Chlorothalonil

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1977

Report date:

1977

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mammalian cell mutagenesis tests with Chinese hamster cells and/or mouse fibroblasts are performed according to the method of Schechtman et al. Ouabain is used as the selective agent to detect point mutations in treated cells. This mutant cell is resistant to ouabain and hence forms mutant colonies in the presence of ouabain. Plates, with treated cells are incubated in ouabain containing medium for 14 days for Chinese hamster cells and 25 days for mouse fibroblasts. Ouabain resistant mutant colonies are counted and compared with controls.

GLP compliance:

no

Type of assay:

other: In vitro mammalian cell gene mutation test using the ouabain resistant locus

Test material

Test material information

Constituent 1

Reference substance name:

Chlorothalonil

EC Number:

217-588-1

EC Name:

Chlorothalonil

Cas Number:

1897-45-6

Molecular formula:

C8Cl4N2

IUPAC Name:

tetrachlorobenzene-1,3-dicarbonitrile

Method

Target gene:

Ouabain resistant locus

Species / strain

Species / strain 1

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The growth medium (Microbiological Associates) was Eagle’s minimal essential medium (MEM) supplemented with L-glutamine (2 mM), nonessential amino acids (0.1 mM), penicillin (100 units/mL), streptomycin (100 mg/mL) and 5% heat-inactivated fetal bovine serum.

Species / strain 2

Species / strain / cell type:

other: Mouse BALB/3T3

Details on mammalian cell type (if applicable):

The growth medium (Microbiological Associates) was Eagle’s minimal essential medium (MEM) supplemented with L-glutamine (2 mM), nonessential amino acids (0.1 mM), penicillin (100 units/mL), streptomycin (100 mg/mL) and 10% heat-inactivated fetal bovine serum.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- method of preparation of S9 mix: Aroclor 1254 (500 mg/kg body weight) was inoculated intraperitoneally (IP) in male Fischer 344 rats and 48 hours later animals were sacrificed, livers were excised, maintained sterile at 4°C, rinsed free of excess blood with 0.15 M KCl buffer, and minced and homogenized at 30% w/v in 0.15 M KCl. The homogenate was centrifuged at 9000 x g for 20 minutes at 4°C. This post-mitochondrial enzyme preparation (S-9) was stored frozen in aliquots at 80°C.
- Biochemical properties of subcellular enzyme fractions (S-9) were determined as follows. Aryl hydrocarbon hydroxylase (AHH) activity which follows the conversion of BP to the phenol 3-OH BP was measured according to the procedures of Nebert and Gelboin (2). Protein determination was made according to the procedure of Lowry.

Test concentrations with justification for top dose:

Main test in presence of S9: 0.3 µg/mL
Main test in absence of S9: 0.03 and 0.3 µg/mL

Vehicle / solvent:

- Solvent used: Acetone
- Final concentration: 2.5 µL/mL

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: MNNG

Details on test system and experimental conditions:

Exponentially growing Chinese hamster lung fibroblasts (V-79) and mouse BALB/3T3 clone A31-1 were grown in 25 cm2 flasks and cells were collected by trypsinization. One to 2 x 10^6 cells were suspended in 4 mL whole medium and various concentrations of test chemical were added to the tube. Cell suspensions in the presence of test chemical were incubated with continuous gentle agitation at 37°C for 2 hours in a humidified atmosphere of 5% CO2 in air. The reaction was stopped by centrifugation (1000 x g, 4°C, 10 minutes). Cells were fixed and stained 8-10 days later with Giemsa stain.

When no activation was required, cells were collected in 4 mL whole medium and approximately LD50 concentrations of test chemical were added. MNNG was used as the positive control. Cell suspension was incubated at 37°C for 2 hours in a humidified atmosphere of 5% CO2 in air. The reaction was stopped by centrifugation (100 x g, 4°C, 10 minutes). Cells were seeded in 75 cm2 Falcon flasks and grown for 48 hours (V-79 cells), or 96 hours (3T3 cells) to allow for expression of the mutagenic event. Cells were then collected by trypsinization and plated at a density of 1 x 10^5 cells/dish (for V-79) or 2 x 10^5 cells/dish (for 3T3) for selection of mutant colonies resistant to 1 mM ouabain according to the procedure of Huberman. Plating efficiency assays were run concomitantly in order to determine the number of cells at risk.

For in vitro metabolic activation, 1-2 x 10^6 cells were suspended in a 4 mL reaction pool consisting of phosphate-buffered saline, pH 7.6, 1.8 mM NADPH , 1.4 mM NADH, 7.4 mM glucose-6-phosphate and 1.9 mM NADP. To this was added 100 µL S-9 + test chemical at a dose determined from LD50 cytotoxicity. The reaction mixture was incubated with continuous gentle agitation at 37°C for 2 hours in humidified atmosphere of %. CO2 in air. Positive control was + BP (12.5 µL/mL) and 100 µL S-9. The reaction was stopped by centrifugation (1000 x g,4°C, 10 minutes). AHH activity was measured in the supernatant fluids. Cells were seeded for expression time as previously described. Selection of mutant colonies resistant to 1 mM ouabain was according to the procedure described above. Cells were fixed and stained after 7-9 days for plating efficiency (cells at risk) or 14 days (V-79 mutagenesis ) and 25 days (3T3 mutagenesis), and the number of surviving. colonies in each dish determined .

