Ethyl (S)-2-hydroxypropionate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-06-09 to 2010-12-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted 21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl (S)-2-hydroxypropionate

EC Number:

211-694-1

EC Name:

Ethyl (S)-2-hydroxypropionate

Cas Number:

687-47-8

Molecular formula:

C5H10O3

IUPAC Name:

ethyl 2-hydroxypropanoate

Specific details on test material used for the study:

- Trade name: Purasolv EL
- Batch number: 0909001195
- Appearance: Clear colourless liquid
- Purity: 99.83%
- Storage: at room temperature, in the dark under nitrogen
- Expiry date: 2010-09-14

Method

Target gene:

Thymidine kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/--3.7.2C mouse lymphoma cells, source: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Type and identity of media:
Horse serum
Horse serum (Invitrogen Corporation) was inactivated by incubation at 56°C for at least 30 minutes.

Basic medium
RPMI 1640 Hepes buffered medium (Dutch modification) (Invitrogen Corporation) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Invitrogen), 1 mM sodium pyruvate (Sigma) and 2 mM L-glutamin (Invitrogen Corporation).

Growth medium
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and β-naphtoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

0, 0.3, 1, 3, 10, 33, 100, 333, 1180 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 medium

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

RPMI 1640 medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

-S9, 15 and 5 µg/mL for a 3 and 24 hour treatment period

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

RPMI 1640 medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

+S9, 7.5 µg/mL

Details on test system and experimental conditions:

Test substance preparation
The test substance was dissolved in RPMI 1640 (Hepes buffered medium (Dutch modification) (Invitrogen Corporation, Breda, The Netherlands)) and filter (0.22 µm)-sterilized. PURASOLV® EL concentrations were used within 40 minutes after preparation.

Exposure medium
For 3-hour exposure:
Cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).

For 24-hour exposure:
Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).

Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/ml trifluorothymidine (TFT) (Sigma).

Non-selective medium
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).

Environmental conditions
All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80 – 100% (actual range 68 – 91%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 34.3 – 37.6°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 (in the range of 35.2 - 36.0°C), humidity (with a maximum of 12%) and CO2 percentage (with a maximum of 1%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity. The temporary deviations from the humidity and the temperature are explained in the protocol deviations 1 and 2.

METHOD OF APPLICATION: in medium

DURATION

Mutagenicity test
PURASOLV® EL was tested both in the absence and presence of S9-mix in two independent experiments. Per culture 8 x 106 cells (106 cells/ml for 3 hours treatment) or 5 x 106 cells (1.25 x 105 cells/ml for 24 hours treatment) were used. The cell cultures for the 3 hours treatment were placed in sterile 30 ml centrifuge tubes and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 spm. The cell cultures for the 24 hours treatment were placed in sterile 25 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 PURASOLV® EL in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to PURASOLV® EL in exposure medium for 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.

After exposure, the cells were separated from treatment solutions by 2 centrifugation steps (216 g, 8 min) each followed by removal of the supernatant. The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the final centrifugation step the cells were resuspended in R10 medium. The cells in the final suspension were counted with the coulter particle counter.

Expression period
For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 106 cells (if possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test substance the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

Determination of the mutation frequency
Eight doses of the test substance were selected for the mutation assay, both in the absence and presence of S9-mix.

For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. 1 cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.

For determination of the MF a total number of 9.6 x 105 cells/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 that, the plates for the TFT-selection were stained for 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.


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWTG 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 substance 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 be biologically relevant and will be compared with the historical control data range.
A test substance 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 substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF (controls) + 126.
b) The results are confirmed in an independently repeated test.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no toxicity in experiment 1, no severe toxicity in experiment 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
PURASOLV® EL did not precipitate in the exposure medium up to and including the concentration of 1180 μg/ml (= 10 mM). Since testing up to 0.01 M is recommended in the guidelines, this concentration was used as the highest test substance concentration in the dose range finding test.
The pH and osmolarity of a concentration of 1180 μg/ml were 7.4 and 0.302 Osm/kg respectively (compared to 7.5 and 0.286 Osm/kg in the solvent control).

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 10 to 1180 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.
Table 1 (Tables see attached background information) shows the cell counts of the cultures after 3 hours of treatment with various concentrations of PURASOLV® EL and after 24 and 48 hours of subculture and the calculated suspension growth and the relative suspension growth.
Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 1180 μg/mL compared to the suspension growth of the solvent controls.
Table 2 shows the cell counts of the cultures after 24 hours of treatment with various concentrations of PURASOLV® EL and after 24 hours of subculture and the calculated suspension growth and the relative suspension growth.
In the absence of S9-mix, the relative suspension growth was 38% at the test substance concentration of 1180 µg/ml compared to the relative suspension growth of the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

Any other information on results incl. tables

Tables are given in the attached background information.

Applicant's summary and conclusion

Conclusions:

In conclusion, Ethyl (S)-lactate is considered to be non-mutagenic in the in vitro mammalian cell gene mutation test (OECD 476, nowadays OECD 490) in the presence and absence of mammalian metabolic activation.

Executive summary:

In a mammalian cell gene mutation assay conducted according to OECD Guideline 476 (nowadays OECD 490), L5178Y mouse lymphoma cells cultured in vitro were exposed to Ethyl (S)-lactate (purity 99.83%) in RPMI 1640 medium at concentrations of 0, 0.3, 1, 3, 10, 33, 100, 333 and 1180 µg/mL in the presence and absence of mammalian metabolic activation. The test item was tested up to the highest concentration recommended in the guideline (0.01 M = 1180 µg/mL). Positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background. Based on the results, it can be concluded, that Ethyl (S)-lactate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

This study is classified as acceptable. This study satifies the requirement for Test Guideline OECD 490 for in vitro mutagenicity (mammalian forward gene mutation) data.