2-ethylhexyl 3-mercaptopropionate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 14, 2022 - June 30, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

adopted 29th July, 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl 3-mercaptopropionate

EC Number:

256-589-1

EC Name:

2-ethylhexyl 3-mercaptopropionate

Cas Number:

50448-95-8

Molecular formula:

C11H22O2S

IUPAC Name:

2-ethylhexyl 3-sulfanylpropanoate

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Rat Liver S9 Fraction

Test concentrations with justification for top dose:

Experiment A with 3/20 h treatment/sampling time,
without S9 mix: 50, 100 and 150 μg/mL test item
with S9 mix: 500, 1000 and 2000 μg/mL test item

Experiment B with 20/20 h treatment/sampling time
without S9 mix: 50, 100, 125 and 150 μg/mL test item

Experiment B with 20/28 h treatment/sampling time
without S9 mix: 50, 100, 125 and 150 μg/mL test item

Experiment B with 3/28 h treatment/sampling time
with S9 mix: 500, 1000 and 2000 μg/mL test item

(selected on the basis of a pre-test on cytotoxicity)

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 5E05 cells/dish
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
- Experiment A: 3-hour treatment, harvest 20 hours from the beginning of treatment
- Experiment B: 20-hour treatment, harvest 20 hours from the beginning of treatment / 20 (without S9 mix)- and 3-hour (with S9 mix) treatment, harvest 28 hours
from the beginning of treatment

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: Colchicine (0.2 μg/mL) 2.5-3 hours prior to harvesting
- Methods of slide preparation and staining technique used including the stain used:
Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes plasma free) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.
300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration as well as the negative and positive controls and were equally divided among the duplicates (150 metaphases/slide). Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.
Additionally, the number of polyploid and endoreduplicated cells were scored. The nomenclature and classification of chromosome aberrations were given based upon ISCN, 1985, and Savage, 1976, 1983.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- pretest for selection of concentrations: Relative Increase in Cell Counts (RICC)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Chromatid and chromosome type aberrations (gaps, deletions and exchanges), the number of polyploid and endoreduplicated cells

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.
Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if, in all experimental conditions examined:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data.

Both biological and statistical significance are considered together.
There is no requirement for verification of a clearly positive or negative response.

Statistics:

For statistical analysis the CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The data were checked for a linear trend in number of cells with aberrations (without gaps). with treatment dose using the adequate regression analysis by Microsoft
Excel software.

Results and discussion

Test results

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:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality.
Clear cytotoxicity in the range required by the guideline (55 ± 5 %) was observed at the highest concentrations without metabolic activation in Experiment A and experiment B (between 53 and 55%). In experiment A and in experiment B at the highest recommended concentration with metabolic activation 45% cytotoxicity was observed.

Applicant's summary and conclusion

Conclusions:

In conclusion, Evabopol® 496D did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to the cytotoxic concentrations in the absence and up to the maximum recommended concentration presence of metabolic activation.
Thus, the test item EHMP is considered as being non-clastogenic in this system.

Executive summary:

The test item EHMP, dissolved in DMSO (Dimethyl sulfoxide), was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on cytotoxicity (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473:
Experiment A with 3/20 h treatment/sampling time
without S9 mix: 50, 100 and 150 μg/mL test item
with S9 mix: 500, 1000 and 2000 μg/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 50, 100, 125 and 150 μg/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 50, 100, 125 and 150 μg/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 500, 1000 and 2000 μg/mL test item
Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).
No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.
Clear cytotoxicity in the range required by the guideline (55 ± 5 %) was observed at the highest concentrations without metabolic activation in Experiment A and experiment B (between 53 and 55%). In experiment A and in experiment B at the highest recommended concentration with
metabolic activation 45% cytotoxicity was observed.
In experiment A, no increases in cells carrying structural chromosomal aberrations compared to concurrent controls or in comparison with the range of historical controls were observed, neither in the absence nor in the presence of metabolic activation. All values for aberrant cells were within the historical control range of 2-5 aberrant cells excluding gaps.
In experiment B no increases in cells carrying structural chromosomal aberrations compared to concurrent controls or in comparison with the range of historical controls were observed in the absence (20-hour treatment/28-hour sampling time) nor in the presence of metabolic activation. All values for aberrant cells were within the historical control range of 2-5 aberrant cells excluding gaps.
There were no polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.
In conclusion, EHMP did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to the cytotoxic concentrations in the absence and up to the maximum recommended concentration presence of metabolic activation.
Thus, the test item is considered as being non-clastogenic in this system.