Icosyl acrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro

Three Ames tests, two HPRT-Tests and two Micronucleus-Tests were conducted with the three mixtures representing long-chain alkyl acrylates. The substances showed no mutagenic potential in neither tests (see table 1).

 

Table 1. Data from in vitro genotoxicity testing

Ester	Test	Method	Results	Reference
2-Propenoic acid, C12-14-alkyl esters [84238-60-8]	Ames-Test	OECD TG 471	not mutagenic	BASF SE 2012
2-Propenoic acid, C12-14-alkyl esters [84238-60-8]	HPRT-Test	OECD TG 476	not mutagenic	BASF SE 2012
2-Propenoic acid, C12-14-alkyl esters [84238-60-8]	Micronucleus-Test	OECD TG 487	not mutagenic	BASF SE 2013
2-Propenoic acid, C16-18-alkyl esters [90530-21-5]	Ames-Test	OECD TG 471	not mutagenic	BASF SE 2012
2-Propenoic acid, C18-22-alkyl esters [85085-17-2]	Ames-Test	OECD TG 471	not mutagenic	BASF SE 2012
2-Propenoic acid, C18-22-alkyl esters [85085-17-2]	HPRT-Test	OECD TG 476	not mutagenic	BASF SE 2012
2-Propenoic acid, C18-22-alkyl esters [85085-17-2]	Micronucleus-Test	OECD TG 487	not mutagenic	BASF SE 2012

 

Conclusion

The absence of a mutagenic potential was demonstrated in various in vitro gene mutation and micronucleus tests for the substances in the group. These include C12 – C22 alkyl acrylate esters and the results are therefore representative for all long-chain alkyl acrylate esters.The available studies are considered to be reliable and suitable to cover the endpoint genetic toxicity and fulfil the REACH information requirements.

 

For this endpoint, a data-gap filling for mono-constituents is addressed by a category based read-across approach. In RAAF nomenclature, this approach is described in scenarios 4 (different compounds have qualitatively similar properties) and governed by AE 4.2 and 4.3 (common underlying mechanism, qualitative and quantitative aspects). Here, the common underlying mechanism is a direct electrophilic reaction of the intact ester. The variable part of the category approach is the length or configuration of the side chain of the parent ester and the alcohol metabolite and their impact on physico-chemical properties and consequent biological properties. Despite the variation, the available data support a weight of evidence-based conclusions that there is no genotoxicity concern for all the category members. Overall, this read-across approach is applied with a high level of confidence.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

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)

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

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)

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 prepared from Wistar rats liver (induced with phenobarbital and β-naphthoflavone)

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 2750, 5500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in ultrapure water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

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

Each experiment includes negative controls in order to check for possible contaminants (sterility control) and to determine the spontaneous mutation rate (vehicle control).

Positive controls
With S9 mix: 2-aminoanthracene, 2.5 μg/plate - strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, - strain: Escherichia coli WP2 uvrA
Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine 5 μg/plate, - strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine 10 μg/plate, - strain: TA 98
• 9-aminoacridine 100 μg/plate, - strain: TA 1537
• 4-nitroquinoline-N-oxide 5 μg/plate, - strain: E. coli WP2 uvrA

Evaluation criteria:

The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain
• The sterility controls revealed no indication of bacterial contamination
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above
• Fresh bacterial culture containing approximately 109 cells per mL were used. For approval the titer of viable bacteria was ≥ 108 colonies per mL.

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY
No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate test and in the preincubation up to the highest required concentration.

SOLUBILITY
Test substance precipitation was found from 2750 μg/plate onward with and without S9 mix.

MUTAGENICITY:
A biologically 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 Stearylacrylate, Synative MM SA, Stearyl 1618 is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Conclusions:

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.

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 - 5500 µg/plate (SPT), 33 µg- 5500 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 from 2750 µg/plate onward with and without S9 mix.

Toxicity: No bacteriotoxic effect was observed under all test conditions.

Mutagenicity: A biologically relevant increase in the number of his+ and 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: 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.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

July 22, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-06-20 to 2012-04-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

July 22, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH, Germany

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):

- Type and identity of media:MEM (minimal essential medium) containing Hank’s salts, glutamine, Hepes (25 mM)
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix (Phenobarbital/beta-naphthoflavone induced rat liver of Wistar rats)

Test concentrations with justification for top dose:

