Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

2-Ethylhexyl-S-lactate was negative in two in vitro gene mutation studies in bacteria, in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells, and in a chromosome aberration test with peripheral human lymphocytes.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Ty
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
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:
Dose levels selected to measure mutation frequencies at the TK-locus were:
Experiment 1:
without metabolic activation, 3 h treatment: 0, 10, 33, 100, 150, 200, 210, 220, 240 µg/ml
with metabolic activation, 3 h treatment: 0, 10, 30, 100, 300, 325, 350, 375, 400 µg/ml
Experiment 1:
without metabolic activation, 24 h treatment: 0, 3, 30, 75, 100, 135, 165, 200, 235 µg/ml
with metabolic activation, 3 h treatment: 0, 10, 30, 100, 200, 300, 350, 385, 400 µg/ml
Vehicle / solvent:
- Solvent used: DMSO.
Untreated negative controls:
yes
Remarks:
solvent control is used as negative control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix
Untreated negative controls:
yes
Remarks:
solvent control is used as negative control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix
Details on test system and experimental conditions:
Test substance preparation
The test substance was dissolved in dimethyl sulfoxide (SeccoSolv, Merck Darmstadt, Germany PURASOLV® EHL concentrations were used within approximately 1 hour after preparation. The final concentration of the solvent in the exposure medium was 0.8 % (v/v).

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 68–98 %), containing 5.0 ± 0.5 % CO2 in air, at a temperature of 37.0 ± 1.0 °C (actual range 33.4–37.6 °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 35.0-36.0 °C), humidity (with a maximum of 11 %) 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 deviation from the humidity and from the temperature are explained in the protocol deviations 1 and 2.

METHOD OF APPLICATION: in medium

DURATION
Mutagenicity test
PURASOLV® EHL was tested both in the absence and presence of S9-mix in two independent experiments. Per culture 8.0E+06 cells (1.0E-+6 cells/ml for 3 hours treatment) or 5.0E+06 cells (1.25E+05 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® EHL in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to PURASOLV® EHL 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 4.0E+06 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 × 96-well microtiter plates/concentration) in non selective medium.
For determination of the MF a total number of 9.6E+05 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 9.6E+05 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 (ref. 12).
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.
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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

The pH and osmolarity of a concentration of 500 μg/ml were 7.3 and 0.410 Osm/kg respectively (compared to 7.5 and 0.414 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 500 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® EHL and after 24 and 48 hours of subculture and the calculated suspension growth and the relative suspension growth.

In the absence of S9-mix, the relative suspension growth was 78% at the test substance concentrations of 100 μg/ml compared to the relative suspension growth of the solvent control. Hardly any or no cell survival was observed at test substance concentrations of 300 μg/ml and above.

In the presence of S9-mix, no toxicity in the relative suspension growth was observed up to test substance concentrations of 300 μg/ml compared to the solvent control. Hardly any cell survival was observed at the test substance concentration of 500 μg/ml.

Table 2 shows the cell counts of the cultures after 24 hours of treatment with various concentrations of PURASOLV® EHL 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 5% at the test substance concentrations of 300 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at the test substance concentration of 500 μg/ml.

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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Tables are given in the attached background information.

1.1.     Mutation experiment

The results of the two mutation experiments were within the validity criteria.

Table 3 and 4 show the percentages of cell survival and the mutation frequencies for various concentrations of PURASOLV®EHL. Individual colony counts of cloning and selective plates and cell counts during subculturing are listed in Tables 5-11 of Appendix I.

1.1.1.    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: 3, 10, 30, 100, 125, 150, 175, 200, 225, 250, 275 and 300 μg/ml exposure medium.

With 8% (v/v) S9-mix: 10, 30, 100, 300, 325, 350, 375, 400, 425, 450, 475 and 500 μg/ml exposure medium.

