cyproconazole (ISO); (2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames: +S9 negative, -S9 negative, S. typhimurium: TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA pKM101 and WP2 pKM101, according to OECD TG 471, Sokolowski 2009

- In vitro gene mutation assay: +S9 negative, -S9 negative, Chinese hamster V79 cells, equivalent to OECD TG 476, Miltenburg 1995

- In vitro chromosome aberration test: +S9 negative, -S9 negative, Chinese hamster Ovary (CHO), equivalent to OECD TG 473, Murli 1985

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:

10 Mar 2009 to 07 May 2009

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his- (S. typhimurium), trp- (E. coli)

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

Species / strain / cell type:

E. coli, other: WP2 pKM101

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : Phenobarbital/β-Naphthoflavone induced rat liver S9
- Method of preparation of S9 mix: The S9 is prepared from 8 - 12 weeks old male Wistar rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. and β- Naphthoflavone p.o. each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at -80 °C. Small numbers of the ampoules can be kept at -20 °C for up to one week. The protein concentration in the S9 preparation was 27.8 mg/mL in both experiments.
- Concentration or volume of S9 mix and S9 in the final culture medium: The amount of S9 supernatant was 10% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix: 8mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 5 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
- Quality controls of S9: Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo(a)pyrene.

Test concentrations with justification for top dose:

Pre-Experiment /Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 1, 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

PRE- EXPERIMENT FOR TOXICITY:
Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment I, if the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

EXPERIMENTAL PERFORMANCE:
For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
- 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µLOverlay agar
In the pre-incubation assay 100 JL test solution solvent or positive control, 500 JL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on selective agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37°C in the
dark

DATA RECORDING:
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer to print out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). Due to reduced background growth the colonies were partly counted manually.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- Regular background growth in the negative and solvent control
- The spontaneous reversion rates in the negative and solvent control are in the range of historical data
- The positive control substances should produce a significant increase in mutant colony frequencies

EVALUATION OF THE RESULTS:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli, other: WP2 uvrA pKM101 and WP2 pKM101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation of the test item: Precipitation of the test item was observed in the test tubes in the presence of metabolic activation in both experiments. No visible precipitation was observed on the incubated agar plates with and without metabolic activation in both experiments.

STUDY RESULTS:
- Signs of toxicity: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at the higher concentrations with and without metabolic activation in both experiments shown in table 1 in 'Any other information on results incl. tables'.

- Mean number of revertant colonies per plate: No substantial increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Table 1. Toxic effects, evident as a reduction in the number of revertants

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	2500, 5000	2500, 5000	2500, 5000	1000 - 5000
TA 1537	1000 - 5000	2500, 5000	1000 - 5000	2500, 5000
TA 98	2500, 5000	2500, 5000	1000 - 5000	1000 - 5000
TA 100	2500, 5000	2500, 5000	2500, 5000	2500, 5000
WP2 uvrA pKM101	2500, 5000	2500, 5000	2500, 5000	1000 - 5000
WP2 pKM101	2500, 5000	2500, 5000	1000 - 5000	1000 - 5000

Conclusions:

The test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strains WP2 uvrA pKM101 and WP2 pKM101, according to GLP principles and OECD TG 471.

Results showed that reduced background growth was observed with and without metabolic activation from 1000 up to 5000 µg/plate in experiment I, and from 333 up to 5000 µg/plate in experiment II. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at the higher concentrations with and without metabolic activation in both experiments. No substantial increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Precipitation of the substance was observed in the test tubes in the presence of metabolic activation in both experiments.  No visible precipitation was observed on the incubated agar plates with and without metabolic activation in both experiments. Appropriate reference mutagens (sodium azide, 4-nitro-o-phenylene-diamine, 2-aminoanthracene and methyl methane sulfonate)were used as positive controls. They showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Oct 1990 to 8 Nov 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1997

Deviations:

yes

Remarks:

Cells were treated for an extended period of time in the absence of S9 treatment.

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2000

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED:
The cells have since been recloned to maintain karyotypic stability. This cell line has an average cycle time of 12 to 14 hours with a modal chromosome number of 21.