4 h exposure period: 7.3/ 14.7/ 29.4/ 58.7 / 117.4 / 234.8 / 469.6 / 939.3 / 1878.5 / 3757.0 µg/mL (without S9 mix Exp. IA)
4 h exposure period: 29.4/ 58.7 / 117.4 / 234.8 / 469.6 / 939.3 / 1878.5 / 3757.0 µg/mL (without S9 mix Exp. IB)
4 h exposure period: 1.0 / 2.5 / 6.2 / 15.4 / 38.5 / 96.2 / 240.4/ 601.1 / 1502.8 / 3757.0 µg/mL (without S9 mix Exp IB)
24 h exposure period: 7.3/ 14.7/ 29.4/ 58.7 / 117.4 / 234.8 / 469.6 / 939.3 / 1878.5 / 3757.0 µg/mL (without S9 mix Exp. II)
4 h exposure period: 7.3/ 14.7/ 29.4/ 58.7 / 117.4 / 234.8 / 469.6 / 939.3 / 1878.5 / 3757.0 µg/mL (with S9 mix Exp. I +II)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Remarks:

Without metabolic activation: MMC; Mitomycin C 0.3 Og/mL; Griseofulvin 8.0 Og/mL; With metabolic activation: CPA; cyclophosphamide 15 Og/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with S-9 mix, followeed by a 20 hour period after replacing the S9 mix; treatment medium FBS

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2000

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was assessed via counting the number of clones consisting of 1 cell (c1), 2 cells (c2), 3 - 4 cells (c4), and 5 - 8 cells (c8) among the cells that were scored for the presence of micronuclei. These clusters represented the cells that have divided 1, 2, or 3 times within the experiment. From these data, a proliferation index (PI) was calculated. Only those cultures were evaluated which showed a PI > 1.3, in order to guarantee for a sufficient cell proliferation during treatment and recovery.
PI = (c1x 1) + (c2 x 2) + (c4 x 3) + (c8 x 4)/n
PI Proliferation index
n Sum of cell clones scored (containing at least 500 colonies)
cx Number of clones with x cells (with x: 1, 2, 4, or 8)

Evaluation criteria:

A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical control data and
- either a statistically significant concentration-related increase in three test groups or a significant increase of micronucleated cells in at least one test group is observed.
A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated test groups is in the range of the historical control data and
- no statistically significant concentration-related increase in the number of micronucleated cells is observed.
Statistical significance was confirmed by means of the Chi square test.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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: no
- Effects of osmolality: no

No relevant influence on osmolarity or pH value was observed. In the evaluated slides either in the absence or in the presence of metabolic activation no cytotoxicity measured as reduced proliferation index was observed.

In the absence and presence of S9 mix, no biologically relevant increase in the percentage of micronucleated cells was observed.

The rates of micronucleated cells after treatment with the test item (0.40 - 1.45 %) were close to the solvent control values (0.30 - 1.60 %) and within the range of the laboratory historical solvent control data.

Mitomycin C (0.3 µg/mL), Griseofulvin (8.0 µg/mL) or CPA (15.0 µg/mL) were used as positive controls and showed a distinct increase in the percentage of micronucleated cells.

Summary of results of the chromosome aberration study

Exp.	Preparation interval	Test item concentration in µg/ml	Proliferation Index	Micronucleated Cells* in %
Exposure period 4 hrs without S9 mix
IB	24 hrs	Solvent control^1	2.66	0.30
		Positive control^2	2.28	8.65^S
		15.4	2.75	0.45
		38.5	2.78	0.65
		96.2^P	2.70	0.40
		3757.0	2.68	n.d.
Exposure period 24 hrs without S9 mix
II	24 hrs	Solvent control^1	2.55	0.85
		Positive control^3	2.72	14.05^S
		58.7	2.76	0.70
		939.3^PS	2.75	0.75
		1878.5	2.83	0.50
		3757.0	2.89	0.85
Exposure period 4 hrs with S9 mix
IA	24 hrs	Solvent control^1	1.99	1.60
		Positive control^4	1.53	10.45^S
		117.4	1.69	0.75
		234.8	1.88	0.85
		469.6^P	1.74	1.45
		3757.0^P	1.79	n.d.
II	24 hrs	Solvent control^1	1.66	1.00
		Positive control^4	1.40	5.80^S
		939.3	1.57	0.90
		1878.5	1.69	0.90
		3757.0	1.65	0.75

* The number of micronucleated cells was determined of each test group in a sample of 2000 cells

P = Precipitation occurred at the end of treatment

PS = Phase separation (waxy drops) occurred at the end of treatment

S = Number of micronucleated cells statistically significantly higher than corresponding control values

1 = THF 0.5 % (v/v)

2 = Mitomycin C 0.3 µg/mL

3= Griseofulvin 8.0 µg/mL

4 = CPA 15.0 µg/mL

n.d. = Not determined

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and presence of metabolic activation. Therefore, the test item is considered to be non-mutagenic in this in vitro test system when tested up to precipitating or the highest required concentration.

