(8α,9R,8'''α,9'''R)-1,1'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(methylene)]bis(6'-methoxycinchonan-1-ium-9-ol) dibromide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic, Ames test with S. typhimurium and E. coli, OECD TG 471, Thompson 2020

Not mutagenic, Hprt test with Chinese hamster ovary cells, OECD TG 476, Woods 2020

Not clastogenic, not aneugenic, in vitro micronucleus test with human lymphocytes, OECD TG 487, Morris 2020

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start: 29 October 2019, End: 29 November 2019

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 using the Hprt and xprt genes)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

functionally hemizygous hypoxanthine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO-K1) cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1 cells were obtained from the European Collection of Cell Cultures. Cells were stored at -196 to -150 °C, in heat-inactivated foetal calf serum (HiFCS) containing 10% dimethyl sulphoxide (DMSO). The cells are screened periodically for the absence of mycoplasma contamination. The modal chromosome number is 20 and the cell doubling time is ca. 12 hours.
The following media were used: H0 = Ham’s Nutrient Mixture F12 (containing 1 mM L glutamine), supplemented with 50 ng/mL amphotericin B / 20 IU/mL penicillin / 20 μg/mL streptomycin; H10 = H0 medium supplemented with 10% heat inactivated fetal calf serum
The selective medium, in which only HPRT deficient cells will grow, consisted of H10 supplemented with 6-TG at a final concentration of 10 µg/mL. All cell cultures were maintained at 34 to 39 °C in an humidified atmosphere of 5% CO2 in air.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction was prepared from male Sprague-Dawley derived rats, dosed with phenobarbital/5,6-benzoflavone to stimulate mixed-function oxidases in the liver. S9 mix contained: S9 fraction (10% v/v), glucose-6-phosphate (6.9 mM), NADP (1.4 mM) in H0.

Test concentrations with justification for top dose:

Preliminary toxicity test: 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL; No precipitate was observed by eye at the end of treatment at concentrations of up to 2000 µg/mL and this was the highest concentration plated for determination of relative survival (RS) in both the absence and presence of S9 mix.
Main experiment (without S9 mix, 3 hours): 9.5, 95, 475, 950, 1100, 1250, 1400, 1550, 1700, 1850 and 2000 µg/mL (cultures assessed for determination of the mutant phenotype were 9.5, 1100, 1250, 1550, 1700, 1850 and 2000 µg/mL); Precipitate was seen by eye at the end of treatment at 2000 µg/mL.
Main experiment (with S9 mix, 3 hours): 9.5, 95, 475, 950, 1100, 1250, 1400, 1550, 1700, 1850 and 2000 µg/mL (cultures assessed for determination of the mutant phenotype were 9.5, 95, 475, 950, 1100, 1250 and 1400 µg/mL); Precipitate was observed by eye at the end of treatment at concentrations of 1400 µg/mL and above. Therefore 1400 µg/mL was the maximum concentration continued.

Vehicle / solvent:

Dimethyl sulfoxide (1% v/v)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Details on test system and experimental conditions:

Preliminary toxicity test: A cell suspension was prepared at 3 x 10E+05 cells/mL. Aliquots of 20 mL of this suspension were dispensed into 150 cm2 flasks, one flask per concentration of test item and two for the vehicle controls both in the absence and in the presence of S9 mix. Eight concentrations of the test item were used. Two additional flasks were prepared to determine average cell density across all flasks at the beginning of the treatment period; this was subsequently used to calculate the adjusted cloning efficiency. The cells were incubated for approximately 24 hours at between 34 and 39 °C, in an atmosphere of 5% CO2 in air, prior to exposure to the test item on Day 1. Prior to treatment, the medium was replaced with 16 mL of fresh medium and aliquots of 4 mL of H0 or S9 mix as appropriate followed by 200 µL of test item (at 100 times the desired final concentration) or vehicle. The flasks were returned to the incubator for a further three hours.
At the end of the exposure period the cells were harvested. Samples were taken from each culture, the cells counted and the cell density calculated. Three flasks per culture were seeded with 200 cells each (survival flasks). The plates were incubated at between 34 and 39 °C, in a humidified atmosphere of 5% CO2 in air, for seven days, after which time colonies growing in the plate were fixed and stained in a methanol:Giemsa solution (4:1 v/v). Colonies were counted and the Day 1 relative survival was calculated.

