Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 June 2018 - 06 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Deviations did not affect the result of the study. The OECD 471 guideline criteria were met.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

yes

Remarks:

Deviations did not affect the result of the study. The guideline criteria were met.

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Deviations did not affect the result of the study. The guideline criteria were met.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

EC Number:

268-417-2

EC Name:

3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

Cas Number:

68084-04-8

Molecular formula:

C13H19N

IUPAC Name:

3-(4-methylpent-3-en-1-yl)cyclohex-3-ene-1-carbonitrile

Constituent 2

Reference substance name:

4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

EC Number:

244-530-2

EC Name:

4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

Cas Number:

21690-43-7

Molecular formula:

C13H19N

IUPAC Name:

4-(4-methylpent-3-en-1-yl)cyclohex-3-ene-1-carbonitrile

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test Item: Reaction mass of 3-(4-methyl-3pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile
- Public Name: Azuril
- EC number: 915-371-2
- CAS number: n/a
- Old/other identifiers: EC 244-530-2, EC 268-417-2, EC 244-530-2, CAS 68084-04-8, CAS 21690-43-7
- Batch/Lot number: A170421E
- Appearance: Clear, almost colourless liquid
- Purity**: 99.35%
- Expiry date: 06 June 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Room temperature (15-25°C, ≤70 RH%), under inert gas, protected from humidity (tightly closed container)
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Identification and Receipt: The test item of a suitable chemical purity was supplied by the Sponsor

- Dilutions: Made in the testing laboratory using Dimethyl sulfoxide (DMSO)

- Analytical determination: stabilityility and homogeneity was not performed because of the character and the short period of study.
- Purity conversion: not applied

Method

Target gene:

S. typhimurium TA98 (hisD3052 mutation)
S. typhimurium TA100 (hisG46 mutation)
S. typhimurium TA1535 (hisG46 mutation)
S. typhimurium. TA1537 (hisC3076 mutation)
E. coli WP2 uvrA (trpE mutation)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

NA

Metabolic activation:

with and without

Metabolic activation system:

Cofactor-supplemented post-mitochondrial S9 fraction

Test concentrations with justification for top dose:

Concentrations were selected based on the results of the preliminary tests.
100 mg/mL stock solution was prepared in DMSO, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was in case of Salmonella typhimurium strains 1581 μg test item/plate, in case of Escherichia coli WP2 uvrA strain 5000 μg test item/plate.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: DMSO and Distilled water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine (NPD)

Details on test system and experimental conditions:

METABOLIC ACTIVATION SYSTEM
Test bacteria were exposed to the test item in the presence of a cofactor-supplemented post-mitochondrial S9 fraction.

INDUCTION OD LIVER ENZYMES
Male Wistar rats were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before sacrifice when food was removed. After euthanasia was performed, the induction of liver enzymes used for preparation S9 used was initiated.

PREPARATION OF RAT LIVER HOMOGENATE S9 FRACTION
On Day 4, the rats were euthanized and the livers were removed, weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-3 mL portions, frozen quickly and stored at -80 ± 10 ºC.
The mean protein concentration of the S9 fraction used was determined to be 30.45 g/L.
The biological activity in the Salmonella assay of S9 was characterized using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The S9 batch was considered functioning suitable for the study.

CONCENTRATIONS
Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Concentration Range Finding Test.

PRELIMINARY COMPATIBILITY TEST:
Due to the better biocompatibility, DMSO was selected as vehicle for the study.

PRELIMINARY CONCENTRATION RANGE FINDING TEST:
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item, in the absence and presence of metabolic activation.

TEST ITEM CONCENTRATIONS IN THE MUTAGENICITY TESTS:
Based on the results of the preliminary tests, 100 mg/mL stock solution was prepared in DMSO, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was in case of Salmonella typhimurium strains 1581 μg test item/plate, in case of Escherichia coli WP2 uvrA strain 5000 μg test item/plate.

CONTROLS:
Strain-specific positive and negative (solvent) controls, both with and without metabolic activation were included in each test. In addition, an untreated control was used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects.

PROCEDURE FOR EXPOSURE IN THE INITIAL TEST AND COMPLEMENTARY INITIAL MUTATION TEST
Molten top agar was prepared and kept at 45°C. The equivalent number of minimal glucose agar plates (three plates per test item concentration and for each control) was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.

PROCEDURE FOR EXPOSURE IN THE CONFIRMATORY MUTATION TESTS AND COMPLEMENTARY CONFIRMATORY MUTATION TEST
A pre-incubation procedure was performed as a Confirmatory Mutation Test and a Complementary Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed.
For the pre-incubation method, bacteria were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
Before the overlaying, 50 µL of the test item formulation or its vehicle (or 50 µL of positive reference controls or their solvents), 100 µL of the overnight culture of bacterial cells and 0.5 mL of the S9 mix (activated test conditions) or phosphate buffer pH 7.4 (non-activated test conditions) were placed in direct contact within the appropriate tubes. The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.

