Condensation products of fatty acids, tall oil with 2-amino-2-ethylpropanediol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

Condensation products of fatty acids, tall oil with 2-amino-2-ethylpropanediol

EC Number:

946-010-7

Molecular formula:

None

IUPAC Name:

Condensation products of fatty acids, tall oil with 2-amino-2-ethylpropanediol

Test material form:

liquid: viscous

Specific details on test material used for the study:

Identification: Alkaterge E
Chemical name (IUPAC, synonym or trade name): 4-Ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol
CAS number: 68140-98-7
Molecular formula: C23H43NO2
Molecular weight: 365.60
Appearance: Brown viscous liquid
Batch: D598F47BC1
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 05 April 2020 (retest date)

Method

Target gene:

thymidine kinase (TK) locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Rat S9, obtained from Trinova Biochem GmbH, Giessen (Germany) is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).

Test concentrations with justification for top dose:

In the first experiment, Alkaterge E was tested up to concentrations of 38 µg/mL in the absence and presence S9-mix. In the absence of S9-mix, relative total growth (RTG) was reduced to 7% and 36% at the concentrations of 25 and 38 µg/mL, respectively. In the presence of S9-mix, no toxicity was observed at the dose level of 38 µg/mL. Alkaterge E precipitated in the culture medium at the dose level of 38 µg/mL. In the second experiment, Alkaterge E was tested up to concentrations of 25 µg/mL in the absence of S9-mix.

Vehicle / solvent:

Ethanol

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Test System L5178Y/TK+/--3.7.2C mouse lymphoma cells. Source American Type Culture Collection, (ATCC, Manassas, USA) (2001)

Stock cultures of these cells are stored in liquid nitrogen (-196°C). The cultures are checked for mycoplasma contamination. Cell density will be preferably kept below 1 x 106 cells/mL.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Alkaterge E precipitated in the exposure medium at concentrations of 38 μg/mL and above. Alkaterge E was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test item concentration for the dose-range finding test was 150 μg/mL.

Dose-range Finding Test
In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of from 4.7 to 150 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and with a range of from 4.3 to 136 µg/mL in the presence of S9-mix with a 3 hour treatment period. In the absence of S9-mix, the relative suspension growth was 16% at the test item concentration of 38 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 75 μg/mL and above. In the presence of S9-mix, the relative suspension growth was 5% at the test item concentration of 136 μg/mL compared to the relative suspension growth of the solvent control. Due to a technical error, the dose level of 150 µg/mL was not used for cytotoxicity determination. The relative suspension growth was 36% at the test item concentration of 19 μg/mL compared to the relative suspension growth of the solvent control. No or hardly any cell survival was observed at the test item concentrations of 38 μg/mL and above.

First Mutagenicity Test
Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test:
Without S9-mix: 0.074, 0.15, 0.3, 0.6, 1.2, 2.4, 4.8, 9.5, 15, 19, 25 and 38 μg/mL exposure medium.
With S9-mix: 0.074, 0.15, 0.3, 0.6, 1.2, 2.4, 4.8, 9.5, 19 and 38 μg/mL exposure medium.

Evaluation of toxicity
In the absence of S9-mix, the dose levels of 0.074 to 15 μg/mL showed no cytotoxicity or showed an inconsistent RSG (0.074 µg/mL). Therefore, the dose levels of 0.074, 0.3, 1.2 and 9.5 µg/mL were not regarded relevant for mutation frequency measurement. In the presence of S9-mix, no cytotoxicity was observed. Therefore, the dose levels of 0.074 and 0.15 µg/mL were not regarded relevant for mutation frequency measurement. The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.15, 0.6, 2.4, 4.8, 15, 19, 25 and 38 μg/mL exposure medium.
With S9-mix: 0.3, 0.6, 1.2, 2.4, 4.8, 9.5, 19 and 38 μg/mL exposure medium.

In the absence of S9-mix, the relative total growth was 7% and 36% at the concentrations of 25 and 38 µg/mL, respectively.In the presence of S9-mix, no toxicity was observed up to and including the highest tested dose level.

Evaluation of the mutagenicity
No significant increase in the mutation frequency at the TK locus was observed after treatment with Alkaterge E either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the Alkaterge E treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second Mutagenicity Test
To obtain more information about the possible mutagenicity of Alkaterge E, a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period. Based on the results of the dose-range finding test, the following dose levels were selected for mutagenicity testing: 0.63, 1.25, 2.5, 5, 10, 15, 20, 25, 30 and 40 µg/mL exposure medium. Further investigation showed that at a concentration of 30 µg/mL Alkaterge E already precipitated in the exposure medium.

Evaluation of toxicity
The dose levels of 30 and 40 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-locus were: 0.63, 1.25, 2.5, 5, 10, 15, 20 and 25 µg/mL exposure medium. The relative total growth of the highest test item was 14% compared to the total growth of the solvent controls.

Evaluation of mutagenicity
No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item.

Applicant's summary and conclusion

Conclusions:

Alkaterge E is not mutagenic in the mouse lymphoma L5178Y test system.

Executive summary:

The objective of this study was to evaluate the mutagenic potential of Alkaterge E by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions. The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.  The study procedures described in this report were based on OECD guideline No. 490. Alkaterge E was a brown viscous liquid. The vehicle of the test item was ethanol. The concentrations analyzed for the dose formulation samples at intermediate and high concentration level were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). For the dose formulation samples at low concentration level the mean accuracy were slightly above the target concentration (i.e. 118% of target). A small response at the retention time of the test item was observed in the chromatograms of the vehicle group, however,this response was only 0.025-12% of the Group Low Samples. Dose formulation samples were stable when stored at room temperature under normal laboratory light conditions for at least 4 hours.

In the first experiment, Alkaterge E was tested up to concentrations of 38 µg/mL in the absence and presence S9-mix. The incubation time was 3 hours. In the absence of S9-mix, relative total growth (RTG) was reduced to 7% and 36% at the concentrations of 25 and 38 µg/mL, respectively. In the presence of S9-mix, no toxicity was observed at the dose level of 38 µg/mL. Alkaterge E precipitated in the culture medium at the dose level of 38 µg/mL. In the second experiment, Alkaterge E was tested up to concentrations of 25 µg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 14%. The mean mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.  It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Alkaterge E did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, Alkaterge E did not induce a significant increase in the mutation frequency.

In conclusion, Alkaterge E is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.