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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

 The Bacterial Reverse Mutation Assay (according to OECD Guidelines for Testing of Chemicals No.: 471) with 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol showed, that the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol is considered non-mutagenic in this bacterial 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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
Initial Mutation Test: 1600/ 500/ 160/ 50/ 16/ 5/ 1.6/ 0.5 µg/plate
Confirmatory Mutation Test and Complementary Plate Incorporation Test: 5000/ 1600/ 500/ 160/ 50/ 16/ 5/ 1.6/ 0.5/ 0.16 µg/plate

- Due to the strong inhibition observed in the Initial Mutation Test in the case of Salmonella typhimurium TA1537 in the absence of exogenous metabolic activation (-S9 Mix); the examined concentration range was modified in the subsequent Confirmatory Mutation Test and the following concentration levels were investigated at this experimental part (S. typhimurium TA1537):
-S9 Mix: 50, 16, 5, 1.6, 0.5 and 0.16 μg/plate;
+S9 Mix: 1600; 500; 160; 50; 16 and 5 μg/plate.
- Inhibition was not noticed in the Initial Mutation Test in the case of Escherichia coli WP2 uvrA in the examined concentration range; therefore in the Confirmatory Mutation Test in the case of Escherichia coli WP2 uvrA the following concentration levels were investigated in the absence of and presence of exogenous metabolic activation (±S9 Mix):
±S9 Mix: 5000, 1600, 500, 160, 50 and 16 μg/plate.
- In the Complementary Plate Incorporation Test the Escherichia coli WP2 uvrA was investigated with the following concentration levels:±S9 Mix: 5000, 1600, 500, 160 and 50 μg/plate.
- The examined concentration levels were not modified in the case of S. typhimurium TA98, TA100 and TA1535 strains.
Vehicle / solvent:
- vehicle for the test item: dimethyl sulfoxide (DMSO)
- vehicle for positive and negative (vehicle) controls: DMSO, Ultrapure water (prepared in the testing laboratory)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylene-diamine; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION
plate incorporation in experiment 1 (initial mutation test + complementary plate incorporation test), pre-incubation method in experiment 2 (confirmatory mutation test)

Each assay was conducted with and without metabolic activation (±S9 Mix). In parallel with the Confirmatory Mutation Test, the plate incorporation test was completed (Complementary Plate Incorporation Test) with investigation of additional concentration levels in the case of Escherichia coli WP2 uvrA, because of the lacking inhibition in the already examined concentration range. The concentrations, including the controls, were tested in triplicate (positive and negative controls were run concurrently).

CONCENTRATION RANGE FINDING and CYTOTOXICITY DETERMINATION
The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate.

EXPERIMENTAL PERFORMANCE
The standard plate incorporation procedure was performed as an initial mutation test and Complementary Plate Incorporation Test. Bacteria (cultured in Nutrient broth No. 2.) were exposed to the test item, both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45 °C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per controls or concentration levels). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared fresh and added to the overlay (45 °C).
The content of the tubes:
Top agar 2000 μL
Vehicle or solution of test item or reference controls 100 μL
Over-night culture of test strain 100 μL
Phosphate buffer (pH: 7.4) or S9 Mix 500 μL
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 Mix were added to each overlay tube. The entire test consisted of non-activated and activated test conditions (without S9 Mix and with addition of S9 Mix) and each of them with the addition of negative and positive controls.
For the pre-incubation method, before overlaying the test item, the bacterial culture and the S9 Mix or phosphate buffer was added into the appropriate tubes, providing direct contact between bacteria and the test item (in its solvent). These tubes were gently mixed and incubated for 20 min at 37 ºC using a shaker. After the incubation the content of the tubes was added to the molten top agar prior to pouring onto the surface of minimal agar plates.
After solidification the plates were inverted and incubated at 37 °C for approximately 48 hours in the dark.
Evaluation criteria:
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.
Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
The mean values and appropriate standard deviations and mutation rates (mean revertants divided by mean spontaneous revertants) were calculated.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The reported data of this mutagenicity assay show, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol is considered non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

Five bacterial strains were used to investigate the mutagenic potential of 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). The results of the Initial Mutation Test were completed with Complementary Plate Incorporation Test performed in E. coli WP2 uvrA. Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently.

In the performed experiments (in the case of Initial Mutation Test together with its completion) all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled.

No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

Sporadic increases in revertant colony numbers compared to the vehicle control values within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

In the performed experiments inhibitory effect of the test item (absent revertants, decreased number of revertant colony numbers and/or affected background lawn development) was observed in all examined Salmonella typhimurium strains. The 16 μg/plate was found to be the lowest cytotoxic concentration, observed in Confirmatory Mutation Test in the case of Salmonella typhimurium TA1537 strains, in the absence of exogenous metabolic activation (-S9 Mix).

In the Informatory Toxicity Test and Complementary Plate Incorporation Tests) microdrops (precipitate as a colloid-chemical phenomenon) were noticed down to and including the concentration level of 160 μg/plate in absence (-S9 Mix) and at the concentration levels of 5000 and 1600 μg/plate in the presence of exogenous metabolic activation (+S9 Mix). In the Initial Mutation Test microdrops were noticed at the concentration level of 1600 μg/plate in the presence of exogenous metabolic activation (+S9 Mix).

The obtained microdrops (precipitate) did not interfere with the scoring in any case.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification