2-[(octadecylcarbamoyl)oxy]ethyl acrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic potential of 2-Propenoic acid, 2-[[(octadecylamino)carbonyl]oxy]ethyl ester.

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: 2-Propenoic acid, 2-[[(octadecylamino)carbonyl]oxy]ethyl ester
Physical state/Appearance: White solid
CAS Number: 78433-08-6
Purity: >98%

Target gene:

Salmonella typhimurium
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor

Escherichia coli
WP2uvrA trp-; uvrA-: base-pair substitution

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:

Experiment 1
The maximum concentration was 5000 microg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 microg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Experiment 2

The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 µg/plate.

Six test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

dimethyl formamide

Untreated negative controls:

yes

Remarks:

untreated control

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
Test Item Preparation and Analysis
The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in dimethyl formamide, therefore, this solvent was selected as the vehicle.

The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl formamide by mixing on a vortex mixer and sonication for 30 minutes at 40 °C on the day of each experiment. The 50 mg/mL formulation was kept warm to conserve a doseable suspension. Formulated concentrations were adjusted to allow for the stated water/impurity content (2%) of the test item. Dimethyl formamide is considered an acceptable vehicle for use in this test system (Maron et al., 1981). Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10-4 microns.

All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. This exception is considered not to affect the purpose or integrity of the study.

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at and above 1500 microg/plate because of test item precipitation. Several further manual counts were also required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As Experiment 1 was deemed negative (please see results section), Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at and above 500 microg/plate because of test item precipitation.

Evaluation criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). A test item precipitate (white and particulate in appearance) was initially noted from 150 µg/plate (pre-incubation method) and 500 g/plate (plate incorporation method), this observation did not prevent the scoring of revertant colonies.
There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method). A small, statistically significant increase in WP2uvrA revertant colony frequency was observed in the presence of S9-mix at
15 µg/plate in the first mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 15 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.7 times the concurrent vehicle control.

Conclusions:

2-Propenoic acid, 2-[[(octadecylamino)carbonyl]oxy]ethyl ester was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 28 November 2017 to 01 March 2018. Report Issued: 30 July 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.

The details of the donors used are:

Preliminary toxicity test: Female aged 25 years
Main experiment: Male, aged 26 years

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital / beta-naphthaflavone induced rat liver S9.

Test concentrations with justification for top dose:

4(20)-hour without S9: 0, 4, 8, 12, 16, 24, 32 and 64 µg/mL
4(20)-hour with S9 (2%): 0, 4, 8, 12, 16, 24, 32 and 64 µg/mL
24-hour without S9: 0, 4, 8, 12, 16, 24, 32 and 64 µg/mL

The doses were selected based on the results from a preliminary toxicity study.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION:
In culture medium.

CELL CULTURE:
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

DURATION
- Preincubation period: approximately 48 hours.
- Exposure duration:
4-hour exposure to test substance without S9 followed by a 20-hour treatment free incubation period
4-hour exposure to test substance with S9 followed by a 20-hour treatment free incubation period
20-hour exposure to test substance without S9.

SPINDLE INHIBITOR (cytogenetic assays):
Colcemid 0.1 µg/mL 2.5 hours before the required harvest time.

STAIN (for cytogenetic assays):
5% Giemsa for 5 minutes

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
After incubation with colcemid, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

NUMBER OF CELLS EVALUATED:
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated


DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was determined in a preliminary toxicity test by observation of heamolysis in cells and mitotic indices.

Key result

Species / strain:

lymphocytes: derived from the blood of human volunteers

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 3.91 to 1000 μg/mL. The maximum dose was the maximum practical dose level.

A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 31.25 μg/mL in the exposure groups in the absence of metabolic activation (S9) and at and above 62.5 μg/mL in the exposure group in the presence of S9 (with precipitate observed at and above 31.25 μg/mL in the blood cultures).

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to the maximum dose, 1000 μg/mL, for all three exposure groups. The test item induced no evidence of toxicity in any of the exposure groups.

The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level which was determined to be 64 μg/mL for all three exposure groups.

Main Test

The qualitative assessment of the slides determined that precipitate was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the maximum dose level of test item, 64 μg/mL in all three exposure groups.

Precipitate observations were made at the end of exposure in blood-free cultures and noted at 32 μg/mL in the 4(20)-hour exposure groups in the presence and absence of S9 and at and above 24 μg/mL in the 24-hour continuous exposure group.

The mitotic index data for the Main Experiment confirmed the qualitative observations in that no dose-related inhibition of mitotic index was observed in any of the exposure groups.

