reaction products of 2-Propenoic acid (2 moles) and Neopentylglycol hydroxypivalate (1 mole)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All the three required in vitro genotoxicity studies were performed on the registered substance. Negative results were obtained in the In vitro gene mutation study in bacteria (OECD 471), in the In vitro cytogenicity / micronucleus study (OECD 487) and in the In vitro gene mutation study in mammalian cells (OECD 476, HPRT). Based on these results, the registered substance is considered to be not mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2016 - 06 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

n/a

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

The selected dose-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate.
Since the test item only induced a moderate emulsion in the final treatment medium and a slight toxicity in the preliminary test, the highest dose-level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice: test item was soluble in the vehicle at 100 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); without S9 mix: direct plate incorporation method; with S9 mix: preincubation method.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn

NUMBER OF REPLICATIONS: 3

Rationale for test conditions:

Since the test item only induced a moderate emulsion in the final treatment medium and a slight toxicity in the preliminary test, the highest dose-level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines.

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

The selected dose-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains, in both mutagenicity experiments with and without S9 mix.

Evaluation criteria:

In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.

The test item is considered to have shown mutagenic activity in this study if:
- a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose-level,
- and/or a reproducible dose-response relationship is evidenced.

The test item is considered to have shown no mutagenic activity in this study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose-levels,
- nor any evidence of a dose-response relationship is noted.

Statistics:

no

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate in both experiments without S9 mix and >= 2500 µg/plate in the first experiment with S9 mix (using the direct plate incorporation method). In the second experiment with S9 mix (using the pre incubation method), no emulsion was noted at any dose-levels.

RANGE-FINDING STUDY: see attached
Since the test item only induced a moderate emulsion in the final treatment medium and a slight toxicity in the preliminary test, the highest dose-level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines.

RESULTS OF CYTOTOXICITY and GENOTOXICITY: see attached
In the absence of S9 mix, a moderate to strong toxicity (decrease in the number of revertants) was noted in the TA 1537 strain at dose-levels >= 2500 µg/plate and at 5000 µg/plate in the first and second experiments, respectively.
In the presence of S9 mix, a moderate to strong toxicity was noted at 5000 µg/plate in the TA 1537 strain in the first experiment, then at dose-levels >= 2500 µg/plate in the TA 98 strain and at 5000 µg/plate in the TA 102 strain in the second experiment.
No other noteworthy toxicity was noted towards the other strains or test conditions.
The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, in either experiment, either with or without S9 mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see attached

Conclusions:

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium.

 

The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B.13/14).

 

Methods

A preliminary toxicity test was performed to define the dose-levels of the test iem, diluted in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).

 

Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

Results

Since the test item only induced a moderate emulsion in the final treatment medium and a slight toxicity in the preliminary test, the highest dose-level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines.

 

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

 

The selected dose-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains, in both mutagenicity experiments with and without S9 mix.

 

A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate in both experiments without S9 mix and >= 2500 µg/plate in the first experiment with S9 mix (using the direct plate incorporation method). In the second experiment with S9 mix (using the pre-incubation method), no emulsion was noted at any dose-levels.

In the absence of S9 mix, a moderate to strong toxicity (decrease in the number of revertants) was noted in the TA 1537 strain at dose-levels

>= 

 2500 µg/plate and at 5000 µg/plate in the first and second experiments, respectively.

In the presence of S9 mix, a moderate to strong toxicity was noted at 5000 µg/plate in the TA 1537 strain in the first experiment, then at dose-levels

>= 

 2500 µg/plate in the TA 98 strain and at 5000 µg/plate in the TA 102 strain in the second experiment.

No other noteworthy toxicity was noted towards the other strains or test conditions.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, in either experiment, either with or without S9 mix.

 

These results met the criteria of a negative response.

 

Conclusion

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains,either in the presence or absence of a rat liver metabolizing system.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2016 - 05 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

26 September 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable (not a gene mutation assay).

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing 10% inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
L5178Y TK+/- cells are an established cell line recommended by international regulations for in vitro mammalian cell gene mutation test and for in vitro micronucleus test. Indeed, they are suitable to reveal chemically induced micronuclei. The average cell cycle time is approximately 10-12 hours.
L5178Y TK+/- cells were obtained from ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (Marne-La-Vallée, France).

