(2RS,3RS)-3-(2-chlorophenyl)-2-(4-fluorophenyl)-[(1H-1,2,4-triazol-1-yl)methyl]oxirane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative results were obtained in the Ames Test with four S. typhimurium strains and one E. coli strain.

Point mutations in mammalian cells were assessed in Chinese hamster ovary cells (HPRT assay). In this study, no mutagenic effects were observed either with or without metabolic activation.

In the chromosome aberration test using Chinese hamster ovary cells, no increase in mutation frequency was noted in the presence or absence of metabolic activation.

DNA damage and repair, investigated in vitro using the UDS test in primary rat hepatocytes, showed no influence of epoxiconazole.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct. 1988 - March 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

adopted May 26, 1983

Deviations:

no

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
- Type and source of cells: CHO cell line, cell bank of CCR
- Suitability of cells: The CHO cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation
rate (doubling time 16 - 20 h in stock cultures) and a high plating efficiency of untreated cells (as a rule more than 50 %) both necessary for the appropriate performance of the study, recommend the use of this cell line.

For cell lines:
- Absence of Mycoplasma contamination: not specified
- Number of passages if applicable: not specified
- Methods for maintenance in cell culture: The cells were subcultured twice weekly. Seeding was done with about 1 x 10^6 cells per flask in 15 mL of DMEM/F12 (mixture 1:1) medium supplemented with 10 % fetal calf serum.
- Doubling time: 16 - 20 h
- Modal number of chromosomes: 20
- Periodically checked for karyotype stability: not specified

MEDIA USED
- Type and composition of media: DMEM/F12 (mixture 1:1) medium supplemented with 10 % fetal calf serum
- CO2 concentration: 11 %
- Humidity level: humdified incubator (not further specified)
- Temperature: 37°C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : liver of Aroclor 1254-induced Wistar rats
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.3 mg/mL in the cultures. The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 5 mM NADP, in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
- concentration or volume of S9 mix in the final culture medium: 20 µL/mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): the metabolic capability was checked by positive controls

Test concentrations with justification for top dose:

7-h interval: 10.0; 50.0; 100.0; 140.0 µg/ml
24-h interval: 1.0; 5.0; 10.0; 50.0; 100.0; 140.0 µg/ml
30-h interval: 10.0; 50.0; 100.0; 140.0 µg/ml

In a pre-experiment for toxicity the colony forming ability of the CHO cells was reduced to about 40 % after treatment with the highest concentration (140.0 µg/mL) in the absence and presence of S9 mix. Higher concentrations precipitated in the culture medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative non-toxicity for the cells.
- Percentage of solvent in the final culture medium: The final concentration of the solvents in the culture medium did not exceed 1 % v/v.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): approx. 0.6 - 1.5 x 10^5 cells/chamber
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): 5, 21 and 27 h after the start of the treatment colcemid was added (0.2 µg/mL culture medium) to the cultures for a duration of 2 h (7 h interval) or 3 h (24 h and 30 h interval).
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): After treatment with the spindle inhibitor, the cells on the slides in the chambers were treated with hypotonic solution (0.4 % KCl) at 37°C for 20 min. Afterwards the cells were fixed with 3:1 absolute methanol:glacial acetic acid. All two slides per group were prepared. After fixation the cells were stained with giemsa.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 100 well spread metaphases per slide (200 per test group) were scored.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations.
- Determination of polyploidy: not specified
- Determination of endoreplication: not specified

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Evaluation criteria:

The test article was classified as mutagenic if it induced either a significant dose-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points.

A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant positive response at any one of the test points was considered non-mutagenic in this system.

Statistics:

A statistical evaluation of the results was not necessary to perform. The aberration rates of the test groups after treatment with the test article were in the range of the control values.

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not specified
- Data on osmolality: not specified
- Possibility of evaporation from medium: not specified
- Water solubility: not specified
- Precipitation: Higher concentrations than 140 μg/mL resulted in test substance precipitation.
- Definition of acceptable cells for analysis: Only metaphases with characteristic chromosome number of 20 +/- 1 were included in the analysis.

