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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-chloro-phenyl)-3-cyclopropyl-1-[1,2,4]triazol-1-yl-butan-2-ol

Cas Number:

94361-06-5

Molecular formula:

C15H18ClN3O

IUPAC Name:

2-(4-chloro-phenyl)-3-cyclopropyl-1-[1,2,4]triazol-1-yl-butan-2-ol

Method

Target gene:

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

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain 3

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

Controls

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

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.