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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471 (in vitro Bacterial reverse mutation test): Negative (±S9)

OECD 487 (in vitro Micronucleus Test): Negative (±S9)

OECD 490 (in vitro mouse lymphoma assay): Negative (±S9)

BlueScreen (in vitro human lymphoblastoid TK6 cells (GLuc-T01) assay): Negative (±S9)

Link to relevant study records

Reference

Reference 1

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

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.

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.

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

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

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 / 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

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.

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

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

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

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

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

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-07-13 to 2019-04-30

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)

Deviations:

yes

Remarks:

deviation was considered without any impact on the validity or integrity of the current study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Emerald Kalama Chemical Ltd (UK); Lot# A170421E
- Expiration date of the lot/batch: 2019-06-06
- Purity test date: 2018-02-07

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature; Protected from humidity; Under nitrogen atmosphere
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: The test item was diluted in the vehicle at concentrations of:
- 378.60 mg/mL for the preliminary cytotoxicity test,
- 22.716 mg/mL for the first experiment and for 24-hour treatment of the second experiment,
- 7.572 mg/mL for the 3-hour treatments of the second experiment.

The stock solutions and their dilutions were prepared within 4 hours before use, and then kept at room temperature until use.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Clear, pale yellow liquid

Purity: 92%

Target gene:

mouse cell line L5178Y TK+/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC (American Type Culture Collection, Manassas, USA)
- Suitability of cells: L5178Y TK+/- cells are an established cell line recommended by international regulations for in vitro mammalian cell gene mutation test and for in vitro micronucleus test. Indeed, they are suitable to reveal chemically induced micronuclei.
- Cell cycle length, doubling time or proliferation index: average cell cycle time is approximately 10-12 hours.
- Methods for maintenance in cell culture if applicable: cells were stored in a cryoprotective medium (10% horse serum and 10% dimethylsulfoxide (DMSO)) at -80°C or in liquid nitrogen (depending on the batch; see § Study plan adherence), and each batch of frozen cells was checked for the absence of mycoplasma and for the modal chromosome number.
- Modal number of chromosomes: 40

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cell cultures were grown at 37°C in a humidified atmosphere of 5% CO2/95% air in culture medium. The culture medium was RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL). This medium was supplemented by heat inactivated horse serum at 10% (v/v).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: Not specified
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver post-mitochondrial fraction (S9 fraction)

Test concentrations with justification for top dose:

Preliminary cytotoxicity test: To assess the cytotoxicity of the test item, eight dose levels (one culture/dose level) were tested both with and without metabolic activation as follows: 0.016, 0.041, 0.10, 0.26, 0.64, 1.6, 4 and 10 mM.

Main Experiment:
Short treatments without S9 mix (-S9):
0.002, 0.005, 0.009, 0.019, 0.038, 0.075, 0.15 and 0.3 mM (first experiment)
0.0025, 0.0050, 0.0100, 0.0125, 0.0188, 0.025, 0.0375, 0.050, 0.075 and 0.10 mM (second experiment)

Continuous treatments without S9 mix (-S9):
0.001, 0.002, 0.005, 0.009, 0.019, 0.038, 0.075 and 0.15 mM (first experiment)
0.0031, 0.0063, 0.0125, 0.02, 0.025, 0.03, 0.0375 and 0.05 mM (second experiment)

Treatments with S9 mix (+S9):
0.005, 0.009, 0.019, 0.038, 0.075, 0.15, 0.3 and 0.6 mM (first experiment)
0.0063, 0.0125, 0.0188, 0.025, 0.0375, 0.050, 0.075, 0.10, 0.15 and 0.20 mM (second experiment)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: According to available solubility data, the vehicle used for the preparation of test item dose formulations and the treatment of vehicle control cultures was dimethylsulfoxide (DMSO).

Note: positive controls were dissolved in water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 3 x 10^5 cells/mL

DURATION
- Exposure duration: 3 hours (± S9) and 24 hours (-S9)
- Expression time (cells in growth medium): 24 or 27 hours depending on the treatment and recovery period

SPINDLE INHIBITOR (cytogenetic assays): Colchicine

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2 (Duplicate)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
After the final cell counting, the cells were washed with culture medium containing 10% inactivated horse serum and 1% pluronic acid. The cells were suspended in 49.5% culture medium containing 10% inactivated horse serum, 50% PBS and 0.5% pluronic acid, before being fixed. Following the fixation, the cells were kept at 5°C for at least an overnight period.

Depending on the observation at the end of the recovery period (presence or absence of precipitate and/or cytotoxicity), at least three dose levels of the test item-treated cultures were selected for spreading on slides. Cells were dropped onto clean glass slides. The slides were air dried before being stained for approximately 15 min in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above.

NUMBER OF CELLS EVALUATED: 1000 mononucleated cells per culture

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 2000 mononucleated cells per dose

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Analysis was performed by microscopic evaluation, on the basis of the recommendations of Miller et al. (1995) (e), according to the following criteria:
• micronuclei should be located within the cytoplasma of the cell,
• micronuclei should be clearly surrounded by a nuclear membrane,
• micronuclei should be round or oval in shape,
• the micronucleus area should be less than one-third of the area of the main nucleus,
• micronuclei should be non-refractile (can be distinguished from artefacts such as staining particles),
• micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
• micronuclei should have similar staining intensity to that of the main nuclei,
• micronuclei may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary,
• only mononucleated cells with a number of micronuclei ≥ 5 will be scored to exclude apoptosis and nuclear fragmentation.

Number of cells with micronuclei and number of micronuclei per cell were given separately for each treated and control culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Population Doubling (PD)
- Any supplementary information relevant to cytotoxicity: The cytotoxicity induced by a treatment was evaluated by the decrease in the PD, when compared to the vehicle control (Mean % PD of the vehicle control set to 100%).

Decrease in PD (%) = 100 - Mean PD as % of control

Rationale for test conditions:

The objective of this study was to evaluate the potential of the test material, to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/-.

The micronuclei observed in the cytoplasm of interphase cells may originate from acentric fragments (chromosome fragments lacking a centromere) or whole chromosomes that are unable to migrate with the rest of the chromosomes during the anaphase of cell division. The assay thus has the potential to detect the activity of both clastogenic and aneugenic chemicals. The test was performed in the presence and absence of a rat liver metabolizing system (S9 mix).

Evaluation criteria:

Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:

• a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test,
• for at least one dose level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
• a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose levels.

Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met.

The biological relevance of the results was always taken into account when evaluating results.

Statistics:

For each condition of the cytogenetic experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures.

This comparison was performed using the X2 test, unless treated culture data are lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance. This statistical analysis was performed using a validated Excel sheet.

To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose levels. This statistical analysis was performed using SAS Enterprise Guide software.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
Preliminary Cytotoxicity Test: At the highest dose level of 10 mM, the pH of the culture medium was approximately 7.4 (as for the vehicle control) and the osmolality was 344 mOsm/kg H2O (377 mOsm/kg H2O for the vehicle control). Therefore, this dose level could be selected as the highest dose level for the main experiment, and none of the tested dose levels was considered to produce extreme culture conditions.

- Effects of osmolality:
Preliminary Cytotoxicity Test: At the highest dose level of 10 mM, the pH of the culture medium was approximately 7.4 (as for the vehicle control) and the osmolality was 344 mOsm/kg H2O (377 mOsm/kg H2O for the vehicle control). Therefore, this dose level could be selected as the highest dose level for the main experiment, and none of the tested dose levels was considered to produce extreme culture conditions.

- Water solubility:
Preliminary Cytotoxicity Test: An emulsion was observed in the culture medium at the end of the 3-hour treatment periods, at dose levels  0.64 mM.

- Precipitation:
Preliminary Cytotoxicity Test: A precipitate was observed in the culture medium at 10 mM at the end of the 3-hour treatment periods, and at dose levels  4 mM at the end of the 24-hour treatment period.

