3-((3,4-dicyanophenyl)sulfonyl)-N-(2-hydroxypropyl)propane-1-sulfonamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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:

The experimental phase of this study was performed between the 5th May 2010 and 28th May 2010. The final report was issued 18th August 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella typhimurium
Tryptophan for Escherichia coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolising system ( liver S9)

Test concentrations with justification for top dose:

The test concentrations were determined from a preliminary toxicity assay and were 0, 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: dimethyl sulphoxide
Justification for choice of solvent/vehicle: The test material wasinsoluble in sterile distilled water at 50 mg/l but was fully soluble in dimethyl sulphoxide at the same concentration in solubility checks. Dimethyl sulphoxide was thereforre selected as the vehicle.

Untreated negative controls:

yes

Remarks:

spontaneous mutation rate of TA100

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Untreated negative controls:

yes

Remarks:

spontaneous mutation rate of TA 1535

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of WP2uvrA

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Untreated negative controls:

yes

Remarks:

spontaneous mutation of TA98

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 mix

Untreated negative controls:

yes

Remarks:

spontaneous mutation of TA1537

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Untreated negative controls:

yes

Remarks:

spontaneous mutation rate of TA 100

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1535

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of WP2uvrA

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Untreated negative controls:

yes

Remarks:

spontaneous mutation rate of TA98

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix

Untreated negative controls:

yes

Remarks:

spontaneous mutation rate of 1537

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Untreated negative controls:

yes

Remarks:

spontaneous mutation of TA100

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar, direct plate incorporation method for experiment 1 and pre-incubation for experiment 2

DURATION
- Preincubation period: 10 hours
- Exposure duration: Approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plating

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance is considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance wasl not the only determining factor for a positive response.A test material is considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Yes, as recommened by UKEMS (Kirkland D J, (Ed) (1989) Statistical Evaluation of Mutagenicity Test Data UKEMS sub-committee on Guidelines for Mutagenicity Testing. Report Part III - Cambridge University Press)

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate. No test material precipitate was observed on the plates of any of the doses tested in either the presence or absence of S9-mix.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. A small, statistically significant increase in Escherichia coli strain WP2uvrA- revertant colony frequency was observed in the presence of S9 at 5000 ug/plate in the second Experiment. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 5000 ug/plate were within the in-house historical untreatedtvehicle control range for the tester strain and the fold increase was only 1.47 times the concurrent vehicle control.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Conclusions:

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

Executive summary:

Introduction

The test item was tested using a protocol designed to be compatible with OECD Guidelines for Testing of Chemicals No. 471 (1997) "Bacterial Reverse Mutation Test" and Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008.The method conforms to the guidelines published by the major Japanese regulatory regulatory authorities including METI, MHLW and MAFF.

Methods

Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2uvrA were treated in two separate experiments 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 for the experiments was 50 to 5000 µg/plate.

 

 Results

The vehicle (dimethyl sulphoxide) control plates gave 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 or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was therefore tested upto the maixmum recomended 5000 ug/plate.  No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 mix.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation.

 

 

Conclusion

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

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 30th March 2017. Experimental completion date 21st July 2017. Report issued: 03 November 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro chromosomal aberration

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

Cells For each experiment: sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.

The details of the donors used are:

Preliminary Toxicity Test: female, aged 27 years
Main Experiment: female, aged 24 years
Main Experiment Repeat: female, aged 26 years

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

4(20)-Hour without S9 0,64, 128, 256,512, 758, 896, 1024
4(20)-Hour with S9 (2%) 0, 32, 64, 96,128, 256, 384, 512
24 hour without S9 0,12, 24, 32, 64, 96,128, 256

No correction for purity was made. The test item was relatively toxic to human lymphocytes and, therefore, the maximum recommended dose level could not be achieved and therefore adjusting for the small difference in purity value would not have affected the outcome of the test.

Vehicle / solvent:

Dimethyl Sulphoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

SPINDLE INHIBITOR (cytogenetic assays): Demecolcine (Colcemid 0.1 µg/mL) 2.5 hours before required harvest time.

