Glycerides, C8-18 and C18-unsatd., reaction products with ethylene oxide and glycerol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacterial reverse mutation assay:
Key study: Experimental results: OECD guideline 471 and EU method B.14. GLP study.
The test material was considered to be non-mutagenic under the conditions of this test.

In vitro chromosome aberrations assay:
Key study: Experimental results: OECD guideline 473 and EU method B.10. GLP study.
The test material was shown to be non-clastogenic to human lymphocytes in vitro.

In vitro mammalian cell gene mutation assay:
Key study: Experimental results: OECD guideline 476 and EU method B.17. GLP study.
It is concluded that the test substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the study.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 12 August 1996 to 20 September 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline 471 and EU method B.14. GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Experiment 1: 0, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 0, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without metabolic activation

Migrated to IUCLID6: (TA100 and TA1535)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation

Migrated to IUCLID6: (TA1537)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation

Migrated to IUCLID6: (TA98)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (TA1538)

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (all strains)

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation). Known aliquots (0.1 ml) of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by 2.0 ml of molten, trace histidine supplemented, top agar at 45 ºC, 0.1 ml of the appropriately diluted test material or vehicle control and either 0.5 ml of the S9 liver rnicrosome mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates. All of the plates were incubated at 37 ºC for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

NUMBER OF REPLICATIONS: Triplicates

DETERMINATION OF CYTOTOXICITY
- Method: The plates were examined for a thinning of the background lawn.

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at subtoxic dose levels. In the event of the two experiments giving conflicting or equivocal results, then a third experiment may be performed to confirm the correct response.

To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between two and five fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was the maximum recommended dose.

Statistics:

All data are statistically analysed using the methods recommended by the UKEMS and normally Dunnett's method of linear regression is used to evaluate the result.

Species / strain:

other: all tested strains

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at a dose level of 5000 µg/plate to the majority of strains of Salmonella tested)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The dose range of the test material used in the preliminary toxicity study was 0, 50, 150, 500, 1500 and 5000 µg/plate. The test material exhibited toxicity at and above 1500 µg/plate to the strain of Salmonella used (TA100).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material caused a visible reduction in the growth of the bacterial lawn at a dose level of 5000 µg/plate to the majority of strains of Salmonella tested.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic adivation. The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test material using the Ames plate incorporation method at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 15 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using a dose range of 50 to 5000 µg/plate, fresh cultures of the bacterial strains and fresh test material formulations. An extra dose was included in Experiment 1 to allow for the toxicity

of the test material. The method used conforms with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471 and also with Method B14 in Commission Directive 92/69/EEC.

The vehicle (dimethyl sulphoxide) control plates produced counts of revertant colonies within the normal range.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without the metabolising system.

The test material caused a visible reduction in the growth of the bacterial lawn at a dose level of 5000 µg/plate to the majority of strains of Salmonella tested. The test material was tested up to the maximum recommended dose of 5000 µg/plate.

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic adivation. The test material was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14th July 1997 to 08th October 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline 473 and EU method B.10. GLP 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 (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: blood was drawn from a volunteer

Details on mammalian cell type (if applicable):

Cells were grown in Eagle´s minimal essential medium at 37 ºC with 5% CO2 in air.

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Experiment 1 (20-hour harvest): 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ml.

Experiment 2 (20-hour harvest): Without S9: 78.13, 156.25, 312.5, 625 and 937.5 µg/ml.

Experiment 2 (20-hour harvest): With S9: 156.25, 312.5, 625, 1250 and 1875 µg/ml.

Experiment 2 (44-hour harvest): Without S9: 312.5, 625 and 937.5 µg/ml.

Experiment 2 (44-hour harvest): With S9: 625, 1250 and 1875 µg/ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Minimal Essential Media (MEM)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Migrated to IUCLID6: at 500 µg/ml

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Migrated to IUCLID6: at 25 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: After 48 hours incubation at 37 ºC, 5% C02 in humidified air, the cells of the with-S9 cultures were centrifuged and all but 1 ml of the culture medium removed, reserved and replaced with 8.0 ml of MEM. 1.0 ml of the appropriate solution of vehicle or test material was added to each culture or 0.1 ml of the positive control. 1 ml of 10% S9 in standard co-factors was added to the with-S9 cultures. All the cultures were then returned to the incubator.

After 4 hours of treatment at 37 ºC the with-S9 cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 16 hours (20-hour harvest) or 40 hours (44-hour harvest).