Main mutation test: The procedure for the main experiments was the same as that for the preliminary experiments, with the following exceptions: positive control cultures were included for all experiments; duplicate cultures were prepared for all cultures (quadruplicate cultures for vehicle controls); and following preparation of survival flasks, 2 x 10E+06 cells from each culture were seeded into 150 cm2 flasks containing 30 mL H10 and incubated for seven days to allow expression of the mutant phenotype. The cultures were sub-cultured during the expression period and after a total of seven days, were harvested. For each culture, three flasks were seeded with 200 cells each, to determine cloning efficiency and five flasks with 5 x 10E+05 cells each in selective medium to determine cloning efficiency. The flasks were returned to the incubator for approximately seven days at between 34 and 39 °C in a humidified atmosphere of 5% CO2 in air. At the end of this incubation period, colonies growing in the flasks were fixed and stained in a methanol:Giemsa solution (4:1 v/v) and counted.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase in mean mutant frequency compared with the concurrent negative control
b) the increase in mean mutant frequency is concentration-related when evaluated with an appropriate trend test
c) any of the results (mean mutant frequency) are outside the distribution of the historical negative control data (above the upper 95% confidence limit)
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
a) none of the test concentrations exhibits a statistically significant increase in mean mutant frequency compared with the concurrent negative control
b) there is no concentration-related increase in mean mutant frequency when evaluated with an appropriate trend test
c) all results (mean mutant frequency) are inside the distribution of the historical negative control data (within the 95% confidence limits).
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

The statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution following the methods described by Arlett et al. (1989). Tests were conducted for a linear concentration-response relationship of the test item, for non-linearity and for the comparison of positive control and treated groups to vehicle control.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test: The osmolality and pH of the substance in medium were measured by analysing samples of H10 media, dosed at 1% (v/v), with either the vehicle (DMSO) or a test substance formulation at 200 mg/mL (to give a final concentration of 2000 μg/mL). No fluctuations in osmolality of the medium of more than 50 mOsmol/kg and no fluctuations in pH of more than 1.0 unit were observed for this formulation compared with the vehicle control. The maximum final concentration tested in the preliminary toxicity test was 2000 μg/mL as this is the standard limit concentration within this test system.
Prior to treatment with the substance, cultures established concurrently to those used in this experiment were assessed to ascertain the cell density. The cell concentration was confirmed to be 1.2 x 10E+06 cells/mL (i.e. 12 x 10E+06 cells treated per concentration, 24 x 10E+06 cells for the vehicle control). This cell concentration was used in the calculation of the adjusted cloning efficiency.
The substance was initially dosed at concentrations up to 2000 µg/mL. No precipitate was observed by eye at the end of treatment at concentrations of up to 2000 µg/mL and this was the highest concentration plated for determination of relative survival (RS) in both the absence and presence of S9 mix. Exposure to the substance for 3 hours at concentrations from 15.63 to 2000 µg/mL in both the absence and presence of S9 mix resulted in RS values from 80% and 4% and from 93% and 1% respectively.

Main Mutation Test - 3-hour Treatment in the Absence of S9 Mix: Prior to treatment, cultures established concurrently to those used in this experiment were assessed to ascertain the cell density. The cell concentration was confirmed to be 1.5 x 10E+06 cells/mL (i.e. 30 x 10E+06 cells treated per concentration, 60 x 10E+06 cells for the vehicle control). This cell concentration was used in the calculation of the adjusted cloning efficiency.
Cultures were exposed to the substance at concentrations from 9.5 to 2000 µg/mL. Precipitate was seen by eye at the end of treatment at 2000 µg/mL. Exposure to the substance resulted in mean RS values from 101 to 28%. Cultures treated at 9.5, 1100, 1250, 1550, 1700, 1850 and 2000 µg/mL were selected for expression of mutant frequency. No significant increases in mutant frequency were observed after exposure. None of the treated groups induced mean mutant frequencies above the laboratory historical 95% confidence limits and tests for both a linear trend and non-linearity were applied across all treatment groups, neither of which was statistically significant. Therefore, this experiment is concluded to be clearly negative.
EMS, the positive control, induced a significant increase in mean mutant frequency compatible with the laboratory’s historical positive control data demonstrating the sensitivity of the test system.

