Ethyl (S)-2-hydroxypropionate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The potential of Ethyl (S)-lactate to induce genotoxicity was tested in an in vitro chromosomal aberration test conducted according to OECD 473 and in an in vitro cell gene mutation assay conducted according to OECD 476 (nowadays OECD 490). Regarding the bacterial reverse gene mutation assay the assessment is based on suitable data derived with the source substance Ethyl lactate. For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Based on the results, it can be concluded that the target substance Ethyl (S)-lactate is considered to not induce genetic toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see Table 2

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see Table 2

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see Table 2

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see Table 2

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see Table 2

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The results of the dose range-finding study conducted in the presence and absence of rat liver microsomes indicate that no appreciable toxicity was observed up to 10000 µg per plate.

Table 2: Results of Salmonella mutagenicity assay with ethyl lactate

Average Revertants Per Plate**± Standard Deviation
Liver Microsomes: None
Dose (µg/plate)	TA98	TA100	TA1535	TA1537	TA1538
0*	14 ± 5	96 ± 20	20 ± 3	5 ± 1	13 ± 3
667	19 ± 2	111 ± 6	20 ± 6	5 ± 1	13 ± 3
1000	9 ± 3	104 ± 14	22 ± 2	7 ± 3	14 ± 4
3333	14 ± 2	111 ± 12	19 ± 1	5 ± 1	19 ± 2
6667	17 ± 3	107 ± 11	17 ± 2	8 ± 2	12 ± 2
10000	20 ± 7	99 ± 6	18 ± 5	5 ± 2	11 ± 2
Positive control	519 ± 53	485 ± 12	281 ± 7	241 ± 23	599 ± 37
Liver Microsomes: Rat Liver S-9
Dose (µg/plate)	TA98	TA100	TA1535	TA1537	TA1538
0*	23 ± 3	105 ± 12	12 ± 4	9 ± 3	22 ± 2
667	34 ± 7	99 ± 14	15 ± 5	6 ± 2	20 ± 8
1000	21 ± 1	102 ± 9	13 ± 6	8 ± 3	22 ± 7
3333	27 ± 6	102 ± 5	13 ± 3	7 ± 3	21 ± 6
6667	28 ± 5	105 ± 13	13 ± 3	12 ± 5	16 ± 5
10000	28 ± 3	102 ± 4	12 ± 3	7 ± 2	21 ± 3
Positive control	950 ± 36	2358 ± 118	300 ± 4	167 ± 7	1387 ± 125

*Vehicle control with dionized, distilled grade water

** Mean of 3 plates

Conclusions:

Under the experimental conditions reported, Ethyl lactate did not cause gene mutations in an Ames Test (equivalent to OECD Guideline 471). Therefore, the test item is considered to be non-mutagenic in this bacterial reverse gene mutation assay.

Executive summary:

In a reverse gene mutation assay (equivalent to OECD Guideline 471) in bacteria, strains TA98, TA100, TA1535, TA1537 and TA1538 of S. typhimurium were exposed to Ethyl lactate at concentrations of 667, 1000, 3333, 6667 and 10000 µg/plate in the presence and absence of mammalian metabolic activation (plate co-incubation).

Ethyl lactate was tested up to the limit concentration of 10000 µg/plate. There was no dose-related increase in the number of revertants in any of the test strains with and without activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for OECD test guideline 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-07-02 to 2010-09-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Name of test material: Purasolv EL
- Batch number of test material: 0909001195
- Purity: 99.83%
- Description: clear colourless liquid
- Storage: at room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Purasolv EL was dissolved in RPMI 1640 medium. Purasolv EL concentrations were used within 2.5 hours after preparation and filter (0.22 µm)-sterilized. The pH and the osmolarity of the culture medium containing the highest tested concentration were recorded.

Target gene:

n.a.

Species / strain / cell type:

lymphocytes: peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media:
Culture medium: Culture medium consisted of RPMI 1640 (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine (2mM) (Invitrogen Corporation), penicillin/streptomycin (50 U/mL and 50 U/mL respectively) (Invitrogen Corporation) and 30 U/mL heparin (Sigma, Zwijdrecht, The Netherlands).