MEDIA USED:
The CHO cells were grown in McCoy's 5a culture medium which was supplemented with 10% fetal calf serum (FCS), 1% L-glutamine, and 1% penicillin and streptomycin, at about 37°C, in an atmosphere of about 5% CO2 in air.

Cytokinesis block (if used):

0.1 mg/mL colcemid

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : The S9 fraction was derived from the liver of male Sprague-Dawley rats which had been previously treated with Aroclor 1254.

Test concentrations with justification for top dose:

- Range finding assay: up to 2050 μg/ml
- The mutagenicity test without S9: 60.1, 100, 150, 200 μg/mL
- The mutagenicity test with S9: 45, 59.9, 99.9 and 150 μg/mL

Vehicle / solvent:

- Solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE:
Single cultures were used for the negative control, solvent control, and the positive controls. In the test without metabolic activation. One day after culture initiation, the cultures were treated with the test substance. 2.5 hours before harvest culture medium was exchanged and supplemented with 0.1 mg/mL colcemid. In the test with metabolic activation cultures were initiated by seeding approximately 1.5 x 10^6 cells/75 cm^2 culture flasks into 10 mL of medium. One day thereafter the cultures were incubated for two hours in the presence of the test substance and the S9-mix in McCoy’s 5a medium without foetal calf serum. After exposure the cells were washed and reefed with complete McCoy’s 5a medium. Incubation was continued for an additional 7.84 hours with 0.1 mg/mL colcemid present during the last 2.5 hours of incubation. The metaphase cells were then harvested and prepared for cytogenetic analysis. Chromosomal aberrations were analysed for the cultures treated at the four highest doses from which results could be obtained. Whenever possible one hundred well spread metaphase figures with 21 + 2 centromeres per culture in the negative control and in the treated groups were examined for chromosome aberrations.


TREATMENT AND HARVEST SCHEDULE:
Monolayer cultures were treated for 17.25 hours in the absence of S9 and harvested for slide preparation 20 hours after beginning of treatment. In the presence of S9 treatment was for 2 hours. Cells were harvested 10 hours after beginning of treatment.

Evaluation criteria:

The following factors were taken into account in the evaluation of the chromosomal aberrations data:
- The overall chromosomal aberration frequencies.
- The percentage of cells with any aberrations.
- The percentage of cells with more than one aberration.
- Any evidence for increasing amounts of damage with increasing dose, i.e., a positive dose response

Statistics:

Statistical analysis employed the Fisher's Exact Test with an adjustment for multiple comparisons (Sokal and Rohlf, 1981) to compare the percentage of cells with aberrations in each treatment group with the results from the pooled solvent and negative controls (the solvent and negative controls were statistically evaluated for similarity prior to the pooled evaluation). Test article significance was established where p<0.01. All factors as stated previously were taken into account and the final evaluation of the test article was based upon scientific judgement.

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:

valid

Positive controls validity:

valid

Additional information on results:

The range-finding assay in the absence of S9 resulted in complete toxicity at 683 µg/mL and above. At the concentration of 205 µg/mL a severe cell cycle delay was noted together with cytotoxicity and delayed cell cycle progression was also noted at 68.3 µg/mL. For the chromosome aberration test a 20 hour harvest time and concentrations from 60 to 500 µg/mL were selected. In the presence of S9 complete toxicity was seen at 205 µg/mL and above. No evidence of cell cycle delay and cytotoxicity was noted at the lower concentrations. A 10 hours harvest time and concentrations from 15 to 200 µg/mL were selected for the chromosome aberration test. In the chromosome aberration test without S9 cultures treated at 300 to 500 µg/mL could not be analysed due to toxicity. Moderate to slight cytotoxicity was also seen at 200 and 150 µg/ml. No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed. Due to cytotoxicity the concentration of 200 µg/mL could not be analysed in the experiment with metabolic activation. Less severe signs of cytotoxicity were also evident at concentrations below 200 µg/mL. There was a 15% reduction in the monolayer at 150 µg/mL, floating debris and a slight reduction in visible mitotic cells were observed in cultures dosed with 100 µg/mL and 60 µg/mL. There was also no significant increase in cells with chromosomal aberrations observed at the concentrations analysed in this part of the experiment.

