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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

410-400-0

EC Name:

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

Cas Number:

88671-89-0

Molecular formula:

C15H17ClN4

IUPAC Name:

2-(4-chlorophenyl)-2-[(1H-1,2,4-triazol-1-yl)methyl]hexanenitrile

Test material form:

solid

Remarks:

amber

Method

Species / strain

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

Controls

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

Results and discussion

Test results

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.

Any other information on results incl. tables

The results are in Tables 1 to 5 below.

Applicant's summary and conclusion

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.