The cells were stored in a cryoprotective medium (10% horse serum and 10% dimethylsulfoxide (DMSO)) at -80°C and each batch of frozen cells was checked for the absence of mycoplasma.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Since the test item was found to be severely cytotoxic in the preliminary test, the highest dose levels selected for the main experiments were based on the level of cytotoxicity, according to the criteria specified in the international regulations (see attached document "Test concentrations with justification for top dose").

Vehicle / solvent:

Vehicle used: dimethylsulfoxide
Justification for choice: According to available solubility data, the vehicle used for the preparation of test item dose formulations and the treatment of vehicle control cultures was dimethylsulfoxide (DMSO).
The test item was diluted in the in the vehicle at concentrations of:
- 500 and 10 mg/mL for the preliminary cytotoxicity tests,
- 10 and 1.25 mg/mL for the first experiment,
- 1.5 mg/mL for the second experiment,
- 2.5 mg/mL for the third and fourth experiments.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine (without S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION:
Preliminary toxicity test:

Without S9 mix
3 h treatment + 24 h recovery
24 h treatment + 0 h recovery

With S9 mix 3 h treatment + 24 h recovery

Main cytogenetic experiments
In four independent experiments, at least seven dose levels of the test item were tested in duplicate (two cultures/dose level), both with and without metabolic activation, using treatment durations as follows:

Without S9 mix
3 h treatment + 24 h recovery
24 h treatment + 0 h recovery

With S9 mix 3 h treatment + 24 h recovery


NUMBER OF CELLS EVALUATED: 2000 mononucleated cells per dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling

Evaluation criteria:

The biological relevance of the results was always taken into account when evaluating results.

Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test,
- for at least one dose level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical/reference range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose levels.

Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met.

When the highest analyzable dose level did not exhibit about 55% cytotoxicity (in case of cytotoxic items), or when results remain inconclusive, additional confirmatory experiments may have been needed.

Statistics:

For each condition of the cytogenetic experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures.
This comparison was performed using the X2 test, unless treated culture data are lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance. This statistical analysis was performed using a validated Excel sheet.

To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose levels. This statistical analysis was performed using SAS Enterprise Guide software.

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: no
- Definition of acceptable cells for analysis: Analysis was performed under a microscope (1000 x magnification), on the basis of the recommendations of Miller et al. (1995), according to the following criteria:
* micronuclei should be clearly surrounded by a nuclear membrane,
* the micronucleus area should be less than one-third of the area of the main nucleus,
* non-refractility of the micronuclei,
* micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
* micronuclei should be located within the cytoplasma of the cell,
* only mononucleated cells with a number of micronuclei = 5 should be scored to exclude apoptosis and nuclear fragmentation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see document attached

RESULTS OF CYTOTOXICITY:
Short treatment without S9 mix: 3 h treatment + 24 h recovery
1st experiment: A marked to severe cytotoxicity was induced at dose levels = 1.56 µg/mL, as shown by a 65 to 100% decrease in the PD.
2nd experiment: Only a slight cytotoxicity was induced at the highest selected dose levels (i.e. 2 µg/mL), as shown by a 27% decrease in the PD.
3rd experiment: A slight to severe cytotoxicity was induced at dose levels = 1 µg/mL, as shown by a 28 to 100% decrease in the PD
4th experiment: A marked to severe cytotoxicity was induced at dose levels = 1.875 µg/mL, as shown by a 60 to 100% decrease in the PD.

Continuous treatment without S9 mix: 24 h treatment + 0 h recovery
1st experiment: A marked to severe cytotoxicity was induced at dose levels = 3.13 µg/mL, as shown by a 79 to 100% decrease in the PD
2nd experiment: A slight to severe cytotoxicity was induced at dose levels = 2.5 µg/mL, as shown by a 27 to 100% decrease in the PD.
3rd experiment: A slight to severe cytotoxicity was induced at dose levels = 1.25 µg/mL, as shown by a 28 to 100% decrease in the PD.
4th experiment: A slight to severe cytotoxicity was induced at dose levels = 1.875 µg/mL, as shown by a 34 to 100% decrease in the PD.