Table 1. Chromosome aberrations in CHO cells in vitro (2nd study) Experiment without S9-mix

Concentration (µg/mL)	% cells with aberrations (excl. gaps)	simple aberrations (breaks, fragments)	complex aberrations (chr and ctd exchanges)	Others
0	0.5	1
60.1	1.5	1	2
100	0.5		1
150	1.0		2
200 MMC	0.5 32*	1 1	10

Experiment with S9-mix

Concentration (µg/mL)	% cells with aberrations (excl. gaps)	simple aberrations (breaks, fragments)	complex aberrations (chr and ctd exchanges)	Others
0	0.0
45.0	0.5		1
59.9	2.0		4
99.9	0.0
150 CP	1.0 44*	1 6	2 9

Conclusions:

The test substance was considered negative for inducing chromosomal aberrations in Chinese hamster ovary cells under both the metabolic activation and non-activation conditions of this assay.

Executive summary:

The objective of this in vitro assay was to evaluate the ability of the test substance to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation, according to GLP principles and OECD TG 473. The test substance was dissolved in dimethyl sulfoxide at a stock concentration of 205 mg/mL. Concentrations of 0.0683 µg/mL to 2050 µg/mL in a half-log series were tested in range-finding assays with and without metabolic activation. The following concentrations were tested in the mutagenicity test: 60.1, 100, 150, 200 µg/mL (without metabolic activation) and 45, 59.9, 99.9 and 150 µg/mL (with metabolic activation). Monolayer cultures were treated for 17.25 hours in the absence of S9 and harvested for slide preparation 20 hours after beginning of treatment. In the presence of S9 treatment was for 2 hours.

Results showed that complete cytotoxicity was observed in the cultures dosed with 683 and 2050 µg/mL, and severe cell cycle delay was evident in the culture dosed with 205 µg/mL, with cell cycle delay persisting in the culture dosed with 68.3 µg/mL in the range- finding assay without activation. Complete cytotoxicity was observed in the cultures dosed with 205, 683, and 2050 µg/mL, and no cell cycle delay was evident in the cultures analysed in the range- finding assay with activation. Based on these results, replicate cultures of CHO cells were incubated with 60.1 µg/mL to 500 µg/mL of the test article solution in a 20 hour non-activation aberrations assay, and with 15.0 µg/mL to 200 µg/mL in a 10 hour aberrations assay with metabolic activation. No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed. The sensitivity of the cell culture for induction of chromosomal aberrations was shown by the increased frequency of aberrations in the cells exposed to the positive control agents (mitomycin C and cyclophosphamide).

In conclusion, the test substance was considered negative for inducing chromosomal aberrations in Chinese hamster ovary cells under both non-activation and activation conditions of this assay.

 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 Jan 1985 to 30 Jan 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

2000

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: The V79 cell line has been used for many years in in vitro experiments with success, due to the high proliferation rate (doubling time 12-16 h in stock cultures) and high plating efficiency of untreated cells (70-90 %)

For cell lines:
- Cell cycle length, doubling time or proliferation index : doubling time 12-16 h in stock cultures
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes, stable

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Two days old logarithmically growing stock cultures more than 50 % confluent were trypsinised and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.2 % in Ca-Mg-free salt solution. The Ca-Mg-free salt solution was composed as follows (per litre): NaCl 8000 mg; KCl 400 mg; glucose 1000 mg; NaHCO3 350 mg. The "saline G" was composed
(per litre): NaCl 8000 mg; KCl 400 mg; glucose 1100 mg; Na2HPO4 · 7H2O 290 mg; KH2PO 4 150 mg. The cells were incubated at 37°C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : The S9 liver microsomal fraction is obtained from the liver of 8-12 weeks old male Wistar rats, strain CFHB (weight ca. 150 -200 g) which had been given a single i.p. injection of 500 mg/kg bw Aroclor 1254 in olive oil 5 days previously.
- Method of preparation of S9 mix : The livers of 5 animals are removed, washed in 150 mM KCl and homogenised. The homogenate, diluted 1:3 in KCl is centrifuged cold at 9000 g for 10 minutes. The supernatant which contains microsomes is frozen in ampoules of 2 or 5 mL and stored in liquid nitrogen.
- Concentration or volume of S9 mix and S9 in the final culture medium: Before the experiment an appropriate quantity of S9 supernatant is thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.3 mg/mL in the cultures. The concentration in the S9 preparation is between 30 and 45 mg/mL. The composition of the cofactor solution is concentrated to yield the following concentrations in the S9 mix: 8 mM MgCl 2; 33 mM KCl; 5 mM glucose-6-phosphate; 5 mM NADP; 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

