Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs was tested for mutation in five histidine-required strains of Salmonella typhimurium, both in the absence and in the presence of metabolic activation S-9. All the test strains were tested at concentrations up to 1200 µg/plate as the maximum test dose.

The mean number of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. No

Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs treatments of the test strains resulted in increased revertant numbers that could be considered indicative of any test article mutagenic activity. No signs of toxicity were apparent for any of the test plates, either with or without S-9. It was concluded that Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and WP2 uvrA) , when tested under the conditions employed in this study.

Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs was tested for its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. The study procedures described in this report were based on the most recent OECD 490 guideline. In the absence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency. In conclusion, 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions.

Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs was tested for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix) in accordance to OECD 487. The possible clastogenicity and aneugenicity was tested in two independent experiments. The test item Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments. In conclusion, Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

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)

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Identification: Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl]derivs
Batch (Lot) Number: 190708588
Expiry date: 31 July 2020 (expiry date)
Physical Description: Transparent liquid
Purity/Composition: See Certificate of Analysis, UVCB
Storage Conditions: At room temperature protected from light

Test Facility test item number: 210500/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name (IUPAC, synonym or trade name): 2-Propenoic acid, ester with C12-16-alkyl glycidyl ether
CAS number: 68071-40-9
EC number 614-257-7
Molecular weight: ≥ 314.46 - ≤ 370.57

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in the international OECD guideline. The objective of this study was to evaluate Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of the test item was tested in two independent experiments.

Cytokinesis block (if used):

Cytotoxicity in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).

Metabolic activation:

with and without

Metabolic activation system:

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

S9-mix was prepared immediately before use (Appendix 6 in attached study report) and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Life Technologies).

The above solution was filter (0.22 m)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.

Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Test concentrations with justification for top dose:

In the first cytogenetic assay, the test item was tested up to 100 µg/mL and 156 µg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction, respectively. The test item precipitated in the culture medium at the dose level of 156 µg/mL and appropriate toxicity was reached at the dose level of 100 µg/mL.

In the second cytogenetic assay, the test item was tested up to 70 µg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

Vehicle / solvent:

The vehicle for the test item was dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany, batch K50270931 925, Expiry date 30 June 2021).

No correction was made for the purity/composition of the test item.

A solubility test was performed based on visual assessment. The test item formed a clear colourless solution in dimethyl sulfoxide.

Test item concentrations were used within 1 hour after preparation.

The final concentration of the solvent in the culture medium was 1.0% (v/v).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBERS OF CULTURES:
- Duplicate cultures per concentration was used.

FIRST CYTOGENETIC ASSAY:
In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose-range finding test. The test item was tested in the absence and presence of S9-mix.
Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 46 ± 2 hours and thereafter exposed to selected doses of the test item for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B (Sigma) was added to the cells simultaneously with the test item at the 24 hours exposure time. A vehicle control was included at each exposure time.
The highest tested concentration was determined by the solubility of the test item in the culture medium.
The test item precipitated at concentrations of 156 µg/mL and upwards. The lymphocytes were cultured in duplicate at the 3 h exposure time and appropriate positive controls were included.
After 3 hours exposure to the test item in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasine B (5 µg/mL) and incubated for another 24 hours (1.5 times normal cell cycle). The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately.
Cytotoxicity of the test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index). No cytotoxicity was observed in the duplicate cultures of the 3 h exposure time in the presence of S9-mix and the slides were scored for micronuclei. The pilot study (short term exposure period, presence of S9-mix) was used as the first cytogenetic assay.
Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.

CYTOGENETCI ASSAY !A:
Since toxicity was observed at the 3 h exposure time in the absence of S9-mix an additional experiment was performed. Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of the test item for 3 hours in the absence of S9-mix. After 3 hours exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasin B (5 µg/mL) and incubated for another 24 hours. Appropriate vehicle and positive controls were included in the first cytogenetic assay.

CYTOGENETIC ASSAY 2:
To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix.
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of the test item with cytochalasin B (5 µg/mL) for 24 hours in the absence of S9-mix. Appropriate vehicle and positive controls were included in the second cytogenetic assay.