Main Mutation Test - 3-hour Treatment in the Presence of S9 Mix: Prior to treatment, cultures established concurrently to those used in this experiment were assessed to ascertain the cell density. The cell concentration was confirmed to be 1.5 x 10E+06 cells/mL (i.e. 30 x 10E+06 cells treated per concentration, 60 x 10E+06 cells for the vehicle control). This cell concentration was used in the calculation of the adjusted cloning efficiency.
Cultures were exposed to the substance at concentrations from 9.5 to 2000 µg/mL. Precipitate was observed by eye at the end of treatment at concentrations of 1400 µg/mL and above. Therefore 1400 µg/mL was the maximum concentration continued. Exposure to the substance resulted in mean RS values from 88 to 62%. Cultures treated at 9.5, 95, 475, 950 1100, 1250 and 1400 µg/mL were selected for expression of mutant frequency. No significant increases in mutant frequency were observed after exposure. None of the treated groups induced mean mutant frequencies above the laboratory historical 95% confidence limits and tests for both a linear trend and non-linearity were applied across all treatment groups, neither of which was statistically significant. Therefore, this experiment is concluded to be clearly negative.
3MC, the positive control, induced a significant increase in mean mutant frequency compatible with the laboratory’s historical positive control data demonstrating the sensitivity of the test system and efficacy of the S9 metabolic fraction.

Summary of results

		3-hour exposure, without S9 mix			3-hour exposure, with S9 mix
Test item	Conc (µg/mL)	Mean relative survival (%)	Mean mutant frequency (per 10E+06 viable cells)	95% conf. intervals	Mean relative survival (%)	Mean mutant frequency (per 10E+06 viable cells)	95% conf. intervals
DMSO solvent	0	100	10.73	1.4 -15.4	100	9.42	0.0 - 15.7
Test subst.	9.5	95	12.35		86	9.12
	95	85	NA		88	6.89
	475	83	NA		85	9.54
	950	49	NA		62	8.36
	1100	80	12.33		80	10.74
	1250	70	13.89		68	5.66
	1400	101	NA		77 b	8.11
	1550	60	8.91		NA c	NA
	1700	51	10.48		NA c	NA
	1850	45	7.04		NA c	NA
	2000	28 b	9.35		NA c	NA
Ethyl methanesulphonate	250	82	88.73***	38.9 - 128.9	Not tested	Not tested
3-methylcholanthrene	5	Not tested	Not tested		82	79.23***	24.0 - 87.7
b) precipitate observed at the end of treatment c) precipitate observed at the end of treatment, cultured discarded *** p<0.001; all other cultures p >0.05; treated groups were compared to the vehicle control using one-tailed Dunnett's tests for an increase and the positive control was compared to the vehicle control using a one-tailed t-test for an increase

Conclusions:

The substance was not mutagenic in this valid in vitro mammalian cell mutation study.

Executive summary:

The substance was tested under GLP for mutagenic potential in an in vitro mammalian cell mutation assay to OECD TG 476. This test system is based on detection and quantitation of forward mutation at the functionally hemizygous hypoxanthine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO-K1) cells. Two independent tests, one in the absence of exogenous metabolic activation (S9 mix) and one in the presence of S9 mix, were conducted.
The vehicle was dimethyl sulfoxide (DMSO), in which the test item dissolved at up to 200 mg/mL. The highest final concentration used in the preliminary toxicity experiment was standard limit concentration 2000 µg/mL recommended in the current guideline. No precipitate was observed by eye at the end of treatment at up to 2000 µg/mL. Cytotoxicity was measured as Day 1 relative survival (RS). After exposure to the substance at concentrations from 15.63 to 2000 µg/mL RS values ranged from 80 to 4% and from 93 to 1%, in the absence and presence of S9 mix respectively.
In the main mutation experiment in the absence of S9 mix, cells were exposed to concentrations from 9.5 to 2000 µg/mL. Precipitate was observed by eye at the end of treatment at 2000 µg/mL. Mean RS values ranged from 101 to 28% relative to the vehicle control. The substance did not induce a statistically significant increase in mean mutant frequency. None of the treated groups induced mean mutant frequencies above the laboratory historical 95% confidence limits and tests for both a linear trend and non-linearity were applied across all treatment groups, neither of which was statistically significant. The positive control, ethyl methanesulphonate, induced a significant increase in mean mutant frequency demonstrating the correct functioning of the assay. The criteria for a clearly negative response were therefore met in this treatment.
In the main mutation experiment in the presence of S9 mix, cells were exposed to concentrations from 9.5 to 2000 µg/mL. Precipitate was observed by eye at the end of treatment at concentrations of 1400 µg/mL and above. Therefore 1400 µg/mL was the maximum concentration continued. Mean RS values ranged from 88 to 62% relative to the vehicle control. The substance did not induce a statistically significant increase in mean mutant frequency. None of the treated groups induced mean mutant frequencies above the laboratory historical 95% confidence limits and tests for both a linear trend and non-linearity were applied across all treatment groups, neither of which was statistically significant. The positive control, 3-methylcholanthrene, induced a significant increase in mean mutant frequency demonstrating the correct functioning of the assay and the efficacy of the S9 metabolic fraction. The criteria for a clearly negative response were therefore met in this treatment

