Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The read across substance 2-Propenoic acid, C12-14-alkyl esters (mixture of CAS no. 2156-97-0 and 21643-42-5) was tested in three in vitro studies (Ames test, Micronucleus test, HPRT) and was found to be negative in all tests.

Based on these results 2 -Propenoic acid, dodecyl ester is also considered to be negative in genetic toxicity tests.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
July 22, 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Cell line and storage
The V79 cell line is a permanent cell line derived from the Chinese hamster and has a
− high proliferation rate (doubling time of about 12 - 14 hours),
− high plating efficiency (≥ 90%),
− stable karyotype (modal number of 22 chromosomes).

Stocks of the V79 cell line (1-mL portions) were maintained at -196°C in liquid nitrogen using 7% (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for the cytogenetic experiments was checked for

− mycoplasma contamination,
− karyotype stability,
− plating efficiency (=colony forming ability) incl. vital staining.


Culture media
MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with
− 10% (v/v) fetal calf serum (FCS)
− 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL)
− 1% (v/v) amphotericine B (250 μg/mL)
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix (Phenobarbital/beta-naphthoflavone iduced rat liver of Wistar rats)
Test concentrations with justification for top dose:
1st experiment:
with and without S9 mix (4 hours exposure)
1.25 μg/mL, 2.50 μg/mL, 5.00 μg/mL, 10.00 μg/mL, 20.00 μg/mL, 40.00 μg/mL, 80.00 μg/mL

2nd experiment:
without S9 mix (24 hours exposure)
0.39 μg/mL , 0.78 μg/mL , 1.56 μg/mL , 3.13 μg/mL , 6.25 μg/mL, 12.50 μg/mL
with S9 mix (4 hours exposure)
6.25 μg/mL,12.50 μg/mL, 25.00 μg/mL, 50.00 μg/mL, 100.00 μg/mL

3rd experiment:
without S9 mix (24 hours exposure)
3.13 μg/mL, 6.25 μg/mL, 12.50 μg/mL, 25.00 μg/mL, 50.00 μg/mL, 100.00 μg/mL
with S9 mix (4 hours exposure)
12.50 μg/mL, 25.00 μg/mL, 50.00 μg/mL, 100.00 μg/mL, 200.00 μg/mL, 400.00 μg/mL
Vehicle / solvent:
- Ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
Without metabolic activation: 500 and 600 μg/mL ethyl methanesulfonate, With metabolic activation: 2.5 μg/mL cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

Three independent experiments were performed with different expsoure times and test concentrations.
In Experiment I the exposure period was 4 hours with and without metabolic activation.
In Experiment II the exposure period was 24 hours without S9 mix and 4 hours with metabolic activation.
In Experiment III the exposure period was 24 hours without S9 mix and 4 hours with metabolic activation

The cells were prepared 24 hours after start of treatment with the test item.

DURATION
- Exposure duration: 4 or 24 h

STAIN (for cytogenetic assays): Wrights solution (modified May-Grünwald solution)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: at least 1 000 cells per culture, means at least 2 000 cells per dose group

DETERMINATION OF CYTOTOXICITY
For additional information about the cytotoxic potential of the test substance cells were seeded in flasks (2.5 x 10E5 cells per 25 cm2 flask) about 24 – 30 hours prior to exposure. The cells were treated similar to the slides using the same media and test substance concentrations. At the end of recovery period single cell suspensions were prepared from each test group (all test groups, except the positive controls) and the cells were counted using a cell counter. Based on these data the relative increase in cell count (RICC) was calculated.

OTHER EXAMINATIONS:
pH value
Changes in the pH were recorded by a change in the color of the indicator in the culture medium (phenol red: no color change from pH 6.7 - 8.3). The pH was measured, at least for the two top doses and for the vehicle control with and without S9 mix.
Osmolarity
Osmolarity was measured, at least for the top dose and for the vehicle control with and without S9 mix.
Solubility
Test substance precipitation was checked immediately after start of treatment of the test cultures (macroscopically) and at the end of treatment (macroscopically/ microscopically).
Evaluation criteria:
Acceptance criteria
The in vitro micronucleus assay is considered valid if the following criteria are met:
• The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells.
• The number of cells containing micronuclei in the vehicle control was within the range of the historical negative control data
• The positive control substances both with and without S9 mix induced a significant increase in the number of micronucleated cells

Assessment criteria
A test substance is considered "positive" if the following criteria are met:
• A significant, dose-related and reproducible increase in the number of cells containing micronuclei.
• The number of micronucleated cells exceeds both the value of the concurrent vehicle control and the range of the historical negative control data.
A test substance generally is considered "negative" if the following criteria are met:
• The number of micronucleated cells in the dose groups is not significant increased above the concurrent vehicle control value and is within the range of the historical negative control data
Statistics:
The statistical evaluation of the data was carried out using the MUVIKE program system (BASF SE). The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not influenced
- Effects of osmolality: not influenced
- Water solubility:
- Precipitation: not observed up to the highest applied test substance concentration

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC), decrease in proliferation index (PI) or non-scorable slides was observed at least at the highest applied test substance concentrations in the 1st and 3rd Experiment.

