Reaction mass of hentriacontan-16-one and pentatriacontan-18-one and tritriacontan-16-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames:

One study in 2018 shows negative to S. typhimurium (TA98, 100, 1535, 1537) and E. coli (WP2uvrA) strains.

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:

From 2017-09-11 to 2017-11-30

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:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch No.: FK650468
Purity: not specific

Target gene:

histidine locus in Salmonella typhimurium, and tryptophan locus of Escherichia coli (E. coli) 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: Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535: 2016, TA1537: 2016, TA98: 2017, TA100: 2016; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA: 2008)]
- Culturation: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth and incubated in a shaking incubator (37 ± 1°C, 150 rpm),
until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/mL).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose-range Finding Test: 1.7, 5.4, 17, 52, 164, 512, 1000 and 1250 μg/plate
First Mutation Experiment: 1.7, 5.4, 17, 52 and 164 μg/plate
Second Mutation Experiment: 15, 27, 48, 86, 154 and 275 μg/plate

Vehicle / solvent:

Vehicle used: xylene

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

TA1535, TA1537, TA100, TA98 and WP2urA with S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

TA1537 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA100 without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

WP2uvrA without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable):1E+09 cells/mL

DURATION
- Exposure duration: 48 ± 4 h.

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were
examined to determine the toxicity.
OTHER EXAMINATIONS:
- Precipitate: Evidence of test item precipitate on the plates were evaluated.

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following 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 whencompared against relevant historical control data generated at the lab.
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 xperiment will be repeated.

Statistics:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants intester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent 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 tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat 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.

Key result

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

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the presence of S9-mix at the highest tested concentration

Vehicle 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:

no cytotoxicity

Vehicle 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

Remarks:

in the presence of S9-mix at the highest tested concentration

Vehicle 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:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose-range Finding Test/First Mutation Experiment
- Precipitate: Precipitation on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 52 μg/plate and 164 μg/plate and above at the end of the incubation period in the dose range finding test and the first mutation experiment, respectively.
- Toxicity: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
- Mutagenicity: No increase in the number of revertants was observed upon treatment with test item under all conditions tested.
Second Mutation Experiment
- Precipitate: Precipitation on the plates was observed at the start and at the end of the incubation period at concentrations of 154 μg/plate and upwards, except in the tester strains TA1535, TA1537 and TA98 in the presence of S9-mix, where precipitation was only observed at the highest dose level of 275 μg/plate at the end of the incubation period.
- Toxicity: Cytotoxicity, as evidenced by a reduction of the bacterial background lawn was observed in tester strains TA1537 and TA100 in the presence of S9-mix at the highest tested concentration.
In tester strain TA100 in the presence of S9-mix, fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, due to the low mean number of revertant colonies in the solvent control group (52), the mean numbers of 48 and 52 revertant colonies are not considered to be biological relevant.
In the other tester strains, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in the absence and presence of S9-mix.
- Mutagenicity: In the second mutation assay, no increase in the number of revertants was observed upon treatment with test item under all conditions tested.

Conclusions:

The test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of test item and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

In the dose-range finding test, the test item was tested up to concentrations of 1250 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Test item precipitated on the plates at dose levels of 52 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose-range finding test were reported as part of the first mutation assay.

Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 1.7 to 164 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the top dose of 164 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 15 to 275 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item was tested up to or beyond a precipitating dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn was observed in tester strains TA1537 and TA100 in the presence of S9-mix at the highest tested concentration. In the other tester strains, no toxicity was observed at any of the dose levels tested.

Test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In conclusion, based on the results of this study it is concluded that test item 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)

Justification for classification or non-classification

Negative result in Ames study.

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, this substance should not be classified as mutagen.