Results and discussion

Test results

Test results 1

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

other: BALB/3T3, Clone A31

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:

The cytotoxicity in the preliminary test in V79 cells was determined at concentrations of 30, 10, 3, 1 and 0.3 µg/mL. The test substance is toxic, even at 0.3 µg/mL. Mutation frequency in the presence of test chemical is not significant even though the spontaneous mutation frequency is ten times higher than it should be. Addition of an exogenous source of liver enzyme (i.e., S-9) did not affect the ability of the test agent to increase the frequency of ouabain resistant mutation. As mentioned in an earlier report, the spontaneous mutation frequency is much too high in this specific clone of V-79, the studies were repeated using a different cell line, namely BALB/ 3T3 clone A31-1.

It was observed that even at 0.03 µg/mL with 2 hours exposure there was approximately 33% death. This indicates that BALB/ 3T3 was about 10 times more sensitive to the cytotoxic effects of the test substance than were the V-79 cells.

At 0.03 µg/mL, the test substance was not effective in inducing ouabain resistant mutants without activation using BALB/3T3 as the target cells. BP was used as the positive control. BP, in the presence of exogenous mammalian S-9 enzyme fraction was converted to 11.5 nM of 3-OH BP in 2 hours incubation. This activating system was effective in inducing mutation frequency of 17.1 x 10^-6. The test chemical failed to increase the mutation frequency above the spontaneous level.

It must be pointed out that the presence of the S-9 preparation allowed us to increase the concentration of the test substance that could be evaluated. A concentration of 0.3 µg/mL (10 times higher than could be used without activation or in the presence of heat inactivated S- 9) was evaluated and yet this higher dose still failed to significantly increase the mutation frequency at the ouabain resistant locus.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this bioassay, the test substance did not induce point mutations at the ouabain resistant locus in both V-79 clone 8 Chinese hamster cells or BALB/3T3 clone A subline 1 cells either with or without the addition of an exogenous source of liver enzyme (S-9) for metabolic activation of the test agent.

Executive summary:

Mammalian cell mutagenesis tests with Chinese hamster cells and/or mouse fibroblasts are performed according to the method of Schechtman et al. Ouabain is used as the selective agent to detect point mutations in treated cells. This mutant cell is resistant to ouabain and hence forms mutant colonies in the presence of ouabain. Plates, with treated cells are incubated in ouabain containing medium for 14 days for Chinese hamster cells and 25 days for mouse fibroblasts. Ouabain resistant mutant colonies are counted and compared with controls. Positive controls and negative controls are included in each experiment. N-methyl-N-nitro-N-nitrosoguanidine (MNNG) serves as the positive control without activation and Benzo (a) pyrene (BP) serves as the positive control with activation. Acetone is the negative control without activation and S-9 alone, heat-inactivated S-9 + BP, are the negative controls without activation.

The cytotoxicity in the preliminary test in V79 cells was determined at concentrations of 30, 10, 3, 1 and 0.3 µg/mL. The test substance is toxic, even at 0.3 µg/mL. Mutation frequency in the presence of test chemical is not significant even though the spontaneous mutation frequency is ten times higher than it should be. Addition of an exogenous source of liver enzyme (i.e., S-9) did not affect the ability of the test agent to increase the frequency of ouabain resistant mutation. As mentioned in an earlier report, the spontaneous mutation frequency is much too high in this specific clone of V-79, the studies were repeated using a different cell line, namely BALB/ 3T3 clone A31-1.

It was observed that even at 0.03 µg/mL with 2 hours exposure there was approximately 33% death. This indicates that BALB/ 3T3 was about 10 times more sensitive to the cytotoxic effects of the test substance than were the V-79 cells.

At 0.03 µg/mL, the test substance was not effective in inducing ouabain resistant mutants without activation using BALB/3T3 as the target cells. BP was used as the positive control. BP, in the presence of exogenous mammalian S-9 enzyme fraction was converted to 11.5 nM of 3-OH BP in 2 hours incubation. This activating system was effective in inducing mutation frequency of 17.1 x 10^-6. The test chemical failed to increase the mutation frequency above the spontaneous level.

It must be pointed out that the presence of the S-9 preparation allowed us to increase the concentration of the test substance that could be evaluated. A concentration of 0.3 µg/mL (10 times higher than could be used without activation or in the presence of heat inactivated S- 9) was evaluated and yet this higher dose still failed to significantly increase the mutation frequency at the ouabain resistant locus.

Under the conditions of this bioassay, the test substance did not induce point mutations at the ouabain resistant locus in both V-79 clone 8 Chinese hamster cells or BALB/3T3 clone A subline 1 cells either with or without the addition of an exogenous source of liver enzyme (S-9) for metabolic activation of the test agent.