Rationale for test conditions:

The study consisted of 6 phases:
- Preliminary Compatibility Test
- Preliminary Range Finding Test (Informatory Toxicity Test)
- Initial Mutation Test
- Complementary Initial Mutation Test
- Confirmatory Mutation Test
- Complementary Confirmatory Mutation Test.

See NOTE 1 and NOTE 2 in "any other information" box.

Evaluation criteria:

General criteria for test validity: :
- the number of revertant colonies of the negative (solvent) and positive controls were in the relevant historical control range in all tester strains of the main tests;- at least five analysable concentrations (furthermore, positive and negative controls) were presented in all strains of the main tests.

Criteria for a Positive Response:
- A test item was considered mutagenic if: - a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

Criteria for and increase biological relevant:
- the number of reversions was more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions was more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

Criteria for a Negative Response:
- The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting.
Visual examination (precipitations or signs of growth inhibition) of the plates was also performed and recorded.

The mean number of revertants per plate, the standard deviation and the mutation factor (MF)* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.

* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no mutagenic potential

Any other information on results incl. tables

NOTE 1: Because no cytotoxicity was observed in the Initial Mutation Test, an additional experiment (Complementary Initial Mutation Test) was performed in Escherichia coli WP2 uvrA strain with and without metabolic activation during the Experimental Period II to complete the data. The Initial Mutation test in case of this strain was considered invalid.

NOTE 2: In the Confirmatory Mutation Test using the pre-incubation method, excessive cytotoxicity was observed in all examined Salmonella typhimurium strains without metabolic activation at several concentrations. In this case, the number of analyzable doses did not meet the recommendations of the test guidelines, thus they were considered invalid. Therefore, an additional experiment (Complementary Confirmatory Mutation Test) without metabolic activation will be performed in these strains in an additional experimental period (Experimental Period III) to complete the data.

Applicant's summary and conclusion

Conclusions:

The test material Azuril [Reaction mass of 3-(4-methyl-3pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3ene-1-carbonitrile] did not show mutagenic activity on the applied bacterial strains under the test conditions used in this study.

Executive summary:

The aim of this study was to evaluate the mutagenic potential of the test item Azuril [Reaction mass of 3-(4-methyl-3pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3ene-1-carbonitrile] and the ability to induce reverse mutations at selected loci of four strains of Salmonella typhimurium (TA1535, TA100, TA1537, TA98 N) and the Escherichia coli WP2 uvr strain (a total of five bacterial strains) in the presence and absence of activated rat liver S9 fraction. The study was performed according the OECD 471 Guideline, EPA Health Effects Test Guidelines, OPPTS 870.5100 and Commission Regulation (EC) No. 440/2008, B.13/14, and under GLP conditions.

 

The study consisted of 6 phases:

• Preliminary Compatibility Test
• Preliminary Range Finding Test (Informatory Toxicity Test)
• Initial Mutation Test
• Complementary Initial Mutation Test
• Confirmatory Mutation Test
• Complementary Confirmatory Mutation Test.

 

In the Preliminary Compatibility Test, Dimethyl sulfoxide (DMSO) was selected as vehicle due to the better biocompatibility. The test item was dissolved in DMSO at a concentration of 100 mg/mL. Concentrations of 10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate were examined in the Range Finding Test in Salmonella typhimurium TA98 and TA100 tester strains. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 0.5, 1.581, 5, 15.81, 50, 158.1, 500 and 1581 μg/plate, in the Complementary Initial Mutation Test were 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate in the Confirmatory Mutation Test in case of Salmonella typhimurium strains were 0.5, 1.581, 5, 15.81, 50, 158.1, 500 and 1581 μg/plate, in case of Escherichia coli WP2 uvrA strain were 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate. The test item concentrations in the Complementary Confirmatory Mutation Test were 0.05, 0.1581, 0.5, 1.581, 5, 15.81, 50 and 158.1 μg/plate.

 

In the Initial Mutation Tests and Confirmatory Mutation Tests, the number of revertant colonies did not show any biologically relevant increase compared to the solvent control. No dose-related trends and no indication of any treatment-related effect were recorded. No precipitate was observed in the main tests in all examined bacterial strains with and without metabolic activation. Inhibitory, cytotoxic effect of the test item was noted in all Salmonella typhimurium strains with and without metabolic activation.

 

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

 

Based on the results of this study, the test item Azuril [Reaction mass of 3-(4-methyl-3pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3ene-1-carbonitrile] did not induce reverse mutations at selected loci of four strains of Salmonella typhimurium (TA1535, TA100, TA1537, TA98 N) and the Escherichia coli WP2 uvr strain (a total of five bacterial strains) in the presence and absence of activated rat liver S9 fraction.