- Definition of acceptable cells for analysis: Analysis was performed by microscopic evaluation, on the basis of the recommendations of Miller et al. (1995) (e), according to the following criteria:
• micronuclei should be located within the cytoplasma of the cell,
• micronuclei should be clearly surrounded by a nuclear membrane,
• micronuclei should be round or oval in shape,
• the micronucleus area should be less than one-third of the area of the main nucleus,
• micronuclei should be non-refractile (can be distinguished from artefacts such as staining particles),
• micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
• micronuclei should have similar staining intensity to that of the main nuclei,
• micronuclei may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary,
• only mononucleated cells with a number of micronuclei ≥ 5 will be scored to exclude apoptosis and nuclear fragmentation.

Number of cells with micronuclei and number of micronuclei per cell were given separately for each treated and control culture.

RANGE-FINDING/SCREENING STUDIES: Preliminary Cytotoxicity Test:
Following the 3-hour treatment without S9 mix, a moderate to severe cytotoxicity was observed at dose levels ≥ 0.016 mM, as shown by a 46 to 100% decrease in the PD.
Following the 24-hour treatment without S9 mix, a slight to severe cytotoxicity was observed at dose levels ≥ 0.016 mM, as shown by a 31 to 100% decrease in the PD.
Following the 3-hour treatment with S9 mix, a moderate to severe cytotoxicity was observed at dose levels ≥ 0.041 mM, as shown by a 57 to 100% decrease in the PD.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: See Table 3 and 4
- Negative (solvent/vehicle) historical control data: See Tables 3 and 4

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Population Doubling (PD)

Main Experiment 1:

Short treatments without S9 mix (-S9):
Cytotoxicity
In the first experiment, a severe cytotoxicity was induced at dose levels ≥ 0.075 mM, as shown by a 99 to 100% decrease in the PD. In the second experiment, a moderate to severe cytotoxicity was induced at dose levels ≥ 0.0125 mM, as shown by a 49 to 100% decrease in the PD.

Micronucleus analysis
The dose levels selected for micronucleus analysis were as follows:
- 0.009, 0.019 and 0.038 mM for the first experiment, the latter inducing only a 28% decrease in the PD but higher dose levels being too cytotoxic,

- 0.0050, 0.0100 and 0.0125 mM for the second experiment, the latter inducing only a 49% decrease in the PD but higher dose levels being too cytotoxic.

In the first experiment, no statistically significant increase in the frequency of micronucleated cells was observed at any of the analysed dose levels relative to the corresponding vehicle control (p>0.05). No dose response relationship was demonstrated by the linear regression (p>0.05) and none of the analysed dose levels showed frequencies of each replicate culture above the vehicle control historical range.

Since the recommended level of cytotoxicity was not reached, a second experiment was undertaken using a narrower range of dose levels.

In the second experiment, no statistically significant increase in the frequency of micronucleated cells was observed at any of the analysed dose levels relative to the corresponding vehicle control (p>0.05). No dose-response relationship was demonstrated by the linear regression (p>0.05) and none of the analysed dose levels showed frequencies of each replicate culture above the vehicle control historical range.

Although none of the selected dose levels induced the recommended level of cytotoxicity, considering the narrow dose levels spacing used in the second experiment, the results were considered as suitable to allow a reliable interpretation, and the overall results are considered to meet the criteria for a negative response.

Continuous treatments without S9 mix (-S9):
Cytotoxicity
In the first experiment, a slight to severe cytotoxicity was induced at dose levels ≥ 0.019 mM, as shown by a 34 to 100% decrease in the PD.

In the second experiment, a moderate to severe cytotoxicity was induced at dose levels ≥ 0.0125 mM, as shown by a 42 to 100% decrease in the PD.

Micronucleus analysis
The dose levels selected for micronucleus analysis were as follows:
- 0.005, 0.009 and 0.019 mM for the first experiment, the latter inducing only a 34% decrease in the PD but higher dose levels being too cytotoxic,

- 0.0063, 0.0125 and 0.02 mM for the second experiment, the latter inducing the recommended level of cytotoxicity (i.e. 59% decrease in the PD).

In the first experiment, no statistically significant increase in the frequency of micronucleated cells was observed at any of the analysed dose levels relative to the corresponding vehicle control (p>0.05), and no dose-response relationship was demonstrated by the linear regression (p>0.05).

However, the frequencies of both replicate culture at the highest analysed dose level of 0.019 mM were slightly above the vehicle control historical range (i.e. 7.0‰ versus [0.0-5.0] for the vehicle control). These results did not meet the criteria for a negative or positive response.

In order to check the reproducibility of this increase and to reach the recommended level of cytotoxicity, a second experiment was undertaken using a narrower range of dose levels.

In the second experiment, no statistically significant increase in the frequency of micronucleated cells was observed in the second experiment, at any of the analysed dose levels relative to the corresponding vehicle control (p>0.05). No dose-response relationship was demonstrated by the linear regression (p>0.05) and, unlike what was observed in the first experiment, none of the analysed dose levels showed frequencies of each replicate culture above the vehicle control historical range.

Since the increase in the frequency of micronucleated cells observed in the first experiment was not reproduced in the second experiment despite the use of a narrower range of dose levels, this increase is considered not to be biologically relevant, and the overall results are considered to meet the criteria for a negative response.

Treatments with S9 mix (+S9):
Cytotoxicity
In the first experiment, a slight to severe cytotoxicity was induced at dose levels ≥ 0.075 mM, as shown by a 34 to 80% decrease in the PD.

In the second experiment, a moderate to severe cytotoxicity was induced at dose levels ≥ 0.0188 mM, as shown by a 47 to 100% decrease in the PD.

Micronucleus analysis
The dose levels selected for micronucleus analysis were as follows:
- 0.038, 0.075 and 0.15 mM for the first experiment, the latter inducing only a 45% decrease in the PD but higher dose levels being too cytotoxic,

- 0.0063, 0.0125 and 0.0188 mM for the second experiment, the latter inducing the recommended level of cytotoxicity (i.e. 57% decrease in the PD).

In the first experiment, no statistically increase in the frequency of micronucleated cells was observed at any of the analysed dose levels relative to the corresponding vehicle control (p>0.05) and no dose response relationship was demonstrated by the linear regression (p>0.05).

However, the frequencies of both replicate culture at the highest analysed dose level of 0.15 mM were slightly above the vehicle control historical range (i.e. 4.0‰ versus [0.0-3.5] for the vehicle control). These results did not meet the criteria for a negative or positive response.

In order to check the reproducibility of this increase and to reach the recommended level of cytotoxicity, a second experiment was undertaken using a narrower range of dose levels. In the second experiment, no statistically significant increase in the frequency of micronucleated cells was observed at any of the analysed dose levels relative to the corresponding vehicle control (p>0.05). No dose-response relationship was demonstrated by the linear regression (p>0.05) and none of the analysed dose levels showed frequencies of each replicate culture above the vehicle control historical range.

Since the increase in the frequency of micronucleated cells observed in the first experiment was not reproduced in the second experiment despite the use of a narrower range of dose levels, this increase is considered not to be biologically relevant, and the overall results in the presence of metabolic activation are considered to meet the criteria for a negative response.