RANGE-FINDING/SCREENING STUDIES: In the pre-experiment on toxicity (colony forming ability) in the absence and presence of S9 mix treatment the highest concentration (140.0 µg/mL) reduced distinctly the colony forming ability. Higher concentrations precipitated in the culture medium.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : see tables 1 - 3
- Results from cytotoxicity measurements: The mitotic index was reduced by about 60 % after treatment with the highest concentration (140.0 µg/mL) at fixation intervals 7 and 24 h.
- Genotoxicity results
o Definition for chromosome aberrations, including gaps : not specified
o The following dose levels were evaluated: 7-h interval: 140.0 µg/mL; 24-h interval: 10.0, 50.0 and 140 µg/mL; 30-h interval: 140 µg/mL
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen : not specified

HISTORICAL CONTROL DATA
- Positive historical control data: not specified
- Negative (solvent/vehicle) historical control data: not specified

Tab. 1: Mutagenicity data (summary of results), fixation interval: 7 h after start of treatment

article	number of cells analysed	conc. per mL	S9 mix	% abberant cells
				incl. gaps	excl. gaps	exchanges
Solvent control	200	0.0 µg	-	4.00	3.00	0.50
Test article	200	140.0 µg	-	4.00	2.50	0.00
Solvent control	200	0.0 µg	+	6.00	1.50	0.00
Test article	200	140.0 µg	+	8.50	1.50	0.00

Tab. 2: Mutagenicity data (summary of results), fixation interval: 24 h after start of treatment

article	number of cells analysed	conc. per mL	S9 mix	% abberant cells
				incl. gaps	excl. gaps	exchanges
Negative control	200	0.0 µg	-	4.00	2.50	1.00
Solvent control	200	0.0 µg	-	1.50	1.00	0.00
Positive control EMS	200	0.72 mg	-	10.50	9.00	6.50
Test article	200	10.0 µg	-	0.50	0.50	0.50
Test article	200	50.0 µg	-	2.00	1.50	0.00
Test article	200	140.0 µg	-	3.50	2.50	0.00
Negative control	200	0.0 µg	+	4.00	2.50	1.00
Solvent control	200	0.0 µg	+	5.50	3.50	0.50
Positive control CPA	200	4.2 µg	+	20.00	17.00	10.00
Test article	200	10.0 µg	+	4.00	2.50	0.50
Test article	200	50.0 µg	+	4.50	3.50	1.00
Test article	200	140.0 µg	+	5.50	3.00	1.00

Tab. 3: Mutagenicity data (summary of results), fixation interval: 30 h after start of treatment

article	number of cells analysed	conc. per mL	S9 mix	% abberant cells
				incl. gaps	excl. gaps	exchanges
Solvent control	200	0.0 µg	-	0.50	0.50	0.00
Test article	200	140.0 µg	-	2.00	1.50	0.00
Solvent control	200	0.0 µg	+	4.00	3.50	1.50
Test article	200	140.0 µg	+	2.00	1.00	0.50

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - July 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Version / remarks:

1986

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: UDS

Species / strain / cell type:

hepatocytes:

Remarks:

primary hepatocytes of male rats

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: primary hepatocytes of male Wistar rats
- Suitability of cells: The population of freshly isolated hepatocytes is essentially non-dividing. Their isolation can be done by standard procedures thus rendering a very standardizable test substrate with a low semi-conservative DNA replication. Male rats were used as the source of hepatocytes, because female rat-liver cells lack certain activating enzymes.

MEDIA USED
- Type and composition of media: Williams medium E supplemented with Hepes (2.38 mg/mL), Glutamin (0.29 mg/mL), Penicillin (100 units/mL), Insulin (0.50 µg/mL), Streptomycin (0.10 mg/mL), Fetal Calf Serum (100 µl/mL); pH adjusted to 7.6
- CO2 concentration: 5 %
- Humidity level: humdified incubator (not further specified)
- Temperature: 37°C

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

Experiment I : 0.15; 0.50; 1.50; 5.00; and 15.00 µg/mL
Experiment II: 1.50; 5.00; 15.00; 50.00; and 150.00 µg/mL

The test concentrations were chosen according to the results of a preliminary experiment for the evaluation of the toxicity of the test substance.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The solvent was chosen according to its solublity properties and its relative nontoxicity for the cells.

- Percentage of solvent in the final culture medium: The solvent did not exceed 1 % in the culture medium.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 x 10^5 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 18 h

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The exposure period (18 h) was terminated by rinsing the cultures twice with PBS to remove all traces of free radiolabel. A hypotonic solution of 1 % sodium citrate was added to each culture for 10 minutes to swell the nuclei for better grain quantification. The cells on the cover slips were then fixed by three changes of methanol:acetic acid (3:1 v/v) for 15 minutes each, rinsed with 96 % ethanol, and air dried. The cover slips were mounted cell surface up on glass slides and coated with ILFORD K-2 photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 7 days at 4°C. The photographic emulsion was then developed with KODAK D-19 at room temperature, fixed in TETENAL and stained with 0.4 % aceto orcein.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): The number of silver grains above the nucleus were counted automatically. In addition, the mean number of grain counts of one nuclear-sized cytoplasm area adjacent to the nucleus was counted. At least two slides per concentration and 50 cells per slide were evaluated. Heavily labelled S-phase
cells were excluded from counting.

