α-n-butyl-α-(4-chlorophenyl)-1H-1,2,4-triazole-1-propanenitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reliable in vitro bacterial and mammalian mutagenicity studies and an in vitro cytogenicity study are available with myclobutanil. Myclobutanil was negative in all assays. 

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:

01-October-1984 to 10-December-1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: Chinese hamster ovary cells obtained from Dr. s. Wolff's laboratory, University of California, San Francisco, USA
- Suitability of cells: recommended in guideline
- Normal cell cycle time (negative control): 12 - 14

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: McCoy's 5a medium supplemented with 10% fetal calf serum, L-glutamine, and antibiotics

Cytokinesis block (if used):

Colcemid is used at 0.1 µg/mL for 2.5 hours.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- The in vitro metabolic activation system comprises rat liver enzymes and an energy-producing system necessary for their function (NADP and isocitric acid). The enzymes are contained in a
preparation of liver microsomes (S9 fraction) from rats treated previously with Arochlor, to induce enzymes capable of transforming chemicals to more active forms.
- The S9 fraction made from male rats "induced" with Arochlor 1254, was purchased from commercial suppliers. The samples were kept frozen at -80°C and thawed immediately before use. The liver fraction was then added to a "core" reaction mixture to form the activation system described below:
NADP (sodium salt): 1.5 mg
Isocitric Acid: 2.7 mg
Homogenate (S9 fraction): 15 microliters

Test concentrations with justification for top dose:

Based on a range finding assay concentrations of 25 µg/mL through 75 µg/mL and 20 µg/mL through 50 µg/mL were analyzed in the nonactivation and activation assays, respectively.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Cyclophosphamide (CP) was dissolved in water and used at final concentrations of 25 and 50 µg/mL.

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 17.25 hours without metabolic activation; 2 hours with metabolic activation
- Harvest time after the end of treatment (sampling/recovery times): about 20 hours without metabolic activation; about 10 hours with metabolic activation

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure: 0.1 mg/mL colcemid for 2.5 hours before cell harvest
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): 5% Giemsa
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 100 cells per slide were scored

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: cell cycle delay

Evaluation criteria:

The objective is to establish whether the test article or its metabolites can interact with cells to induce gross chromosomal breaks. Chemically induced lesions may result in breaks in chromatin that are either repaired by the cell in such a way as to be undetectable, or can result in visible damage. Aberrations are a consequence of failure or mistakes in repair processes that result in lack of rejoining of breaks, or rejoining in abnormal configurations.
The following factors were taken into account in evaluation:
i. The estimated number of breaks involved in production of the different types of aberrations observed.
ii. The percentage of cells with at least one aberration.
iii. The frequency of cells with more than one aberration.
iv. Any evidence for increasing amounts of damage with increasing dose, i.e., a positive dose response.
v. The estimated number of breaks involved in production of the different types of aberrations observed, i.e, complex aberrations may have more significance than simple breaks.

Statistics:

Statistical analysis employs the Fisher's Exact Test to compare the percentage of cells with aberrations in treated cells with pooled results from solvent and negative controls. The difference is considered significant where p

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE FINDING/SCREENING STUDIES:
without metabolic activation: There was a 88% reduction in monolayer confluency with no dividing cells present at 340 µg/mL and 1.02 mg/mL. At 102 µg/mL there was a 25% reduction in the cell monolayer and very few observable mitotic cells. Results were analyzed from 10.2 µg/mL to 102 µg/mL. The test article caused a large degree of cell cycle delay at 34 µg/mL and 102 µg/mL. A delayed fixation time with a dose range between 25 µg/mL and 200 µg/mL was selected for the nonactivation assay.

With metabolic activation: There was complete toxicity at 102 µg/mL through 1.02 mg/mL. At 34
µg/mL there was only a slight reduction in monolayer confluency with no decrease in observable mitotic cells. There was no cell cycle delay at 10.2 µg/mL and 34 µg/mL and a 10 hour fixation time with a dose range of 20 µg/mL to 90 µg/mL was chosen for the activation assay.

The test substance was tested up to levels of toxicity.

The results are in Tables 1 to 5 below.

Conclusions:

The test item, is considered negative for inducing chromosomal aberrations in Chinese Hamster ovary cells under both the metabolic activation and nonactivation conditions of this assay.