The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

Table 3. Preliminary cytotoxicity test
Treatment condition	Treatment	Cell concentration used for treatment ( x 10^4cells/mL)	Post-treatment cell count ( x 10^4cells/mL)	PD	PD as % of control	Decrease in PD
Without S9 Mix:   3h-Treatment + 24-h Recovery	Vehicle Control	30	84.5	1.5	100
	Test Item (mM)
	0.016	30	32.0	0.1	6	94 (too cytotoxic)
	0.041	30	52.5	0.8	54	46
	0.10	30	4.7	#	#	#
	0.26	30	1.5	#	#	#
	0.64 E	30	0.2	#	#	#
	1.6 E	30	0.0	#	#	#
	4 E	30	0.0	#	#	#
	10 P + E	30	0.0	#	#	#
Without S9 Mix:   24h-Treatment + 0-h Recovery	Vehicle Control	30	76.5	1.4	100
	Test Item (mM)
	0.016	30	57.0	0.9	69	31
	0.041	30	21.6	#	#	#
	0.10	30	9.9	#	#	#
	0.26	30	0.0	#	#	#
	0.64	30	0.0	#	#	#
	1.6	30	0.0	#	#	#
	4 P	30	0.0	#	#	#
	10 P	30	0.0	#	#	#
With S9 Mix:   3h-Treatment + 24-h Recovery	Vehicle Control	30	82.0	1.5	100
	Test Item (mM)
	0.016	30	77.5	1.4	94	6
	0.041	30	43.3	0.5	36	64 (too cytotoxic)
	0.10	30	34.0	0.2	12	88 (too cytotoxic)
	0.26	30	39.0	0.4	26	74 (too cytotoxic)
	0.64 E	30	29.3	#	#	#
	1.6 E	30	46.0	0.6	43	57
	4 E	30	20.0	#	#	#
	10 P + E	30	6.0	#	#	#

PD: Population Doubling

Vehicle control: DMSO

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment

P: precipitate was noted in the culture medium at the end of treatment

E: emulsion was noted in the culture medium at the end of treatment

Table 4. First experiment - Cytotoxicity
Treatment	Cell concentration used for treatment ( x 10^4cells/mL)	Culture	Post-treatment cell count ( x 10^4cells/mL)	Mean PD	Mean PD as % of control	Decrease in PD (%)
Short treatment without S9 mix (3-h treatment + 24-h recovery)
Vehicle Control	30	C1	79.5	1.4	100
		C2	83.0
Test Item (mM)
0.002	30	C1	82.0	1.6	111	None
		C2	100
0.005	30	C1	79.0	1.9	98	2
		C2	81.0
0.009	30	C1	74.0	1.2	86	14
		C2	68.0
0.019	30	C1	64.0	1.2	81	19
		C2	70.0
0.038	30	C1	61.0	1.0	72	28
		C2	62.5
0.075	30	C1	23.2	0.0	1.0	99 (too cytotoxic)
		C2	37.3
0.15	30	C1	8.1	#	#	#
		C2	21.0
0.3	30	C1	1.1	#	#	#
		C2	0.7
Positive controls
MMC (1 μg/mL )	30	C1	35.3	0.3	18	82
		C2	36.3
COL (0.5 μg/mL)	30	C1	18.7	#	#	#
		C2	22.6
Continuous treatment without S9 mix (24-h treatment + 0-h recovery)
Vehicle Control	30	C1	95.0	1.5	100
		C2	70.5
Test Item (mM)
0.001	30	C1	80.0	1.5	100	None
		C2	86.0
0.002	30	C1	88.0	1.4	98	2
		C2	75.0
0.005	30	C1	77.5	1.3	90	10
		C2	72.5
0.009	30	C1	101.0	1.6	109	None
		C2	79.5
0.019	30	C1	44.0	1.0	66	34
		C2	73.5
0.038	30	C1	49.7	0.4	25	75 (too cytotoxic)
		C2	27.5
0.075	30	C1	19.8	#	#	#
		C2	17.5
0.15	30	C1	28.0	#	#	#
		C2	14.9
Positive Controls
MMC (1 μg/mL )	30	C1	31.5	0.1	8	92
		C2	33.5
COL (0.5 μg/mL)	30	C1	25.5	#	#	#
		C2	24.2
With S9 mix (3-h treatment + 24-h recovery)
Vehicle Control	30	C1	129.0	2.2	100
		C2	139.0
Test Item (mM)
0.005	30	C1	134.0	2.0	91	9
		C2	101.0
0.009	30	C1	114.0	2.1	98	2
		C2	146.0
0.019	30	C1	135.0	2.3	105	None
		C2	155.0
0.038	30	C1	123.0	2.1	96	4
		C2	130.0
0.075	30	C1	84.0	1.4	66	34
		C2	77.0
0.15	30	C1	60.0	1.2	55	45
		C2	76.5
0.3	30	C1	38.3	0.4	20	80 (too cytotoxic)
		C2	42.7
0.6	30	C1	52.5	0.9	41	59
		C2	58.5
Positive Controls
CPA (6 μg/mL )	30	C1	76.0	1.7	77	23
		C2	113.0

PD: population doubling

Vehicle control : DMSO

MMC: Mitomycin

CCOL: Colchicine

C1: Culture 1

C2: Culture 2

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment

 

Table 5. Second experiment - Cytotoxicity
Treatment	Cell concentration used for treatment ( x 10^4cells/mL)	Culture	Post-treatment cell count ( x 10^4cells/mL)	Mean PD	Mean PD as % of control	Decrease in PD (%)
Short treatment without S9 mix (3-h treatment + 24-h recovery)
Vehicle Control	30	C1	136.0	2.0	100
		C2	109.0
Test Item (mM)
0.0025	30	C1	90.0	1.8	87	13
		C2	113.0
0.0050	30	C1	92.0	1.7	83	17
		C2	100.0
0.0100	30	C1	97.5	1.5	75	25
		C2	75.5
0.0125	30	C1	64.5	1.0	51	49
		C2	59.0
0.0188	30	C1	50.3	0.5	24	76 (too cytotoxic)
		C2	34.0
0.025	30	C1	50.5	0.8	38	62 (too cytotoxic)
		C2	52.0
0.0375	30	C1	46.0	0.5	23	77 (too cytotoxic)
		C2	37.0
0.050	30	C1	8.0	#	#	#
		C2	14.0
0.075	30	C1	3.9	#	#	#
		C2	11.7
0.10 E	30	C1	6.9	#	#	#
		C2	6.8
Positive controls
MMC (1 μg/mL )	30	C1	38.0	0.3	13	87
		C2	33.7
COL (0.5 μg/mL)	30	C1	23.6	#	#	#
		C2	30.0
Continuous treatment without S9 mix (24-h treatment + 0-h recovery)
Vehicle Control	30	C1	72.5	1.2	100
		C2	62.5
Test Item (mM)
0.0031	30	C1	66.5	1.1	92	8
		C2	63.5
0.0063	30	C1	66.0	1.2	96	4
		C2	68.5
0.0125	30	C1	50.5	0.7	57	43
		C2	46.3
0.020	30	C1	37.3	0.5	41	59
		C2	47.3
0.025	30	C1	33.3	0.3	28	72 (too cytotoxic)
		C2	42.3
0.03	30	C1	50.5	0.7	58	42
		C2	47.3
0.0375	30	C1	40.3	0.4	32	68 (too cytotoxic)
		C2	38.3
0.05	30	C1	4.8	#	#	#
		C2	33.3
Positive controls
MMC (1 μg/mL )	30	C1	31.8	#	#	#
		C2	27.0
COL (0.5 μg/mL)	30	C1	20.2	#	#	#
		C2
With S9 mix (3-h treatment + 24-h recovery)
Vehicle Control	30	C1	116.0	1.9	100
		C2	101.5
Test Item (mM)
0.063	30	C1	87.5	1.7	92	8
		C2	109.0
0.0125	30	C1	76.0	1.5	79	21
		C2	90.0
0.0188	30	C1	50.0	0.8	43	57
		C2	54.0
0.025	30	C1	41.0	1.0	53	47
		C2	78.0
0.0375	30	C1	33.8	0.5	26	74 (too cytotoxic)
		C2	50.0
0.050	30	C1	53.5	0.9	46	54
		C2	55.5
0.075	30	C1	26.3	0.4	22	78 (too cytotoxic)
		C2	53.0
0.10 E	30	C1	29.0	0.3	14	86 (too cytotoxic)
		C2	42.7
0.15 E	30	C1	21.2	#	#	#
		C2	22.2
0.20 E	30	C1	22.8	#	#	#
		C2	27.3
Positive Controls
CPA (6 μg/mL)	30	C1	60.0	1.2	62	38
		C2	74.0