Evaluation criteria:

A test article was classified as positive if it induced either a statistically significant concentration-related increase in radiolabel incorporation expressed as grains per nucleus or a reproducible and statistically significant positive response for at least one of the test points.

A test article producing neither a statistically significant concentration-related increase in radiolabel incorporation expressed as grains per nucleus nor a statistically significant and reproducible positive response at any one of the test points was considered non-effective in this system.

Statistics:

A statistical evaluation of the results was not necessary to perform as the number of nuclear and net grain counts of the groups treated with the test article were in the range of the corresponding controls.

Species / strain:

hepatocytes: primary hepatocytes of male rats

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not specified
- Data on osmolality: not specified
- Possibility of evaporation from medium: not specified
- Water solubility: not specified
- Precipitation: Concentrations higher than 125.00 µg/mL precipitated in the culture medium.

RANGE-FINDING/SCREENING STUDIES (if applicable): A pre-experiment was performed to determine the appropriate concentration range for the main experiments. The following concentrations were tested:
0.30; 1.00; 2.00; 3.00; 10.00; 20.00; 30.00; 100.00; 200.00 and 300.00 µg/ml. Concentrations higher than 125.00 µg/ml precipitated in the culture medium. In the pre-experiment treatment with concentrations higher than
30.00 µg/ml yielded toxic effects evidenced by a reduction of neutral red uptake. Up to the highest concentration tested no total suppression of neutral red uptake was obtained.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : see tables 2 and 3
- Signs of toxicity : In experiment I concentrations above 15.00 µg/mL yielded strong toxic effects evidenced by suppression of neutral red uptake and morphological alterations of the hepatocytes leading to unscorable slides. In experiment II toxic effects were also observed after treatment with concentrations higher than 15.00 µg/mL. However, up to the highest concentration tested (150 µg/mL) the toxic effects in experiment II were less dramatic and the resulting slides could be taken for scoring.

HISTORICAL CONTROL DATA
- Positive historical control data: not specified
- Negative (solvent/vehicle) historical control data: not specified

In the two independent experiments, after treatment with the test article, no reproducible concentration-dependent increase in the number of nuclear grain counts and net grain counts up to the highest evaluated concentration was observed. In experiment I the nuclear grain counts of the controls ranged from 5.83 to 12.18 and the net grain counts were in the range of -2.03 to -0.21. After treatment with the test article the values obtained were in the range of 5.57 to 7.62 for the nuclear grains and -3.28 to -l.92 for the net grains, respectively. In experiment II the nuclear grain counts of the control ranged from 3.92 to 4.15 and the net grain counts were in the range of -2.51 to 0.48. After treatment with the test article the values obtained were in the range of 3.08 to 4.57 for the nuclear grains and -2.35 to 0.28 for the net grains, respectively. The different toxicity levels of the test article in the main experiments may be due to different functional integry, different metabolic activity, and different activity of detoxification pathways of the cells obtained from the individual animals.

Tab. 1: Summary table of toxicity data

Concentration (µg/mL)	Experiment I		Experiment II
	Neutral Red adsorption OD 540 nm	Percentage of viable cells relative to the controls	Neutral Red adsorption OD 540 nm	Percentage of viable cells relative to the controls
Solvent	1.046	100	1.310	100
2.23 (2AAF)	0.285	27	0.531	41
0.00	1.373	132	1.520	116
0.05	0.875	84	1.145	87
0.15	0.913	87	1.253	96
0.50	1.089	104	1.411	108
1.50	0.706	67	1.046	80
5.00	0.774	74	0.975	74
15.00	0.767	73	0.884	67
50.00	0.294	28#	0.533	41
150.00	0.099	91#	0.604	46

# - slides not scorable due to toxicity

Tab. 2: Summary of results, Experiment I

Treatment	Grains per nucleus (Mean* +/- Standard deviation)	Grains per cytoplasm area (Mean* +/- Standard deviation)	Net grains per nucleus (Mean* +/- Standard deviation)
Negative control: medium	5.83        +/-          3.36	7.86     +/-          3.30	-2.03    +/-          2.11
Solvent control: DMSO	12.18      +/-          4.92	12.39   +/-          4.12	-0.21   +/-          3.45
Positive control: 2AAF (2.23 µg/mL)	38.82      +/-          21.67	6.35     +/-          4.32	32.47   +/-          19.87
1. Concentration (0.15 µg/mL)	7.62        +/-          5.53	9.73    +/-         5.27	-2.11   +/-         5.03
2. Concentration (0.50 µg/mL)	7.27        +/-          3.65	10.13  +/-          3.48	-2.86   +/-          2.66
3. Concentration (1.50 µg/mL)	6.60        +/-          3.61	9.88     +/-          4.40	-3.28    +/-          2.62
4. Concentration (5.00 µg/mL)	6.65        +/-          3.52	9.77    +/-          4.09	-3.12   +/-          3.14
5. Concentration (15.00 µg/mL)	5.57        +/-          2.54	7.49    +/-          2.62	-1.92   +/-          2.09