Test concentrations with justification for top dose:

20, 50, 100 or 200 μg/mL of the test substance

Vehicle / solvent:

- Solvent used: Dimethyl sulfoxide (DMSO).
- Percentage of solvent in the final culture medium: 1%

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE:
24 h after subculturing the medium was replaced with medium containing 2 % FCS and the test substance, without S9 mix and with 20 µL/mL S9 mix. After 4 h this medium was replaced with normal medium after rinsing with "saline G". Treatment was performed with each 4 concentrations of the test substance without and with S9 mix. The toxicity of the test substance was determined in a pre-experiment by establishing the concentration related plating efficiency. According to these data the concentration range was chosen.
Treatment scheme:
Day 1: Subculturing of a log-phase culture:
a) About 400 cells in 5 mL medium/25 cm2 plastic flask for plating efficiency; in duplicate per experimental point,
b) 1x10^6 cells in 30 mL medium/175 cm2 plastic flask for the mutagenicity test, 1 flask per experimental point
Day 2: Treatment of a) and b);
Day 5: Subculturing of b) in 175 cm2 plastic flasks
Day 8: Fixation and staining of colonies in: a) flasks = plating efficiency and dose relationship
Day 9: Subculturing of b) in five 80 cm2 plastic flasks containing selective medium: mutant selection (about 6x10^5 cells/flask); subculturing of b) in two 25 cm2 flasks for plating efficiency (about 500 cells/flask)
Day 16: Fixation and staining of colonies in b) - derived flasks seeded on day 9
All incubations are done at 37°C.

Evaluation criteria:

The evaluation of the results was performed as follows:
- The test substance is classified as mutagenic if it induces for at least one of the test substance concentrations reproducibly a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment
- The test substance is classified as mutagenic if there is a reproducible concentration related increase in the mutant frequency. Such evaluation may be considered independently of the enhancement factor for the induced mutants.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Concentrations of the test substance higher than 200 µg/mL produced precipitations. Up to 200 µg/mL the test substance did not induce a marked cell mortality. In two independently performed experiments no mutagenic activity could be detected under the experimental conditions described in the test report

Table 1. Summary of results of gene mutation test in V79 cells  

	without S9				with S9
	plating efficiency after treatm.	plating efficiency after express.	mutants per plate	mutants per 10^6 cells	plating efficiency after treatm.	plating efficiency after express.	mutants per plate	mutants per 10^6 cells
1st experiment
DMSO Pos. control 20 µg/mL 50 µg/mL 100 µg/mL 200 µg/mL	98 92 101 95 95 86	86 82 88 103 101 97	28.8 194.3 19.2 21.4 27.0 12.2	70.7 428.6 49.8 45.2 48.8 24.0	97 56 92 90 96 86	84 94 88 97 100 95	7.6 52.4 9.0 18.0 14.0 13.8	27.9 154.8 23.2 38.2 30.7 32.2
2nd experiment
DMSO Pos. control 20 µg/mL 50 µg/mL 100 µg/mL 200 µg/mL	101 89 95 105 100 83	78 76 86 83 99 87	7.2 112.8 16.2 12.2 9.4 13.0	24.7 394.2 54.4 38.2 23.7 39.2	91 25 96 94 94 75	83 102 93 85 85 104	14.6 49.2 11.4 9.4 9.6 12.4	43.8 93.5 31.7 21.3 29.4 27.7

Conclusions:

It is concluded that under the conditions of the experiments the test substance is not mutagenic in V79 Chinese hamster cells in vitro.