PD: population doubling

Vehicle control : DMSO

MMC: Mitomycin

CCOL: Colchicine

C1: Culture 1

C2: Culture 2

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment

E: Emulsion was noted in the culture medium at the end of treatment

Table 6. First experiment - Cytogenetic Results
Treatment	Mean PD as % of Control	Culture	No. of Cells Analysed	Number of Cells with n Micronuclei					Total Micronucleated Cells		Frequency of Micronucleated Cells (‰)
				n = 1	n = 2	n = 3	n = 4	n = 5	Per culture	Per dose	Per culture	Per dose
Short treatment without S9 mix (3-h treatment + 24-h recovery)
Vehicle Control	100	C1	1000	2	0	0	0	0	2	4	2.0	2.0
		C2	1000	2	0	0	0	0	2		2.0
Test Item (mM)
0.002	111	C1
		C2
0.005	98	C1
		C2
0.009	86	C1	1000	3	0	0	0	0	3	4	3.0	2.0
		C2	1000	1	0	0	0	0	1		1.0
0.019	81	C1	1000	1	0	0	0	0	1	3	1.0	1.5
		C2	1000	2	0	0	0	0	2		2.0
0.038	72	C1	1000	2	0	0	0	0	2	5	2.0	2.5
		C2	1000	3	0	0	0	0	3		3.0
0.075	1	C1
		C2
0.15	#	C1
		C2
0.3	#	C1
		C2
Positive Controls
MMC (1 µg/mL)	18	C1	1000	120	25	4	0	0	149	267	149.0	133.5 ***
		C2	1000	94	22	2	0	0	118		118.0
COL (0.5 µg/mL)	#	C1	1000	63	6	0	0	0	69	134	69.0	67.0 ***
		C2	1000	57	8	0	0	0	65		65.0
Continuous treatment without S9 mix (24-h treatment + 0-h recovery)
Vehicle Control	100	C1	1000	3	0	0	0	0	3	6	3.0	3.0
		C2	1000	3	0	0	0	0	3		3.0
Test Item (mM)
0.001	100	C1
		C2
0.002	98	C1
		C2
0.005	90	C1	1000	2	0	0	0	0	2	5	2.0	2.5
		C2	1000	3	0	0	0	0	3		3.0
0.009	109	C1	1000	1	0	0	0	0	1	3	1.0	1.5
		C2	1000	2	0	0	0	0	2		2.0
0.019	66	C1	1000	7	0	0	0	0	7	14	7.0	7.0
		C2	1000	7	0	0	0	0	7		7.0
0.038	25	C1
		C2
0.075	#	C1
		C2
0.15	#	C1
		C2
Positive Controls
MMC (1 µg/mL)	8	C1	1000	37	13	0	0	0	50	91	50.0	45.5 ***
		C2	1000	33	8	0	0	0	41		41.1
COL (0.5 µg/mL)	#	C1	1000	72	7	1	0	0	80	148	80.0	74.0 ***
		C2	1000	64	4	0	0	0	68		68.0
With S9 mix (3-h treatment + 24-h recovery)
Vehicle Control	100	C1	1000	2	0	0	0	0	2	4	2.0	2.0
		C2	1000	2	0	0	0	0	2		2.0
Test Item (mM)
0.005	91	C1
		C2
0.009	98	C1
		C2
0.019	105	C1
		C2
0.038	96	C1	1000	2	0	0	0	0	2	4	2.0	2.0
		C2	1000	2	0	0	0	0	2		2.0
0.075	66	C1	1000	1	0	0	0	0	1	4	1.0	2.0
		C2	1000	3	0	0	0	0	3		3.0
0.15	55	C1	1000	4	0	0	0	0	4	8	4.0	4.0
		C2	1000	4	0	0	0	0	4		4.0
0.3	20	C1
		C2
0.6	41	C1
		C2
Positive Controls
CPA (6 µg/mL)	77	C1	1000	39	3	1	0	0	43	77	43.0	38.5 ***
		C2	1000	32	2	0	0	0	34		34.0

PD: population doubling

Statistics: 2 x 2 contingency table: ***: p < 0.001

Vehicle control: DMSO

MMC: Mitomycin C

COL: Colchicine

CPA: Cyclophosphamide

C1: Culture 1

C2: Culture 2

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment

 

Table 7. Second experiment - Cytogenetic Results
Treatment	Mean PD as % of Control	Culture	No. of Cells Analysed	Number of Cells with n Micronuclei					Total Micronucleated Cells		Frequency of Micronucleated Cells (‰)
				n = 1	n = 2	n = 3	n = 4	n = 5	Per culture	Per dose	Per culture	Per dose
Short treatment without S9 mix (3-h treatment + 24-h recovery)
Vehicle Control	100	C1	1000	3	0	0	0	0	3	3	3.0	1.5
		C2	1000	0	0	0	0	0	0		0.0
Test Item (mM)
0.0025	87	C1
		C2
0.0050	83	C1	1000	1	0	0	0	0	1	3	1.0	1.5
		C2	1000	1	1	0	0	0	2		2.0
0.0100	75	C1	1000	0	0	0	0	0	0	1	0.0	0.5
		C2	1000	1	0	0	0	0	1		1.0
0.0125	51	C1	1000	0	0	0	0	0	0	1	0.0	0.5
		C2	1000	1	0	0	0	0	1		1.0
0.0188	24	C1
		C2
0.025	38	C1
		C2
0.0375	23	C1
		C2
0.050	#	C1
		C2
0.075	#	C1
		C2
0.10 E	#	C1
		C2
Positive Controls
MMC (1 µg/mL)	13	C1	1000	31	1	0	0	0	32	41	32.0	20.5 ***
		C2	1000	9	0	0	0	0	9		9.0
COL (0.5 µg/mL)	#	C1	1000	8	0	0	0	0	8	18	8.0	9.0 **
		C2	1000	10	0	0	0	0	10		10.0
Continuous treatment without S9 mix (24-h treatment + 0-h recovery)
Vehicle Control	100	C1	1000	1	0	0	0	0	1	2	1.0	0.5
		C2	1000	1	0	0	0	0	1		1.0
Test Item (mM)
0.0031	92	C1
		C2
0.0063	96	C1	1000	1	1	0	0	0	1	4	1.0	2.0
		C2	1000	3	0	0	0	0	3		3.0
0.0125	57	C1	1000	0	0	0	0	0	0	1	0.0	0.5
		C2	1000	1	0	0	0	0	1		1.0
0.02	41	C1	1000	0	0	0	0	0	0	1	0.0	0.5
		C2	1000	1	0	0	0	0	1		1.0
0.025	28	C1
		C2
0.03	58	C1
		C2
0.0375	32	C1
		C2
0.05	#	C1
		C2
Positive Controls
MMC (1 µg/mL)	#	C1	1000	18	2	0	0	0	20	40	20.0	20.0 ***
		C2	1000	20	0	0	0	0	20		20.0
COL (0.5 µg/mL)	#	C1	1000	33	0	0	0	0	33	52	33.0	26.0 ***
		C2	1000	19	0	0	0	0	19		19.0
With S9 mix (3-h treatment + 24-h recovery)
Vehicle Control	100	C1	1000	0	0	0	0	0	0	1	0.0	0.5
		C2	1000	1	0	0	0	0	1		1.0
Test Item (mM)
0.0063	92	C1	1000	1	0	0	0	0	1	2	1.0	1.0
		C2	1000	1	0	0	0	0	1		1.0
0.0125	79	C1	1000	2	0	0	0	0	2	4	2.0	2.0
		C2	1000	2	0	0	0	0	2		2.0
0.0188	43	C1	1000	0	0	0	0	0	0	1	0.0	0.5
		C2	1000	1	0	0	0	0	0		1.0
0.025	53	C1
		C2
0.0375	26	C1
		C2
0.050	46	C1
		C2
0.075	22	C1
		C2
0.10 E	14	C1
		C2
0.15 E	#	C1
		C2
0.20 E	#	C1
		C2
Positive Controls
CPA (6 µg/mL)	62	C1	1000	27	0	0	0	0	27	63	27.0	31.5 ***
		C2	1000	36	0	0	0	0	36		36.0