* - Mean of 100 cells

Tab. 3: Summary of results, Experiment II

Treatment	Grains per nucleus (Mean* +/- Standard deviation)	Grains per cytoplasm area (Mean* +/- Standard deviation)	Net grains per nucleus (Mean* +/- Standard deviation)
Negative control: medium	3.92        +/-          2.02	3.44     +/-          1.60	0.48     +/-          1.85
Solvent control: DMSO	4.15        +/-          2.35	6.66     +/-          2.50	-2.51   +/-          2.25
Positive control: 2AAF (2.23 µg/mL)	10.69      +/-          5.76	4.94     +/-          2.16	5.75     +/-          4.86
1. Concentration (1.50 µg/mL)	4.14        +/-          2.79	4.71   +/-          2.39	-0.57   +/-          1.97
2. Concentration (5.00 µg/mL)	4.57        +/-          2.68	6.92    +/-          3.13	-2.35   +/-          2.91
3. Concentration (15.00 µg/mL)	4.40        +/-          2.70	4.12     +/-          2.38	0.28     +/-          2.15
4. Concentration (50.00 µg/mL)	3.86        +/-          2.84	4.06    +/-          2.06	-0.20   +/-          2.75
5. Concentration (150.00 µg/mL)	3.08        +/-          1.88	3.67    +/-          1.61	-0.59   +/-          1.60

* - Mean of 100 cells

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec. 1989 - May 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

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:

hgprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Chinese hamster ovary (CHO) cells, clone CHO-K1-BH4; isolated by Dr. A.W. Hsie (Oak Ridge National Laboratory, Oak Ridge, Tennessee)
- Suitability of cells: given
- Normal cell cycle time (negative control): not specified

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: not specifed
- Methods for maintenance in cell culture: not specifed
- Cell cycle length, doubling time or proliferation index : doubling time 11 - 14 h
- Modal number of chromosomes: not specifed
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media: Ham's Nutrient Mixture F12 supplemented with L-glutamine, antibiotics, and fetal bovine serum (8 % by volume)
- CO2 concentration: 5 % +/- 1.5 %
- Humidity level: humidified atmosphere (not further specified)
- Temperature: 37°C +/- 1.5°C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : liver of Aroclor 1254-induced rats
- method of preparation of S9 mix: The S9 fraction was mixed with the reaction mixture (final concentration in cultures: 1 mM NADP (sodium salt), 5 mM glucose-6-phosphate, 2 mM CaCl2, 6.6 mM KCl, 2 mM MgCl2, 2 mM phosphate) immediately before use.
- concentration or volume of S9 mix and S9 in the final culture medium: 20 µL/mL (S9 homogenate)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Before use in the assay, each lot of S9 homogenate was tested when purchased. S9 at various concentrations was tested against reference chemicals such as benzo(a)pyrene or 3-methylcholanthrene. The optimum S9 concentration was selected based on induction of HGPRT- mutants in CHO cells, and this amount of S9 was used in all subsequent assays with that particular lot of S9.

Test concentrations with justification for top dose:

0.05, 0.1, 0.4, 0.6, 0.8, and 1.0 mg/mL (determined in a preliminary range-finding cytotoxicity assay with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: solubility of the test material

- Percentage of solvent in the final culture medium: 1 %

Untreated negative controls:

yes

Remarks:

only in the cytotoxicity assays

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

other: 5-Bromo-2'-deoxyuridine (50 µg/mL, without S9 mix)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 2
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 3 x 10^6 cells per tissue culture flask (75 cm²)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 d
- Selection time (if incubation with a selective agent): 7 - 10 d
- Selective agent: thioguanine (4 µg/mL; 24 mM)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency, relative survival (RS)

Evaluation criteria:

Criteria for a positive response:
- The 95% confidence level must be met
- The mutant frequency must meet or exceed 15 x 10^6
- A dose-related or toxicity-related increase in mutant frequency
- Significant mutagenic activity in one culture of a dose level should be confirmed in the replicate culture of the same dose level
- A mutagenic dose-response in one mutation assay should be confirmed in the second mutation assay
- an increase in mutant frequency in one trial for a treated culture near the highest testable toxicity, as defined previously, with the number of mutant colonies being more than twice the value needed to indicate a significant response

A test article will be evaluated as nonmutagenic in two assays only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to toxicity causing about 10 % to 15 % survival or extends to a concentration at least 75 % of that causing excessive toxicity.