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The histidine dependent strains are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally, due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The last alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named uvrB-. In the strains TA 98 and TA100 the R-factor plasmid pKM101 carries the ampicillin resistance marker (Mortelmans and Zeiger, 2000). Strain WP2 (Green and Muriel 1976) and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent (Trp+) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excisable repair damage). Such a repair-deficient strain may be more readily mutated by agents. The E. coli strain WP2 uvrA pKM101 is constructed by introduction of the R-factor plasmid pKM101.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

The S9 Microsomal fraction (Sprague-Dawley) was purchased from Moltox and stored at approximately -196 °C in a liquid nitrogen freezer; Lot No. 4123 was used in this study and the protein level was adjusted to 20 mg/mL.

Test concentrations with justification for top dose:

The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended concentration level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
The concentration range used for Experiment 2 was determined by the results of Experiment 1 and was 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. To allow for the toxicity of the test item, nine test item concentrations per bacterial strain were selected in the second mutation test following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoantracene

Remarks:

N-ethyl-N'-nitrosoguanidine, 9-aminoacridine, 4-nitroquinoline-1oxide were used without metabolic activation; 2-aminoanthracene, benzo[a]pyrene were used with metabolic activation

Details on test system and experimental conditions:

Top agar was prepared using 0.6% Bacto agar (lot number 8255817 07/2023) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 51673 09/2019 and 51956 10/2019).
Precultures - A culture of each of the bacterial strains was prepared by inoculating nutrient broth with the appropriate coded stock culture and incubated, with shaking, for approximately 10 hours at 37 ± 3 °C. The bacterial cell count for each culture was determined by viable count analysis on nutrient agar plates on the day.
Main test, Experiment 1, without metabolic activation - 0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlaid onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
Main test, Experiment 1, with metabolic activation - The procedure was the same as described previously (see 3.5.2.2) except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.
Incubation, Experiment 1 - All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates wee viewed microscopically for evidence of thinning (toxicity).
Main test, Experiment 2, without metabolic activation: 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 30 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
Main test, Experiment 2, with metabolic activation: The procedure was the same as described previously (see Experiment 1) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 30 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.
Incubation, Experiment 2: All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Evaluation criteria:

If exposure to a test item produces a reproducible increase, in one or more concentration, in mean revertant colony numbers of at least twice that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship in at least one strain with or without metabolic activation system, it will be considered to exhibit mutagenic activity in this test system (Mortelmans and Zeiger 2000). No statistical analysis was performed.
If exposure to a test item does not produce an increase in mean revertant colony numbers, it will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis was performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used will usually be Dunnett’s test followed, if appropriate, by trend analysis (Mahon et al, 1989). Biological significance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below twice those of the concurrent vehicle controls (as described above) will not be considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director will use his/her scientific judgment.

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity occurred in the experiment with pre-incubation only.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True 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:

cytotoxicity

Remarks:

Toxicity occurred in the experiment with pre-incubation only.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity occurred in the experiment with pre-incubation only.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True 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:

cytotoxicity

Remarks:

Toxicity occurred in the experiment with pre-incubation only.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True 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:

cytotoxicity

Remarks:

Toxicity occurred in the experiment with pre-incubation only.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the first mutation test (plate incorporation method), there was no visible reduction in the growth of the bacterial background lawns of any of the tester strains dosed at any concentration in either the absence or presence of metabolic activation (S9-mix). There were substantial reductions (<0.5 fold) in revertant colony numbers observed in TA100, TA98 and TA1537 at 5000 μg/plate. A concentration of 5000 μg/plate was selected as the maximum concentration in the second mutation test.
Treatment with the substance in Experiment 2 (pre-incubation) resulted in toxicity, either by a reduction in the growth of the background lawn or a reduction in the number of spontaneous revertant colonies (below factor of 0.5 fold compared to concurrent control) in all strains in the absence and presence of metabolic activation. In the absence of metabolic activation, toxicity was observed from 150 μg/plate in TA1535, from 500 μg/plate in TA98 and TA1537 and 1500 μg/plate in TA100 and WP2uvrA pKM101. In the presence of metabolic activation, toxicity was observed in all of the tester strains from 1500 μg/plate.
No test item precipitate was observed on the plates at any of the concentrations tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1 and 2.
There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any concentration of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) or Experiment 2 (pre-incubation method).
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within or close to the normal range. 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 substance was considered to be negative (i.e. non-mutagenic) in the bacterial reverse mutation assay.