PD: population doubling

Statistics: 2 x 2 contingency table: **: p < 0.01; ***: p < 0.001

Vehicle control: DMSO

MMC: Mitomycin C

COL: Colchicine

CPA: Cyclophosphamide

C1: Culture 1

C2: Culture 2

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment

E: emulsion was noted in the culture medium at the end of treatment

Table 8. Historical Data – Experiments without S9 Mix
Parameter	Frequency of Micronucleated cells in 1000 cells
Treatment Conditions	3 hours treatment + 24 hours recovery			24 hours treatment + 0 hours recovery
Control Items	Vehicle Control	MMC (1 µg/mL)	COL (0.5 µg/mL)	Vehicle Control	MMC (1 µg/mL)	COL (0.5 µg/mL)
N	50	50	50	28	28	28
Mean	1.7	133.0	22.9	2.1	46.4	34.5
SD	1.0	67.5	13.3	1.1	19.8	17.5
Lower CL 95%	1.4	113.8	19.1	1.6	38.7	27.7
Upper CL 95%	1.9	152.2	26.6	2.5	54.1	41.2
5^thPercentile	0.5	23.0	7.5	0.0	11.5	14.5
Median	1.5	138.3	20.0	2.0	49.5	27.0
95^th Percentile	4.0	238.5	54.5	3.5	72.5	65.5
Min	0.0	18.0	5.5	0.0	8.0	10.0
Max	4.5	306.0	65.0	5.0	97.5	75.0

COL: Colchicine

MMC: Mitomycin C

CL: Confidence Limit

Max: Maximal Value

SD: Standard Deviation

Min: Minimal Value

N – number of values

 

Table 9. Historical Data – Experiments with S9 Mix
Parameter	Frequency of Micronucleated cells in 1000 cells
Treatment Conditions	3 hours treatment + 24 hours recovery
Control Items	Vehicle Control	CPA (6 µg/mL)
N	67	67
Mean	1.5	101.9
SD	0.9	49.3
Lower CL 95%	1.3	89.8
Upper CL 95%	1.7	113.9
5^thPercentile	0.5	26.0
Median	1.5	105.5
95^thPercentile	3.0	184.5
Min	0.0	14.0
Max	3.5	251.0

CPA: Cyclophosphamide

CL: Confidence Limit

Max: Maximal Value

SD: Standard Deviation

Min: Minimal Value

N – number of values

Conclusions:

The test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile]) did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

A Key OECD Guideline 487 in vitro study was conducted to evaluate the potential of the test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile) to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/-.

 

After a preliminary cytotoxicity test, the test material diluted in dimethylsulfoxide (DMSO), was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

 

Without S9 Mix (-S9): First and Second Experiment: 3 h treatment + 24 h recovery and 24 h treatment + 0 h recovery.

 

With S9 Mix (+S9): First and Second Experiment: 3 h treatment + 24 h recovery

 

Each treatment was coupled to an assessment of cytotoxicity at the same dose levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.

 

After the final cell counting, the cells were washed and fixed. Then, cells from at least three dose levels of the test item treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

 

Since the test item was found to be cytotoxic in the preliminary test, the highest dose level selected for the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international regulations.

 

The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

An emulsion was observed in the culture medium at the end of the 3-hour treatment periods at dose levels ≥ 0.10 mM in the second experiment.

 

Short treatments without S9 mix

With a treatment volume of 0.5% (v/v) in culture medium, the dose levels selected for the treatments were as follows:

- 0.002, 0.005, 0.009, 0.019, 0.038, 0.075, 0.15 and 0.3 mM for the first experiment,

- 0.0025, 0.0050, 0.0100, 0.0125, 0.0188, 0.025, 0.0375, 0.050, 0.075 and 0.10 mM for the second experiment.

 

Cytotoxicity

In the first experiment, a severe cytotoxicity was induced at dose levels ≥ 0.075 mM.

In the second experiment, a moderate to severe cytotoxicity was induced at dose levels ≥ 0.0125 mM.

 

Micronucleus analysis

The dose levels selected for micronucleus analysis were as follows:

- 0.009, 0.019 and 0.038 mM for the first experiment, the latter inducing only a 28% decrease in the PD but higher dose levels being too cytotoxic,

- 0.0050, 0.0100 and 0.0125 mM for the second experiment, the latter inducing only a 49% decrease in the PD but higher dose levels being too cytotoxic.

 

In the first experiment, no statistically significant increase in the frequency of micronucleated cells was observed at any of the analysed dose levels relative to the corresponding vehicle control. No dose response relationship was demonstrated by the linear regression and none of the analysed dose levels showed frequencies of each replicate culture above the vehicle control historical range.

 

Since the recommended level of cytotoxicity was not reached, a second experiment was undertaken using a narrower range of dose levels, and confirmed the results obtained in the first one.

 

Although none of the selected dose levels induced the recommended level of cytotoxicity in either experiment despite the use of a narrower range of dose levels, since both assays met the criteria for a negative response, the results were considered as suitable to allow a reliable interpretation.

 

Continuous treatments without S9 mix

With a treatment volume of 0.5% (v/v) in culture medium, the dose levels selected for the treatments were as follows:

- 0.001, 0.002, 0.005, 0.009, 0.019, 0.038, 0.075 and 0.15 mM for the first experiment,

- 0.0031, 0.0063, 0.0125, 0.02, 0.025, 0.03, 0.0375 and 0.05 mM for the second experiment.

 

Cytotoxicity

In the first experiment, a slight to severe cytotoxicity was induced at dose levels ≥ 0.019 mM.

In the second experiment, a moderate to severe cytotoxicity was induced at dose levels ≥ 0.0125 mM.

 

Micronucleus analysis

The dose levels selected for micronucleus analysis were as follows:

- 0.005, 0.009 and 0.019 mM for the first experiment, the latter inducing only a 34% decrease in the PD but higher dose levels being too cytotoxic,

- 0.0063, 0.0125 and 0.02 mM for the second experiment, the latter inducing the recommended level of cytotoxicity.

 

In the first experiment, no statistically significant increase in the frequency of micronucleated cells was observed at any of the analysed dose levels relative to the corresponding vehicle control, and no dose response relationship was demonstrated by the linear regression.

 

However, the frequencies of both replicate culture at the highest analysed dose level of 0.019 mM were slightly above the vehicle control historical range. These results did not meet either the criteria for a negative or positive response.

 

In order to check the reproducibility of this increase and to reach the recommended level of cytotoxicity, a second experiment was undertaken using a narrower range of dose levels.

 

In the second experiment, no statistically significant increase in the frequency of micronucleated cells was observed in the second experiment, at any of the analysed dose levels relative to the corresponding vehicle control. No dose-response relationship was demonstrated by the linear regression and none of the analysed dose levels showed frequencies of each replicate culture above the vehicle control historical range.

 

Since the increase in the frequency of micronucleated cells observed in the first experiment was not reproduced in the second experiment despite the use of a narrower range of dose levels, this increase is considered not to be biologically relevant, and the overall results are considered to meet the criteria for a negative response.

 

Treatments with S9 mix

With a treatment volume of 0.5% (v/v) in culture medium, the dose levels selected for the treatments were as follows:

- 0.005, 0.009, 0.019, 0.038, 0.075, 0.15, 0.3 and 0.6 mM for the first experiment,

- 0.0063, 0.0125, 0.0188, 0.025, 0.0375, 0.050, 0.075, 0.10, 0.15 and 0.20 mM for the second experiment.

 

Cytotoxicity

In the first experiment, a slight to severe cytotoxicity was induced at dose levels ≥ 0.075 mM.

In the second experiment, a moderate to severe cytotoxicity was induced at dose levels ≥ 0.0188 mM.

 

Micronucleus analysis

The dose levels selected for micronucleus analysis were as follows:

- 0.038, 0.075 and 0.15 mM for the first experiment, the latter inducing only a 45% decrease in the PD but higher dose levels being too cytotoxic,

- 0.0063, 0.0125 and 0.0188 mM for the second experiment, the latter inducing the recommended level of cytotoxicity.

 

In the first experiment, no statistically increase in the frequency of micronucleated cells was observed at any of the analysed dose levels relative to the corresponding vehicle control and no dose-response relationship was demonstrated by the linear regression.