PREPARATION OF SLIDES:
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for
10 - 30 min with 6.7% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).


METHODS FOR MEASUREMENT OF CYTOTOXICITY
A minimum of 500 cells (with a maximum deviation of 5%) per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).

To prevent bias, all slides were randomly coded before examination of micronuclei and scored. An adhesive label with Charles River Den Bosch study identification number and code was stuck over the marked slide. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately. Since the lowest concentration of-C and CP resulted in a positive response the highest concentration was not examined for the presence of micronuclei.

Due to cytotoxicity the number of examined bi- or mononucleated cells in the positive control groups might be <1000. However, when an expected statistical significant increase was observed, this has no effect on the study integrity. The following criteria for scoring of binucleated cells were used (1 - 2, 6):

- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.

The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).

The following criteria for scoring micronuclei were adapted from Fenech, 1996 (1):
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.

Rationale for test conditions:

All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 28 - 94%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.4 - 37.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Evaluation criteria:

An in vitro micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analyzed by the Chi-square test (one-sided, p < 0.05).

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data. A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:

a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:

a) one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Key result

Species / strain:

lymphocytes: See test report

Remarks:

See test report

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See test report

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See test report

Vehicle controls validity:

valid

Remarks:

In accordance to HCD

Untreated negative controls validity:

valid

Remarks:

In accordance to HCD

True negative controls validity:

valid

Remarks:

In accordance to HCD

Positive controls validity:

valid

Remarks:

In accordance to HCD

Additional information on results:

Dose range finding/first cytogenicity assay:
At a concentration of 156 µg/mL the test item precipitated in the culture medium. At the 3 h exposure time, blood cultures were treated in duplicate with 19.5, 39, 78 and 156 µg test item/mL culture medium with and without S9-mix (first cytogenetic assay).
At the 24 hour exposure time single blood cultures were treated with 4.9, 9.8, 19.5, 39, 78 and 156 µg test item/mL culture medium without S9-mix (dose-range finding test).
In the absence of S9-mix no appropriate dose levels could be selected for scoring of micronuclei since at the concentration of 78 µg/mL not enough cytotoxicity was observed (45%), whereas the next higher concentration of 156 µg/mL was too toxic for scoring (71%). The experiment was repeated in cytogenetic assay 1A.
In the presence of S9 mix, the following dose levels were selected for the scoring of micronuclei:
With S9-mix: 39, 78 and 156 µg test item/mL culture medium (3 hours exposure time, 27 hours harvest time). In the presence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei (see attached study report).

Cytogenetic assay 1A:
Based on the results of the dose-range finding test the following dose levels were selected for the first cytogenetic assay:
Without S9-mix: 10, 50, 70, 80, 90, 100, 125 and 156 µg/mL culture medium (3 hours exposure time, 27 hours harvest time).
The following dose levels were selected for scoring of micronuclei:
Without S9-mix: 10, 70 and 100 µg/mL culture medium (3 hours exposure time, 27 hours harvest time).
In the absence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei (see attached study report). The test item did induce a statistically significant increase in the number of mononucleated cells with micronuclei. However, this result was within the 95% control limits of the distribution of the historical solvent control database and was therefore considered to be have no biological relevance.

Cytogenetic assay 2:
To obtain more information about the possible clastogenicity and aneugenicity of the test item, a second cytogenetic assay was performed in which human lymphocytes were exposed for 24 hours in the absence of S9-mix. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix 1, 10, 60, 70, 80, 90 and 100 µg/mL culture medium (24 hours exposure time, 24 hours harvest time).
The following dose levels were selected for the scoring of micronuclei:
Without S9-mix: 10, 60 and 70 µg test item/mL culture medium (24 hours exposure time, 24 hours harvest time).
The test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei (see attached study report).

Conclusions:

The test item Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs was tested for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix) in accordance to OECD 487. The possible clastogenicity and aneugenicity was tested in two independent experiments.

The test item Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

In conclusion, Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described.

Executive summary:

The test item Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs was tested for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix) in accordance to OECD 487. The possible clastogenicity and aneugenicity was tested in two independent experiments.