Statistics:

The statistical tables provided by Kastenbaum and Bowman (1970) were used to determine whether the results at each dose level were significantly different from the negative controls at 95 % or 99 % confidence levels.
confidence levels.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Treatment medium pH remained at about pH 7.2 for concentrations up to the maximum applied concentration of 1.0 mg/mL.
- Data on osmolality: Osmolality measurements of nonactivation treatment media showed the negative (media) control to be 289 mOsm/kg, the 1-% dimethyl sulfoxide vehicle control to be 445 mOSm/kg, the low test article concentration of 0.05 mg/mL to be 445 mOSm/kg and the high test article concentration of 1.0 mg/mL to be 435 mOsm/kg.
- Precipitation and time of the determination: Insoluble white precipitate was observed at concentrations of 0.25 mg/mL and higher.

RANGE-FINDING/SCREENING STUDIES (if applicable): The rangefinding cytotoxicity assays showed that the test article was nontoxic to CHO cells in cultures at all dose levels (0.00195 mg/mL to 1.0 mg/mL) without S9 metabolic activation. With metabolic activation, cytotoxicity increased with increasing concentrations up to the highest soluble concentration of 0.125 mg/mL. The test article became less toxic at the insoluble concentrations of 0.25, 0.5, and 1.0 mg/mL.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The mutant frequency of the vehicle control was within the historical range of the laboratory; the positive control indicated that the test system is able to detect known mutagens.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data (50 µg/mL 5-bromo-2'-deoxyuridine) -without S9 mix :
Mean (+/- SD): 121.6 +/- 27.9 x10^-6
Range: 38.7 to 165.6 x10^-6
Number of experiments: 50
Number of controls: 59

- Positive historical control data (5 µg/mL 3-methylcholanthrene) - with S9 mix :
Mean (+/- SD): 370.0 +/- 173.3 x10^-6
Range: 152.3 to 941.6 x10^-6
Number of experiments: 50
Number of controls: 61

- Negative (solvent/vehicle) historical control data - without S9 mix:
Mean (+/- SD): 3.9 +/- 2.9 x10^-6
Range: 0 to 16.8 x10^-6
Number of experiments: 50
Number of controls: 88

- Negative (solvent/vehicle) historical control data - with S9 mix:
Mean (+/- SD): 2.9 +/- 2.1 x10^-6
Range: 0 to 10.0 x10^-6
Number of experiments: 50
Number of controls: 86

Table 1: Mutant frequencies with metabolic activation

Test group/ Epoxiconazole concentration (mg/L)	Mutant frequency (10^-6cells)
	Experiment I		Experiment II
	Culture 1	Culture 2	Culture 1	Culture 2
0.05	19.0**	10.7	3.7	4.4
0.1	10.5	15.7	7.2	6.1
0.4	18.5**	22.5*	3.9	5.7
0.6	10.7	10.0	4.7	5.6
0.8	7.7	15.4	10.1#	2.9
1	18.2**	13.9	9.4##	7.4
Vehicle control	Experiment I		Experiment II
1% DMSO	15.3		5.9
	12.2		8.0
	8.4		2.3
Positive control	Experiment I		Experiment II
3-Methylcholanthrene, 5 µg/mL	301.4*		297.9*
	335.3*		290.1*

* Kastenbaum-Bowmann test p <= 0.01 and mutant frequency >= 15x10^-6

** Kastenbaum-Bowmann test p <= 0.05 and mutant frequency >= 15x10^-6

# Kastenbaum-Bowmann test p <= 0.01 but mutant frequency is within acceptable background range (< 15x10^-6)

## Kastenbaum-Bowmann test p <= 0.05 but mutant frequency is within acceptable background range (< 15x10^-6)

Table 2: Mutant frequencies without metabolic activation

Test group/ Epoxiconazole concentration (mg/L)	Mutant frequency (10^-6cells)
	Experiment I		Experiment II
	Culture 1	Culture 2	Culture 1	Culture 2
0.05	7.3	0.4	1.1	2.9
0.1	2.4	8.7	2.5	4.9
0.4	1.2	3.5	2.6	0.4
0.6	3.9	4.0	2.1	4.6
0.8	2.6	3.0	3.2	4.3
1	8.5	4.9	1.8	3.4
Vehicle control	Experiment I		Experiment II
1% DMSO	5.6		3.8
	7.0		2.5
	2.8		3.8
Positive control	Experiment I		Experiment II
5 -Bromo-2´-deoxyuridine, 50 µg/mL	163.2*		119.0*
	170.8*		137.6*

