2-hydroxy-3-(prop-2-enoyloxy)propyl 2-methyl-2-propylhexanoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Ames test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 26, 2015 to June 3, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Neodecanoic acid, 1-ester with 1,2,3-propanetriol mono-2-propenoate

Molecular formula:

C16H28O5

IUPAC Name:

Neodecanoic acid, 1-ester with 1,2,3-propanetriol mono-2-propenoate

Constituent 2

Reference substance name:

Neo undecanoic acid, 1-ester with 1,2,3-propanetriol mono-2-propenoate

Molecular formula:

C17H30O5

IUPAC Name:

Neo undecanoic acid, 1-ester with 1,2,3-propanetriol mono-2-propenoate

Constituent 3

Reference substance name:

glycerol 1,3-dineodecanoate

Molecular formula:

C23H44O5

IUPAC Name:

glycerol 1,3-dineodecanoate

Constituent 4

Reference substance name:

Sum of other constituents, each of them at <1%

Molecular formula:

Not available

IUPAC Name:

Sum of other constituents, each of them at <1%

Test material form:

liquid

Specific details on test material used for the study:

Batch no.: JBGJ0045R
Purity: 100% (UVCB)
Appearance: clear yellowish liquid

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium LT2

Remarks:

TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

- The following nominal concentrations were tested in the first experiment: 500 µg/plate, 150 µg/plate, 50 µg/plate, 15 µg/plate and 5 µg/plate.
- The following nominal concentrations were tested in the second experiment: 500 µg/plate, 250 µg/plate, 125 µg/plate, 63 µg/plate, 32 µg/plate, 16 µg/plate and 8 µg/plate.
(As demanded in the guideline, the last turbid concentration will be tested as top concentration. In this case, the highest concentration in the first treatment was 500 µg/plate.)

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene diamine, 2-amino-anthracene

Details on test system and experimental conditions:

Eight hours before the start of each experiment, the nutrient broth was inoculated. For the incubation of strains TA97a, TA98, TA100, TA102; ampicilline was added to the nutrient broth (25 mg/L), for the incubation of strain TA102, tetracycline was added (2 mg/L) in addition to ampicilline. TA1535 was incubated without the addition of antibiotics. The flasks were incubated at 37 ± 1°C for 8h. On the day of the test, the overnight cultures were checked for growth. The incubation time was 48h at 37 ± 1°C. The plate incorporation method was used in this experiment (cfr "Any other information on materials and methods incl. tables").

Rationale for test conditions:

Pre-test results

Evaluation criteria:

The colonies were counted visually, the numbers were recorded. A spreadsheet software was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants (“Rev. abs.”, mean revertants less mean spontaneous revertants) were also calculated. A test substance is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least 1 strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Statistics:

Mean values and standard deviations

Results and discussion

Test results

Key result

Species / strain:

other: Salmonella typhimurium LT2

Remarks:

TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

First Experiment:
5 concentrations of the test substance, dissolved in DMSO (ranging from 5 to 500 µg/plate) were used. Five genetically changed strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 (genetically manipulated) and TA1535) were exposed to the test substance both in the presence and in the absence of a metabolic activation system (S9-mix, rat liver S9-mix induced by Aroclor 1254) for 48h, using the plate incorporation method. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test substance did not show any mutagenic effects in the first experiment. The test substance showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.
Second Experiment:
To verify the results of the first experiment, a second experiment was performed, using 7 concentrations of the test substance (ranging from 8 to 500 µg/plate) and a modification in study performance (pre-incubation method). The test substance did not show mutagenic effects in the second experiment, either. The test substance showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.
The study was considered valid.
In conclusion, the test substance did not show mutagenic effects towards Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535. Therefore, no concentration-effect relationship could be determined. The test substance was considered as “not mutagenic under the conditions of the test”.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was considered as not mutagenic (reverse mutation assay).

Executive summary:

An in vitro reverse mutation assay was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. Two experiments were performed. In the first experiment, 5 concentrations of the test substance, dissolved in DMSO (ranging from 5 to 500 µg/plate) were used. 5 strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 (genetically manipulated) and TA1535) were exposed to the test substance both in the presence and in the absence of a Aroclor induced rat liver S9-mix metabolic activation system for 48 h, using the plate incorporation method. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test substance showed no precipitates on the plates in all tested concentrations. To verify the results of the first experiment, a second experiment was performed, using 7 concentrations of the test substance (ranging from 8 to 500 µg/plate) and a modification in study performance (pre-incubation method). The test substance did not show mutagenic effects in the second experiment, either. The test substance also showed no precipitates on the plates in all tested concentrations. Further, in both the experiments, no signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were also in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. The study was therefore considered valid.Therefore, the test substance was considered to be non-mutagenic under the conditions of the reverse mutation assay(Geitlinger, 2015)