In the first cytogenetic assay, the test item was tested up to 100 µg/mL and 156 µg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction, respectively. The test item precipitated in the culture medium at the dose level of 156 µg/mL and appropriate toxicity was reached at the dose level of 100 µg/mL. In the second cytogenetic assay, the test item was tested up to 70 µg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly and the test is therefore considered valid.

The test item Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

In conclusion, Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Sep 2019 to 11 Nov 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

Identification: Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl]derivs
Batch (Lot) Number: 190708588
Expiry date: 31 July 2020 (expiry date)
Physical Description: Transparent liquid
Purity/Composition: See Certificate of Analysis, UVCB
Storage Conditions: At room temperature protected from light

Test Facility test item number: 210500/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name (IUPAC, synonym or trade name): 2-Propenoic acid, ester with C12-16-alkyl glycidyl ether
CAS number: 68071-40-9
EC number 614-257-7
Molecular weight: ≥ 314.46 - ≤ 370.57

Target gene:

L5178Y mouse lymphoma cells (L5178Y/TK+/--3.7.2C) are used because they are sensitive indicators of mutagenic activity of a broad range of chemical classes. The TK mutational system is able to detect base pair alterations, frame shift mutations and small deletions and clastogenic effect.

Cells deficient in thymidine kinase (TK), due to the forward mutation (TK+/- to TK-/-) are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT). TK deficient cells cannot incorporate the analogue into its phosphorylated derivative (nucleotide); the nucleotides needed for cellular metabolism are obtained solely from de novo synthesis. In the presence of TK, TFT is converted into nucleotides, which is lethal to the cells. Thus, cells that are able to proliferate in culture medium containing TFT are mutated, either spontaneously or by the action of the test item, to a TK deficient phenotype. Furthermore, by applying the TFT-selection procedure it is possible to discriminate between two different classes of TFT-resistant mutants (small and large colonies). The large colonies are believed to be the result of mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. The small colonies are believed to be the result of chromosomal damage to the TK and adjacent genes.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10-4 M hypoxanthine (Sigma), 2 x 10-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10-5 M thymidine (Sigma) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life technologies). The above solution was filter (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
The concentration of the S9-fraction in the exposure medium was 4% (v/v).

Test concentrations with justification for top dose:

In the first experiment, the test item was tested up to concentrations of 25 and 90 μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 16 and 12% in the absence and presence of S9-mix, respectively.

In the second experiment, the test item was tested up to concentrations of 35 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 11%.

Vehicle / solvent:

The vehicle for the test item was dimethyl sulfoxide (DMSO, Merck Darmstadt, Germany, CAS 67-68-5, batch K50270931 925, Expiry date 30 June 2021).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Cleansing:
Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10-4 M hypoxanthine (Sigma), 2 x 10-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10-5 M thymidine (Sigma) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.

Dose-range Finding Test:
In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 106 cells (106 cells/mL for 3 hour treatment) or 6 x 106 cells (1.25 x 105 cells/mL for 24 hour treatment) with a number of test item concentrations increasing by approximately half log steps. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. The test item was tested in the absence and presence of S9-mix.
Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 625 μg/mL exposure medium. For the 3 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation
step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium (R10-medium). For the 24 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions
by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium (R10-medium). The cells in the final suspension were counted with the coulter particle counter.The surviving cells of the 3 hour treatment were subcultured twice to determine cytotoxicity. After 24 hour of subculturing, the cells were counted and subcultured again for another 24 hours, after that the cells were counted. The surviving cells of the 24 hour treatment were
subcultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25 x 105 cells/mL were counted no subculture was performed. The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose-range for the mutagenicity tests.

Mutagenicity Test:
Seven to eight doses of the test item were tested in the mutation assay. The test item was tested in the presence of S9-mix with a 3 hour treatment period and in the absence of S9-mix with 3 and 24 hour treatment periods. The highest doses that were tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the solvent control. Also some intermediate doses were tested.