OTHER:
The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells.
Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei.

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.
Conclusions:
Under the experimental conditions chosen here, the conclusion is drawn that the test substance has not the potential to induce micronuclei (clastogenic and / or aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.
Executive summary:

The test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Three independent experiments were carried out with and without the addition of liver S9 mix from induced rats (exogeneous metabolic activation).

Based on the observations and the toxicity data of a previously performed pretest for a HPRT study the following concentrations were tested. The test groups printed in bold type were evaluated.

1st Experiment

4 hours exposure; 24 hours harvest time; without S9 mix

0; 1.25; 2.50; 5.00; 10.00; 20.00; 40.00; 80.00 µg/mL

4 hours exposure; 24 hours harvest time; with S9 mix

0; 1.25; 2.50; 5.00; 10.00; 20.00; 40.00; 80.00 µg/mL

2nd Experiment

24 hours exposure; 24 hours harvest time; without S9 mix

0; 0.39; 0.78; 1.56; 3.13; 6.25; 12.50 µg/mL

4 hours exposure; 24 hours harvest time; with S9 mix

0; 6.25; 12.50; 25.00; 50.00; 100.00 µg/mL

3rd Experiment

24 hours exposure; 24 hours harvest time; without S9 mix

0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 µg/mL

4 hours exposure; 24 hours harvest time; with S9 mix

0; 12.50; 25.00; 50.00; 100.00; 200.00; 400.00 µg/mL

A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group.

The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei.

Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC), decrease in proliferation index (PI) or non-scorable slides was observed at least at the highest applied test substance concentrations in the 1st and 3rd Experiment.

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Thus, under the experimental conditions described, the test substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
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)
Remarks:
(from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz
Type of assay:
bacterial reverse mutation assay
Target gene:
his, trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix pepared from rat livers (induced with phenobarbital and β-naphthoflavone)
Test concentrations with justification for top dose:
0; 33; 100; 333; 1000; 2500 and 5000 μg/plate, with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, acetone was used as vehicle,
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see remark
Remarks:
With S9 mix: 2-aminoanthracene (2-AA); Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Standard plate test with and without S9 mix, Preincubation test with and without S9 mix

DURATION
- Preincubation period: 20 min
- Exposure duration: 48-72 h
Evaluation criteria:
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.
Statistics:
not required
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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
Additional information on results:
TEST CONDITIONS:
Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY:
Precipitation of the test substance was found at 5000 μg/plate with and without S9 mix.

TOXICITY:
A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 5000 μg/plate.

MUTAGENICITY:
A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard
plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella thyphimurium / Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Conclusions:
Under the experimental conditions chosen here, it is concluded that the test substance is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.
Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

Strains: TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA

Dose Range: 33 µg - 5000 µg/plate (SPT), 33 µg - 5000 µg/plate (PIT)

Test Conditions: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

Solubility: Precipitation of the test substance was found at 5000 µg/plate with and without S9 mix.

Toxicity: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 5000 µg/plate.

Mutagenicity: A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

Conclusion: Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium / Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May - Sep 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (wistar rat, liver induced with phenobarbital and β-naphthoflavone)
Test concentrations with justification for top dose:
Test 1: 0; 0.78; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 μg/mL (without S9 mix, 4-hour ); 0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 μg/mL (with S9 mix, 4-hour)
Test 2: 0; 0.78; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 μg/mL (without S9 mix, 24 hour); 0; 1.56; 3.13; 6.25; 12.50; 25.0; 50.0; 100.0; 200.0 μg/mL (with S9 mix, 4-hour)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Due to the insufficient solubility of the test substance in water, Ethanol was selected as the vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical data are available. The final concentration of the vehicle Ethanol in the culture medium was 1% (v/v).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
see remarks
Remarks:
Without metabolic activation: 300 μg/mL ethyl methanesulfonate; With metabolic activation: 1.25 μg/mL 7.12-Dimethylbenz[a]anthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h / 24 h
- Expression time (cells in growth medium): 6-8 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

CYTOTOXICITY:
Cloning efficiency (CE) (pre-experiment)
The procedure for the determination of the cloning efficiency in the pre-experiment was similar to that described for the determination of the cloning efficency 1 (CE1) in the main experiments, excepting that every dose group contained only two cultures.

Cloning efficiency 1 (CE1; survival)
For the determination of the influence of the test substance directly after the exposure period, about 200 cells per dose group were seeded in 25 cm² flasks in duplicate using 5 mL Ham's F12 medium incl. 10% (v/v) FCS. After an attachment period of 20 – 24 hours, the cells were
treated with the vehicle, test substance or positive control for 4 hours or 24 hours. After exposure, the cells were rinsed several times with HBSS. Then, the cells were cultured in 5 mL Ham's F12 medium incl. 10% (v/v) FCS.