Executive summary:

The test item, dissolved in THF, was assessed for its potential to induce micronuclei in V79 cells of the Chinese hamster in vitro in three independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. IB	Exp. II	Exp. IA & II
Exposure period	4 hours	24 hours	4 hours
Recovery	20 hours	-	20 hours
Preparation interval	24 hours	24 hours	24 hours

In each experimental group two parallel cultures were analyzed and 1000 cells per culture were scored for micronuclei.

The highest applied concentration (3757.0 µg/mL; approx. 10 mM) was chosen with regard to the molecular weight and the purity (93.7 %) of the test item and with respect to the OECD Guideline No. 487.

Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurence of test item precipitation.

In the absence and presence of S9 mix no cytotoxicity was observed up to the highest applied concentration.

No mutagenicity was observed at the concentrations evaluated.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in the percentage of micronucleated cells.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and presence of metabolic activation.

Therefore, the test item is considered to be non-mutagenic in this in vitro test system when tested up to precipitating or the highest required concentration.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

May - Sep 2012

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-04-17 to 2012-08-17

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)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

dated May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

pre-experimant: 29.7 - 3800 µg/mL
Experiment I (with and without S9 mix): 14.9, 29.7, 59.4, 118.8, 237.5, 475.0 µg/mL
Experiment II (without S9-mix): 59.4, 118.8, 237.5, 475.0, 950.0, 1900.0 µg/mL / with S9-mix: 3.7, 7.4, 14.9, 29.7, 59.4, 118.8 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone (pre-test); tetrahydrofurane (main-test)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

other: see remarks

Remarks:

without metabolic activation: EMS; ethylmethane sulfonate (0.150 mg/mL = 1.2 mM); with metabolic activation: DMBA; 7,12-dimethylbenz(a)anthracene (DMBA; 7,12-dimethylbenz(a)anthracene) (1.1 µg/mL= 4.3 µM)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

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

SELECTION AGENT (mutation assays): 6-thioguanine 11 µg/mL

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance was considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation.

Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

No relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in any of the experimental parts with and without metabolic activation. The concentration range was limited by the solubility of the test item in aqueous medium. Precipitation of the test item was noted at 59.4 μg/mL and above in the first experiment with and at 475.0 μg/mL without metabolic activation. In the second experiment precipitation was noted at 29.7 μg/mL and above with and at 1900 μg/mL without metabolic activation. Increasing turbidity but no precipitation was observed at the lower concentrations of the second experiment without metabolic activation. The observed turbidity in the presence of 10% fetal bovine serum during 24 hour treatment indicates protein aggregate formation.

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The highest concentration applied in the pre-experiment was 3800 µg/mL (equal to 10 mM). The maximum concentration of the main experiments was limited by precipitation of the test item in aqueous media. The test item was dissolved in acetone (pre-experiment) and THF (experiment I and II).

No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation.

Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2011-11-23 to 2012-01-12

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE

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 of Phenobarbital/ß-naphtoflavone induced rats.

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2750 and 5500 μg/plate (with and without S9 mix)

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see remarks

Remarks:

2-aminoanthracene (2-AA) (with S9 mix; for all strains)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see remarks

Remarks:

without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (for strains: TA 1535, TA 100), 4-nitro-o-phenylenediamine (NOPD) (for strain: TA 98) , 9-aminoacridine (AAC) (for strain: TA 1537), 4-nitroquinoline-N-oxide (4-NQO) (for E.coli WP2 uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Standard plate test with and without S9 mix (experiment 1), Preincubation test with and without S9 mix (experiment 2)

DURATION
- Preincubation period: 20 min (exp.2)
- Exposure duration: 48 - 72 h

Evaluation criteria:

Toxicity detected by a
• decrease in the number of revertants
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
• reduction in the titer is recorded for all test groups both with and without S9 mix in all experiments.

Acceptance criteria
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data.
• Fresh bacterial culture containing approximately 10 e9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10 e8 colonies per mL.

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

SOLUBILITY:

Precipitation of the test substance was found from 2750 μg/plate onward with and without S9 mix.

TOXICITY:

A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2750 μg/plate onward

MUTAGENICITY:

A biologically 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 biologically 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 or nearby 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 Behenylacrylate (Acrylate 22 45%) is not mutagenic in the Salmonella typhimurium/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 - 5500 µg/plate (SPT), 33 µg - 5500 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 from 2750 µg/plate onward with and without S9 mix.

Toxicity: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2750 µg/plate onward.