Treatment of the Cells:
Per culture 8 x 106 cells (106 cells/mL for 3 hour treatment) or 6 x 106 cells (1.25 x 105 cells/mL for 24 hour treatment) were used. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. Solvent and positive controls were included and the solvent control was tested in duplicate. In the first experiment, cell cultures were exposed for 3 hours to the test item in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to the test item in exposure medium for 24 hours in the absence of S9-mix. For the 3 hour treatment, cell cultures were exposed to the test item in exposure medium in
the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium (R10-medium).
For the 24 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium (R10-medium). The cells in the final suspension were counted with the coulter particle counter.

Determination of the Mutation Frequency:
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium. For determination of the mutation frequency (MF) a total number of 9.6 x 105 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

Evaluation criteria:

Any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range. The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should bebiologically relevant and will be compared with the historical control data range.

A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study. A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TSolubility:
The test item precipitated in the exposure medium at concentrations of 313 μg/mL and above. The concentration used as the highest test item concentration for the dose-range finding test was 625 μg/mL.

Dose-range Finding Test:
In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 20 to 313 μg/mL in the absence and presence of S9-mix with a 3 hour treatment period and a test item concentration range of 20 to 625 μg/mL in the absence of S9-mix with a 24 hour treatment period. Table 1 shows the cell counts of the cultures from the 3 hours of treatment with various concentrations of the test item after 24 and 48 hours of subculture, the calculated suspension growth and the relative suspension growth. In the absence of S9-mix, the relative suspension growth was 62% at the test item concentration of 20 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 39 μg/mL and above. In the presence of S9-mix, the relative suspension growth was 58% at the test item concentration of 78 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 156 μg/mL and above. Table 2 shows the cell counts of the cultures after 24 hours of treatment with various concentrations of the test item and after 24 hours of subculture and the calculated suspension growth and the relative suspension growth. The relative suspension growth was 18% at the test item concentration of 39 μg/mL compared to the relative suspension growth of the solvent. No cell survival was observed at the test item concentrations of 78 μg/mL or above.

First Mutagenicity Test:
Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test:
Without S9-mix: 0.5, 1, 2, 5, 10, 20, 25, 30, 35 and 40 μg/mL exposure medium.
With S9-mix: 2, 5, 10, 20, 40, 80, 90, 100, 110, 120 and 130 μg/mL exposure medium.
Evaluation of toxicity:
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.5, 1, 2, 5, 10, 20 and 25 μg/mL exposure medium.
With S9-mix: 2, 5, 10, 20, 40, 80 and 90 μg/mL exposure medium.
In the absence of S9-mix (Table 3), the relative total growth of the highest test item concentration was 16% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test item concentration was 12% compared to the total growth of the solvent controls.
Evaluation of the mutagenicity:
No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second Mutagenicity Test:
To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period. Based on the results of the dose-range finding test, the following dose levels were selected for mutagenicity testing: 0.5, 1, 2, 5, 10, 20, 25, 30, 35, 40 and 45 μg/mL exposure medium.
Evaluation of toxicity:
The dose levels selected to measure mutation frequencies at the TK-locus were:
0.5, 1, 2, 10, 20, 25, 30 and 35 μg/mL exposure medium.
The relative total growth of the highest test item was 11% compared to the total growth of the solvent controls; see Table 4.
Evaluation of mutagenicity:
No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Conclusions:

The objective of this study was to evaluate the mutagenic potential of Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix).
The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. The study procedures described in this report were based on the most recent OECD 490 guideline.

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

In conclusion, 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions.

Executive summary:

The objective of this study was to evaluate the mutagenic potential of Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs

by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix).

The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. The study procedures described in this report were based on the most recent OECD 490 guideline.

In the first experiment, the test item was tested up to concentrations of 25 and 90 μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 16 and 12% in the absence and presence of S9-mix, respectively. In the second experiment, the test item was tested up to concentrations of 35 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 11%. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly and the test is therefore considered valid.

In conclusion, 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Aug - 15 Sep 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Aug 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Version / remarks:

June 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYÉI, Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in a tightly closed container, protected from light and humidity.