STAIN (for cytogenetic assays): 5% Giemsa for five minutes

NUMBER OF REPLICATIONS: Duplicate cultures

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The lymphocytes were re-suspended in several mL of fresh fixative ( fresh methanol/glacial acetic acid (3:1 v/v)) before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 (150 per duplicate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

- OTHER:
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm. The pH and osmolality of the test item was investigated in a separate study.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The dose range for the Preliminary Toxicity Test was 7.81 to 2000 μg/mL. The maximum dose was the maximum recommended dose level. A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at 2000 μg/mL in the all three exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 500 μg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 125 μg/mL. The test item induced marked evidence of toxicity in all of the exposure groups.


The selection of the maximum dose level for the Main Experiment was based on toxicity for all three exposure groups.

The dose levels of the controls and the test item are given in the table below:

Group	Final concentration of test substance (μg/mL)
4(20)-hour without S9	0*, 64*, 128*, 256*, 512, 768, 896, 1024, MMC 0.4*
4(20)-hour with S9 (2%)	0*, 32, 64, 96*, 128*, 256*, 384*, 512, CP 2*
24-hour without S9	0*, 12, 24*, 32*, 64*, 96*, 128, 256, MMC 0.1*

* dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test. There were metaphases suitable for scoring present at 512 μg/mL in the 4(20)-hour with and without S9 and up to 128 μg/mL in the 24-hour continuous exposure group.

No precipitate was observed at the end of exposure in the blood cultures in any of the exposure groups. The mitotic index data for the Main Experiment confirmed the qualitative observations in that a dose-related inhibition of mitotic index was observed in all three exposure groups.

In the 4(20)-hour exposure group in the absence of S9, 32%, 64% and 84% mitotic inhibition was achieved at 128, 256 and 512 μg/mL, respectively. Above this concentration, no metaphases were present for scoring. Therefore, the maximum concentration chosen for metaphase analysis was 256 µg/mL because this dose level only slightly exceeded the toxicity limit as stated in OECD 473 (55±5%).

In the 4(20)-hour exposure group the presence of S9, a plateau of inhibition of mitotic index of 36%, 40% and 54% was observed at 128, 256 and 384 µg/mL, respectively. There were metaphases present at 512 µg/mL but there were insufficient numbers and they were not very scorable due to the excessive toxicity of the test item. Therefore, the maximum concentration chosen for metaphase analysis was 384 µg/mL because it achieved optimum toxicity as defined by the guideline (55±5%).

In the 24-hour exposure group, a moderate plateau of toxicity was observed with an inhibition of mitotic index of 42%, 36% and 23% at 64, 96 and 128 µg/mL, respectively. Above this concentration there were no metaphases present. Therefore, the maximum concentration selected for metaphase analysis was 96 µg/mL because this dose and 64 µg/mL approached optimum toxicity.

Conclusions:

The test substance induced a statistically significant increase in the frequency of cells with chromosomes aberrations, in both the absence or presence of a liver enzyme metabolising system. The test item is therefore considered to be clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction

The purpose of the study was to assess the potential chromosomal mutagenicity of the test substance on the metaphase chromosomes of normal human lymphocytes. The test method was OECD guideline for Testing of Chemicals No 473 "In vitro Mammmalian Chromsome Aberration Test" adopted 29 July 2016.

Method

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

Results

The test substance induced a statistically significant increase in the frequency of cells with chromosomes aberrations, in both the absence or presence of a liver enzyme metabolising system.

Conclusion

The test susbtance was considered to be clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 23rd March 2017. Experimental completion date 19th April 2017. Report date 11th December 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

The purity of the test substance was less than 99% and not adjusted for purity as stated in the study plan. The deviation is not considered to have any impact on the outcome of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

yes

Remarks:

The purity of the test substance was less than 99% and not adjusted for purity as stated in the study plan. The deviation is not considered to have any impact on the outcome of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

Thymidine kinase TK +/- locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

4-hour exposure without S9: 0, 80, 160, 240, 320, 400 and 480 µg/mL
4-hour exposure with S9 (2%): 0, 40, 80, 160, 240, 320 and 400 µg/mL
24-hour exposure without S9: 0, 20, 40 ,60, 80, 100 and 120 µg/mL

Test concentrations were based on the results of a preliminary toxicity test.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

The study was designed to assess the potential mutagenicity of the test substance on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Following a preliminary toxicity test,using concentrations of 0, 7.81, 15.63, 31.25, 62.5,125,250, 500,1000 and 2000 µg/mL the following concentrations were selected for the main test:

4 hours + and - S9 test: 0, 20, 40, 80,160, 240, 320, 400 and 480 µg/mL.