Cloning efficiency 2 (CE2; viability)
For the determination of the mutation rate after the expression period, about 200 cells were separated during the transfer into selection medium (after 7 – 9 days) and seeded in two flasks (25 cm2) containing 5 mL Ham's F12 medium incl. 10% (v/v) FCS each.
In all cases, after seeding the flasks were incubated for 5 - 8 days to form colonies. These colonies were fixed, stained and counted.
The absolute and relative cloning efficiencies (%) were calculated for each test group.
Evaluation criteria:
A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.

Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 106 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.
Statistics:
An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective vehicle control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.
Species / strain:
Chinese hamster Ovary (CHO)
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
Additional information on results:
In the 1st and 3rd Experiment in the absence and presence of metabolic activation, the highest concentrations tested for gene mutations were clearly cytotoxic. However, in the 2nd Experiment in the absence and presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration. Therefore, a 3rd Experiment was performed which showed clearly reduced colony numbers at least at the highest concentrations.
Conclusions:
In the absence and the presence of metabolic activation, the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.
Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, all of them with and without the addition of liver S9 mix from phenobarbital- and ß-naphthoflavone-induced rats (exogeneous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested. Test groups printed in bold type were evaluated in this study:

1st Experiment

without S9 mix (4 -hour exposure period)

0; 0.78; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 µg/mL

with S9 mix (4 -hour exposure period)

0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 µg/mL

2nd Experiment

without S9 mix (24 -hour exposure period) (unexpected weak cytotoxicity - not valid)

0; 0.31; 0.63; 1.25; 2.50; 5.00; 10.00; 20.00 µg/mL

with S9 mix (4 -hour exposure period) (unexpected weak cytotoxicity - not valid)

0; 2.50; 5.00; 10.00; 20.00; 40.00; 80.00 µg/mL

3rd Experiment

without S9 mix (24 -hour exposure period)

0; 0.78; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 µg/mL

with S9 mix (4 -hour exposure period)

0; 1.56; 3.13; 6.25; 12.50; 25.0; 50.0; 100.0; 200.0 µg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 and 24 hours in the absence of metabolic activation and for 4 hours or in the presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6 -thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line.

Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations.

In this study, in the 1st and 3rd Experiment in the absence and presence of metabolic activation, the highest concentrations tested for gene mutations were clearly cytotoxic. However, in the 2nd Experiment in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration. Therefore, a 3rd Experiment was performed which showed clearly reduced colony numbers at least at the highest concentrations.

On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two independent experiments.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and presence of metabolic activation.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

AMES Test (read-across):

The test substance 2-Propenoic acid, C12-14-alkyl esters (mixture of CAS no. 2156-97-0 and 21643-42-5) was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. (strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA, dose range: 33 μg - 5 000 μg/plate (SPT) 33 μg - 5000 μg/plate (PIT) test conditions: standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found at 5000 μg/plate with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 5000 μg/plate. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

HPRT (read-across):

The substance 2-Propenoic acid, C12-14-alkyl esters (mixture of CAS no. 2156-97-0 and 21643-42-5) was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, all of them with and without the addition of liver S9 mix from phenobarbital- andβ-naphthoflavone induced rats (exogenous metabolic activation) Doses from 0 – 200 µg/mL were tested. Cells were treated with the test substance for 4 and 24 hours in the absence of metabolic activation and for 4 hours or in the presence of metabolic activation. Subsequently, cells were cultured for 6-8 days and then selected in 6- thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations. In the 1st and 3rd Experiment in the absence and presence of metabolic activation, the highest concentrations tested for gene mutations were clearly cytotoxic. However, in the 2nd Experiment in the absence and presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration. Therefore, a 3rd Experiment was performed which showed clearly reduced colony numbers at least at the highest concentrations. On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two independent experiments. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in theabsence and presence of metabolic activation.

In vitro MNT (read-across):

The substance 2-Propenoic acid, C12-14-alkyl esters (mixture of CAS no. 2156-97-0 and 21643-42-5) was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Three independent experiments were carried out with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). Concentrations from 0 – 400 µg/mL were tested. A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group. The vehicle controls gave frequencies of micronucleated cells within the historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei. Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC), decrease in proliferation index (PI) or non-scorable slides was observed at least at the highest applied test substance concentrations in the 1st and 3rd Experiment. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system. Thus, under the experimental conditions described, the test item is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No adverse findings on genotoxicity were observed in the Ames test (OECD 471), the HPRT test (OECD 476) and the in vitro micronucleus test in mammalian cells (OECD 487). As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EC) No. 2017/776.