Evaluation of toxicity

In the absence of S9-mix, no dose level with a cell survival below 40% was reached; therefore this part of the experiment was rejected. The following dose range was selected for mutation experiment 1A: 10, 33, 100, 150, 175, 200, 210, 220, 230, 240, 250 and 260 µg/ml. The dose levels of 100 to 210 μg/ml and 220 to 240 µg/ml showed similar cytotoxicity. Therefore, the dose levels of 175 and 230 µg/ml were not regarded relevant for mutation frequency measurement. The dose levels of 250 and 260 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

In the presence of S9-mix, the dose levels of 400 to 425 μg/ml showed similar cytotoxicity. Therefore, the dose level of 425 µg/ml was not regarded relevant for mutation frequency measurement. The dose levels of 450 to 500 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

The dose levels selected to measure mutation frequencies at the TK-locus were:

Without S9-mix: 10, 33, 100, 150, 200, 210, 220 and 240 μg/ml exposure medium.

With S9-mix: 10, 30, 100, 300, 325, 350, 375 and 400 μg/ml exposure medium.

In the absence of S9-mix (Table 3), the relative total growth of the highest test substance concentration of 240 µg/ml was reduced by 65% and the dose level of 220 µg/ml was reduced by 74 % compared to the total growth of the solvent controls (see protocol deviation 3).

In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 90% compared to the total growth of the solvent controls.

Evaluation of the mutagenicity

No significant increase in the mutation frequency at the TK locus was observed after treatment with PURASOLV®EHL either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the PURASOLV®EHL treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

1.1.2.    Second mutagenicity test

To obtain more information about the possible mutagenicity of PURASOLV®EHL, a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period and in the presence of 12 % (v/v) S9-mix with a 3 hour treatment period.

Based on the results of the dose range finding test and experiment 1, the following dose levels were selected for mutagenicity testing.

Without S9-mix: 1, 3, 10, 30, 75, 100, 135, 165, 200, 235, 265, 300 and 335 µg/ml exposure medium.

With 12 % (v/v) S9-mix: 10, 30, 100, 200, 300, 350, 365, 385, 400, 425, 450 and 475 μg/ml exposure medium.

Evaluation of toxicity

In the absence of S9-mix, the dose levels of 1 to 75 μg/ml showed no cytotoxicity. Therefore, the dose levels of 1 and 10 µg/ml were not regarded relevant for mutation frequency measurement. The dose levels of 265 to 335 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

In the presence of S9-mix, the dose levels of 300 to 365 μg/ml showed similar cell growth delay. Therefore, the dose level of 365 µg/ml was not regarded relevant for mutation frequency measurement. The dose levels of 425 to 475 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-locus were:

Without S9-mix: 3, 30, 75, 100, 135, 165, 200 and 235 µg/ml exposure medium.

With S9-mix: 10, 30, 100, 200, 300, 350, 385 and 400 μg/ml exposure medium.

In the absence of S9-mix (Table 4), the relative total growth of the highest test substance was reduced by 92 % compared to the total growth of the solvent controls.

In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 70 % compared to the total growth of the solvent controls (see protocol deviation 3).

Evaluation of mutagenicity

No significant increase in the mutation frequency at the TK locus was observed after treatment with PURASOLV®EHL either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the PURASOLV®EHL treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Conclusions:
Interpretation of results (migrated information):
negative

PURASOLV® EHL (2-ethylhexyl-S-lactate) is not mutagenic in the mouse lymphoma L5178Y test system.
Executive summary:

In a mammalian cell gene mutation assay (thymidine kinase (TK) locus), L5178Y mouse lymphoma cells cultured in vitro were exposed to PURASOLV®EHL (2-ethylhexyl-S-lactate, purity 99 %) at concentrations up to 500 and 335 µg/ml in, respectively, the presence and absence of mammalian metabolic activation (phenobarbital and β-naphtoflavone induced rat liver S9-mix).

PURASOLV®EHL was tested up to cytotoxic concentrations. Positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satifies the requirement for test guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation).