Target gene:

his operon, tryp operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats, treated with phenobarbital and β-naphthoflavone

Test concentrations with justification for top dose:

Based on a range-finding study (performed in tester strains TA 98 and TA 100; doses applied: 5, 16, 50, 160, 500, 1600, 5000 μg/plate), the following concentrations were used in the main experiments:
First and second experiment (all strains): 1.6, 5, 16, 50, 160, 500, 1200 μg/plate with and without metabolic activation. Test items that are cytotoxic at concentrations <5 mg/plate should be tested up to a cytotoxic concentration. Therefore, in this test the highest test item concentration was determined to be 1200 μg/plate due to bacterial toxicity of the test item (noticed in the Informatory Toxicity Test).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in DMSO, it was selected as the vehicle.

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine (NPD); 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (Range Finding Test and first experiment); preincubation (second experiment)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number and inspection of the bacterial background lawn

Evaluation criteria:

Acceptance criteria
The study was considered valid if:
- the phenotypes of the tester strains could be confirmed
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains
- the tester strain culture titers are in the 10^9 cells/mL order
- the batch of S9 used in this study shows the appropriate biological activity
- the reference mutagens show an increase of at least: 3-fold in induced revertant colonies over the mean value of the respective vehicle control
- at least five analyzable concentrations were presented in all strains of the main tests (a minimum of three non-toxic dose levels is required to evaluate assay data)

Evaluation criteria
When the test substance shows a biologically relevant and dose-related increase in the number of revertant colonies of more than two times (TA 100) or three times (TA 98, TA 1535, TA 1537 and WP2 uvrA) compared to that of the solvent control, the response is judged to be positive. Additionally, the positive response should be reproducible for at least one of the dose groups and should occur in at least one strain with or without metabolic activation. The biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is therefor not regarded as necessary

The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

Mean values and standard deviations were calculated.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Number of revertant colonies decreased to less than half compared to solvent control in exp. 1 at 1200 μg/plate (97%) with S9 and in exp. 2 at 500 μg/plate (60%) / 1200 μg/plate (76%) without S9 and at 500 μg/plate (52%)/ 1200 μg/plate (84%) with S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Number of revertant colonies decreased to less than half compared to solvent control in exp.1 at 500 μg/plate (70%)/ 1200 μg/plate (92%) without S9 and at 500 μg/plate (97%) with S9 and also in exp. 2 (see remarks on results).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Number of revertant colonies decreased to less than half compared to solvent control in exp.1 at 1200 μg/plate (85%) without S9 and at 500 μg/plate (79%)/ 1200 µg/plate (100%) with S9 and also in exp. 2 (see remarks on results).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Number of revertant colonies decreased to less than half compared to solvent control in exp.1 at 500 μg/plate (70%)/ 1200 μg/plate (90%) without S9 and at 1200 µg/plate (100%) with S9 and also in exp. 2 (see remarks on results).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Number of revertant colonies decreased to less than half compared to solvent control in exp. 1 at 1200 μg/plate (60%) with S9 and in exp. 2 at 1200 μg/plate (79%) without S9 and at 1200 μg/plate (84%) with S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Number of revertant colonies decreased to less than half compared to solvent control in exp. 2 at 500 μg/plate (83%) / 1200 μg/plate (100%) without S9 and at 500 μg/plate (80%)/ 1200 μg/plate (100%) with S9.

Table 1:Summary of test results (experiment 1; Initial Mutation Test, Plate Incorporation Method)