Executive summary:

The ability of the substance to induce reverse mutations, either directly or after metabolic activation, in the plate incorporation test (Experiment 1) and the pre-incubation test (Experiment 2), using the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strain WP2uvrApKM101 was tested under GLP to OECD TG 471. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within or close to the normal range. The maximum concentration of the test item in the first experiment was selected as the maximum recommended concentration of 5000 μg/plate. In the first mutation test (plate incorporation method), there was no visible reduction in the growth of the bacterial background lawns of any of the tester strains dosed at any concentration in either the absence or presence of metabolic activation (S9-mix). There were substantial reductions (<0.5 fold) in revertant colony numbers observed in TA100, TA98 and TA1537 at 5000 μg/plate. A concentration of 5000 μg/plate was selected as the maximum concentration in the second mutation test.
The treatment with the substance in Experiment 2 (pre-incubation) resulted in toxicity, either by a reduction in the growth of the background lawn or a reduction in the number of spontaneous revertant colonies (below factor of 0.5 fold compared to concurrent control) in all strains in the absence and presence of metabolic activation. In the absence of metabolic activation, toxicity was observed from 150 μg/plate in TA1535, from 500 μg/plate in TA98 and TA1537 and 1500 μg/plate in TA100 and WP2uvrA pKM101. In the presence of metabolic activation, toxicity was observed in all of the tester strains from 1500 μg/plate.
No test item precipitate was observed on the plates at any of the concentrations tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1 and 2. There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any concentration of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) or Experiment 2 (pre-incubation method). 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.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

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:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Following genotoxic insult, chromosome aberrations induced within a cell population give rise to chromosome fragments termed "acentric fragments" (AF). After DNA replication and nuclear division, these fragments or even whole chromosomes, can be excluded from the daughter nuclei and form small secondary nuclei (micronuclei-MN), either alone or in conjunction with other AF (Stopper and Müller, 1997). In its simplest form, the micronucleus test provides a measure of chromosome loss or breakage, and if more mechanistic information is required complementary techniques, such as fluorescence in situ hybridisation (FISH) with centromeric DNA probes, or immunolabelling of kinetochores can be performed (Miller et al., 1998). These techniques can determine the origin of MN and therefore are valuable tools for the differentiation of aneuploidy-inducers and clastogens.

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Sufficient whole blood was drawn from the peripheral circulation of a human non-smoking female, aged 24 years (preliminary toxicity test), a non-smoking female, aged 30 years (main experiment, without S9 mix) and a male, aged 24 years (main experiment, without S9 mix repeat and with S9 mix).

Cytokinesis block (if used):

Cytochalasin B (cytoB) was formulated in DMSO and added to all cultures, after washing, at the end of the exposure period at a final concentration of 4.5 μg/mL for a period of 24 hours.

Metabolic activation:

with and without

Metabolic activation system:

The Rat S9 Microsomal fraction was purchased from Moltox. Lot No. 4061 was used in the Preliminary Toxicity Test and Lot No 4127 was used for the 4-hour with S9 of the Main Experiment.

Test concentrations with justification for top dose:

Preliminary toxicity test: The concentration range of test item used was 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μg/mL.
Main experiment: The concentration range of test item used for the three exposure groups of the main experiment was 0, 250, 500, 1000, 1200, 1400, 1600, 1800 and 2000 μg/mL.
Main experiment repeat: The concentration range of test item used was 0, 250, 500, 1000, 1200, 1400, 1600, 1800 and 2000 μg/mL.
The maximum concentration in all tests was the highest recommended concentration according to the test guideline.

Vehicle / solvent:

The test item was insoluble in Minimal Essential Medium at 20 mg/mL but was soluble in dimethyl sulphoxide (DMSO) at 200 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine

Remarks:

Mitomycin C, demecolcine only without S9 mix, cyclophosphamide only with S9 mix

Details on test system and experimental conditions:

Cell cultures - Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Culture conditions - Duplicate lymphocyte cultures (A and B) and quadruplicate for the vehicle were established for each concentration level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 – 9.10 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.70 - 0.75 mL heparinized whole blood
4-hour exposure with S9 mix - After 44 - 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment. After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the 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 24 hours at approximately 37 ºC in 5% CO2 in humidified air with Cytochalasin B at a final concentration of 4.5 μg/mL.
4-hour exposure without S9 mix - After 44 - 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL. After 4 hours at approximately 37 ºC, the 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 reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.
24-hour exposure without S9 mix - The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After 44 - 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 24 hours. The extended exposure detailed above does not follow the suggested cell treatment schedule in the Guideline. The OECD guideline permits modified treatment times where justified. This design avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response (Whitwell et al. 2019). Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format. The Preliminary Toxicity Test was performed using the exposure conditions as described for the Main Experiment but using single cultures for the test item concentration levels and duplicate cultures for the vehicle controls, whereas the Main Experiment used duplicate cultures for the test item and quadruplicate cultures for the vehicle controls.
Preliminary toxicity test - Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The concentration range of test item used was 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods. Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate concentration levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity (cytostasis) and for selection of the concentration levels for the exposure groups of the Main Experiment.
Main experiment - Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The concentration range of test item used for the three exposure groups of the main experiment was 0, 250, 500, 1000, 1200, 1400, 1600, 1800 and 2000 μg/mL. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Main experiment repeat, 4-hour exposure without S9 mix - Repeat of the 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The concentration range of test item used was 0, 250, 500, 1000, 1200, 1400, 1600, 1800 and 2000 μg/mL. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Cell harvest - At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.
Preparation of microscope slides - The lymphocytes were re-suspended in several mL of fresh fixative 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 with gentle warming. Each slide was permanently labeled with the appropriate identification data.
Staining - When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Evaluation criteria:

See under "Any other information on materials and methods"

Statistics:

When there is no indication of any increase at all concentration levels tested then statistical analysis may not be necessary. In all other circumstances comparisons were made between the appropriate vehicle control and each individual concentration level, using Chi-squared Test on observed numbers of cells with micronuclei (Hoffman et al., 2003). A statistically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 when compared to its concurrent control. The concentration-relationship was assessed using a linear regression model. An arcsine square-root transformation was applied to the percentage of binucleated cells containing micronuclei (excluding positive control(s)). A linear regression model was applied to these transformed values with concentration values fitted as the explanatory variable. The F-value from the model was assessed at the 5% statistical significance level.

Key result

Species / strain:

lymphocytes: obtained from healthy human donours

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test - 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 all three exposure groups. Hemolysis was observed following exposure to the test item at 2000 μg/mL in the 24-hour exposure group only. Hemolysis is an indication of a toxic response by the erythrocytes and not indicative of any genotoxic response to the lymphocytes. Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 2000 μg/mL in all three exposure groups. The test item induced toxicity at 2000 μg/mL.
Main test - The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring at the maximum concentration of test item, 2000 μg/mL in all three exposure groups of the main experiment. Precipitate of test item was observed in the parallel blood-free cultures at the end of the exposure period at 2000 μg/mL in the 4-hour exposure in the absence of S9 and in the 24-hour exposure group and at and above 1600 μg/mL in the 4-hour exposure in the presence of S9. No marked toxicity was observed at 2000 μg/mL in the 4-hour exposure in the absence of S9. In the presence of S9 a modest concentration-related inhibition of CBPI was observed, and 33% cytostasis was observed at 1600 μg/mL, the lowest precipitating concentration. In the 24-hour exposure group the toxicity was more marked and 36%, 42% and 59% cytostasis was demonstrated at 1600, 1800, and 2000 μg/mL, respectively. The maximum concentration selected for analysis of binucleate cells was 2000 μg/mL, the maximum recommended concentration for the 4-hour exposure group in the absence of S9 and in the 24-hour exposure group. In the 4-hour exposure group in the presence of S9 the maximum concentration selected for analysis of the binucleate cells was the lowest precipitating concentration, 1600 μg/mL. The vehicle control cultures for all three exposure groups had frequencies of binucleate cells with micronuclei which were considered acceptable for addition to the laboratory’s historical
negative control data and were within the 95% control limits. The positive control items induced statistically significant increases in the frequency of cells with micronuclei which were compatible with the laboratory’s historical positive control data base. The cytotoxicity achieved by the positive control items did not exceed the limits of acceptability recommended in the OECD 487 guideline. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, in the presence of metabolic activation or in the 24-hour exposure group in the absence of metabolic activation. The results were within the distribution of the historical vehicle control data (within 95% control limits). There was no concentration-related increase when evaluated with a trend test. Hence, both of these conditions afforded a clearly negative result. In the 4-hour exposure group in the absence of S9 there was a small but statistically significant increase in the frequency of binucleate cells with micronuclei at 1600 μg/mL and 2000 μg/mL. These increases were well within the 95% control limit of the laboratory historical control data for a vehicle and were not concentration related when evaluated with a trend test. They were therefore considered to be of no toxicological significance. However, a repeat of the 4-hour exposure group in the absence of S9 was performed as indicated by the OECD test guideline for the assay.
Main experiment repeat - The qualitative assessment of the slides determined that the toxicity was similar to that
observed in the previous experiment and that there were binucleate cells suitable for scoring at the maximum concentration of test item, 2000 μg/mL. Precipitate of test item was observed in the parallel blood-free cultures at the end of the exposure period at 2000 μg/mL. Modest toxicity was demonstrated at the maximum concentration, 2000 μg/mL with 24% cytostasis. The maximum concentration selected for evaluating the binucleate cells for micronuclei was the maximum recommended concentration, 2000 μg/mL. The vehicle control cultures had frequencies of binucleate cells with micronuclei which were considered acceptable for addition to the laboratory historical negative control data and were within the 95% control limits. The positive control item induced statistically significant increases in the frequency of cells with micronuclei which were compatible with the laboratory historical positive control data base. The cytotoxicity achieved by the positive control items did not exceed the limits of acceptability recommended in the OECD 487 guideline. Thus, the sensitivity of the assay was validated. The test item did demonstrate very small but statistically significant increases in the frequency of binucleate cells with micronuclei when compared with the vehicle control at 1000 μg/mL and 1600 μg/mL. Although these increases were statistically significant they were within the 95% control limits of the laboratory historical control data for a vehicle and were therefore considered to be of no toxicological significance. The concentration response was very weakly statistically significant. These increases were considered of no toxicological significance since they remained within the laboratory’s historical vehicle control range and the 24 hour treatment without S9 which tested to the maximum recommended concentration was clearly negative. The 4 hour exposure in the absence of metabolic activation was therefore considered to be negative overall.

