Fatty acids, C16-18 and C18-unsatd., reaction products with glycidyl neodecanoate and 7-oxabicyclo[4.1.0]hept-3-ylmethyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic or genotoxic

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 97

Details on mammalian cell type (if applicable):

S. typhimurium TA 97a

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from liver of rats treated with Arochlor 1254

Test concentrations with justification for top dose:

5000 μg/plate, 1500 μg/plate, 500 μg/plate, 150 μg/plate and 50 μg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene Diamine 2-Amino-Anthracene, 2-Aminoanthracene

Details on test system and experimental conditions:

Two valid experiments were undertaken, first using the plate incorporation method and then using the preincubation method, according to guideline procedures.

Evaluation criteria:

The substance was considered to have mutagenic potential if a reproducible increase in the number of revertant colonies on the place exceeded an increase factor of 2 in at least one strain. A concentration-related increase over the range tested is also taken as sign of mutagenic activity.

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

ZEF 6099/100 showed no precipitates on the plates at any of the concentrations in either assay. 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 item ZEF 6099/100 showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

Conclusions:

Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Test concentrations with justification for top dose:

5 μL/mL, 0.63 μL/mL, 0.31 μL/mL, 0.16 μL/mL, 0.08 μL/mL and 0.04 μL/mL
A pre-test was performed with the sample substance to determine the concentrations needed for the experiment.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Stocks of cells were checked for mycoplasma contamination and stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

No significant increase of the mutation frequency or dose dependent upward trend in mutation frequency was observed in any of the trials within this study.

Conclusions:

In an OECD 473 guideline study, the test item did not induce mutations in the HPRT locus of V79 cells in the absence and presence of metabolic activation. The substance is not considered mutagenic under the conditions of this study.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Remarks:

primary cultures of human peripheral lymphocytes from nonsmoking volunteers ages 23-35

Cytokinesis block (if used):

Cytochalasin B, 6 µg/ml final concentration.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from livers of male rats treated with Arochlor 1254

Test concentrations with justification for top dose:

Pre Experiment- 5, 2.5, 1.25, 0.63, 0.31 μL/mL
Experiment 1 With metabolic activation - 0.5, 0.4, 0.3, 0.2, 0.1 μL/mL
Experiment 1 Without metabolic activation- 0.8, 0.6, 0.4, 0.2, 0.1 μL/mL
Experiment 2 Without metabolic activation- 0.6, 0.55, 0.5, 0.4, 0.2, 0.1 μL/mL

When the test item is a substance of unknown or variable composition, a complex reaction product or of biological origin (UVCB), testing may be started at a higher concentration to increase the amount of each of the test item components.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other:

Details on test system and experimental conditions:

Data are expressed as CBPI, or Cytokinesis Block Proliferation Index and calculated as the sume of mononucleated*1, binucleated*2 and multinucleated*3 cells divided by the total number of cells. Cytotoxicity was calculated as reduction in CBPI compared to the CBPI of the concurrent solvent control. The number of binucleated cells with and without micronuclei in each treatment group was compared with the solvent control value.

Rationale for test conditions:

According to OECD TG 487, the maximum concentration of the test item should be 2 µl/mL, 2 mg/mL or 10 mM, whichever is the lowest. When cytotoxicity occurs, the highest concentration should aim to produce 55 ± 5% cytotoxicity. When the test item is a UVCB, testing may be started at a higher concentration to increase the amount of each of the test item components. When solubility is a limiting factor, the maximum concentration, if not limited by cytotoxicity, should be the lowest concentration at which turbidity or minimal precipitate is visible in the cultures. For insoluble or particulate materials specific adaptation may be needed. For the test item ZEF 6099/100 (UVCB), the maximum concentration according to these demands was 5 µL/mL.

Evaluation criteria:

The test item is considered to have genotoxic effects if:
• At least one test concentration shows a statistically significant increase of micronucleated cells compared to the concurrent solvent control.
• In at least one experimental condition a dose-related increase of micronucleated cells can be observed.
• Any of the results lies outside the range of the historical laboratory control data for solvent controls

Statistics:

The number of binucleated cells with micronuclei in each treatment group was compared with the solvent control. Statistical significance was tested using Fisher’s exact test at the five per cent level (p ≤ 0.05)

Key result

Species / strain:

lymphocytes:

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:

There was no increase in the number of micronucelated cells after exposure to the test substance.

Remarks on result:

other: not genotoxic

The proper concentration ranges were difficult to ascertain due to inconsistent cytotoxicity.

In the pre-experiment, cytotoxicity was observed at rates of 14.3 -25.1 at the two highest concentrations (2.5 and 5 microliter/ml), but complete toxicity was observed in the mid-dose of 1.25 microliter/ml. Lower concentrations resulted in cytotoxicity between 21.9 and 24.3%.

This can possibly be attributed to the fact that the test item in high concentration (neat and the first dilution) did not dissolve well in the test medium and as a consequence did not interact homogeneously with the cultivated lymphocytes in the aqueous cell culture medium. Therefore, in the first and the second main experiment (experiment I without and with metabolic activation, experiment II without metabolic activation – extended exposure) a different range of concentrations was tested.

In the experimental part of the study without metabolic activation, the 3 highest test item concentrations suggested mild precipitation (turbidity) and these were evaluated for genotoxicity. In the experimental part with metabolic activation, the highest tested concentration (0.8 µL/mL) revealed complete cytotoxicity. Therefore, the following three concentrations were scored for the presence of micronuclei. A cytotoxicity of 55±5% was not achieved, in spite of a narrow spacing of the concentrations. As no relation between test item concentration and proportion of binucleated cells was observed, this fact is considered noncritical.

No increase of the number of binucleated cells with micronuclei was detected at the evaluated concentrations and a second experiment (experiment II without metabolic activation, extended exposure) was performed.

In the second experiment, concentrations were: 0.6 microliters/ml, 0.55, 0.5, 0.4, 0.2 and 0.1 microliters/ml. Four concentrations were chosen for evaluation based on whether sufficient binucleated cells were available. At the two highest evaluated concentrations of the test item, the proportion of binucleated cells was higher than in the concurrent solvent control, but not statistically significant increased. The values obtained for these test item concentrations (0.5 and 0.6 µL/mL) lay above the historical laboratory control data for the solvent DMSO but still inside the 99.7% control range. No increase of the number of binucleated cells with micronuclei was detected at the concentrations of 0.2 and 0.1 µL/mL.

In conclusion, it can be stated that under the experimental conditions reported, the test item ZEF 6099/100 did not show genotoxic activity in this in vitro test for the induction of micronuclei.

Conclusions:

The test substance did not induce the formation of micronuclei in primary human lymphocytes in vitro, with and without metabolic activation.. It is considered not genotoxic under the conditions of the OECD 487 assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The genotoxicity of the substance was investigated in several in vitro assays, including the bacterial Ames Assay, mammalian micronucleus in human peripheral lymphocytes, and mammalian mutagenicity (HPRT locus). All tests were negative for genetic toxicity.

Justification for classification or non-classification

The substance is not mutagenic or clastogenic, and hence, the criteria for genetic toxicity in Regulation EC No. 1272/2008 are not met. The substance is not classified for mutagenicity, and fails to qualify for mutagenicity in a CMR evaluation