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Frameshift type		Base-pair substitution type
		TA1537	TA98	TA100	TA1535	WP2 uvrA
–	Solvent control (distilled water)	-	-	97.3 ± 6.66	9.3 ± 2.08	22.7 ± 1.53
	Solvent control (DMSO)	6.7 ± 2.31	15.3 ± 3.21	92.7 ± 11.72	11.0 ± 4.36	21.7 ± 2.08
	Untreated control	5.7 ± 1.15	12.3 ± 1.53	96.0 ± 7.00	11.0 ± 3.00	23.7 ± 3.51
	1.6	6.0 ± 1.73	12.3 ± 4.16	86.3 ± 7.37	13.0 ± 6.56	30.0 ± 3.61
	5	9.0 ± 4.36	14.7 ± 3.21	94.0 ± 12.53	13.0 ± 4.58	30.3 ± 2.52
	16	7.3 ± 4.16	16.7 ± 4.73	83.7 ± 3.79	9.3 ± 1.53	30.7 ± 1.53
	50	5.3 ± 2.52	13.0 ± 3.61	77.3 ± 7.57	11.7 ± 3.51	26.3 ± 3.51
	160	5.7 ± 1.53	14.7 ± 4.62	50.7 ± 17.79	7.3 ± 2.08	27.7 ± 5.51
	500	2.0 ± 1.00 B	9.0 ± 3.46	28.0 ± 2.65	6.0 ± 3.61	27.7 ± 2.31
	1200	0.7 ± 0.58 B	7.0 ± 4.00	7.0 ± 1.73	1.7 ± 1.15	11.0 ± 3.61
	Positive controls (unit/plate)	9AA (50 µg)	4-NPD (4 µg)	SAZ (2 µg)	SAZ (2 µg)	MMS (2 µL)
	Mean No. of colonies/plate (average of 3 plates)	385.3 ± 68.86	318.0 ± 17.09	1450.7 ± 56.76	1069.3 ± 148.02	1162.7 ± 68.97
+	Solvent control (DMSO)	7.0 ± 3.00	20.0 ± 3.61	96.7 ± 8.62	12.7 ± 4.73	28.3 ± 3.21
	Untreated control	6.3 ± 2.52	23.7 ± 4.04	129.3 ± 6.43	10.0 ± 2.65	28.7 ± 3.79
	1.6	8.3 ± 2.08	18.7 ± 4.73	121.7 ± 7.51	11.7 ± 6.43	20.3 ± 1.15
	5	8.7 ± 4.62	24.7 ± 4.04	128.0 ± 16.52	12.0 ± 2.65	20.3 ± 3.06
	16	7.0 ± 2.00	18.7 ± 5.51	126.3 ± 22.28	11.3 ± 3.21	21.3 ± 7.77
	50	8.3 ± 1.15	16.0 ± 2.65	115.3 ± 8.50	8.7 ± 2.08	26.7 ± 1.53
	160	6.7 ± 5.51	15.0 ± 2.00	114.3 ± 4.73	8.0 ± 3.00	33.0 ± 6.00
	500	5.0 ± 2.65	15.3 ± 4.73	66.0 ± 9.00	2.7 ± 1.53	25.3 ± 6.35
	1200	0.0 ± 0.00 B	0.7 ± 0.58	2.7 ± 0.58	0.0 ± 0.00	11.3 ± 3.21
	Positive controls (µg/plate)	2AA (2)	2AA (2)	2AA (2)	2AA (2)	2AA (50)
	Mean No. of colonies/plate (average of 3 plates)	140.0 ± 14.42	1373.3 ± 124.28	1957.3 ± 367.07	160.0 ± 14.73	154.7 ± 6.11

SD = Standard Deviation

9AA = 9-aminoacridine

4-NPD = 4-nitro-1,2-phenylene-diamine

SAZ = sodium azide

MMS = methyl-methanesulfonate

2AA = 2-aminoanthracene

B = Reduced background lawn development

Table 2: Summary of test results (experiment 2; Confirmatory Mutation Test, Pre-Incubation

Method)