 

However, the frequencies of both replicate culture at the highest analysed dose level of 0.15 mM were slightly above the vehicle control historical range. These results did not meet either the criteria for a negative or positive response.

 

In order to check the reproducibility of this increase and to reach the recommended level of cytotoxicity, a second experiment was undertaken using a narrower range of dose levels.

In the second experiment, no statistically significant increase in the frequency of micronucleated cells was observed at any of the analysed dose levels relative to the corresponding vehicle control. No dose response relationship was demonstrated by the linear regression and none of the analysed dose levels showed frequencies of each replicate culture above the vehicle control historical range.

Since the increase in the frequency of micronucleated cells observed in the first experiment was not reproduced in the second experiment despite the use of a narrower range of dose levels, this increase is considered not to be biologically relevant, and the overall results in the presence of metabolic activation are considered to meet the criteria for a negative response.

 

Under the experimental conditions of the study, the test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile), did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-07-03 to 2018-12-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

yes

Remarks:

Deviations had no impact on the study on the results or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Emerald Kalama Chemical Limited (Dans Road WA8 0RF Widnes Cheshire, United Kingdom); Lot# A170421E
- Expiration date of the lot/batch: 2019-06-06
- Purity test date: 2018-02-07

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25 °C, ≤ 70 % relative humidity (RH)), under inert gas, protected from humidity (tightly closed container)
- Stability under test conditions: Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.
- Solubility and stability of the test substance in the solvent/vehicle: Based on the result of the preliminary solubility test, the 200 mg/mL concentration using Dimethyl sulfoxide (DMSO) was suitable for this study.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Clear, pale yellow liquid

Target gene:

tk+/- (thymidine kinase) locus in L5178Y cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection (Manassas, Virginia, USA); Lot# 60797977

- Suitability of cells: Cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK-/--mutants and checked for the absence of mycoplasma.

- Methods for maintenance in cell culture if applicable: For each experiment, one or more vials was thawed rapidly, cells were diluted in RPMI-10 medium and incubated at 37 ± 0.5°C in a humidified atmosphere containing approximately 5% CO2 in air. When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than four times before used in the main study).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: 3 types of RPMI 1640 medium (Table 1); 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital (PB) and β-naphthoflavone (BNF) induced post-mitochondrial fraction (S9 fraction)

Test concentrations with justification for top dose:

Treatment concentrations for the mutation assays were selected on the basis of the result of a short preliminary toxicity test. The highest concentration tested in the preliminary test was 2000 µg/mL (the recommended maximum concentration). The highest selected concentration for the main tests was based on the observed cytotoxicity. A total of 7 concentrations with metabolic activation were selected in Assay 1 and a total of 8 concentrations without metabolic activation were selected for Assay 1 (Table 2) and with and without metabolic activation for Assay 3 (Table 3).

Note: In the Assay 2 with and without metabolic activation an unexpected infection was observed on the plates / cultures. The observed effect caused major decrease in the cell numbers (even for untreated and negative controls), therefore the Assay 2 was stopped and it was considered to be invalid. Therefore, an additional experiment (Assay 3) was performed to provide fully valid, interpretable data. The experimental conditions and concentrations were the same as in the Assay 2 with and without metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Dimethyl sulfoxide)

- Justification for choice of solvent/vehicle: Based on the available information (trial formulation of the test item performed in an in vivo study at the Test Facility [17/181-001P]) Distilled water at 200 mg/mL concentration was not a suitable vehicle for this study. However, based on the result of the preliminary solubility test, the 200 mg/mL concentration using Dimethyl sulfoxide (DMSO) was suitable for this study. Thus, DMSO was selected for vehicle (solvent) of this study. This vehicle (solvent) was compatible with the survival of the cells and the S9 activity.

Note: Dimethyl sulfoxide was used for vehicle (solvent) of the test item and Dimethyl sulfoxide was used for vehicle (solvent) of the positive control chemicals

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 10^7 cells were placed in each of a series of 75 cm^2 sterile flasks

DURATION
- Exposure duration: Assay 1 (3 hours ±S9); Assay 2 (+S9: 3 hours; -S9: 24 hours)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 or 14 days (Due to technical reasons in Assay 3 the long treatment (24-hour treatment without metabolic activation) mutagenicity and viability microplates were incubated two days longer than the recommendation of the OECD No. 490 Guideline, however it was in accordance with the Study Plan, therefore this fact was not considered to be as a deviation from the Study Plan. Since this experiment was considered to be valid and the results considered to be negative, this fact had no impact on the results or integrity of the study)

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 (Duplicate cultures)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Plating for Survival
Cultures of cell density 2 x 10^5 cells/mL, were further diluted to 8 cells/mL. Using a multi-channel pipette, 0.2 mL of the final concentration of each culture were placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37ºC ± 0.5°C containing approximately 5% (v/v) CO2 in air for two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

Plating for Viability
At the end of the expression period, the cell density in the selected cultures was determined and adjusted to 1 x 10^4 cells/mL with RPMI-20 for plating for a viability test. Samples from these cultures were diluted to 8 cells/mL. Using a multi-channel pipette, 0.2 mL of the final concentration of each culture was placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37ºC ± 0.5°C containing approximately 5% (v/v) CO2 in air for approximately two weeks (12 days or 14 days). Wells containing viable clones were identified by eye using background illumination and counted.

Plating for 5-trifluorothymidine (TFT) resistance
At the end of the expression period, the cell concentration was adjusted to
1 x 10^4 cells/mL. TFT (300 µg/mL stock solution) was diluted 100-fold into these suspensions to give a final concentration of 3 µg/mL. Using a multi-channel pipette, 0.2 mL of each suspension was placed into each well of four, 96-well microplates (384 wells) at 2 x 10^3 cells per well.

Microplates were incubated at 37ºC ± 0.5°C containing approximately 5% (v/v) CO2 in air for approximately two weeks (12 days or 14 days) and wells containing clones were identified by eye and counted. In addition, scoring of large and small colonies was performed to obtain information on the possible mechanism of action of the test item, if any.

NUMBER OF CELLS EVALUATED: number of mutants per 10^6 viable cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG)
- Any supplementary information relevant to cytotoxicity: RTG was given as the percentage value of the treated cultures compared to the corresponding negative (vehicle/solvent) control value.

Rationale for test conditions:

While many mammalian cell gene mutation systems are available, however the mouse lymphoma assay (MLA), employing the tk+/- (thymidine kinase) locus in L5178Y cells, has the advantage of detecting both gene mutations and chromosome aberrations. The principle of this assay is based on placing cells under selective pressure so that only mutant cells are able to survive. The tk locus is autosomal and the L5178Y cell line is heterozygous (tk+/-) for the gene that produces the enzyme thymidine kinase. This enzyme is a salvage enzyme for nucleic acid breakdown products but if a toxic base analogue (5-trifluorothymidine) is present in the medium, the enzyme will incorporate the analogue into the cells. Thus, the cells will not survive unless the enzyme is rendered inactive, by mutation. Resistance to 5-trifluorothymidine (TFT) results in a lack of thymidine kinase (TK) activity so the mutants (tk-/-) are unable to incorporate the toxic analogue and therefore survive in its presence.

Two types of TFT-resistant mutant colonies occur and these are designated as large colonies (normal-growing) and small (slow-growing) colonies. Molecular analysis has indicated that the large colonies tend to represent events within the gene (base-pair substitutions and deletions), whereas small colony mutants often involve large genetic changes frequently visible as chromosome aberrations. Thus, in this assay, gene mutations within the tk gene (11 to 13 kilobases) and chromosomal events involving the gene may be detected. The TK system has a high spontaneous mutant frequency and high numbers of cells can be treated and sampled, therefore it is statistically robust.

Evaluation criteria:

The test item was considered to be clearly positive (mutagenic) in this assay if all the following criteria were met:

1. At least one concentration exhibited a statistically significant increase (p<0.05) compared with the concurrent negative (vehicle) control and the increase was biologically relevant (i.e. the mutation frequency at the test concentration showing the largest increase was at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle/solvent) control value).

2. The increases in mutation frequency were reproducible between replicate cultures and/or between tests (under the same treatment conditions).