Executive summary:

This in vitro assay evaluated the ability of a myclobutanil to induce chromosome aberrations in the Chinese Hamster Ovary (CHO) cell line with and without an in vitro metabolic activation system. CHO cells were grown in McCoy's 5a medium supplemented with fetal bovine serum, penicillin, streptomycin and L-glutamine. The cells were incubated at 37°C with 5% CO2.
The maximum dose to be tested was determined based upon the solubility of the test item and any relevant toxicity information available on the test item. A preliminary rangefinding assay was conducted to determine toxicity and any effects on cell cycle kinetics which might be caused by the test item. The determination of dose levels and fixation times for the aberrations test was determined based upon observations of toxicity and cell cycle delay in the rangefinding assay. For the aberrations assay, cells were seeded at 1.0 to 1.5E6 cells/T75 flask. One day after seeding the culture medium was replaced with either fresh culture medium or medium containing the metabolic activation system. The cells were dosed with the test article and incubation was continued for 2 (+S9) to 17.5 (-S9) hours. The medium containing the test item was then removed, cells were washed twice with PBS, fresh culture medium was replaced, and incubation was continued for 2.5 to 18 hours with colcemid present the final 2.5 hours. The cells were then harvested according to standard harvest procedures.
The results of the assays were obtained by scoring 200 metaphase cells from the top four surviving dose levels. Concentrations of 25 µg/mL through 75 µg/mL and 20 µg/mL through 50 µg/mL were analyzed in the nonactivation and activation assays, respectively. The test article caused no increase in chromosomal aberrations at the concentrations tested and was considered negative under both the nonactivation and activation conditions of this assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25-April-1983 to 26-January-1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

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):

The study was carried out with a subclone of the CHO cell line designated CHO-K1-BH4

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Liver S-9 homogenate fraction from Aroclor 1254 induced male Sprague-Dawley rats.
- With metabolic activation, the assay was initially performed using an S-9 concentration of approximately 1 mg protein (Lowry) per ml final concentration in culture medium. If the test compound exhibited no adverse effect both without and with activation at 1 mg/ml S-9, then the Gene Mutation Assay was repeated with a range of S-9 concentrations (0.3 and 2.0 mg/ml S-9).

Test concentrations with justification for top dose:

Myclobutanil was tested for mutation induction with metabolic activation at concentrations ranging from 120 to 175 µg/ml and without metabolic activation at concentrations ranging from 25 to 90 µg/ml.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

100 µg/ml ethyl methanesulfonate (EMS) in the absence of activation
7 µg/ml 7,12-dimethylbenzanthracene (DMBA) in the presence of metabolic activation.

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5x10E5 cells per plate
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 18 to 20 hours without S9 and 5 hours with S9

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 8 days
- Selection time (if incubation with a selective agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: 10 mM 6-thioguanine for 7 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2 x 10E5

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: survival, plating efficiency

Evaluation criteria:

Tests are performed either with metabolic activation or without metabolic activation. An evaluation of either no adverse effect or an adverse effect is made independently with metabolic activation and without metabolic activation, regardless of the result of either evaluation. The following Assay Evaluation Criteria are applied in making these evaluations :
- An adverse effect must exhibit a reproducible, significant increase in the Mutant Frequency and be accompanied by a real increase in the average no. of colonies per selection plate, compared to that of the solvent control.
- Duplicates to confirm the reproducibility of the result may be in the sane test, if 2 different concentrations of the test compound produce a significant increase in the Mutant Frequency. If the increase in the Mutant Frequency is at only one test compound concentration, then the result must be reproduced in an independent test before accepting it as evidence for an adverse effect.
- A conclusive result of no adverse effect, must evaluate the test compound to its limits of solubility or over a range of toxicities from >75% survival to <20% survival relative to the solvent control. If a test compound is relatively nontoxic, the maximum treatment concentration will be 1000 µg per mL for a pure material.

Statistics:

The Mutant Frequency at each concentration of test compound is compared to that of both the simultaneous solvent controls and the historical negative control for this laboratory. The results of tests performed at different times are not pooled but analyzed independently.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

The CHO/HGPRT Gene Mutation Assay for myclobutanil included five Range Finding Tests and six Mutation Tests. The results are in Tables I to XI.

Conclusions:

Myclobutanil was tested for mutagenic activity at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells, both with or without metabolic activation, following the US EPA 870.5100 guideline. Myclobutanil did not induce mutation at the HGPRT locus in CHO cells when tested from 25 to 90 µg/mL without metabolic activation. These treatments resulted in 104% to 23% cell survival. With a metabolic activation system, myclobutanil did not induce mutations when tested from 120 to 175 µg/mL, which yielded 100% to 19% cell survival, respectively.

