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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Germ cell mutagenicity data are not available for CIL4 HCl. For the similar substance CIL4 (i.e. the non-salified form) the following information is available.

 

CIL4 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed, both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. The test item was used as a solution in dimethylsulfoxide (DMSO).

 

Overall, increases in the revertant number were observed with TA1535 tester strain. Using the plate incorporation method (Main Assay I), these increases slightly exceeded two-fold the concurrent negative control in the presence of metabolic activation; whereas, using the pre-incubation method (Main Assay II), the number of revertant colonies reached twice the negative control in the absence of S9 metabolism.

In all cases, the results for TA1535 are very close to the limits for biological and statistical significance; therefore, they reveal a very slight mutagenic activity of CIL4 on this specific Salmonella strain.

 

So, since bacteria did not seem to be an appropriate test system for CIL4, anin vitromicronucleus assay has been performed.

 

The test item (CIL4) was assayed for the ability to induce micronuclei in Chinese hamster V79 cells, followingin vitrotreatment in the presence and absence of S9 metabolic activation.

As a result of this assay it was concluded that the test item does not induce micronuclei in Chinese hamster V79 cells afterin vitrotreatment.

 

Even if there is no concern for potential of CIL4 to have clastogenic effects on genetic material, noin vitrogene mutation studies in mammalian cells are available; therefore, data are inconclusive for the classification of the substance CIL4 and, consequently, for CIL4 HCl.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 1997

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

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

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 / cell type:

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

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.

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

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

other information

Study period:

from 10.04.2014 to 17.09.2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

July 2010

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) Step 4 version (9 November 2011).

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Internal name: CIL4

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Chinese hamster V79 cells were obtained from Dr. J. Thacker, MRC Radiobiology Unit, Harwell, UK. This cell line, V79 4(H) can be traced back directly to the original V79 isolate prepared by Ford and Yerganian (1958). The karyotype, generation time and plating efficiency have been checked in this laboratory. The cells are checked at regular intervals for the absence of mycoplasmal contamination.
Permanent stocks of the V79 cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.

Metabolic activation:

with and without

Metabolic activation system:

Species: Rat Strain: Sprague Dawley Tissue: Liver Inducing Agents Phenobarbital – 5,6-Benzoflavone Producer: MOLTOX, Molecular Toxicology, Inc. Batch Number: 3192

Test concentrations with justification for top dose:

Based on the preliminary solubility assay, dose levels of 1.00, 0.571, 0.327, 0.187, 0.107, 0.0609, 0.0348, 0.0199, 0.0114 and 0.00650 mM were used for
the first main experiment. Since negative results were obtained, a second experiment was performed using a continuous treatment in the absence of S9
metabolism. Since no cytotoxicity was observed, dose levels of 1.00, 0.500, 0.250, 0.125, 0.0625, 0.0313, 0.0156, 0.00781 and 0.00391 mM were used for
the second main experiment.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine

Details on test system and experimental conditions:

Approximately 20 hours before treatment, an appropriate number of flasks for the experiment was prepared from a single pool of cells. Each 25 sq.cm. flask
was seeded with 500,000 cells in EMEM complete medium. The composition of the treatment media was as indicated in the Study Protocol.

Treatment medium in the presence of S9 metabolism

Test item or control solution 0.05 mL
S9 mix 0.50 mL
EMEM complete 4.45 mL

Treatment medium in the absence of S9 metabolism
Test item or control solution 0.05 mL
EMEM complete 4.95 mL

Treatment medium in the absence of S9 metabolism
(Experiment 2: test item and vehicle control cultures)
Test item or control solution 0.025 mL
EMEM complete 4.975 mL

In the first experiment, both in the absence and presence of S9 metabolism, the cultures were incubated for 3 hours. At the end of treatment, the medium
was removed and the flasks were washed twice with Phosphate Buffered Solution (PBS). Fresh culture medium was added and the cultures were incubated for further 21 hours (Recovery Period) before harvesting. At the same time Cytochalasin B was added to achieve a final concentration of 3 μg/mL.

Statistics:

For the statistical analysis, a modified chi-squared test was used to compare the number of cells with micronuclei in control and treated cultures.

Assay results

Following treatment with the test item, no increase in the incidence of micronucleated cells over the concurrent control value was observed in the

presence or absence of S9 metabolism, at any concentration in any experiment. Marked increases in the incidence of micronucleated cells were observed

following treatments with the positive controls Cyclophosphamide, Mitomycin-C and Colchicine, indicating the correct functioning of the test system.

Statistical analysis

For the statistical analysis, a modified chi-squared test was used to compare the number of cells with micronuclei in control and treated cultures.

Following treatment with 5-(4-CHLOROBUTYL)-1-CYCLOHEXYLTETRAZOLE, no statistically significant increase in the incidence of micronucleated

cells was observed in the absence or presence of S9 metabolism in any experiment.

Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide,

Mitomycin-C and Colchicine, indicating the correct functioning of the test system.

Conclusions:

On the basis of these results, it is concluded that 5-(4-CHLOROBUTYL)-1-CYCLOHEXYLTETRAZOLE does not induce micronuclei in Chinese hamster V79 cells after in vitro treatment, under the reported experimental conditions.