DURATION
- Fixation time (start of exposure up to fixation or harvest of cells): Experiment 1: harvest time: 20 h; Experiment 2: harvest time: 20 and 44 h

SPINDLE INHIBITOR (cytogenetic assays): Demecolcine (Colcemid 0.1 µg/ml) two hours before the required harvest time.
STAIN (for cytogenetic assays): 5% Gurrs Giemsa R66 for 5 minutes

NUMBER OF REPLICATIONS: Duplicate cultures

NUMBER OF CELLS EVALUATED: Where possible the first 100 consecutive well-spread metaphases from each culture were counted.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index: a total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared with the concurrent vehicle control value using Fisher's Exact test or Chi-squared test.

Species / strain:

lymphocytes: blood was drawn from a volunteer

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(a dose-related increase in toxicity was observed in both the with and without activation treatment groups)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: A dose-related increase in toxicity was observed in both the with and without activation (S9) treatment groups. The without S9 treatment was more toxic with no scorable metaphases at and above 1250 µg/ml. In the with-S9 dose group treatment there were no scorable metaphases at and above 2500 µg/ml.

Experiment 2: A similar toxic response was observed for the with-S9 treatment, to that in Experiment 1. However, the test material was more toxic at 625 µg/ml in the 20 hours without-S9 treatment, with mitotic inhibition of 94% (mitotic index 6% of control). Therefore, the three lower doses 78.1 3, 156.25 and 31 2.5 µg/ml were selected for metaphases analysis. For the 44-hour time-point the same high dose levels were selected for analysis as at the earlier time-point.

Remarks on result:

other: other: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test material induced no statistically significant increases in the frequency of cells with aberrations or polyploid cells in either the presence or absence of a liver enzyme metabolising system. The test material was shown to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Duplicate cultures of lymphocytes, treated with the test material were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used, ie. 4 hours exposure with the addition of an induced rat liver homogenate metabolising system at 10% in standard co-factors with cell harvest after 16 and 40-hour expression periods and a 20 and 44-hour continuous exposure in the absence of activation. In Experiment 1 the dose range for evaluation was selected from a series of 8 dose levels on the basis of toxicity.

The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 92/69/EEC.

All vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control treatments gave significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material induced no statistically significant increases in the frequency of cells with aberrations or polyploid cells. The test material was shown to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 12 December 2011 to 07 February 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline 476 and EU method B.17. GLP 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 (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine-kinase locus (TK-locus)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. Cell density was preferably kept below 1 x 10E06 cells/ml.

Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10 medium containing 10-4 M hypoxanthine, 2 x 10-7 M aminopterine and 1.6 x 10-5 M thymidine to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Experiment 1: Without S9-mix: 0.1, 1, 3, 10, 33, 100, 200 and 250 μg/ml
With S9-mix: 1, 3, 10, 33, 100, 300, 375 and 425 μg/ml

Experiment 2: Without S9-mix: 1, 3, 10, 33, 100, 160, 200 and 235 μg/ml
With S9-mix: 1, 3, 10, 33, 100, 300, 400 and 450 μg/ml

Experiment 2A: Without S9-mix: 1, 3, 10, 33, 100, 160, 200, 235, 270 and 333 μg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The test substance was tested both in the absence and presence of S9-mix in two independent experiments. Per culture 8 x 10E06 cells (10E06 cells/ml for 3 hours treatment) or 5 x 10E06 cells (1.25 x 10E05 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 75 cm2 culture flasks at 37.0 ± 1.0°C.

DURATION
- Exposure duration: First experiment: 3 hours in the absence and presence of S9-mix. Second experiment: 24 hours in the absence of S9-mix and 3 hours in the presence of S9-mix.
- Expression time (cells in growth medium): 2 days after the treatment period.

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Single treated cultures

NUMBER OF CELLS EVALUATED:
For determination of the mutation frequency (MF) a total number of 9.6 x 10E05 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 10E05 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for cloning efficiency (CE) day2 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) 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:
The cloning efficiency was determined by dividing the number of empty wells by the total number of wells. This value obtained is the P(0), the zero term of the Poisson distribution:
P(0) = number of empty wells/total number of wells
The cloning efficiency (CE) is therefore:
CE = -ln P(0)/number of cells plated per well
The relative survival (RS) in each treatment group was determined by comparing cloning efficiencies in treatment and control cultures:
RS = [CE(test)/CE(controls)] x 100
The Relative Total Growth (RTG) was also calculated as the product of the cumulative relative suspension growth (RSG) and the relative cloning efficiency for each culture:
RTG = RSG x RSday2/100

Suspension Growth (SG) =
[Day 0 cell count/ (1.25 x 10E05) *] x [Day 1 cell count/(1.25 x 10E05) *] x [Day 2 cell count]
* Or appropriate cell concentration
Relative Suspension Growth (RSG) = SG (test) / SG (controls) x 100
RSday2 = CE day2 (test) / CE day2 (controls) x 100
The growth rate, as an indicator of optimally growing cultures, was calculated for the solvent control cultures:
Growth Rate (GR) for the 3 hours treatment=
[Day 1 cell count/(1.25 x 10E05) *] x [Day 2 cell count/(1.25 x 10E05) *]
Growth Rate (GR) for the 24 hours treatment=
[Day 0 cell count/(1.25 x 10E05) ] x [Day 1 cell count/(1.25 x 10E05) *] x [Day 2 cell count/(1.25 x 10E05) *]
* Or appropriate cell concentration

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 IWGT 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 (Moore et al, 2006. Environmental and Molecular Mutagenesis 47: 1-5).