Experiments with S9 mix
1st experiment: A marked to severe cytotoxicity was induced at dose levels = 12.5 µg/mL, as shown by a 73 to 100% decrease in the PD.
2nd experiment: No noteworthy cytotoxicity was induced at any of the selected dose levels in this experiment.
3rd experiment: A slight to severe cytotoxicity was induced at dose levels = 5 µg/mL, as shown by a 33 to 100% decrease in the PD.
4th experiment: A slight to severe cytotoxicity was induced at dose levels = 7.5 µg/mL, as shown by a 27 to 100% decrease in the PD.

Conclusions:

Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK^+/-.

 

The study was performed in compliance with the principles of Good Laboratory Practice and the study design was based on the OECD guideline No. 487, adopted 26 September 2014.

 

Methods

After a preliminary toxicity test, the test item diluted in dimethylsulfoxide (DMSO), was tested in four independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

 

Without S9 mix	3 h treatment + 24 h recovery
	24 h treatment + 0 h recovery
With S9 mix	3 h treatment + 24 h recovery

 

Each treatment was coupled to an assessment of cytotoxicity at the same dose levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.

Then, after the final cell counting, the cells were washed and fixed. Then, cells from at least three dose levels of the test item-treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). When the slide analysis was undertaken, micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).

Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

 

Results

Since the test item was found to be severely cytotoxic in the preliminary test, the highest dose levels selected for the main experiments were based on the level of cytotoxicity, according to the criteria specified in the international regulations.

 

The mean Population Doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

First of all, no emulsion or precipitation was observed in the culture medium at the end of the treatment periods, in any experiments, up to the highest tested dose level of 100 µg/mL.

Short treatment without S9 mix: 3 h treatment + 24 h recovery

With a treatment volume of 1% (v/v) in culture medium, the dose levels selected for the first experiment were 0.10, 0.20, 0.39, 0.78, 1.56, 3.13, 6.25 and 12.5 µg/mL.

 

A marked to severe cytotoxicity was induced at dose levels = 1.56 µg/mL, as shown by a 65 to 100% decrease in the PD. The dose levels selected for micronucleus analysis were 0.20, 0.39 and 0.78 µg/mL, the latter inducing only a 9% decrease in the PD but higher dose levels being too cytotoxic.

No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. Moreover, no dose-response relationship was demonstrated by the linear regression and all frequencies remained within the vehicle control historical range [0.0-3.5‰]. These results met the criteria of a negative response.

 

Since the highest analyzable dose level did not exhibit about 55% cytotoxicity in the first experiment, a second one was undertaken under the same experimental conditions but using a narrower range of dose levels, as follows:0.125, 0.25, 0.5, 0.75, 1, 1.25, 1.5 and 2 µg/mL. Only a slight cytotoxicity was induced at the highest selected dose levels (i.e. 2 µg/mL), as shown by a 27% decrease in the PD.

 

Since the highest analyzable dose level did not exhibit about 55% cytotoxicity in the second experiment, the corresponding slide analyses were not performed and a third experiment was undertaken under the same experimental conditions but using a modified range of dose levels, as follows: 0.156, 0.313, 0.625, 1, 1.25, 2.5, 3.75 and 6 µg/mL. A slight to severe cytotoxicity was induced at dose levels = 1 µg/mL, as shown by a 28 to 100% decrease in the PD. Nevertheless, as for the second experiment, none of the selected dose levels induced the recommended level of cytotoxicity and the corresponding slide analyses were therefore not undertaken.

 

A fourth experiment was then undertaken using more dose levels and an even narrower range, as follows: 0.313, 0.469, 0.625, 0.938, 1.25, 1.5, 1.875, 2.5, 3.75 and 5 µg/mL. A marked to severe cytotoxicity was induced at dose levels = 1.875 µg/mL, as shown by a 60 to 100% decrease in the PD.

The dose levels selected for micronucleus analysis were 0.938, 1.25 and 1.5 µg/mL, the latter inducing only a 15% decrease in the PD but higher dose levels being too cytotoxic.

No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. Moreover, no dose-response relationship was demonstrated by the linear regression andnone of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle control historical range [0.0-3.5‰]. These results met the criteria of a negative response.

 

Despite the use of an even narrower range of dose levels in this fourth experiment, none of the selected dose levels induced the recommended level of cytotoxicity. However, considering the narrow dose levels spacing used in this fourth experiment and the negative results obtained in both independent experiments analyzed (first and fourth), the overall available results were considered as suitable to allow a reliable interpretation.