PURASOLV®EHL (2-ethylhexyl-S-lactate) is not a mutagen in L5178 mouse lymphoma cells.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: cultured 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:
phenobarbital and β-naphtoflavone induced rat liver S9-mix
Test concentrations with justification for top dose:
Dose range finding test: 0, 10, 33, 100, 333 and 1000 µg/ml (3 h exposure time) and 0, 10, 33, 100, 333, 1000 and 2023 µg/ml (24 and 48 h exposure time). The highest tested concentration was determined by the solubility of PURASOLV®EHL in the culture medium at the 3 h exposure time. At the 24 and 48 h exposure time, PURASOLV®EHL was tested beyond the limit of solubility to obtain adequate toxicity data.
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 had an inhibition of the mitotic index of 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control.
First cytogenic assay: with and without S9-mix: 0, 100, 125, 150, 175, 200, 235, 270 and 300 µg/ml culture medium (3h exposure time, 24 h fixation time)
Cytogenetic assay 1A: with S9-mix: 0, 150, 200, 250, 270, 275, 280, 285, 290, 295 and 300 µg/ml culture medium (3 h exposure time, 24 h fixation time).
Second cytogenetic assay:
Without S9-mix: 0, 30, 100, 125, 150, 175, 200, 225, 250, 275 and 300 µg/ml culture medium (24 and 48 h exposure time, 24 and 48 h fixation time).
With S9-mix: 0, 150, 200, 250, 270, 280, 290, 300, 310, 320 and 330 µg/ml culture medium (3 h exposure time, 48 h fixation time).
Cytogenic assay 2A:
Without S9-mix: 0, 150, 200, 250, 300, 310, 320, 330, 340 and 350 µg/ml culture medium (48 hours exposure time, 48 hours fixation time)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO. PURASOLV®EHL was dissolved in demethyl sulfoxide of spectroscopic quality (SeccoSolv, Merck, Darmstadt, Germany). PURASOLV®EHL concentrations wer used within 2.5 hours after preparation. The final concentration of the solvent in the culture medium was 1.0% (v/v).
- Justification for choice of solvent/vehicle: Not indicated.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION:
- Exposure duration: Dose range finding test: 3, 24 and 48 h in the absence of S9-mix or 3h in the presence of S9-mix. First cytogenetic assay: 3 h in the presence and absence of S9-mix. Second cytogenetic assay: 24 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 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.
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 dose-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.
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

All results are presented in Tables 1–10 in the attached background material (NOTOX Project 494584).

Conclusions:
PURASOLV®EHL (2-ethylhexyl-S-lactate) is not clastogenic in human lymphocytes.
Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberration), primary peripheral human lymphocytes cultures were exposed to PURASOLV®EHL (2-ethylhexyl-S-lactate, purity 99 %) at concentrations of 0, 100, 150, 200, 225, 250, 270 and 300 µg/ml without metabolic activation and 0, 150, 270, 280, 300 and 330 µg/ml with metabolic activation (phenobarbital and β-naphtoflavone induced rat liver S9 -mix).

PURASOLV®EHL was tested up to cytotoxic concentrations. Positive controls induced the appropriate response. There was no evidence of chromosome aberration induced over background.

This study is classified as acceptable. This study satifies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data.

PURASOLV®EHL (2-ethylhexyl-S-lactate) is not clastogenic in human lymphocytes.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
April 2003-May 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Default
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 from Aroclor induced rat liver
Test concentrations with justification for top dose:
Dose range finding test (only tested in the tester strains TA100 and Wp2uvrA): 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of 5 % (v/v) S9-mix.
Main test:
Experiment 1 (tester strain TA1535, TA1537 and TA98): 3, 10, 33, 100, 333, 1000, 2000 and 3330 µg/plate in the absence and presence of 5 % (v/v) S9-mix.
Experiment 2 (tester strain TA98, TA100, TA1535 and TA1537): 33, 100, 333, 1000 and 2000 µg/plate in the absence and presence of 10% (v/v) S9-mix.: 33, 100, 333, 1000, 2000 and 3330 µg/plate in the absence and presence of 10 % (v/v) S9-mix, and tester strain Wp2uvrA: 33, 100, 333, 1000 and 3330 µg/plate in the absence and presence of 10 % (v/v) S9-mix.
Experiment 3 (tester strain TA100): 100, 333, 1000, 1333, 1666, 2000 µg/plate in the presence of 10 % (v/v) S9 mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO of spectroscopic quality (Merck)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
used for strain TA 1535 without S9 mix
Positive control substance:
9-aminoacridine
Remarks:
used for strain TA1537 without S9 mix
Positive control substance:
other: daunamocyn
Remarks:
used for strain TA98 without S9 mix
Positive control substance:
methylmethanesulfonate
Remarks:
used for strain TA100 without S9 mix
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
used for strain WP2uvrA without S9 mix
Positive control substance:
other: 2-aminoanthracene
Remarks:
used for all strains with S9 mix
Positive control substance:
benzo(a)pyrene
Remarks:
all S9-batches were characterize with the metabolic activation requiring positive control; benzo(a)pyrene in tester strain TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Pre-incubation period: none
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: the revertant colonies (histidine independent c.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or maually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth and other: to determine the toxicity of 2-ethylhexyl lactate, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
Evaluation criteria:
A test substance is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation.
b) The positive response should be reproducible in at least one indepently repeated experiment.
Statistics:
No formal hypothesis testing was done.
The reviewer considers the analyses used (see evaluation criteria) to be appropriate.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Remarks:
TA100 with S9-mix at 2000 µ/plate
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS (precipitation and cytotoxicity)
Dose range finding test:
Precipitate: The test substance precipitated in the top agar at concentrations of 1000 µg/plate and upwards. Precipitation of 2-ethylhexyl lactate on the plates was observed at the start and at the end of the incubation period at concentrations of 3330 and 5000 µg/plate.
Cytotoxicity: To determine the toxicity of 2-ethylhexyl lactate, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined and are given in Table 1. All other concentrations, not mentioned in Table 1, showed no reduction in the bacterial background lawn or in the number of revertant colonies.