Executive summary:

The test substance was assessed for its mutagenic potential in the mammalian HGPRT system using Chinese hamster cell line V79, according to GLP principles and OECD TG 476. The cells were exposed to the test substance both without and with metabolic activation for four hours at concentrations of 20, 50, 100, and 200 µg/mL without S9 mix and with S9 mix. Concentrations of the test substance higher than 200 µg/mL produced precipitations.
Results showed that up to 200 µg/mL the test substance did not induce a marked cell mortality. In two independently performed experiments no mutagenic activity could be detected under the experimental conditions. The clearly enhanced mutation rates after treatment with the positive control substances ethyl-methane sulfonate and 9,10- dimethyl-1,2-benzanthracene demonstrated the sensitivity of the test system.

Therefore, it can be stated that a mutagenic activity of the test substance could not be detected in the in vitro gene mutation test, HGPRT test, using cells of Chinese hamster cell line V79.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- In vivo micronucleus test in mouse, negative, equivalent to OECD TG 474, Taalman 1985

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 Dec 1984 to 19 Dec 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

Swiss

Remarks:

random

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: not reported
- Housing: Animals were group-housed up to 20 mice per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days

IN-LIFE DATES: 3 Dec 1984 to 19 Dec 1984

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: DMSO

Frequency of treatment:

Single dose

Post exposure period:

Test group: 24, 48 and 72 hours.
Control animals: 24 hours

Dose / conc.:

16.7 mg/kg diet

Remarks:

Low dose

Dose / conc.:

55.7 mg/kg diet

Remarks:

Mid dose

Dose / conc.:

167 mg/kg diet

Remarks:

High dose

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: Cyclophosphamide (CP)
- Route of administration: intraperitoneal injection
- Doses: 100 mg/kg

Tissues and cell types examined:

Bone marrow, Polychromatic erythrocytes (PCEs) and the number of mature erythrocytes (RBCs)

Details of tissue and slide preparation:

BONE MARROW EXTRACTION:
Animals were killed with CO2 or by cervical dislocation and the adhering soft tissue and epiphyses of both tibiae were removed. The marrow was flushed from the bone and transferred to centrifuge tubes containing 3 mL fetal calf serum (one tube for each animal).

DETAILS OF SLIDE PREPARATION:
Following centrifugation to pellet the tissue, the supernatant was drawn off, the cells resuspended in a drop of serum, and the suspension spread on slides and air-dried. The slides were then fixed in methanol, stained in May-Gruenwald solution followed by Giemsa, and rinsed in deionized water (Schmid, 1975).

METHOD OF ANALYSIS:
One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The frequency of PCEs versus mature RBCs was determined by scoring the number of mature erythrocytes (RBCs) observed in the optic fields while scoring the first 1000 PCEs for micronuclei. For control of bias, all slides were coded prior to scoring.

Evaluation criteria:

The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by colour of polychromatic and normochromatic erythrocytes (bluish-grey and red, respectively).

Statistics:

Two-tailed student's t-test

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Animals exposed to test substance showed no significant increase in micronucleus frequency at any dose level, kill-time or sex. In addition no increase in the numbers of micronuclei was observed when the data for both sexes were pooled. The positive control, cyclophosphamide, induced a large and statistically significant increase in the frequencies of micronuclei (p ≤ 0.02 for males, p ≤ 0.01 females).