Mutagenicity: A biologically 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro

 

Bacterial reverse mutation assay

2-Propenoic acid, C12-14-alkyl esters was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e.Salmonella typhimuriumandEscherichia coli, in a reverse mutation assay.

Strains: TA 1535, TA 100, TA 1537, TA 98 andE.coliWP2 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 theSalmonella typhimurium/Escherichia colireverse mutation assay in the absence and the presence of metabolic activation.

 

2-Propenoic acid, C16-18-alkyl esters was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e.Salmonella typhimuriumandEscherichia coli, in a reverse mutation assay.

Strains: TA 1535, TA 100, TA 1537, TA 98 andE.coliWP2 uvrA

Dose Range: 33 µg – 5500 µg/plate (SPT), 33 µg – 5500 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 from 2750 µg/plate onward with and without S9 mix.

Toxicity: No bacteriotoxic effect was observed under all test conditions.

Mutagenicity: A biologically relevant increase in the number of his⁺and 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 theSalmonella typhimurium/Escherichia colireverse mutation assay in the absence and the presence of metabolic activation (BASF SE, 2012).

 

In another Ames test, the mixture 2-Propenoic acid, C18-22-alkyl esters was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e.Salmonella typhimuriumandEscherichia coli, in a reverse mutation assay.

Strains: TA 1535, TA 100, TA 1537, TA 98 andE.coliWP2 uvrA

Dose Range: 33 µg – 5500 µg/plate (SPT), 33 µg – 5500 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 from 2750 µg/plate onward with and without S9 mix.

Toxicity: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2750 µg/plate onward.

Mutagenicity: A biologically 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 theSalmonella typhimurium/Escherichia colireverse mutation assay in the absence and the presence of metabolic activation (BASF SE, 2012).

 

HPRT-Test in mammalian cells

The mixture 2-Propenoic acid, C12-14-alkyl esters was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cellsin 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:

1^stExperiment

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

2^ndExperiment

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

3^rdExperiment

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 1^stand 3^rdExperiment in the absence and presence of metabolic activation, the highest concentrations tested for gene mutations were clearly cytotoxic. However, in the 2^ndExperiment in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration. Therefore, a 3^rdExperiment 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, 2-Propenoic acid, C12-14-alkyl esters is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and presence of metabolic activation (BASF SE, 2012).

 

In another HPRT-Test, 2-Propenoic acid, C18-22-alkyl esters was investigated for its potential to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration applied in the pre-experiment was 3800 µg/mL (equal to 10 mM). The maximum concentration of the main experiments was limited by precipitation of the test item in aqueous media. The test item was dissolved in acetone (pre-experiment) and THF (experiment I and II). No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, 2-Propenoic acid, C18-22-alkyl esters is considered to be non-mutagenic in this HPRT assay (BASF SE, 2012).

 

Micronucleus-Test

The mixture 2-Propenoic acid, C12-14-alkyl esters was assessed for its potential to induce micronuclei in V79 cellsin 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 pre-test for a HPRT study the following concentrations were tested. The test groups printed in bold type were evaluated.

1^stExperiment

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

2^ndExperiment

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; 25.00; 50.00; 100.00 µg/mL

3^rdExperiment

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 1^stand 3^rdExperiment. 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) underin vitroconditions in V79 cells in the absence and the presence of metabolic activation (BASF SE, 2013).

 

In another Micronucleus assay, the mixture 2-Propenoic acid, C18-22-alkyl esters, dissolved in THF, was assessed for its potential to induce micronuclei in V79 cells of the Chinese hamsterin vitroin three independent experiments.

1^stExperiment

4 hours exposure period, 20 hours recovery, 24 hours preparation interval, with S9 mix

4 hours exposure period, 20 hours recovery, 24 hours preparation interval, without S9 mix

2^ndExperiment

4 hours exposure period, 20 hours recovery, 24 hours preparation interval, with S9 mix

24 hours exposure period, no recovery, 24 hours preparation interval, without S9 mix

In each experimental group two parallel cultures were analyzed and 1000 cells per culture were scored for micronuclei. The highest applied concentrations (3757.0 µg/mL; approx. 10 mM) was chosen with regard to the molecular weight and the purity (93.7 %) of the test item and with respect to the OECD Guideline No. 487. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of test item precipitation. In the absence and presence of S9 mix no cytotoxicity was observed up to the highest applied concentration. No mutagenicity was observed at the concentrations evaluated. Appropriate mutagens were used as positive controls. They induced statistically significant increases in the percentage of micronucleated cells. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei in V79 cells (Chinese hamster cell line)in vitroin the absence and presence of metabolic activation. Therefore, the test item is considered to be non-mutagenic in thisin vitrotest system when tested up to precipitating or the highest required concentration.

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.