For the 24 hours S9 test the following concentrations were used 0,10,20,40,60,80,100,120 and 240 µg/mL.

Ethylmethansuphonate and cyclophosphamide were used as positive controls. Two independent experiments were performed. In experiment 1, cells were treated for 4-hours in either the absence or presence of metabolic activation (rat liver S9). In experiment 2 the exposure groups were 24-hours in the absence of metabolic activation. The positive controls responded as expected indicating that the test system was functioning correctly.

There was no marked change in pH when the test material was dosed into media and osmolarity did not incease by more than 50 mOsm.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

Statistics:

The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). The statistical package used indicates the presence of statistically significant increases and linear-trend events.An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed in preliminary toxicity test

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Results

Preliminary Test

The dose range of the test item used in the preliminary toxicity test was 7.81 to 2000 μg/mL.

There was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in all of the three exposure groups when compared to the concurrent vehicle control groups. Precipitate of the test item was observed at 2000 μg/mL in all three of the exposure groups at the end of the exposure periods. Therefore, the dose levels selected for the man test were based on test item induced toxicity.

Main Test

There was evidence of marked dose related toxicity following exposure to the test item in all of the three exposure groups, as indicated by the %RSG and RTG values. There was evidence of marked reductions in viability (%V) in the 4-hour exposure in the absence of metabolic activation, indicating that residual toxicity had occurred in this exposure group. Acceptable levels of toxicity were seen with the positive control substances. The concentrations of 480 μg/mL in the 4-hour exposure in the presence of metabolic activation and 240 μg/mL in the 24-hour exposure were not plated out for 5-TFT resistance and viability due to excessive toxicity. The concentration of 480 μg/mL in the 4-hour exposure in the absence of metabolic activation was plated out for 5-TFT resistance and viability, however was later discarded from analysis due to post treatment toxicity. Precipitate of the test item was not observed in the main test.

 

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

 

The test item induced statistically significant and dose related (linear-trend) increases in both 4-hour exposures. The GEF value was exceeded at the test item concentration of 400 μg/mL in both 4-hour exposure groups. A small but statistically significant linear trend increase can be observed in the 24-hour exposure group. However the GEF value is not exceeded so cannot be regarded as mutagenic in this exposure.

Conclusions:

The test item induced increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor, in both 4-hour exposure groups, consequently it is considered to be mutagenic in this assay.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the tyymidine kinase, TK+/-, locus of the L5178Y mouse lymphoma cell line. The method used was designed to be compatible with the OECD guidelines for the testing of Chemicals No 490 " In vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" and the method B17 of commission regulation (EC) No. 440/2008.

Method

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (acetone), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

Results

The maximum dose level used in the Mutagenicity Test was limited by test item induced toxicity. The test item induced statistically significant and dose related (linear-trend) increases in both 4-hour exposures. The Global Evaluation Factor (GEF) value was exceeded at the test item concentration of 400 µg/mL in both 4-hour exposure groups.

Conclusion

The test item induced increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor, in both 4-hour exposure groups, consequently it is considered to be mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

An Ames study gave a negative (non-mutagenic) test result, however, a Chromosome Aberration Test in CHL cells and a mouse lymphoma test both gave positive results in the presence and absence of S9 metabolic activation.

The reason for the positive responses in the in vitro studies is unknown and there are no alerts for genotoxicity with the structure.  The test material was subsequently found to be negative for gentoxity in an in vivo mouse micronucleus test and on this basis it is considered that there are no mutangenic concerns with the substance.