Table 1. Mean percentage (±SD) of micronucleated PCEs in mouse bone marrow

Test substance (concentration)	Sex	24 hours	48 hours	72 hours
167 mg/kg	Males Females Mean	0.32 ± 0.12 0.10 ± 0.06 0.21 ± 0.07	0.28 ± 0.11 0.06 ± 0.05 0.16 ± 0.06	0.40 ± 0.40 0.18 ± 0.08 0.24 ± 0.11
55.7 mg/kg	Males Females Mean	0.30 ± 0.14 0.16 ± 0.05 0.23 ± 0.07	0.16 ± 0.11 0.06 ± 0.02 0.11 ± 0.06	0.25 ± 0.07 0.10 ± 0.00 0.17 ± 0.04
16.7 mg/kg	Males Females Mean	0.14 ± 0.05 0.26 ± 0.14 0.20 ± 0.07	0.44 ± 0.20 0.26 ± 0.14 0.35 ± 0.12	0.24 ± 0.14 0.18 ± 0.04 0.21 ± 0.07
Negative control Vehicle (DMSO)	Males Females Mean	0.10 ± 0.05 0.34 ± 0.06 0.22 ± 0.05
Positive control Cyclophosphamide (100 mg/kg)	Males Females Mean	1.94 ± 0.41* 1.56 ± 0.25** 1.75 ± 0.24**

*) p ≤ 0.02 **) p ≤ 0.01

Conclusions:

The test substance did not induce any significant increase in micronucleus frequencies in bone marrow polychromatic erythrocytes of the mouse. Therefore, the test substance was considered inactive under the conditions of this assay

Executive summary:

The test substance was evaluated for its ability to induce micronuclei in polychromatic erythrocytes (PCE’s) of male and female Swiss mouse bone marrow in a study following GLP principles and performed similar to OECD TG 474. Dose selection was based upon the LD50 (200 mg/kg for male mice and 218 mg/kg for female mice), using 80% of the combined LD50 for both sexes. Groups of 5 animals per sex per dose were received a single dose of 167.0, 55.7 and 16.7 mg/kg bw via oral gavage. The test substance was dissolved in Dimethylsulfoxide (DMSO). The positive control received an intraperitoneal injection of Cyclophosphamide (CP) at 100 mg/kg. Groups of animals were killed 24, 48 and 72 hours after administration of the test substance, the vehicle control and the positive control.

Results showed that animals exposed to the test substance showed no significant increase in micronucleus frequency at any dose level, kill time or sex. Also, no elevation of the numbers of micronuclei was observed if the data for the sexes are pooled. The positive control compound, cyclophosphamide (CP), induced large and statistically significant increases in the frequencies of micronuclei (p 0.02 ≤ males, p ≤ 0.01 females).

The test substance did not induce any significant increase in micronucleus frequencies in bone marrow polychromatic erythrocytes of the mouse. The test substance showed no mutagenicity under the conditions of this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

All available data was assessed and the studies representing the worst-case effects were included as key studies. Other studies are included as supporting information. The key study is considered to be worst-case and was selected for the CSA. For in vitro genetic toxicity, three key studies and six supporting studies are available. For in vivo genetic toxicity, one key study and two supporting studies are available.

 

In vitro genetic toxicity

The key study (Sokolowski 2009) was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strains WP2 uvrA pKM101 and WP2 pKM101, according to GLP principles and OECD TG 471. 

Results showed that reduced background growth was observed with and without metabolic activation from 1000 up to 5000 µg/plate in experiment I, and from 333 up to 5000 µg/plate in experiment II. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at the higher concentrations with and without metabolic activation in both experiments. No substantial increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Precipitation of the substance was observed in the test tubes in the presence of metabolic activation in both experiments.  No visible precipitation was observed on the incubated agar plates with and without metabolic activation in both experiments. Appropriate reference mutagens (sodium azide, 4-nitro-o-phenylene-diamine, 2-aminoanthracene and methyl methane sulfonate)were used as positive controls. They showed a distinct increase of induced revertant colonies. 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

In the key study (Miltenburger 1985), the test substance was assessed for its mutagenic potential in the mammalian HGPRT system using Chinese hamster cell line V79, according to GLP principles and OECD TG 476. The cells were exposed to the test substance both without and with metabolic activation for four hours at concentrations of 20; 50; 100; and 200 µg/mL without S9 mix and with S9 mix. Concentrations of the test substance higher than 200 µg/ml produced precipitations.

Results showed that up to 200 µg/mL the test substance did not induce a marked cell mortality. In two independently performed experiments no mutagenic activity could be detected under the experimental conditions. The clearly enhanced mutation rates after treatment with the positive control substances ethyl-methane sulfonate and 9,10- dimethyl-l,2-benzanthracene demonstrated the sensitivity of the test system.

