5-(4-chlorobutyl)-1-cyclohexyl-1H-tetrazole hydrochloride (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

other information

Study period:

From 28.11.2013 to 27.01.2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

5-(4-chlorobutyl)-1-cyclohexyl-1H-tetrazole

EC Number:

616-036-0

Cas Number:

73963-42-5

Molecular formula:

C11H19ClN4

IUPAC Name:

5-(4-chlorobutyl)-1-cyclohexyl-1H-tetrazole

Test material form:

solid

Specific details on test material used for the study:

Identity: CIL4
Label name: CIL4 base libera
Batch no. SP117-0110
Expiry date: 04 Dec 2014
Storage conditions: room temperature
RTC number: 13860

Method

Target gene:

Histidine requirement : No Growth on Minimal plates + Biotin.
Growth on Minimal plates + Biotin + Histidine.
Tryptophan requirement : No Growth on Minimal agar plates
Growth on Minimal plates + Tryptophan.
uvrA, uvrB : Sensitivity to UV irradiation.
rfa : Sensitivity to Crystal Violet.
pKM101 : Resistance to Ampicillin.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone

Test concentrations with justification for top dose:

Main Assay I
The test item CIL4 was assayed in Main Assay I, using the plate incorporation method, at a maximum concentration of 5000 μg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 μg/plate.
Toxicity was observed with TA1537 and TA100 tester strains at the highest dose level, in the absence and presence of S9 metabolism. Precipitation of the test item was observed at the end of the incubation period at the highest concentration tested.

Main Assay II
Since a two-fold increase in the number of reventant colonies was noted only with TA1535 at the highest dose level, the result obtained was considered equivocal and a confirmatory experiment was performed. A pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the following dose levels:
TA1535, WP2 uvrA, TA98 (with and without S9) 5000, 2500, 1250, 62.5 and 313 (microg/plate)
TA100, TA1537 (with and without S9) 5000, 2500, 1250, 62.5, 313 and 156 (microg/plate)

Vehicle / solvent:

Solvent/vehicle controls: untreated and solvent vehicle controls will be prepared for each experiment; when the solvent is distilled water, these will be considered to be equivalent and only one set of controls will be performed.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Results and discussion

Any other information on results incl. tables

Solubility

Solubility of the test item was evaluated in a preliminary trial using DMSO and water for injection. These solvents were selected since they are compatible

with the survival of the bacteria and the S9 metabolic activity. The test item was found to be soluble in DMSO at 100 mg/mL. This result permitted a

maximum concentration of 5000 μg/plate to be used in the Main Assay I.

Assay for reverse mutation

Two experiments were performed.

The test item CIL4 was assayed in the Main Assay I at a maximum dose level of 5000 μg/plate and at four lower concentrations spaced at approximately

half-log intervals: 1580, 500, 158 and 50.0 μg/plate. Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, was observed with TA1537 and TA100 tester strains at the highest dose level both in the absence and presence of S9 metabolism.

No toxicity was noted with the remaining tester strains at any concentration tested. Precipitation of the test item, which did not interfere with the scoring,

was observed at the end of the incubation period at the highest concentration.

Dose-related increases in the number of revertant colonies were observed with TA1535 tester strain, at higher concentrations, in the absence and presence of S9 metabolism. These increases reached two-fold the concurrent negative control value, in the presence of metabolic activation, at the highest dose level.

In addition the number of revertant colonies observed fell out the historical control range, indicating a biological significance. Since a two-fold increase

(2.06 fold) in the number of reventant colonies was noted only with TA1535 tester strain at the highest dose level, the result obtained was considered

equivocal and a confirmatory experiment was performed. A pre-incubation step was included for all treatments of Main Assay II. Taking into account

the toxicity observed with TA1537 and TA100 tester strains at the highest concentration, the test item was assayed at the following dose levels:

TA1535, WP2 uvrA, TA98 (with and without S9)5000, 2500, 1250, 62.5 and 313 (μg/plate)

TA100, TA1537 (with and without S9) 5000, 2500, 1250, 62.5, 313 and 156

(μg/plate)

Toxicity was observed with TA1537 and TA100 tester strains at the highest dose level, in the presence of S9 metabolic activation. No toxicity was

noted with the remaining tester strain/treatment condition combinations. Precipitation of the test item was observed at the end of the incubation

period at the highest concentration tested.

Dose-related increases in the number of revertant colonies were observed with TA1535 tester strain, in the absence and presence of S9 metabolism.

At the highest dose level, in the absence of metabolic activation, revertant numbers reached two-fold the concurrent negative control value and fell out the

historical control range indicating a statistical and biological significance. No increases in the number of revertant colonies were noted, with the remaining

tester strains at any concentration tested.

The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these

solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay

system was functioning correctly.

Analysis of results

Criteria for outcome of the assays

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Evaluation

Results show that mean plate counts for untreated and positive control plates fell within laboratory acceptance criteria based on historical control data (confidence interval: men value 2 standard deviations).

The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. No plates were lost through contamination

or cracking. The study was accepted as valid. Increases in revertant numbers were noted only with TA1535 tester strain at the highest dose level. Moreover, using the plate incorporation method (Main Assay I), the observed increase slightly exceeded two-fold (2.06 fold) the concurrent negative control in the presence of S9 metabolism; while, using the pre-incubation method (Main Assay II), the number of revertant colonies reached twice (2.13 fold) the negative control in the absence of S9 metabolism.

These results are considered inconclusive.

Applicant's summary and conclusion

Conclusions:

The results obtained, under the reported experimental conditions, precluded making a definite judgement about the mutagenicity of the test substance, thus the study was considered inconclusive.