 

The overall results (short treatments without S9 mix) were considered to meet the criteria of a negative response.

Continuous treatment without S9 mix:24 h treatment + 0 h recovery

With a treatment volume of 1% (v/v) in culture medium, the dose levels selected for the first experiment were 0.20, 0.39, 0.78, 1.56, 3.13, 5.56, 8.33 and 12.5 µg/mL.

A marked tosevere cytotoxicity was induced at dose levels = 3.13 µg/mL, as shown by a 79 to 100% decrease in the PD. The dose levels selected for micronucleus analysis were 0.39, 0.78 and 1.56 µg/mL the latter inducing only a 19% decrease in the PD but higher dose levels being too cytotoxic.

No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. Moreover, no dose-response relationship was demonstrated by the linear regression and none of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle control reference data range[1.0-6.0‰]. These results met the criteria of a negative response.

           

Since the highest analyzable dose level did not exhibit about 55% cytotoxicity in the first experiment, a second one was undertaken under the same experimental conditions but using a narrower range of dose levels, as follows:0.5, 0.75, 1, 1.5, 2, 2.5, 3.13 and 5 µg/mL.

A slight to severecytotoxicity was induced at dose levels = 2.5 µg/mL, as shown by a 27 to 100% decrease in the PD. The dose levels selected for micronucleus analysis were 2, 2.5 and 3.13 µg/mL, the latter inducingonly a 43% decrease in the PD but the higher dose level being too cytotoxic. The highest selected dose level did not induce the recommended level of cytotoxicity, nevertheless considering the narrow dose levels spacing used in this experiment, the analyzed dose levels were considered as suitable to allow a reliable interpretation.

A statistically significant increase in the frequency of micronucleated cells was observed at the dose level of 2.5 µg/mL relative to the vehicle control. At this dose level, the frequency of micronucleated cells of both replicate cultures was above the corresponding vehicle reference data range(i.e. 7 and 10‰ versus [1.0 -6.0‰] for reference data). Since no dose-response relationship was demonstrated by the linear regression, these results did not meet either the criteria for a positive or a negative response.

 

In order to check the reproducibility of the increase observed in the second experiment, a third experiment was undertaken under the same experimental conditions but using a narrower range of dose levels, as follows: 0.313, 0.625, 1.25, 2.5, 3.75, 5 and 7.5 µg/mL.

A slight to severe cytotoxicity was induced at dose levels = 1.25 µg/mL, as shown by a 28 to 100% decrease in the PD. Since none of the selected dose levels induced the recommended level of cytotoxicity, the corresponding slide analyses were not undertaken.

 

A fourth experiment was then undertaken using more dose levels and an even narrower range, as follows: 0.313, 0.469, 0.625, 0.938, 1.25, 1.5, 1.875, 2.5, 3.75 and 5 µg/mL. A slight to severe cytotoxicity was induced at dose levels = 1.875 µg/mL, as shown by a 34 to 100% decrease in the PD.

The dose levels selected for micronucleus analysis were 1.5, 1.875 and 2.5 µg/mL, the latter inducing only a 34% decrease in the PD but higher dose levels being too cytotoxic.

No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. Moreover, no dose-response relationship was demonstrated by the linear regression andnone of the analyzed dose levels showed frequency ofmicronucleated cellsof both replicate cultures above the corresponding vehicle control reference data range[1.0-6.0‰]. These results met the criteria of a negative response.

Since the statistically significant increase observed in the second experiment was not reproduced in this fourth experiment, it was considered as non-biologically relevant.

Despite the use of an even narrower range of dose levels in this fourth experiment, none of the selected dose levels induced the recommended level of cytotoxicity. However, considering the narrow dose levels spacing used in this fourth experiment and the negative results obtained in the first and fourth experiments, the overall available results were considered as suitable to allow a reliable interpretation.

 

The overall results (continuous treatments without S9 mix) were considered to meet the criteria of a negative response.

 

Experiments with S9 mix

With a treatment volume of 1% (v/v) in culture medium, the dose levels selected for the first experiment were 1.56, 3.13, 6.25, 12.5, 25, 44.4, 66.7 and 100 µg/mL.