 

Conclusions:

The test substance not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolising system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction

The study was undertaken to investigate the effect of the test substance to induce chromosomal aberrations in cultured mammalian cells in vitro. The test method was designed to meet the requirements of OECD Guidelines for Testing of Chemicals No. 473 "In Vitro Mammalian Chromosome Aberration Test" adopted 29 July 2016

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated as follows: 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited on precipitate where the maximum dose was determined to be 64 µg/mL. The dose levels selected for the Main Experiment were as follows:

Group	Test Substance Concentration (µg/mL)
4(20)-hour without S9	0, 4, 8, 12, 16, 24, 32, 64
4(20)-hour with S9 (2%)
24 -hour without S9

Results

All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level.

Conclusion

The test substance was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 23 April 2018 to 15 May 2018. Report Issue: 12 Oct 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Target gene:

Thymidine kinase TK +/- locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Stock cultures were propagated in plastic flasks in RPMI 1640 complete culture medium. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), were used during the course of the study. The cell cultures were incubated at 37 +/- 1.5°C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5 % ambient air.

-Selective Media: RPMI 1640 (complete culture medium) with the addition of 5 µg/mL of trifluorothymidine (TFT).

- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital / Β-naphthaflavone induced rat liver S9

Test concentrations with justification for top dose:

Main Test Doses Applied
4-hours without S9: 0, 0.98, 1.95, 3.91, 7.81, 15.63 and 31.25 µg/mL
4-hours with S9 (2%): 0, 0.98, 1.95, 3.91, 7.81, 15.63 and 31.25 µg/mL
24-hour without S9: 0, 1.95, 3.91, 7.81, 15.63, 31.25 and 62.5 µg/mL

The doses used for the main test were based on the results of a preliminary range-finding test.

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (suspension growth)

NUMBER OF REPLICATIONS: Duplicate for each test concentration and controls

Statistics:

The experimental data was analysed using a dedicated computer program, Mutant 2.40 by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989).

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the concentration levels investigated.

Preliminary Cytotoxicity Test

There was evidence of modest reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in the 4-hour exposure groups in both the absence and presence of metabolic activation, and more marked reductions in the 24-hour exposure group in the absence of metabolic activation. Precipitate of the test item was observed at and above 31.25 μg/mL in the 4-hour exposure groups in both the absence and presence of metabolic activation, and at and above 62.5 μg/mL in the 24-hour exposure group in the absence of metabolic activation, at the end of the exposure periods. Therefore, following the recommendations of the OECD 490 guideline, the maximum dose level in the Mutagenicity test was limited by the onset of test item precipitate in all three of the exposure groups.

Main Test

There was no evidence of any marked dose-related toxicity in cells treated with the test item in the 4-hour exposure groups in both the absence and presence of metabolic activation, and modest reductions in the 24-hour exposure group in the absence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of any reductions in viability (%V) in any of the three exposure groups, indicating that residual toxicity had not occurred. At the end of the exposure periods, precipitate of the test item was observed at and above 31.25 μg/mL in the 4-hour exposure groups in both the absence and presence of metabolic activation, and at and above 62.5 μg/mL in the 24-hour exposure group in the absence of metabolic activation. Therefore, following the recommendations of the OECD 490 guideline, the lowest precipitating dose level of 31.25 μg/ mL for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 62.5 μg/mL for the 24-hour exposure group in the absence of metabolic activation, was plated for viability and 5-TFT resistance. Acceptable levels of toxicity were seen with the positive control substance.

 

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

 

The test item did not induce any toxicologically significant or dose related increases in the mutant frequency x 10-6 per viable cell at any of the dose levels, including the lowest precipitating dose level in all three of the exposure groups.

Conclusions:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it is considered to be non-mutagenic in this assay.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the following guidelines:

•       OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016,

•       Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

Methods

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (acetone), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

Group	Concentration of Test Substance (μg/mL) plated for viability and mutant frequency
4-hour without S9	0.98, 1.95, 3.91, 7.81, 15.63, 31.25
4-hour with S9 (2%)	0.98, 1.95, 3.91, 7.81, 15.63, 31.25
24-hour withourt S9	1.95, 3.91, 7.81, 15.63, 31.25, 62.5

Results

The maximum dose levels in the Mutagenicity Test were limited by the onset of test item precipitate in all three of the exposure groups, as recommended by the OECD 490 guideline. The vehicle control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it is considered to be non-mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

According to the ECHA Guidance on the Application of the CLP Criteria (version 4.1, June 2015), a substance is classified as a germ cell mutagen from a positive result in an in vitro gene mutation study.

2 -Propenoic acid, 2 -[[(octadecylamino)carbonyl]oxy]ethyl ester gave negative responses in the Ames, Chromosome Aberration and Mouse Lymphoma tests and therefore is not classified as mutagenic.