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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

614-257-7

Cas Number:

68071-40-9

Molecular formula:

C18H34O4, C20H38O4 and C22H42O4 (mainly)

IUPAC Name:

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

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

Method

Species / strain

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).

Controls

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.

Results and discussion

Test results

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).

Applicant's summary and conclusion

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.