3. The increase was concentration-related (p < 0.05) as indicated by the linear trend analysis.

The test item was considered clearly negative (non-mutagenic) in this assay if in all experimental conditions examined there was no concentration related response or, if there is an increase in MF, but it did not exceed the GEF. Then, test item was considered unable to induce mutations in this test system.

Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological significance. Caution was exercised with positive results obtained at levels of cytotoxicity lower than 10% (as measured by RTG).

Statistics:

Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel software.

The negative (vehicle/solvent) control log mutant frequency (LMF) was compared to the LMF of each treatment concentration, based on Dunnett's test for multiple comparisons and the data were checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In Assay 1 and 3, there were no large changes in pH after treatment.
- Effects of osmolality: In Assay 1 and 2, there were no large changes in osmolality after treatment.
- Water solubility: In Assay 1 and 3 no insolubility was observed in the final treatment medium at the end of the treatment with and without metabolic activation.
- Precipitation: Insolubility was detected in the preliminary experiment. Precipitate/ minimal amount of precipitate was detected in the final treatment medium at the end of the treatment in 2000 µg/mL concentration with metabolic activation, in the 2000-1000 µg/mL concentration range in case of short treatment without metabolic activation and in 2000 µg/mL concentration in case of long treatment without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: Insolubility and excessive cytotoxicity was detected in the preliminary experiment. Precipitate/ minimal amount of precipitate was detected in the final treatment medium at the end of the treatment in 2000 µg/mL concentration with metabolic activation, in the 2000-1000 µg/mL concentration range in case of short treatment without metabolic activation and in 2000 µg/mL concentration in case of long treatment without metabolic activation.

Excessive cytotoxicity was also detected in the preliminary experiments: no cells survived the expression period in the 2000-125 µg/mL concentration range using the short treatment with and without metabolic activation and in the 2000-62.5 µg/mL concentration range using the long treatment without metabolic activation.

Concentrations up to the cytotoxic range were selected for the main experiments according to the instructions of the relevant OECD No. 490 guideline. At least seven concentrations were selected for the main experiments in each assay.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: See Table 9
- Negative (solvent/vehicle) historical control data: See Table 9.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Relative Total Growth (RTG)
- Other observations when applicable:
Preliminary Test:
Insolubility and excessive cytotoxicity was detected in the preliminary experiment. Excessive cytotoxicity was also detected in the preliminary experiments: no cells survived the expression period in the 2000-125 µg/mL concentration range using the short treatment with and without metabolic activation and in the 2000-62.5 µg/mL concentration range using the long treatment without metabolic activation.

Concentrations up to the cytotoxic range were selected for the main experiments according to the instructions of the relevant OECD No. 490 guideline. At least seven concentrations were selected for the main experiments in each assay.

Mutation Assay 1:
In the presence of S9-mix (3-hour treatment), marked or excessive cytotoxicity was seen at higher concentrations. In Assay 1 with metabolic activation no cells survived the expression period in the samples of 100 µg/mL concentration. Relative total growth of the highest evaluated concentration (80 µg/mL) was 33%. An evaluation was made using data of six concentrations (concentration range of 80-5 µg/mL).

Mutation Assay 2:
In the Assay 2 with and without metabolic activation an unexpected infection was observed on the plates / cultures. The observed effect caused major decrease in the cell numbers (even for untreated and negative controls), therefore the Assay 2 was stopped and it was considered to be invalid. Therefore, an additional experiment (Assay 3) was performed to provide fully valid, interpretable data. The experimental conditions and concentrations were the same as in the Assay 2 with and without metabolic activation.

Mutation Assay 3:
In the presence of S9-mix (3-hour treatment) with metabolic activation marked or excessive cytotoxicity was seen at higher concentrations (relative total growth of the highest tested concentration (90 µg/mL) was 9%, relative total growth of the highest evaluated concentration (85 µg/mL) was 15%). An evaluation was made using data seven concentrations (concentration range of 85-5 µg/mL) in Assay 3.

Table 4. Results of Mutation Test (Assays 1 and 3)
Assay	Concentration (RTG) and MF values	Increase in MF compared to vehicle control (GEF)	Concentration related increase was indicated by the linear trend analysis	Reproducible between cultures	Conclusion
Assay 1 3-hour +S9	80 µg/mL (33%): 124.2*	53.7 (<GEF)	Yes (statistically significant increase in the MF values was seen at the highest evaluated concentration)	Yes	Yes (statistically significant increase in the MF values was seen at the highest evaluated concentration)
	60 µg/mL (81%): 101.8	31.3 (<GEF)
	40 µg/mL (79%): 108.4	37.9 (<GEF)
	20 µg/mL (92%): 82.2	11.7 (<GEF)
	10 µg/mL (77%): 100.9	30.4 (<GEF)
	Vehicle (100%): 70.5
Assay 1 3-hour -S9	50 µg/mL (58%): 71.2	11.7 (<GEF)	No	Yes	Negative (all MF values were well below the GEF, thus showing no biological relevance).
	40 µg/mL (74%): 78.1	18.6 (<GEF)
	20 µg/mL (70%):  74.0	14.5 (<GEF)
	10 µg/mL (89%): 71.8	12.3 (<GEF)
	5 µg/mL (95%): 62.6	3.1 (<GEF)
	Vehicle (100%): 59.5
Assay 3 3-hour +S9	85 µg/mL (15%): 160.3*	68.8 (<GEF)	Yes (statistically significant increase in the MF values was seen at the highest evaluated concentration)	Yes	Negative (all MF values were well below the GEF, thus showing no biological relevance).
	80 µg/mL (33%): 110.1	18.6 (<GEF)
	60 µg/mL (88%):  99.4	7.9 (<GEF)
	40 µg/mL (88%):  88.7	-2.8 (<GEF)
	20 µg/mL (89%): 79.6	-11.9 (<GEF)
	Vehicle (100%): 91.5
Assay 3 24-hour -S9	30 µg/mL (18%): 139.7	28.3 (<GEF)	Yes (No statistically significant increase in the MF values was seen)	Yes	Negative (all MF values were below the GEF, thus showing no biological relevance).
	20 µg/mL (48%): 151.4	40 (<GEF)
	10 µg/mL (91%): 104.1	-7.3 (<GEF)
	5 µg/mL (93%): 104.1	-7.3 (<GEF)
	2.5 µg/mL (102%):  84.9	-26.5 (<GEF)
	Vehicle (100%): 111.4

Notes: Results of the five highest evaluated concentrations are shown in the table. Statistical significance labelled by *.

MF: Mutation Frequency (refers to 106 viable cells), GEF: Global Evaluation Factor (=126 per 106 viable cells)

Table 5. Mutagenicity Results of Assay 1 (3-hour treatment in the presence of S9-mix)
S9 Mix	Treatment period (hours)	Test item or control concentration	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/ total number of wells	Dn^2/var(Dn) ♦	Mutation frequency
+	3	80 μg/mL	517 / 768	159 / 768	92 / 768	6.063	124.2*
		60 μg/mL	615 / 768	67 / 768	86 / 768	2.297	101.8
		40 μg/mL	605 / 768	73 / 768	90 / 768	3.208	108.4
		20 μg/mL	652 / 768	61 / 768	55 / 768	0.364	82.2
		10 μg/mL	635 / 768	68 / 768	65 / 768	2.089	100.9
		5 μg/mL	632 / 768	82 / 768	54 / 768	2.704	105.8
		Vehicle Control	661 / 768	54 / 768	53 / 768	--	70.5
		Untreated Control	654 / 768	69 / 768	45 / 768	--	76.2
		Positive Control (CP: 4 μg/mL)	171 / 768	200 / 768	397 / 768	♦♦ 6.65E-14	1621.4*

In linear trend analysis β²/var (β) = 4.60, significant (at p<0.05).

* = Statistically significant

♦ = Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn) > 5.24 (at p<0.05).

♦♦ = Evaluated by T-test for independent samples (compared to the DMSO vehicle control).