A marked to severe cytotoxicity was induced at dose levels = 12.5 µg/mL, as shown by a 73 to 100% decrease in the PD. The dose levels selected for micronucleus analysis were 1.56, 3.13 and 6.25 µg/mL the latter inducing only a 21% decrease in the PD but higher dose levels being too cytotoxic.

No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. Moreover, no dose-response relationship was demonstrated by the linear regression and none of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle control historical range [0.0-3.5‰]. These results met the criteria of a negative response.

 

Since the highest analyzable dose level did not exhibit about 55% cytotoxicity in the first experiment, a second one was undertaken under the same experimental conditions but using a narrower range of dose levels, as follows: 1.5, 3.13, 6.25, 7.5, 8.33, 10, 12.5 and 15 µg/mL. No noteworthy cytotoxicity was induced at any of the selected dose levels in this experiment.

 

Since none of the analyzable dose levels exhibited about 55% cytotoxicity in the second experiment, the corresponding slide analyses were not performed and a third experiment was undertaken under the same experimental conditions but using a higher range of dose levels, as follows: 2.5, 5, 7.5, 10, 12.5, 15, 20 and 25 µg/mL. A slight to severe cytotoxicity was induced at dose levels = 5 µg/mL, as shown by a 33 to 100% decrease in the PD. Nevertheless, none of the selected dose levels induced the recommended level of cytotoxicity and there were only two analyzable dose levels, i.e. inducing acceptable decrease in the PD. Consequently theslide analyses were not performed.

 

A fourth experiment was then undertaken using more dose levels and an even narrower range, as follows: 1.875, 2.5, 3.75, 5, 6.25, 7.5, 10, 12.5, 15 and 25 µg/mL. A slight to severe cytotoxicity was induced at dose levels = 7.5 µg/mL, as shown by a 27 to 100% decrease in the PD.

The dose levels selected for micronucleus analysis were 6.25, 7.5 and 10 µg/mL, the latter inducingonly a 39% decrease in the PD but higher dose levels being too cytotoxic.

No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. Moreover, no dose-response relationship was demonstrated by the linear regression and all frequencies remained within the vehicle control historical range [0.0-3.5‰]. These results met the criteria of a negative response.

Despite the use of an even narrower range of dose levels in this fourth experiment, none of the selected dose levels induced the recommended level of cytotoxicity. However, considering the narrow dose levels spacing used in this fourth experiment and the negative results obtained in both independent experiments analyzed (first and fourth), the overall available results were considered as suitable to allow a reliable interpretation.

 

The overall results with S9 mix were considered to meet the criteria of a negative response.

 

Conclusion

Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK^+/-mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August - December 2017

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:

2015

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro gene mutation study in mammalian cells

Target gene:

hprt locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co.
- Storage at Covance: as frozen stocks in liquid notrogen.
Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free.
For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37+/-1°C. When the cells were growing well, subcutltures were established in an appropriate number of flasks.

MEDIA USED
- Type and identity of media: RPMI 1640 media containing L-glutamine and HEPES

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Preliminary solubility data indicated that the test substance was soluble in anhydrous analytical grade dimethyl-sulphoxide (DMSO) at concentrations up to at least 505.2 mg/mL. The solubility limit in culture medium was in the range of 315.8 to 631.5 µg/mL, as indicated by precipitation at the higher concentration following 3 hours incubation at 37±1°C. A maximum concentration of 2000 µg/mL was therefore selected for the initial cytotoxicity Range-Finder Experiment in order that treatments were performed up to a precipitating treatment concentration

Range finder 1 (+/-S9): 62.5-125-250-500-1000-2000 mg/ml
Range finder 2 (+/-S9): 0,3906 - 0.7813- 1.563 - 3.125- 6.250-12.5-25-50-100200
Mutation experiment 1(-S9): 0.25-0.5-1-2-4-6-8-10-12.5-15 mg/ml
Mutation experiment 1 (+S9): 5-10-15-20-25-30-35-40-50-60 mg/ml
Mutation experiment 2(-S9): 0.5-1-2-4-5-6-6.5-7-7.5-8-10 mg/ml

Vehicle / solvent:

DMSO
Preliminary solubility data indicated that the test substance was soluble in anhydrous analytical grade dimethyl-sulphoxide (DMSO) at concentrations up to at least 505.2 mg/mL. The solubility limit in culture medium was in the range of 315.8 to 631.5 µg/mL, as indicated by precipitation at the higher concentration following 3 hours incubation at 37±1°C.
Test article stock solutions were prepared by formulating test substance under subdued lighting in DMSO, with the aid of vortex mixing, to give the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 2.5 hours of initial formulation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 3h
- Exposure duration: 7d
- Expression time (cells in growth medium): 7d

NUMBER OF CELLS EVALUATED: At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 105 cells/mL in readiness for plating for 6TG resistance.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: Cloning Efficiency (CE) in any given culture is therefore: CE = P/No of cells plated per well, and as an average of 1.6 cells/well were plated on all survival and viability plates, CE = P/1.6.
Percentage Relative Survival (% RS) in each test culture was determined by comparing plating efficiencies in test and control cultures thus: % RS = [CE (test)/CE (control)] x 100.
To take into account any loss of cells during the 3 hour treatment period, percentage relative survival values for each concentration of test article were adjusted as follows: Adjusted % RS = [% RS x Post-treatment cell concentration for test article treatment] / Post-treatment cell concentration for vehicle control


- OTHER: metabolic activation system
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it is prepared from male Sprague Dawley rats induced with Aroclor 1254. The batches of S-9 were stored frozen in aliquots at <-50°C prior to use (Booth et al., 1980). Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities).
The S-9 mix was prepared in the following way: G6P (180 mg/mL), NADP (25 mg/mL), KCl (150 mM) and rat liver S-9 were mixed in the ratio 1:1:1:2. For all cultures treated in the presence of S-9, an aliquot of the mix was added to each cell culture to achieve the required final concentration of test article in a total of 20 mL. The final concentration of the liver homogenate in the test system was 2%.

Rationale for test conditions:

Acceptance Criteria: The assay was considered valid if the following criteria were met:
1. The MF in the concurrent negative control was considered acceptable for addition to the laboratory historical negative control database,
2. The MF in the concurrent positive controls induced responses that were compatible with those generated in the historical positive control database and give a clear, unequivocal increase in MF over the concurrent negative control,
3. The test was performed with and without metabolic activation,
4. Adequate numbers of cells and concentrations were analysable.

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The MF at one or more concentrations was significantly greater than that of the vehicle control (p=0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p=0.05)
3. The results were outside the historical vehicle control range.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance. Extreme caution was exercised with positive results obtained at levels of RS lower than 10%.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity
In the initial cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 62.5 to 2000 µg/mL (limited by solubility in culture medium). Upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations tested (500 to 2000 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest two concentrations tested (1000 and 2000 µg/mL). The lowest precipitating concentration was retained and the higher concentration discarded. Complete/extreme toxicity was observed at all concentrations tested with only a single concentration in the presence of S-9 giving relative survival (RS) greater than 10% (62.5 µg/mL gave 11% RS).
Based on these data, a second Range-Finder Experiment was performed. In the second cytotoxicity Range-Finder Experiment, ten concentrations were tested in the absence and presence of S-9 ranging from 0.3906 to 200 µg/mL (limited by toxicity). Upon addition of the test article to the cultures, precipitate was observed at the highest concentration tested (200 µg/mL) but no precipitate was observed following the 3 hour treatment incubation period. The highest concentrations to give ¿10% RS were 6.25 µg/mL in the absence of S-9 and 25 µg/mL in the presence of S-9, which gave 29% and 19% RS, respectively.
No marked changes in osmolality or pH were observed in the initial Range-Finder at the highest concentrations analysed (1000 µg/mL, limited by post treatment precipitate) as compared to the concurrent vehicle controls.

In Mutation Experiment 1 ten concentrations, ranging from 0.25 to 15 µg/mL in the absence of S-9 and from 5 to 60 µg/mL in the presence of S-9, were tested. No precipitate was observed at the time of test article addition or following the treatment incubation period. Seven days after treatment, the highest three concentrations in the absence of S-9 (10 to 15 µg/mL) and the highest concentration in the presence of S-9 (60 µg/mL), were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 8 µg/mL in the absence of S-9 and 50 µg/mL in the presence of S-9, which gave 8% and 14% RS, respectively. The toxicity profile observed in the absence of S-9 was steep, with no concentration giving the desired 10 to 20% RS (the next lowest concentration tested, 6 µg/mL, gave 26% RS).