* Kastenbaum-Bowmann test p <= 0.01 and mutant frequency >= 15x10^-6

Table 3: Mean Mutant Colonies and Relative Cloning Efficiencies without metabolic activation; Assay I

DoseLevel	MeanMutantColonies±s.D.	%RelativeCloning Efficiency*
vc	1.00±1.04	-
vc	1.42±1.24	-
vc	0.50±0.90	-
PC	27.00±5.30	88.8
PC	31.50±5.90	99.0
0.05mg/ml	1.42±0.90	104.1
0.05mq/ml	0.08±0.29	117.0
0.1 mg/ml	0.50±0.80	111.1
0.1 mg/ml	1.67±0.98	103.1
0.4 mg/ml	0.25±0.45	111.9
0.4mg/ml	0.75±0.87	114.1
0.6mq/ml	0.83±1.03	114.1
0.6mq/ml	0.75±0.75	99.6
0.8mq/ml	0.50±0.52	102.0
0.8mg/ml	0.58±0.67	104.2
1.0mg/ml	1.67±1.15	105.2
1.0mg/ml	0.83±0.83	92.1

VC - Vehicle Control (1% DMSO)

PC - Positive Control (50 µg/mL BrdU)

* Relative to the average vehicle control cloning efficiency

Table 4: Mean Mutant Colonies and Relative Cloning Efficiencies without metabolic activation; Assay II

DoseLevel	MeanMutantColonies±s.D.	%RelativeCloning Efficiency*
vc	0.83±0.83	-
vc	0.50±0.52	-
vc	1.0±0.95	-
PC	25.83±4.97	95.5
PC	25.83±5.32	82.7
0.05mg/ml	0.25±0.45	100.5
0.05mq/ml	0.67±0.89	99.5
0.1 mg/ml	0.58±0.67	100.8
0.1 mg/ml	1.08±1.31	96.4
0.4 mg/ml	0.50±0.67	84.5
0.4mg/ml	0.09±0.30	89.8
0.6mq/ml	0.42±0.67	85.8
0.6mq/ml	0.92±1.00	87.9
0.8mq/ml	0.67±0.65	90.4
0.8mg/ml	1.00±1.35	101.6
1.0mg/ml	0.42±0.51	101.4
1.0mg/ml	0.83±1.27	106.3

VC - Vehicle Control (1% DMSO)

PC - Positive Control (50 µg/mL BrdU)

* Relative to the average vehicle control cloning efficiency

Table 5: Mean Mutant Colonies and Relative Cloning Efficiencies with metabolic activation; Assay I

DoseLevel	MeanMutantColonies±s.D.	%RelativeCloning Efficiency*
vc	2.75±1.22	-
vc	2.58±1.00	-
vc	1.67±.89	-
PC	58.42±5.68	98.4
PC	61.83±8.62	93.7
0.05mg/ml	4.00±1.65	107.1
0.05mq/ml	2.25±1.71	106.7
0.1 mg/ml	2.25±1.82	108.7
0.1 mg/ml	3.42±2.02	110.6
0.4 mg/ml	3.67±1.61	100.8
0.4mg/ml	3.92±1.62	88.6
0.6mq/ml	2.33±1.37	110.4
0.6mq/ml	1.92±1.16	97.5
0.8mq/ml	1.58±1.16	104.6
0.8mg/ml	3.08±2.02	101.5
1.0mg/ml	3.42±1.73	95.2
1.0mg/ml	2.50±2.11	91.4

VC - Vehicle Control (1% DMSO)

PC - Positive Control (5 µg/mL 3-MCA)

* Relative to the average vehicle control cloning efficiency

Table 6: Mean Mutant Colonies and Relative Cloning Efficiencies with metabolic activation; Assay II

DoseLevel	MeanMutantColonies±s.D.	%RelativeCloning Efficiency*
vc	1.33±1.23	-
vc	1.64±1.36	-
vc	0.55±0.69	-
PC	58.92±6.07	88.8
PC	59.42±7.32	91.9
0.05mg/ml	0.75±0.75	91.9
0.05mq/ml	0.92±0.67	92.8
0.1 mg/ml	1.50±1.00	93.8
0.1 mg/ml	1.33±0.78	98.7
0.4 mg/ml	0.83±0.72	95.6
0.4mg/ml	1.27±1.35	99.9
0.6mq/ml	0.91±0.94	85.9
0.6mq/ml	1.08±1.44	86.8
0.8mq/ml	2.17±1.27	96.1
0.8mg/ml	0.58± 0.79	90.2
1.0mg/ml	1.92±1.78	91.4
1.0mg/ml	1.42±1.62	85.9

