Climbazole

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-05-26 to 2006-06-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD guideline No. 471 and in accordance with GLP.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Climbazole

EC Number:

253-775-4

EC Name:

Climbazole

Cas Number:

38083-17-9

Molecular formula:

C15H17ClN2O2

IUPAC Name:

1-(4-chlorophenoxy)-1-(1H-imidazol-1-yl)-3,3-dimethylbutan-2-one

Method

Target gene:

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not appplicable

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

not appplicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate (S9 mix) obtained from male Sprague-Dawley rats pretreated with Aroclor 1254 (500 mg/kg)

Test concentrations with justification for top dose:

Initial mutagenicity test: 25, 50, 100, 250, 500, 1000, 2000 and 5000 μg/plate;
Second mutagenicity test: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate

Vehicle / solvent:

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the Sponsor’s request and compatibility with the target cells.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (10 µg/plate)

Remarks:

for all Salmonella tester strains, in the presence of metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (1.0 µg/plate)

Remarks:

for WP2 uvrA (pKM101), in the presence of metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

for TA98, in the absence of metabolic activation Migrated to IUCLID6: 1.0 µg/plate

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

for TA100 and TA1535, in the absence of metabolic activation Migrated to IUCLID6: 1.0 µg/plate

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

for TA1537, in the absence of metabolic activation Migrated to IUCLID6: 75 µg/plate

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

for WP2 uvrA (pKM101), in the absence of metabolic activation Migrated to IUCLID6: 1,000 µg/plate

Details on test system and experimental conditions:

Tester strains:
Overnight cultures of the tester strains were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing ~50 mL of culture medium.
To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a resting shaker/incubator at room temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm at 37±2°C approximately 12 h before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of approximately 10^9 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

On the day of its use, minimal top agar, containing 0.8 % agar (w/v) and 0.5 % NaCl (w/v), was melted and supplemented with L-histidine, D-biotin, and L-tryptophan solution to a final concentration of 50 μM each. Top agar not used with S9 or sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar.
Bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (w/v) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (w/v) agar and supplemented with 2.5 % (w/v) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient broth was Vogel-Bonner salt solution supplemented with 2.5 % (w/v) Oxoid Nutrient Broth No. 2 (dry powder). Test article dilutions were prepared immediately before use.


Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 fraction was prepared from male Sprague-Dawley rats induced with a single i.p. injection of Aroclor 1254 (500 mg/kg) five days prior to sacrifice. The S9 was lot prepared and purchased from MolTox (Boone, NC). Upon arrival, the S9 was stored at -60°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to S. typhimurium TA100.
The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose-6-phosphate, 4 mM NADP, 8 mM MgCl2, and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4.

The sham S9 mixture (sham mix), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. To confirm the sterility of the S9 and sham mixes, a 0.5 mL aliquot of each was plated on selective agar.

Initial mutagenicity assay (preincubation method):
The initial mutagenicity assay was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. Vehicle control, positive controls and eight dose levels of the test article were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA (pKM101) on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9.

In the confirmatory mutagenicity assay using the plate incorporation method, 0.5 mL of S9 or sham mix, 100 μL of tester strain and 50 μL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 μL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 h at 37±2°C.


Confirmatory mutagenicity assay (plate incorporation method):

The confirmatory mutagenicity assay was used to evaluate the mutagenic potential of the test article. Seven or eight dose levels of test article along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA (pKM101) on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.

In this assay, 0.5 mL of S9 or sham mix, 100 μL of tester strain and 50 μL of vehicle or test article dilution were added to 13 X 100 mm glass culture tubes pre-heated to 37±2°C. After vortexing, these mixtures were incubated with shaking for 20±2 minutes at 37±2°C. Following the preincubation, 2.0 mL of selective top agar was added to each tube and the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 μL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 h at 37±2°C.

Evaluation criteria:

Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the assay was the preliminary toxicity assay or the plate exhibited toxicity.
The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate.
Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. For the test article to be evaluated positive, it had to cause a dose-related increase in the mean revertants per plate of at least one tester strain.

Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100, and WP2 uvrA (pKM101) were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value.

Statistics:

The use of statistics was limited to the calculation of means and standard deviations.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

see below

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Initial Mutagenicity Assay (Preincubation Method):

 

In the initial mutagenicity assay, consisting of the experimentsExperiments B1, B2, and B4, the maximum dose tested was 5000 μg per plate.

 

In the first experiment (B1), no positive mutagenic responses were observed with any of the tester strains in the absence of S9 activation or with tester strains TA98, TA1535, TA1537 and WP2uvrA (pKM101) in the presence of S9 activation.

Precipitate was observed beginning at 2000 or at 5000 μg per plate. Toxicity was observed beginning at 250, 500, 1000 or 2000 μg per plate. Both plates at 100 μg per plate with tester strain TA100 in the presence of S9 activation were lost due to water damage; therefore, this test condition was retested in another experiment (B2). Based on the findings of the initial mutagenicity assay, the maximum doses plated in the confirmatory mutagenicity assay were 5000 μg per plate with tester strain WP2uvrA (pKM101) and 500 μg per plate with all remaining test conditions.

Due to an unacceptable positive control value in experiment B2, tester strain TA100 in the presence of S9 activation was not evaluated but was retested in experiment B4.

In experiment B4, the dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1,500, and 5,000 μg per plate. No positive mutagenic responses were observed with tester strain TA100 in either the presence or absence of S9 activation. Tester strain TA100 in the absence of S9 activation was retested because the Study Director wrote the instructions incorrectly. Precipitate was observed at 5,000 μg per plate. Toxicity was observed beginning at 150 or 500 μg per plate.

Confirmatory Mutagenicity Assay (Plate Incorporation Method):

 

This assay consisted of the two experiments B3 and B5.

In Experiment B3, Crinipan was tested at concentrations of 50, 100, 250, 500, 1000, 2000, and 5000 μg per plate with tester strain WP2uvrA (pKM101) and 5.0, 10, 25, 50, 100, 250, and 500 μg per plate with all Salmonella tester strains. No positive mutagenic responses were observed. Precipitate was observed beginning at 500, 2000 or at 5000 μg per plate with some test conditions. Toxicity was observed beginning at 500, 2,000 or at 5,000 μg per plate with tester strain TA98 in the absence of S9 activation and tester strain WP2uvrA (pKM101) in the presence and absence of S9 activation. No toxicity was observed with the remaining test conditions; therefore, those test conditions were retested in experiment B5.

 

In experiment B5, Crinipan was tested at concentrations of 1.5, 5.0, 15, 50, 150, 500, 1500, and 5000 μg per plate. No positive mutagenic responses were observed with tester strain TA98 in the presence of S9 activation or with tester strains TA100, TA1535 and TA1537 in the presence or absence of S9 activation. Precipitate was observed at 5,000 μg per plate with some test conditions. Toxicity was observed beginning at 1500 μg per plate.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions employed this study, Crinipan was not mutagenic in the Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 as well as in the Escherichia coli tester strain WP2 uvrA (pKM101), both in the presence and in the absence of metabolic activation by rat S9 mix.

Executive summary:

The test article, crinipan, was tested in the bacterial reverse mutation assay (Ames test) using the Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA (pKM101) in the presence and absence of metabolic activation by rat liver S9 mix. The assay was performed using both the plate incorporation and the preincubation method. Under the conditions of this study, crinipan was concluded to be negative in all tester strains in the presence and absence of metabolic activation.