Based on the toxicity profile observed and the statistically significant increase at the highest concentration tested in the absence of S-9, a confirmatory experiment was performed.
In Mutation Experiment 2, eleven concentrations, ranging from 0.5 to 10 µg/mL, were tested in the absence of S-9. Seven days after treatment, the highest two concentrations tested (8 and 10 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition the lowest concentration tested (0.5 µg/mL) was not selected as there were sufficient non-toxic concentrations to generate a toxicity profile. All other concentrations were selected. The highest concentration analysed was 7.5 µg/mL which gave 10% RS.

Mutation :
Following 3 hour treatment in the absence of S-9 in Mutation Experiment 1, a statistically significant increase in MF compared to the vehicle control, was observed at the highest concentration analysed (8 µg/mL) and a significant linear trend was observed. However, all concentrations were within the range generated by the last twenty experiments performed in this laboratory (1.48 to 8.21 mutants per 106 viable cells at the time of this experiment) and this observation only occurred at a concentration giving excessive toxicity (<10% relative survival (RS)) and so was considered of questionable biological relevance. A confirmatory experiment was performed.
Following 3 hour treatment in the absence of S-9 in Mutation Experiment 2, no statistically significant increases in MF were observed, there was no significant linear trend and the mean MF values were within the range generated by the last twenty studies (1.59 to 6.65 mutants per 106 viable cells at the time of this experiment). Based on current interpretations of data of this type (Thybaud et al, 2007) results that are not reproduced within or between experiments are considered of little or no biological relevance. The isolated increase observed in Experiment 1 was not reproduced in Experiment 2 and therefore is considered not biologically relevant.
Following 3 hour treatment in the presence of S-9, no statistically significant increases in MF were observed, there was no significant linear trend and the mean MF values were within the range generated by the last twenty experiments performed in this laboratory (2.37 to 7.06 mutants per 106 viable cells at the time of this experiment).

Conclusions:

It is concluded that the test substance did not induce biologically relevant increases in mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of toxicity, for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9).

Executive summary:

The test substance was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of two cytotoxicity Range-Finder experiments followed by two Mutation Experiments. Two Range-Finder Experiments were performed as complete/extreme toxicity was observed in the initial experiment. Both cytotoxicity Range-Finder Experiments and the first Mutation Experiment were conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9) and the second Mutation Experiment was conducted in the absence of S-9 only. The second Mutation Experiment was performed as no appropriate limit concentration was achieved in the first Mutation Experiment. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used for each experiment.

In the initial cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 62.5 to 2000 µg/mL (limited by solubility in culture medium). Complete/extreme toxicity was observed at all concentrations tested with only a single concentration in the presence of S-9 giving relative survival (RS) greater than 10% (62.5 µg/mL gave 11% RS). Based on these data a second Range-Finder Experiment was performed.

In the second cytotoxicity Range-Finder Experiment, ten concentrations were tested in the absence and presence of S-9 ranging from 0.3906 to 200 µg/mL (limited by toxicity). The highest concentrations to give>10% RS were 6.25 µg/mL in the absence of S-9 and 25 µg/mL in the presence of S-9, which gave 29% and 19% RS, respectively.

In Mutation Experiment 1 ten concentrations, ranging from 0.25 to 15 µg/mL in the absence of S-9 and from 5 to 60 µg/mL in the presence of S-9, were tested. The highest concentrationsanalysedwere 8 µg/mL in the absence of S-9 and 50 µg/mL in the presence of S-9, which gave 8% and 14% RS, respectively. The toxicity profile observed in the absence of S-9 was steep, with no concentration giving the desired 10 to 20% RS (the next lowest concentration tested, 6 µg/mL, gave 26% RS).

Following 3 hour treatment in the presence of S-9 no statistically significant increases in mutant frequency (MF) were observed, there was no statistically significant linear trend and the mean MF values were within the range generated by the last twenty experiments performed in this laboratory (2.37 to 7.06 mutants per 10^6 viable cells at the time of this experiment).