Lymphocytes cultures: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel, Europe Ltd., United Kingdom) was added.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Rat liver microsomal enzymes prepared from adult male Wistar rats obtained from Charles River (Sulzfeld, Germany)
- method of preparation of S9 mix :
Preparation of S9-fraction: Rats were orally dosed at three consecutive days with a suspension of phenobarbital (80 mg/kg bw) and β-naphtoflavone (100 mg/kg bw) in corn oil (they were denied access to food for 3 to 4 hours preceding each dosing). One day after final exposure (24h), the rats were sedated using oxygen/carbon dioxide and then killed by decapitation. The rats received a limited quantity of food during the night before sacrifice. The livers of the rats were removed aseptically, and washed in cold (0 °C), sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA (Merck). The livers were minced in a blender and homogenised in 3 volumes of phosphate buffer with a Potter homogeniser. The homogenate was centrifuged for 15 min 9000g. The supernatant (S9) was transferred into sterile ampules which were stored in liquid nitrogen (-196 °C) for a maximum of 1 year.
Preparation of S9-mix: S9-mix was prepared immediately before use and kept on ice. S9-mix components contained per mL: 1.63 mg MgCl26H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, UK); 4 µmol HEPES. The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-fraction (batches 10-6 and 10-7) was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium : 1.8% (v/v)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): metabolic capability

Test concentrations with justification for top dose:

Dose range finding test: 0, 10, 33, 100, 333 and 1180 µg/mL (1180 µg/mL = 0.01 M)
First and second cytogenetic assay: 0, 100, 333 and 1180 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 medium (Invitrogen Corporation, Breda, The Netherlands)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

-S9 mix, 0.5 µg/mL (3 h), 0.2 µg/mL (24 h), 0.1 µg/mL (48 h)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation, 10 µg/mL (3 h)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION:
Preincubation period: 48 h
- Exposure duration:
Dose range finding test: 3 h, 24 h and 48 h in the absence of S9-mix or 3 h in the presence of S9-mix.
First cytogenetic assay: 3 h in the presence and absence of S9-mix.
Second cytogenetic assay: 24 h and 48 h in the absence of S9-mix and 3 h in the presence of S9-mix.
- Fixation time (start of exposure up to fixation or harvest of cells): Dose range finding test: 24 h (3 h and 24 h exposure) or 48 h (48 h exposure). First cytogenetic assay: 24 h. Second cytogenetic assay: 24 h (24 h exposure) or 48 h (3 and 48 h exposure).

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 µg/mL medium) (Acros Organics, Belgium)

STAIN (for cytogenetic assays): slides were stained for 10-30 min with 5% (v/v) Giemsa (merck) solution in tap water

NUMBER OF REPLICATIONS: duplicate.

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p< 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dos-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if non of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p< 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

The incidence of aberrant cells (cells with one ore more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Key result

Species / strain:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

All results are presented in Tables 1-8 in the attached background material (NOTOX Project 494586).

Conclusions:

Ethyl (S)-lactate is considered to be not clastogenic in the in vitro mammalian chromosomal aberration test using human lymphocytes, with and without metabolic activation.

Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberration) according to OECD Guideline 473, primary peripheral human lymphocytes cultures were exposed to Ethyl (S)-lactate (purity 99.83%) at concentrations of 0, 100, 333 and 1180 µg/mL with and without metabolic activation.

The test item was tested up to the limit concentration of 1180 µg/mL (0.01 M). Positive controls induced the appropriate response. There was no evidence of chromosome aberration induced over background.

In conclusion, Ethyl (S)-lactate is not clastogenic in human lymphocytes.

This study is classified as acceptable and satisfies the requirement for the in vitro mammalian chromosomal aberration test according to OECD 473.

Reference 3

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

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

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

Target gene:

Thymidine kinase

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

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

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.

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.

Tables are given in the attached background information.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA1538 of S. typhimurium were exposed to Ethyl lactate at concentrations of 667, 1000, 3333, 6667 and 10000 µg/plate in the presence and absence of mammalian metabolic activation (plate co-incubation). Ethyl lactate was tested up to the limit concentration of 10000 µg/plate. There was no dose-related increase in the number of revertants in any of the test strains with and without activation.

In an in vitro chromosome aberration assay conducted according to OECD 473, primary peripheral human lymphocytes cultures were exposed to Ethyl (S)-lactate at concentrations of 0, 100, 333 and 1180 µg/mL with and without metabolic activation. There was no evidence of induced chromosome aberration over background.

In a mammalian cell gene mutation assay conducted according to OED 476 (nowadays OECD 490), cultured L5178Y mouse lymphoma cells were exposed to Ethyl (S)-lactate, 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. There was no evidence of induced mutant colonies over background.

Based on the results, Ethyl (S)-lactate is considered to not induce genetic toxicity.

Justification for classification or non-classification

Based on the available results, the target substance Ethyl (S)-lactate is not considered to be genotoxic and no classification is warranted in accordance with CLP Regulation 1272/2008.