 hours

NUMBER OF REPLICATIONS: 3

PERFORMANCE OF THE ASSAY: Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C

CYTOTOXICITY:
- Cytotoxicity was examined by the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

COLONY COUNTING:
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

In addition to the criteria stated above, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

Acceptability criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at this laboratory.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

In the second experiment a 6.6-fold increase compared to the solvent control was observed at a test concentration of 5000 µg/plate.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRECIPITATION:
- Dose-range finding/first experiment: at the start and at the end of the incubation period at concentrations of 5000 µg/plate.
- Second experiment: at the start and at the end of the incubation period at concentrations of 5000 µg/plate, except in tester strains TA1535, TA1537 and TA98 at the start of the incubation period where no precipitate was observed.
- Third experiment: at the start of the incubation period at concentrations of 1000 µg/plate and upwards and at the end of the incubation period at the top dose level of 5000 µg/plate

CYTOTOXICITY
- Dose-range finding/first experiment: observed in all tester strains in the absence and presence of S9-mix.
- Second experiment: observed in all tester strains in the absence of S9-mix and in tester strains TA100 and WP2uvrA in the presence of S9-mix.
- Third experiment: observed at all dose levels tested.

MUTAGENICITY
- Dose-range finding/first experiment: no increase in the number of revertants observed.
- Second experiment: in tester strain TA1537 in the presence of S9-mix, the test item induced an up to 6.6-fold increase in the number of revertant colonies compared to the vehicle control. The increases observed were above the laboratory historical control data ranges and more than three-fold the concurrent solvent control. No increases were observed in all the other tester strains at any of the dose levels tested.
- Third experiment: no increase in the number of revertants observed.

HISTORICAL CONTROL DATA
- The negative control values and the strain-specific positive control values were within the laboratory historical control data ranges.

Conclusions:

Based on the results of an AMES test, performed according to OECD guideline 471 and GLP principles, C12-16 alkyletherdiol is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

C12-16 Alkyletherdiol was not found to be mutagenic in bacterial reverse mutation assay.