VC - Vehicle Control (1% DMSO)

PC - Positive Control (5 µg/mL 3-MCA)

* Relative to the average vehicle control cloning efficiency

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

only 4 strains of Salmonella typhimurium tested

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 4 strains of Salmonella typhimurium tested

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Arochlor 1254-induced rat liver
- method of preparation of S9 mix: S9 fraction was prepared according to Ames et al. (Ames, B.N.; McCann. J.; Yamasaki, E.: Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mut. Res., 31, 347 - 364 (1975)). The S9 mix was prepared freshly prior to each experiment. 3 volumes of S9 fraction were mixed with 7 volumes of S9 supplement (8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM phosphate buffer (pH 7.4))
- concentration or volume of S9 mix and S9 in the final culture medium: 18.5 % (S9 mix), 5.55 % (S9 fraction)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): the metabolic capability was checked by positive controls

Test concentrations with justification for top dose:

20, 100, 500, 2500 and 5000 µg/plate; top dose according to OECD guideline

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: not specified

- Justification for percentage of solvent in the final culture medium: not specified

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene (10 µg, dissolved in DMSO, with S9 mix); 4-nitro-o-phenylendiamine (10 µg, dissolved in DMSO, without S9 mix); 9-aminoacridine chloride monohydrate (100 µg, dissolved in DMSO, without S9 mix)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3 test plates per dose or per control
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.1 mL bacterial suspension (with >= 10^8 bacteria/mL)
- Test substance added in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Evaluation criteria:

In general a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-rosponse relationship
- reproducibility of the results

Statistics:

no data

Species / strain:

other: TA 1535, TA 100, TA 1537, TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no data
- Data on osmolality: no data
- Possibility of evaporation from medium: no data
- Water solubility: no data
- Precipitation and time of the determination: Incomplete solubility of the test substance in DMSO from about 500 µg/plate onward. Time of determination not specified.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : valid

For all test methods and criteria for data analysis and interpretation:
- Statistical analysis: no data

Ames test:
- Signs of toxicity : No bacteriotoxic effect (reduced his- background growth) was observed.

HISTORICAL CONTROL DATA
- Positive historical control data: no data
- Negative (solvent/vehicle) historical control data: no data

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

only 1 E. coli strain tested

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

trp

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Arochlor 1254-induced rat liver
- method of preparation of S9 mix: S9 fraction was prepared according to Ames et al. (Ames, B.N.; McCann. J.; Yamasaki, E.: Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mut. Res., 31, 347 - 364 (1975)). The S9 mix was prepared freshly prior to each experiment. 3 volumes of S9 fraction were mixed with 7 volumes of S9 supplement (8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM phosphate buffer (pH 7.4))
- concentration or volume of S9 mix and S9 in the final culture medium: 18.5 % (S9 mix), 5.55 % (S9 fraction)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): the metabolic capability was checked by positive controls

Test concentrations with justification for top dose:

20, 100, 500, 2500 and 5000 µg/plate; top dose according to OECD guideline

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: not specified

- Justification for percentage of solvent in the final culture medium: not specified

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene (60 µg, dissolved in DMSO, with S9 mix)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3 test plates per dose or per control
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.1 mL bacterial suspension (with >= 10^8 bacteria/mL)
- Test substance added in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Evaluation criteria:

In general a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-rosponse relationship
- reproducibility of the results

Statistics:

no data

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no data
- Data on osmolality: no data
- Possibility of evaporation from medium: no data
- Water solubility: no data
- Precipitation and time of the determination: Incomplete solubility of the test substance in DMSO from about 2500 µg/plate onward. Time of determination not specified.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : valid

For all test methods and criteria for data analysis and interpretation:
- Statistical analysis: no data

Ames test:
- Signs of toxicity : No bacteriotoxic effect (reduced trp- background growth) was observed.