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

Remarks:

(Experiment 1: at 250 μg/ml in the absence of S9-mix and at concentrations >300 μg/ml in the presence of S9-mix. Experiment 2: at concentrations > 200 μg/ml in the absence of S9-mix)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
The test substance precipitated in the exposure medium at concentrations of 100 μg/ml and above.

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.

In the absence of S9-mix, no toxicity in the relative suspension growth was observed up to test substance concentrations of 100 μg/ml compared to the solvent control the relative suspension growth. Hardly any cell survival was observed at test substance concentrations of 333 μg/ml and above.

In the presence of S9-mix, the relative suspension growth was 10% at the test substance concentration of 500 μg/ml compared to the relative suspension growth of the solvent control.

In the absence of S9-mix, the relative suspension growth was 14% at the test substance concentration of 100 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at test substance concentrations of 333 μg/ml and above.

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. The growth rate over the two-day expression period for cultures treated with ethanol was between 18 and 21 (3 hours treatment) and 82 and 85 (24 hours treatment).

Mutation frequencies in cultures treated with positive control chemicals were increased by 10- and 7-fold for MMS in the absence of S9-mix, and by 14- and 10-fold for CP in the presence of S9-mix, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned
properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
First mutagenicity test:
In the absence of S9-mix, the dose levels of 0.1 to 160 μg/ml showed no cytotoxicity. Therefore, the dose levels of 125 and 160 μg/ml were not regarded relevant for mutation frequency measurement. The dose level of 300 μg/ml was not used for mutation frequency measurement, since this dose level was too toxic for further testing.

In the presence of S9-mix, the dose level of 500 μg/ml was not used for mutation frequency measurement, since this dose level was too toxic for further testing.

Second mutageniticy test:
In the absence of S9-mix, no dose level with a cell survival below 62% was reached and the test substance was only tested up to moderate precipitate, therefore this experiment was rejected. In the repeat experiment (2A), the following dose range was selected: 1, 3, 10, 33, 100, 160, 200, 235, 270
and 333 μg/ml exposure medium. The dose levels of 270 to 333 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing or showed an unexplainable survival.

In the presence of S9-mix, the test substance was tested up to severe precipitate. To minimize the selection of too many dose levels with precipitation in the exposure medium, the dose level of 350 and 425 μg/ml were not selected for mutation frequency measurement.

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that the test substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Executive summary:

This report describes the effects of the test substance on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

The test substance was a clear colourless to slightly yellowish viscous liquid. The test substance was dissolved in ethanol.

In the first experiment, the test substance was tested up to concentrations of 250 and 425 μg/ml in the absence and presence of 8% (v/v) S9-mix, respectively. The incubation time was 3 hours. The test substance was tested up to cytotoxic levels of 81 and 77% in the absence and presence of S9-mix, respectively. The test substance precipitated in the culture medium at dose levels of 100 μg/ml and above.

In the second experiment, the test substance was tested up to concentrations of 235 and 450 μg/ml, but in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. The test substance was tested up to cytotoxic levels of 80 and 22% in the absence and presence of S9-mix, respectively. The test substance precipitated in the culture medium at dose levels of 100 μg/ml and above.

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased by 10- and 7-fold for MMS in the absence of S9-mix, and by 14- and 10-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test substnace did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, the test substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

It is concluded that the test substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro bacterial reverse mutation assay:

Key study: Experimental results: OECD guideline 471 and EU method B.14. GLP study.

The test material was considered to be non-mutagenic under the conditions of this test.

In vitro chromosome aberrations assay:

Key study: Experimental results: OECD guideline 473 and EU method B.10. GLP study.

The test material was shown to be non-clastogenic to human lymphocytes in vitro.

In vitro mammalian cell gene mutation assay:

Key study: Experimental results: OECD guideline 476 and EU method B.17. GLP study.

It is concluded that the test substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the study.

Justification for classification or non-classification

Based on the available information, the substance is not classified for genetic toxicity: negative results in the three in vitro studies.