Conclusions:

The substance was considered to be negative (i.e. non-clastogenic and non-aneugenic) to human lymphocytes in vitro.

Executive summary:

The clastogenic and aneugenic potential of the substance on the nuclei of normal human lymphocytes was studied under GLP to OECD TG 487. Duplicate cultures of human lymphocytes, obtained from healthy human donours and treated with the test item, were evaluated for micronuclei in binucleate cells at up to four concentrations, together with vehicle (quadruplicate cultures) and positive controls (duplicate cultures). Three exposure conditions were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B. The concentrations used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be the maximum recommended concentration. The 4-hour exposure group in the absence of S9 was repeated as a confirmatory experiment due to a small but statistically significant response observed in the initial experiment.
All vehicle (dimethyl sulphoxide) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes and were considered acceptable for addition to the laboratory historical negative control data base. The positive control items induced statistically significant increases in the frequency of cells with micronuclei and with responses that were compatible with the laboratory historical positive control data base. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. There were no statistically significant increases in the frequency of binucleate cells with micronuclei in the 4-hour with S9 exposure group or in the 24-hour exposure group using a concentration range which included the maximum recommended concentration. Furthermore, the micronucleus frequencies of all test item concentrations in these exposures were within the 95% control limit of the historical vehicle control and there was no concentration related relationship. The criteria for a clearly negative response were therefore met in the 4 hour exposure with S9 and the 24 hour exposure. In the 4-hour exposure group without S9, two small but statistically significant increases in the frequency of cells with micronuclei were observed. These increases remained within the 95% control limit of the historical vehicle control and there was no concentration related trend. This treatment condition was repeated, as indicated in the OECD test guideline for the assay. In the repeat 4-hour without S9 exposure experiment, two small but statistically significant increases were again observed however, all micronucleus frequencies remained within the 95% control limits. The trend test for this experiment was statistically significant. These increases were considered of no toxicological significance since they remained within the laboratory’s historical vehicle control range and the 24 hour treatment without S9 which tested to the maximum recommended level was clearly negative. The 4 hour exposure in the absence of metabolic activation was therefore considered to be negative overall.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Since the three reliable and valid in vitro tests gave clearly negative results, in vivo studies were not performed.

Additional information

The mutagenicity and genotoxicity potential of the substance on bacterial and mammalian cells was investigated under GLP in three reliable and valid in vitro studies. 