Following 3 hour treatment in the absence of S-9 a statistically significant increase in MF, compared to the vehicle control, was observed at the highest concentration analysed (8 µg/mL) and a statistically significant linear trend was observed. However, all concentrations were within the range generated by the last twenty experiments performed in this laboratory (1.48 to 8.21 mutants per 10^6 viable cells at the time of Experiment 1) and this observation only occurred at a concentration giving excessive toxicity (<10% RS). Based on the increase in MF and the toxicity observed in this experiment a second Mutation Experiment was performed under the same treatment condition.

In Mutation Experiment 2 eleven concentrations, ranging from 0.5 to 10 µg/mL, were tested in the absence of S-9. The highest concentration analysed was 7.5 µg/mL which gave 10% RS. Following 3 hour treatment in the absence of S-9 in Experiment 2 no statistically significant increases in MF were observed, there was no statistically significant linear trend and the mean MF values were within the range generated by the last twenty experiments performed in this laboratory (1.59 to 6.65 mutants per 10^6 viable cells at the time of this experiment).

Based on current interpretations of data of this type, results that are not reproduced within or between experiments are considered of little or no biological relevance. The isolated increase observed in Experiment 1 was not reproduced in Experiment 2 and therefore is considered not biologically relevant.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

It is concluded that the tesst substance did not induce biologically relevant increases in mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of toxicity, for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo data is available.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

in vitro gene mutation study in bacteria (Chevallier 2017)

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium. The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B.13/14). 

Five strains of bacteria Salmonella typhimuriumwere used: TA 1535, TA 1537, TA 98, TA 100 and TA 102.

Since the test item only induced a moderate emulsion in the final treatment medium and a slight toxicity in the preliminary test, the highest dose-level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines. The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

The selected dose-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains, in both mutagenicity experiments with and without S9 mix.

A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels>= 1250 µg/plate in both experiments without S9 mix and >= 2500 µg/plate in the first experiment with S9 mix (using the direct plate incorporation method). In the second experiment with S9 mix (using the pre-incubation method), no emulsion was noted at any dose-levels.

In the absence of S9 mix, a moderate to strong toxicity (decrease in the number of revertants) was noted in the TA 1537 strain at dose-levels >=  2500 µg/plate and at 5000 µg/plate in the first and second experiments, respectively.

In the presence of S9 mix, a moderate to strong toxicity was noted at 5000 µg/plate in the TA 1537 strain in the first experiment, then at dose-levels >=  2500 µg/plate in the TA 98 strain and at 5000 µg/plate in the TA 102 strain in the second experiment.

No other noteworthy toxicity was noted towards the other strains or test conditions.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, in either experiment, either with or without S9 mix. These results met the criteria of a negative response.

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains,either in the presence or absence of a rat liver metabolizing system.

In vitro cytogenicity / micronucleus study, OECD 487 (Chevallier 2017b):

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/-.

After a preliminary toxicity test, the test item diluted in dimethylsulfoxide (DMSO), was tested in four independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows: 3 h treatment + 24 h recovery (without S9 mix), 24 h treatment + 0 h recovery (without S9 mix) and 3 h treatment + 24 h recovery (with S9 mix).

Each treatment was coupled to an assessment of cytotoxicity at the same dose levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.

Since the test item was found to be severely cytotoxic in the preliminary test, the highest dose levels selected for the main experiments were based on the level of cytotoxicity, according to the criteria specified in the international regulations. First of all, no emulsion or precipitation was observed in the culture medium at the end of the treatment periods, in any experiments, up to the highest tested dose level of 100 µg/mL.

The overall results were considered to meet the criteria of a negative response. Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK +/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

In vitro gene mutation study in mammalian cells / OECD 476 (Hargreaves 2018)

The test substance was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of two cytotoxicity Range-Finder experiments followed by two Mutation Experiments. Two Range-Finder Experiments were performed as complete/extreme toxicity was observed in the initial experiment. Both cytotoxicity Range-Finder Experiments and the first Mutation Experiment were conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9) and the second Mutation Experiment was conducted in the absence of S-9 only. The second Mutation Experiment was performed as no appropriate limit concentration was achieved in the first Mutation Experiment. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). A 3 hour treatment incubation period was used for each experiment.

It is concluded that the test substance did not induce biologically relevant increases in mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of toxicity, for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9).

Justification for classification or non-classification

Based on the available data, no classification for genetic toxicity is required for the registered substance according to the Regulation EC N°1272/2008.