Executive summary:

Myclobutanil was tested for mutagenic activity at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells both with and without Aroclor 1254-induced rat liver S-9 metabolic activation. Without metabolic activation, cells were exposed to the test compound dissolved in dimethyl sulfoxide (DMSO) for 18 to 20 hr at 37 C in Ham's nutrient medium F-12 supplemented with 5% heat-inactivated, dialyzed, fetal calf serum. With S-9 metabolic activation, cells were exposed to the test compound for approximately 5 hr at 37 C in serum-free Ham's nutrient medium F-12. After treatment, cells were grown for approximately 8 days to permit expression of the mutation, and then cultured in 10 µM 6-thioguanine (6TG) to select for HGPRT locus mutants. After incubation for approximately 7 days, the mutant colonies were counted and compared to the results of concurrent and historical negative (solvent and untreated) controls.
Without metabolic activation, myclobutanil did not induce mutations when tested from 25 to 90 µg/mL (20 to 73 µg/mL). These treatment concentrations yielded approximately 104 to 23% cell survival relative to DMSO solvent controls. With a metabolic activating system (1 mg S-9 protein per mL), myclobutanil did not induce mutations when tested from 120 to 175 µg/mL (97 to 142 µg/mL). These treatments produced 100 to 19% cell survival relative to the solvent controls. In addition, 160 µg/mL myclobutanil did not induce mutations when tested using a higher S-9 concentration (2.0 mg 5-9 protein per mL). Therefore, myclobutanil does not induce mutations at the HGPRT locus in CHO cells in culture when tested either in the presence or absence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03-September-2008 to 23-October-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his, trp

Species / strain / cell type:

E. coli WP2 uvr A

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:
- Liver microsomal enzymes (S9 homogenate) were purchased from Molecular Toxicology, Inc., (36.5, 42.8 and 43.3 mg of protein per mL, respectively).
- The homogenate was prepared from male Sprague-Dawley rats that had been injected with Aroclor™ 1254 (200 mg per mL in corn oil) at 500 mg/kg.
- S9 mix contained the components:
H2O: 0.70 mL
1M NaH2PO4/Na2HPO4, pH 7.4: 0.10 mL
0.25M Glucose-6-phosphate: 0.02 mL
0.10M NADP: 0.04 mL
0.825M KCl/0.2M MgCl2: 0.04 mL
S9 Homogenate: 0.10 mL
Total volume: 1.00 mL

Test concentrations with justification for top dose:

The concentrations tested in the initial mutagenicity assay with all tester strains were 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 µg per plate in presence and absence of S9 mix. The highest concentration evaluated in the initial mutagenicity assay was the limit concentration of 5000 µg per plate.
The results of the initial mutagenicity assay were confirmed in an independent mutagenicity
assay at concentrations of 100, 333, 1000, 3330, and 5000 µg per plate in presence and absence
of S9 mix, using three plates per concentration.

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other:

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added preincubation

Evaluation criteria:

Criteria For A Positive Response:
- Tester Strain TA100
For a test item to be considered positive, it must produce at least a 2-fold concentration related and reproducible increase in the mean revertants per plate over the mean revertants per plate of the appropriate vehicle control. A response that does not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) will not be evaluated as positive.
- Tester Strains TA98, TA1535, TA1537, and WP2MvrA
For a test item to be considered positive, it must produce at least a 3-fold concentration related and reproducible increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. A response that does not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) will not be evaluated as positive.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

The results are included in Tables 1 to 5 below.

Conclusions:

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay
Preincubation Method with a Confirmatory Assay with the test item indicate that under the
conditions of this study, the test item did not cause a positive increase in the mean number of
revertants per plate with any of the tester strains either in the presence or absence of microsomal
enzymes prepared from Aroclor™-induced rat liver (S9).

Executive summary:

The objective of this study was to evaluate the test item and/or its metabolites for the ability to induce reverse mutations either in the presence or absence of mammalian microsomal enzymes at 1) the histidine locus in the genome of several strains of Salmonella typhimurium and at 2) the tryptophan locus of Escherichia coli strain WP2uvrA. This assay satisfied the following guidelines: U.S. EPA (1998), EEC (2000), OECD (1997), and MAFF (Shirasu, 1988).
The tester strains used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2uvrA. The initial mutagenicity assay was conducted with eight concentrations of test item in the presence and absence of S9 mix, along with concurrent vehicle control and positive controls using two plates per concentration. In the initial mutagenicity assay, the concentrations tested with all tester strains were 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg per plate in the presence and absence of S9 mix. The results of the initial mutagenicity assay were confirmed in an independent mutagenicity assay at concentrations of 100, 333, 1000, 3330, and 5000 μg per plate in presence and absence of S9 mix, along with concurrent vehicle control and positive controls using three plates per concentration.
The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay Preincubation Method with a Confirmatory Assay with myclobutanil indicate that under the conditions of this study, the test item did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The genotoxicity of myclobutanil was investigated in a comprehensive package of studies. Five in vitro genotoxicity tests were conducted including the Ames test, in vitro cytogenic assay and in vitro forward mutation assay in Chinese hamster ovary cells. The studies generally meet the requirements of current guidelines. Myclobutanil was negative in all in vitro studies.

Justification for classification or non-classification

Based on the available data it is concluded that Myclobutanil does not meet classification criteria for genetic toxicity properties according to the Classification, Labelling and Packaging (CLP) Regulation ((EC) No 1272/2008).