In vitro bacterial reverse mutagenicity
The Ames test to OECD TG 471 studied the mutagenicity potential in the plate incorporation test (Experiment 1) and the pre-incubation test (Experiment 2), using the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strain WP2uvrApKM101.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within or close to the normal range. There were substantial reductions (<0.5 fold) in revertant colony numbers observed in TA100, TA98 and TA1537 at 5000 μg/plate in Experiment 1. The treatment at 5000 μg/plate in Experiment 2 resulted in toxicity, either by a reduction in the growth of the background lawn or a reduction in the number of spontaneous revertant colonies (below factor of 0.5 fold compared to concurrent control) in all strains in the absence and presence of metabolic activation.
No test item precipitate was observed on the plates at any of the concentrations tested in either the presence or absence of S9 mix in Experiments 1 and 2.
There were no biologically relevant increases in the frequency of revertant colonies at any test concentration (with or without S9 mix) recorded in the plate incorporation or the pre-incubation test.
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.

In vitro mammalian cell mutagenicity
An in vitro mammalian cell mutation assay to OECD TG 476 used two independent tests, one in the absence of exogenous metabolic activation (S9 mix) and one in the presence of S9 mix.
The vehicle was dimethyl sulfoxide (DMSO), in which the test item dissolved at up to 200 mg/mL. A preliminary toxicity experiment was performed up to the limit concentration of 2000 µg/mL resulting in no precipitation but leading to reductions in relative survival after exposure to the substance at concentrations from 15.63 to 2000 µg/mL, with RS value ranging from 80 to 4% and from 93 to 1%, in the absence and presence of S9 mix respectively. The main mutation experiment was run with concentrations from 9.5 to 2000 µg/mL (without S9 mix), resulting in precipitate at 2000 µg/mL. Mean RS values ranged from 101 to 28% compared to the vehicle control. The substance did not induce a statistically significant increase in mean mutant frequency and no linearity trend was apparent. The positive control induced a significant increase in mean mutant frequency demonstrating the correct functioning of the assay. The criteria for a clearly negative response were therefore met in this treatment.
The main mutation experiment in the presence of S9 mix was also conducted with concentrations from 9.5 to 2000 µg/mL, resulting in precipitate at concentrations of 1400 µg/mL and above (maximum concentration thus was 1400 µg/mL). Mean RS values ranged from 88 to 62% compared to the vehicle control. The substance did not induce a statistically significant increase in mean mutant frequency. The positive control induced a significant increase in mean mutant frequency demonstrating the correct functioning of the assay and the efficacy of the S9 metabolic fraction. The criteria for a clearly negative response were therefore met in this treatment

In vitro micronucleus (clastogenicity and aneugenicity)
The clastogenic and aneugenic potential of the substance on the nuclei of normal human lymphocytes was studied under GLP to OECD TG 487 using duplicate cultures of human lymphocytes that were treated with the test item and evaluated for micronuclei in binucleate cells at up to four concentrations, together with vehicle (quadruplicate cultures) and positive controls (duplicate cultures). Three exposure conditions were studied: a 4-hour exposure in the presence and absence of S9 mix, a 24-hour exposure in the absence of metabolic activation. Following exposure, cells were washed and incubated for a further 24 hours in the presence of Cytochalasin B. All experiments were conducted up to the maximum concentration of 2000 μg/mL. All vehicle (dimethyl sulphoxide) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes and were considered acceptable for addition to the laboratory historical negative control data base. The positive control items induced statistically significant increases in the frequency of cells with micronuclei and with responses that were compatible with the laboratory historical positive control data base. There were no statistically significant increases in the frequency of binucleate cells with micronuclei in the 4-hour with S9 exposure group or in the 24-hour exposure group using a concentration range which included the maximum recommended concentration. Furthermore, the micronucleus frequencies of all test item concentrations in these exposures were within the 95% control limit of the historical vehicle control and there was no concentration related relationship. The criteria for a clearly negative response were therefore met in the 4 hour exposure with S9 and the 24 hour exposure. In the 4-hour exposure group without S9, two small but statistically significant increases in the frequency of cells with micronuclei were observed with no concentration-related treand, which were within the 95% control limit of the historical vehicle control. This experiment was therefore repeated, producing again two small but statistically significant increases. However, all micronucleus frequencies remained within the 95% control limits. The trend test for this experiment was statistically significant. These increases were considered of no toxicological significance since they remained within the laboratory’s historical vehicle control range and the 24 hour treatment without S9 which tested to the maximum recommended level was clearly negative. The 4 hour exposure in the absence of metabolic activation was therefore considered to be negative overall.

Justification for classification or non-classification

On the basis of the available information obtained in reliable and valid in vitro studies, the substance is not classified for genotoxicity in accordance with Regulation (EC) No. 1272/2008.