Main experiment:
Experiment 1:
Precipitate: 2-Ethylhexyl lactate precipitated in the top agar at concentrations of 2000 µg/plate and upwards. Precipitation of 2-ethylhexyl lactate on the plates was observed at the start and at the end of the incubation period at the concentration of 3330 µg/plate.
Cytotoxicity: The reduction of the bacterial background lawn and the reduction in the number of revertants are presented in Table 2. All other concentrations, not mentioned in Table 2, showed no reduction in the bacterial background lawn or in the number of revertant colonies.

Experminent 2:
Precipitate: 2-Ethylhexyl lactate precipitated in the top agar of 1000 µg/plate and upwards. Precipitation of 2-ethylhexyl lactate on the plates was observed at the start and at the end of the incubation period at the concentration of 3330 µg/plate.
Cytotoxicity: The reduction of the bacterial background lawn and the reduction in the number of revertants are presented in Table 3. All other concentrations, not mentioned in Table 3, showed no reduction in the bacterial background lawn or in the number of revertant colonies.

Experiment 3:
Precipitate: 2-Ethylhexyl lactate precipitated in the top agar at concentrations of 1000 µg/plate and upwards. Precipitation of 2-ethylhexyl lactate on the plates was not observed at the start or at the end of the incubation period.
Cytotoxicity: A slight reduction of the bacterial background lawn was observed at test substance concentrations of 1333 and 1666 µg/plate and a modereate reduction of the bacterial background lawn at 2000 µg/plate.

MUTAGENIC RESPONSE:
The mutagenic response data for the dose-range study and experiment 1, 2 and 3 are given in Tables 4-6 in the attached background material section. Results from the dose-range study and experiment 1 are combined in one table (Table 4).
In experiment 2 in tester strain TA100, 2-ethylhexyl lactate induced an up to 3.1-fold increase in the number of revertant colonies compared to the solvent control in the presence of 10% (v/v) S9-mix. However, this result is not biologically relevant since at the dose resulting in increased number of revertants cytotoxicity was observed (moderately reduced bacterial background lawn). Verification of this result was performed in an additional experiment (experiment 3) in which it was confirmed that an increase in number of revertants was accompanied by cytotoxicity. Further, in this experiment only an up to 1.8 fold increase in the number of revertant colonies compared to the solvent control was found. Taken together, the observed increases in tester strain TA100 (with S9-mix) are considered to be not biologically relevant and it is concluded that 2-ethylhexyl lactate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escheria coli reverse mutation assay.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. Toxicity of 2-ethylhexyl lactate in the dose range finding test (Reduction of the bacterial background lawn and in the number of revertant colonies).

