Isooctyl palmitate

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:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Isooctyl palmitate

EC Number:

215-675-9

EC Name:

Isooctyl palmitate

Cas Number:

1341-38-4

Molecular formula:

C24H48O2

IUPAC Name:

6-methylheptyl hexadecanoate

Test material form:

liquid

Remarks:

a colourless liquid

Specific details on test material used for the study:

The following information concerning identity and composition of the test item was pro-vided by the sponsor.
Name 1341-38-4 Isooctyl palmitate
Batch no. 180418
Appearance clear, colourless liquid
Composition Isooctyl palmitate
Purity 98.7%
Homogeneity homogeneous
Expiry date 17. Apr. 2020
Storage Room Temperature (20 ± 5 °C)

The following additional information was relevant to the conduct of the study, according to
OECD 471:
CAS No. 1341-38-4
EINECS-No. 215-675-9
Stability in solvents H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown

Method

Target gene:

All Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA97a: 5033D, TA98: 5136D, TA100: 5141D, TA102: 5145D, TA1535: 5138D) and were stored as lyophilizates in the refrigerator at 2-8 °C.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 97

Remarks:

TA97a

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The following nominal concentrations were prepared for the experiment 1:
5 µL/plate, 1.5 µL/plate, 0.5 µL/plate, 0.15 µL/plate and 0.05 µL/plate

The following nominal concentrations were prepared for the second experiment:
5 µL/plate, 2.5 µL/plate, 1.25 µL/plate, 0.63 µL/plate, 0.31 µL/plate and 0.16 µL/plate

Vehicle / solvent:

Based on the non-GLP pre-test, acetone was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Controls

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:

Preparations
Different media and solutions were prepared preliminary (exact production dates are doc-umented in the raw data).
On the day of the test, the bacteria cultures were checked for growth visually. The incuba-tion chambers were heated to 37 ±1 °C. The water bath was turned to 43 ±1 °C. The table surface was disinfected.
The S9 mix was freshly prepared and stored at 0 °C.
Experimental Parameters
Experiment 1
Date of treatment 27. Feb. 2019
Concentrations tested 5 / 1.5 / 0.5 / 0.15 / 0.05 µL/plate
Incubation time 48 h
Incubation temperature 37 ±1 °C
Tested strains TA97a, TA98, TA100, TA102, TA1535
Method plate incorporation method

Experiment 2
Date of treatment 12. Mar. 2019
Concentrations tested 5 / 2.5 / 1.25 / 0.63 / 0.31 / 0.16 µL/plate
Incubation time 48 h
Incubation temperature 37 ±1 °C
Tested strains TA97a, TA98, TA100, TA102, TA1535
Method pre-incubation method

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants less mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 97

Remarks:

TA 97a

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

Test results 2

Species / strain:

S. typhimurium TA 98

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

Test results 3

Species / strain:

S. typhimurium TA 100

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

Test results 4

Species / strain:

S. typhimurium TA 102

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

Test results 5

Species / strain:

S. typhimurium TA 1535

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
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the nega-tive controls were in the normal range of the test laboratory (historical data of the laborato-ry see chapter 15, page 39). All positive controls (diagnostic mutagens) showed mutagen-ic effects with and without metabolic activation and were within the historical control data ranges.
Solubility and Toxicity
In the first experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial back-ground lawn was visible and not affected. The number of revertant colonies was not re-duced.
Mutagenicity
No increase of the number of revertant colonies in the treatments with and without meta-bolic activation could be observed. No concentration-related increase over the tested range was found.

Second Experiment
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the nega-tive controls were in the normal range of the test laboratory (historical data of the laborato-ry see chapter 15, page 39). All positive controls (diagnostic mutagens) showed mutagen-ic effects with and without metabolic activation and were within the historical control data ranges.
Solubility and Toxicity
In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial back-ground lawn was visible and not affected. The number of revertant colonies was not re-duced.
Mutagenicity
No increase of the number of revertant colonies in the treatments with and without meta-bolic activation could be observed. No concentration-related increase over the tested range was found.

Therefore, the test item is stated as not mutagenic under the conditions of this experiment.


Therefore, the test item is stated as not mutagenic under the conditions of this experiment.
To verify this result, a further experiment was performed.

Any other information on results incl. tables

In all experiments, no precipitation of the test item1341-38-4 Isooctyl palmitatewas observed at any of the tested concentrations up to 5 µL/plate.

In both experiments, the test item caused no cytotoxicity towards all bacteria strains.

The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 10⁹bacteria/mL.

All of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. All numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory (historical data of the laboratory see chapter15, page39) and were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens.

Since all criteria for acceptability have been met, the study is considered valid.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that 1341-38-4 Isooctyl palmitate is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experi-mental conditions in this study.

Executive summary:

Two valid experiments were performed.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test item1341-38-4 Isooctyl palmitatewas tested in theSalmonella typhimuriumreverse mutation assay with five strains ofSalmonella typhimurium(TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

 

Experiment 1:

In the experiment 1,the test item (dissolved in acetone) was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test itemshowed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Experiment 2:

Based on the first experiment,the test item was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in all bacteria strains using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test itemshowed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertant colonies in all strains, in the presence and absence of metabolic activation.