Therefore, it can be stated that a mutagenic activity of the test substance could not be detected in the in vitro gene mutation test, HGPRT test, using cells of Chinese hamster cell line V79.

 

In the key study (Murli 1985), the objective of this in vitro assay was to evaluate the ability of the test substance to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation, according to GLP principles and OECD TG 473. The test substance was dissolved in dimethyl sulfoxide at a stock concentration of 205 mg/mL. Concentrations of 0.0683 µg/mL to 2050 µg/mL in a half-log series were tested in range-finding assays with and without metabolic activation. The following concentrations were tested in the mutagenicity test: 60.1, 100, 150, 200 µg/mL (without metabolic activation) and 45, 59.9, 99.9 and 150 µg/mL (with metabolic activation). Monolayer cultures were treated for 17.25 hours in the absence of S9 and harvested for slide preparation 20 hours after beginning of treatment. In the presence of S9 treatment was for 2 hours.

Results showed that complete cytotoxicity was observed in the cultures dosed with 683 and 2050 µg/mL, and severe cell cycle delay was evident in the culture dosed with 205 µg/mL, with cell cycle delay persisting in the culture dosed with 68.3 µg/mL in the range- finding assay without activation. Complete cytotoxicity was observed in the cultures dosed with 205, 683, and 2050 µg/mL, and no cell cycle delay was evident in the cultures analysed in the range- finding assay with activation. Based on these results, replicate cultures of CHO cells were incubated with 60.1 µg/mL to 500 µg/mL of the test article solution in a 20 hour non-activation aberrations assay, and with 15.0 µg/mL to 200 µg/mL in a 10 hour aberrations assay with metabolic activation. No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed. The sensitivity of the cell culture for induction of chromosomal aberrations was shown by the increased frequency of aberrations in the cells exposed to the positive control agents (mitomycin C and cyclophosphamide).

In conclusion, the test substance was considered negative for inducing chromosomal aberrations in Chinese hamster ovary cells under both non-activation and activation conditions of this assay.

 

All six of the supporting studies support the findings of the key studies that the test substance is negative for in vitro genetic toxicity. One of those studies was an Ames test (Hoorn 1986), one was a mutagenicity test in yeast cells (Hoorn 1985), two were chromosome aberration tests (Enninga 1995; Saigo 1995), another was a unscheduled DNA synthesis test (Curren 1986), and the final study was a transformation study (Miltenburger 1985). 

 

In vivo genetic toxicity

In the key study (Taalman 1985), the test substance was evaluated for its ability to induce micronuclei in polychromatic erythrocytes (PCE’s) of male and female Swiss mouse bone marrow in a study following GLP principles and performed similar to OECD TG 474. Dose selection was based upon the LD50 (200 mg/kg for male mice and 218 mg/kg for female mice), using 80% of the combined LD50 for both sexes. Groups of 5 animals per sex per dose were received a single dose of 167.0, 55.7 and 16.7 mg/kg bw via oral gavage. The test substance was dissolved in Dimethylsulfoxide (DMSO). The positive control received an intraperitoneal injection of Cyclophosphamide (CP) at 100 mg/kg. Groups of animals were killed 24, 48 and 72 hours after administration of the test substance, the vehicle control and the positive control.

Results showed that animals exposed to the test substance showed no significant increase in micronucleus frequency at any dose level, kill time or sex. Also, no elevation of the numbers of micronuclei was observed if the data for the sexes are pooled. The positive control compound, cyclophosphamide (CP), induced large and statistically significant increases in the frequencies of micronuclei (p 0.02 ≤ males, p ≤ 0.01 females).

The test substance did not induce any significant increase in micronucleus frequencies in bone marrow polychromatic erythrocytes of the mouse. The test substance showed no mutagenicity under the conditions of this assay.

 

Two supporting studies are available which support the findings of the key study that the test substance is negative for in vivo genetic toxicity. One of the studies was a chromosomal aberration test in mice (Ogorek 2000) and the other was a dominant lethal assay in rats (Putman 1991).

Justification for classification or non-classification

Based on the available data classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.