 

Without S9-mix

With S9-mix

 

Dose

(µg/plate)

Bacterial background lawn

Revertant colonies

Dose

(µg/plate)

Bacterial background lawn

Revertant colonies

 

TA100

1000

slight

1000

slight

3330

extreme

microcolonies

3330

extreme

microcolonies

5000

extreme

microcolonies

5000

extreme

microcolonies

WP2uvrA

5000

slight

5000

-¹ No reduction of the bacterial background lawn

-² No reduction in the number of revertant colonies

Table 2. Toxicity of 2-ethylhexyl lactate in the first mutation assay of the main study (Reduction of the bacterial background lawn and in the number of revertant colonies).

 

Without S9-mix

With S9-mix

 

Dose

(µg/plate)

Bacterial background lawn

Revertant colonies

Dose

(µg/plate)

Bacterial background lawn

Revertant colonies

TA1535

2000

extreme

microcolonies

2000

extreme

microcolonies

3330

extreme

microcolonies

3330

extreme

microcolonies

TA 1537

2000

extreme

microcolonies

2000

extreme

microcolonies

3330

absent

complete

3330

extreme

microcolonies

TA98

1000

moderate

 

 

 

2000

extreme

microcolonies

2000

slight

extreme

3330

extreme

microcolonies

3330

extreme

microcolonies

-¹ No reduction of the bacterial background lawn

Table 3. Toxicity of 2-ethylhexyl lactate in the second mutation assay of the main study (Reduction of the bacterial background lawn and in the number of revertant colonies).

 

Without S9-mix

With S9-mix

 

Dose

(µg/plate)

Bacterial background lawn

Revertant colonies

Dose

(µg/plate)

Bacterial background lawn

Revertant colonies

TA1535

1000

slight

 

 

 

2000

absent

complete

2000

extreme

microcolonies

TA1537

2000

extreme

microcolonies

 

 

 

TA 98

1000

slight

moderate

2000

extreme

microcolonies

2000

extreme

microcolonies

2000

extreme

microcolonies

TA100

2000

moderate

2000

moderate

WP2urvA

3330

sight

3330

-¹ No reduction in the number of revertant colonies

-² No reduction of the bacterial background lawn

Conclusions:
Ambiguous results were found for 2-ethylhexyl lactate in the Salmonella typhimurium reverse mutation assay for tester strain TA100 with metabolic activation. Results for other strains, or without metabolic activation were negative.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535 and TA1537 of S. typhimurium and strain WP2uvrA of E. coli were exposed to 2-ethylhexyl lactate in DMSO at concentrations of 3, 10, 33, 100, 333, 1000, 1333, 1666, 2000, 3330 and 5000 µg/plate in the presence and absence of mammalian metabolic activation (plate co-incubation).

2-Ethylhexyl lactate was tested up to the limit concentration of 5000 µg/plate. In one experiment 2-ethylhexyl lactate induced an up to 3.1-fold increase in the number of revertant colonies compared to the solvent control in the presence of 10 % (v/v) S9-mix. However, this result is ambiguous since cytotoxicity was observed at the dose resulting in increased number of revertants (moderately reduced bacterial background lawn) and further a clear dose-related increase in the number of revertant colonies was lacking. Verification of this result was performed in an additional experiment (experiment 3) in which it was confirmed that an increase in number of revertants was accompanied by cytotoxicity. Further, in this experiment only an up to 1.8 fold increase in the number of revertant colonies compared to the solvent control was found. Taken together, the observed increases in tester strain TA100 (with S9-mix) are considered ambigous, but probably not biologically relevant. Since we are not able to see the test plates ourselves to come to an independent judgement and there has not been a study into the histidine auxotrophy of the formed colonies to confirm lack of reverse mutation it must be concluded that results for 2-ethylhexyl lactate are ambigous in the Salmonella typhimurium reverse mutation assay for tester strain TA100. For the other strains of S. typhimurium and E. coli there was no evidence of induced colonies over background.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

2-Ethylhexyl-S-lactate was negative in the in vitro gene mutation tests with bacteria and mammalian cells as well as in an in vitro chromosome aberration test. Therefore, there is no reason to perform further studies.

Justification for classification or non-classification

2-Ethylhexyl-S-lactate was negative in the in vitro gene mutation tests with bacteria and mammalian cells as well as in an in vitro chromosome aberration test. It is therefore concluded that 2-ethylhexyl-S-lactate is not genotoxic and no classifcation is warranted in accordance with the CLP Regulation 1272/2008.