HISTORICAL CONTROL DATA
- Positive historical control data: no data
- Negative (solvent/vehicle) historical control data: no data

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo, no indication of a clastogenic or spindle poisoning effect was observed in the mouse micronucleus test after a single application of epoxiconazole by gavage.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

not specified

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, WIGA, 8741 Sulzfeld, Germany
- Age at study initiation: not specified
- Weight at study initiation: 28 g (mean)
- Assigned to test groups randomly: yes (with the help of an appropriate computer program)
- Fasting period before study: not specified
- Housing: 5 animals/cage during acclimation period; single housing during test period
- Diet: Kliba Haltungsdiät, ad libitum
- Water: drinking water, ad libitum
- Acclimation period: approx. 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: not specified

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: aqueous 0.5 % CMC formulation
- Justification for choice of solvent/vehicle: not specified
- Concentration of test material in vehicle: 25 g/100 mL (high dose group), 5 g/100 mL (mid dose group) or 1 g/100 mL (low dose group)
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw

Details on exposure:

- applied dosage volume: 20 mL/kg bw (vehicle group and dosage groups), 10 mL/kg bw (positive control)


PREPARATION OF DOSING SOLUTIONS:

Frequency of treatment:

one single treatment

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

high dose group: 15
other treatment groups, vehicle control group and positive control group cyclophosphamide: 5
positive control group vincristine: 3 males and 2 females

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide, dissolved in aqua dest.
- Justification for choice of positive control(s): positive control for clastogenicity
- Route of administration: oral
- Doses / concentrations: 20 mg/kg bw in a volume of 10 mL/kg bw

vincristine, dissolved in aqua dest.
- Justification for choice of positive control(s): positive control for spindle poison effects
- Route of administration: intraperitoneal
- Doses / concentrations: 0.15 mg/kg bw in a volume of 10 mL/kg bw

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The doses were selected based on the results of a pretest for the determination of the acute oral toxicity. Herein, the highest applicable dose of 5000 mg/kg bw was survived by all animals but led to signs of toxicity such as irregular respiration, apathy and abdominal position. Therefore, a dose of 5000 mg/kg bw was selected as the highest dose in the present cytogenetic study. 1000 mg/kg bw and 200 mg/kg bw were administered as further doses.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): sacrifice intervals: 16 h (high dose group), 24 h (all groups), 48 h (high dose group)

DETAILS OF SLIDE PREPARATION: The two femora were prepared from the animals and all soft tissues were removed. After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum (37°C; about 2 mL/femur). The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant
was removed except for a few drops and the precipitate was resuspended. 1 drop of this suspension was dropped onto clean microscopic slides using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained. The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in aqua dest. and then placed in fresh aqua dest. for 2 or 3 minutes. They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Corbit-Balsam.

METHOD OF ANALYSIS: In general, 1000 polychromatic erythrocytes from each of the male and female animals of every test group were evaluated and investigated for micronuclei. Normochromatic erythrocytes (= normocytes) were also scored. The following parameters were recorded:
- number of polychromatic erythrocytes
- number of polychromatic erythrocytes containing micronuclei
- number of normochromatic erythrocytes
- number of normochromatic erythrocytes containing micronuclei
- ratio of polychromatic to normochromatic erythrocytes
- number of small micronuclei (d < D/4) and of large micronuclei (d >= D/4) (d = diameter of micronucleus, D = cell diameter)

Statistics:

A detailed statistical evaluation was not necessary to perform as the rate of micronucleated polychromatic erythrocytes after test substance treatment was nearly in the range of the actual control values and within the range of that of the historical control values.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Clinical examination: at 200 mg/kg bw irregular respiration; at 1000 mg/kg bw irregular respiration and apathy, abdominal position (in a few cases); at 5000 mg/kg bw irregular respiration, abdominal position, apathy, staggering (in a few cases), poor general state, 4 animals died; no clinical signs were observed on the day after treatment
- Induction of micronuclei:
vehicle control - 1.6 % polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval;
high dose group (5000 mg/kg bw) - 1.8 %, 2.3 % and 1.3 % polychromatic erythrocytes containing micronuclei were found after 16 h, 24 h and after 48 h, respectively;
mid dose group (1000 mg/kg bw) - 1.5 % polychromatic erythrocytes containing micronuclei were found after the 24-hour sacrifice interval;
low dose group (200 mg/kg bw) - 2.8 % polychromatic erythrocytes containing micronuclei were found after the 24-hour sacrifice interval;
positive control cyclophosphamide - 16.1 % polychromatic erythrocytes containing exclusively small micronuclei after the 24-hour sacrifice interval;
positive control vincristine - 98.6 % polychromatic erythrocytes containing the expected amount of large micronuclei after the 24-hour sacrifice interval;
The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals;
No inhibition of erythropoiesis induced by the treatment of mice with the test substance was detected.
- Ratio of PCE/NCE: The ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The studies cover all endpoints required for mutagenicity and genotoxicity testing. It is concluded that epoxiconazole has no mutagenic or genotoxic potential.

Therefore, no classification according to Regulation (EC) 1272/2008 is warranted.