With or without S9-Mix	Test substance (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Frameshift type		Base-pair substitution type
		TA1537	TA98	TA100	TA1535	WP2 uvrA
–	Solvent control (distilled water)	-	-	94.7 ± 15.37	13.3 ± 5.51	26.3 ± 3.06
	Solvent control (DMSO)	7.7 ± 4.73	18.3 ± 3.51	82.0 ± 6.56	12.7 ± 5.51	20.0 ± 4.36
	Untreated control	8.0 ± 5.29	17.3 ± 5.86	93.0 ± 9.17	14.3 ± 5.13	24.7 ± 7.02
	1.6	8.0 ± 3.00	18.7 ± 4.04	91.3 ± 5.77	11.3 ± 3.06	29.0 ± 4.58
	5	13.0 ± 3.46	15.0 ± 5.20	84.7 ± 12.66	12.7 ± 2.89	25.0 ± 3.46
	16	7.3 ± 4.16	17.0 ± 5.29	71.3 ± 15.01	11.0 ± 5.29	25.3 ± 7.51
	50	5.0 ± 1.73	13.3 ± 3.79	46.7 ± 5.13	8.0 ± 1.00	24.7 ± 5.86
	160	3.0 ± 2.65 SB	10.0 ± 4.36	42.0 ± 7.55	4.3 ± 1.53 SB	31.7 ± 3.51
	500	0.0 ± 0.00 B	7.3 ± 2.08	14.0 ± 5.20 B	1.3 ± 0.58 SB	16.7 ± 3.06
	1200	0.0 ± 0.00 B	4.3 ± 2.08 SB	0.0 ± 0.00 B	0.0 ± 0.00 B	4.3 ± 2.52
	Positive controls (unit/plate)	9AA (50 µg)	4-NPD (4 µg)	SAZ (2 µg)	SAZ (2 µg)	MMS (2 µL)
	Mean No. of colonies/plate (average of 3 plates)	818.0 ± 20.30	276.0 ± 28.00	1149.3 ± 160.86	665.3 ± 20.13	976.0 ± 50.12
+	Solvent control (DMSO)	6.3 ± 0.58	21.0 ± 1.00	98.0 ± 21.00	9.7 ± 1.15	35.0 ± 3.61
	Untreated control	7.7 ± 0.58	21.0 ± 3.46	104.3 ± 2.08	13.0 ± 2.65	27.7 ± 4.73
	1.6	7.3 ± 0.58	19.7 ± 4.04	96.7 ± 21.39	8.0 ± 3.61	32.7 ± 8.96
	5	8.0 ± 3.46	24.3 ± 4.73	95.3 ± 6.43	11.0 ± 3.61	41.3 ± 2.52
	16	5.0 ± 1.73	24.3 ± 2.31	93.0 ± 4.36	12.0 ± 3.61	40.3 ± 2.08
	50	6.3 ± 3.51	25.7 ± 6.03	93.7 ± 19.01	10.0 ± 3.00	41.7 ± 11.55
	160	3.3 ± 1.53	18.0 ± 5.57	92.3 ± 7.64	12.3 ± 3.06	36.3 ± 7.23
	500	0.0 ± 0.00 SB	10.0 ± 1.73	20.0 ± 6.24	4.3 ± 1.53	36.0 ± 4.58
	1200	0.0 ± 0.00 B	3.3 ± 2.08 SB	0.0 ± 0.00	0.0 ± 0.00 B	5.7 ± 0.58
	Positive controls (µg/plate)	2AA (2)	2AA (2)	2AA (2)	2AA (2)	2AA (50)
	Mean No. of colonies/plate (average of 3 plates)	137.0 ± 19.08	1648.0 ± 85.51	1124.7 ± 127.03	165.0 ± 8.19	165.0 ± 25.12

SD = Standard Deviation

9AA = 9-aminoacridine

4-NPD = 4-nitro-1,2-phenylene-diamine

SAZ = sodium azide

MMS = methyl-methanesulfonate

2AA = 2-aminoanthracene

B = Reduced background lawn development

SB = Slightly reduced background lawn development

Conclusions:

Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs was tested for mutation in five histidine-required strains of Salmonella typhimurium, both in the absence and in the presence of metabolic activation S-9. All the test strains were tested at concentrations up to 1200 µg/plate as the maximum test dose.

The mean number of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. No
Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs treatments of the test strains resulted in increased revertant numbers that could be considered indicative of any test article mutagenic activity. No signs of toxicity were apparent for any of the test plates, either with or without S-9.

It was concluded that Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and WP2 uvrA) , when tested under the conditions employed in this study.

Executive summary:

reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs was tested for mutation in five histidine-required strains of Salmonella typhimurium, both in the absence and in the presence of metabolic activation S-9. All the test strains were tested at concentrations up to 1200 µg/plate as the maximum test dose.

The mean number of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. No

Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs treatments of the test strains resulted in increased revertant numbers that could be considered indicative of any test article mutagenic activity. No signs of toxicity were apparent for any of the test plates, either with or without S-9.

It was concluded that Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and WP2 uvrA) , when tested under the conditions employed in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on available data on genetoc toxicity, Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs shall not be classified.