Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Propanoic acid, 2-hydroxy-, 2-ethylhexyl ester, (2S)-
EC Number:
606-097-1
Cas Number:
186817-80-1
Molecular formula:
C11H22O3
IUPAC Name:
Propanoic acid, 2-hydroxy-, 2-ethylhexyl ester, (2S)-
Constituent 2
Reference substance name:
2-ethylhexyl-S-lactate
IUPAC Name:
2-ethylhexyl-S-lactate
Details on test material:
Batch no.: 0805000655
Purity: 99 %

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:
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.
Controlsopen allclose all
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.

Results and discussion

Test results
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'.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.

Categories Display