D_n= Difference of log mutant frequency of dose “n” and that of the vehicle control

var(D_n) = variance of Dn

β = slope of the curve

var(β) = variance of the slope

+ = in the presence of S9-mix

Negative (vehicle) control = 1% (v/v) DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide

Note: Mutation frequency refers to 10⁶viable cells

 

Table 6. Mutagenicity Results of Assay 1 (3-hour treatment in the absence of S9-mix)
S9 Mix	Treatment period (hours)	Test item or control concentration	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/ total number of wells	Dn^2/var(Dn) ♦	Mutation frequency
-	3	50 μg/mL	646 / 768	82 / 768	40 / 768	0.475	71.2
		40 μg/mL	659 / 768	66 / 768	43 / 768	1.043	78.1
		20 μg/mL	665/ 768	58 / 768	45 / 768	0.662	74.0
		10 μg/mL	653 / 768	71 / 768	44 / 768	0.509	78.1
		5 μg/mL	673 / 768	61 / 768	34 / 768	0.035	62.6
		Vehicle Control	676 / 768	53 / 768	39 / 768	--	59.5
		Untreated Control	663 / 768	66 / 768	39 / 768	--	69.7
		Positive Control (NQO: 0.15μg/mL)	323 / 768	206 / 768	239 / 768	♦♦ 6.65E-14	503.6*

In linear trend analysis β²/var (β) = 0.81, not significant.

* = Statistically significant

♦ = Evaluated by Dunnett’s test for multiple comparisons. Significant if D_n²/var(D_n) > 4.97 (at p<0.05).

♦♦ = Evaluated by T-test for independent samples (compared to the DMSO vehicle control).

D_n= Difference of log mutant frequency of dose “n” and that of the vehicle control

var(D_n) = variance of Dn

β = slope of the curve

var(β) = variance of the slope

- = in the absence of S9-mix

Negative (vehicle) control = 1% (v/v) DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide

Note: Mutation frequency refers to 10⁶viable cells

 

Table 7. Mutagenicity Results of Assay 3 (3-hour treatment in the presence of S9-mix)
S9 Mix	Treatment period (hours)	Test item or control concentration	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/ total number of wells	Dn^2/var(Dn) ♦	Mutation frequency
+	3	90 μg/mL	NE	NE	NE	NE	NE
		85 μg/mL	538 / 768	125 / 768	105 / 768	5.703	160.3*
		80 μg/mL	604 / 768	82 / 768	82 / 768	0.566	110.1
		60 μg/mL	627 / 768	62 / 768	79 / 768	0.108	99.4
		40 μg/mL	657 / 768	52 / 768	59 / 768	0.013	88.7
		20 μg/mL	659 / 768	59 / 768	50 / 768	0.265	79.6
		10 μg/mL	667 / 768	64 / 768	37 / 768	0.474	76.0
		5 μg/mL	656 / 768	63 / 768	49 / 768	0.115	83.6
		Vehicle Control	641 / 768	72 / 768	55 / 768	--	91.5
		Untreated Control	638 / 768	67 / 768	63 / 768	--	86.4
		Positive Control (CP: 4 μg/mL)	96 / 768	268 / 768	404 / 768	♦♦ 1.59E-17	1621.4*

In linear trend analysis β²/var (β) = 6.69, significant (at p<0.01).

* = Statistically significant

♦ = Evaluated by Dunnett’s test for multiple comparisons. Significant if D_n²/var(D_n) > 5.48 (at p<0.05).

♦♦ = Evaluated by T-test for independent samples (compared to the DMSO vehicle control).

D_n= Difference of log mutant frequency of dose “n” and that of the vehicle control

var(D_n) = variance of Dn

β = slope of the curve

var(β) = variance of the slope

+ = in the presence of S9-mix

Negative (vehicle) control = 1% (v/v) DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide

NE = Not evaluated since the RTG value of the next concentration was in the appropriate cytotoxicity range.

Note: Mutation frequency refers to 10⁶viable cells

 

Table 8. Mutagenicity Results of Assay 1 (24-hour treatment in the absence of S9-mix)
S9 Mix	Treatment period (hours)	Test item or control concentration	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/ total number of wells	Dn^2/var(Dn) ♦	Mutation frequency
-	24	55 μg/mL	ND	ND	ND	ND	ND
		50 μg/mL	NE	NE	NE	NE	NE
		40 μg/mL	NE	NE	NE	NE	NE
		30 μg/mL	557 / 768	100 / 768	111 / 768	1.089	139.7
		20 μg/mL	568 / 768	95 / 768	105 / 768	1.977	151.4
		10 μg/mL	622 / 768	71 / 768	75 / 768	0.087	104.1
		5 μg/mL	634 / 768	78 / 768	56 / 768	0.085	104.1
		2.5 μg/mL	643 / 768	69 / 768	56 / 768	1.322	84.9
		Vehicle Control	614 / 768	104 / 768	50 / 768	--	111.4
		Untreated Control	632 / 768	92 / 768	44 / 768	--	96.3
		Positive Control (NQO: 0.1μg/mL)	86 / 768	403 / 768	279 / 768	♦♦ 1.75E-16	1216.5*

In linear trend analysis β²/var (β) < 5.63, significant (at p<0.01).

* = Statistically significant

♦= Evaluated by Dunnett’s test for multiple comparisons. Significant if D_n²/var(D_n) > 4.97 (at p<0.05).

♦♦ = Evaluated by T-test for independent samples (compared to the DMSO vehicle control).

D_n= Difference of log mutant frequency of dose “n” and that of the vehicle control

var(D_n) = variance of D_n

β = slope of the curve

var(β) = variance of the slope

- = in the absence of S9-mix

Negative (vehicle) control = 1% (v/v) DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide

ND = No data (Cells did not survive the expression period.)

NE = Not evaluated due to the excessive cytotoxicity.

Note: Mutation frequency refers to 10⁶viable cells

Table 9. Historical Control Data
Mutation Frequency of the Negative Controls (2006-2016)
	Culture medium			Distilled water
Treatments	3h,S9+	3h,S9-	24h,S9-	3h,S9+	3h,S9-	24h,S9-
Average	94.3	103.6	106.4	90.4	96.6	96.3
SD	26.9	35.3	27.4	22.7	19.0	24.6
Min.	39.3	52.6	41.7	33.4	55.1	43.2
Max.	198.5	235.6	179.1	121.8	125.0	141.1
n	84	43	44	26	13	13
Dimethyl sulfoxide (DMSO)
Treatments	3h,S9+	3h,S9-	24h,S9-
Average	97.3	97.3	98.9
SD	33.7	38.5	26.8
Min.	44.2	33.7	47.1
Max.	269.9	261.6	159.4
n	101	57	50
Mutation Frequency of the Positive Controls (2006-2016)
	Cyclophosphamide			4-Nitroquinoline-N-oxide
Treatments	3h,S9+				3h,S9-	24h,S9-
Average	1178.7				722.2	831.9
SD	524.7				330.0	337.2
Min.	196.1				223.5	245.0
Max.	2642.5				1687.3	1577.6
n	106				58	52

h = hour

SD = Standard Deviation

S9+ = experiment with metabolic activation

S9- = experiment without metabolic activation

n = number of cases

Mutation Assay Results

Assay 1 (Presence of S9-mix (3-hour treatment)):

In the presence of S9-mix (3-hour treatment) there was a statistically significant increase in the mutation frequency values in the highest evaluated concentration in Assay 1. No statistically significant increase was observed at further concentrations. Concentration related increase was indicated by the linear trend analysis, however the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF (thus showing no biological relevance) in each case. Reproducibility was seen between replicates. This experiment was considered to be negative.

 

Assay 1 (Absence of S9-mix (3-hour treatment)):

In the absence of S9-mix (3-hour treatment) without metabolic activation no statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. Reproducibility was seen between replicates. No concentration related increase was indicated by the linear trend analysis. This experiment was considered to be negative.

 

Assay 3 (Presence of S9-mix (3-hour treatment)):

In the presence of S9-mix (3-hour treatment) with metabolic activation there was a statistically significant increase in the mutation frequency values in the highest evaluated concentration in Assay 3 (at proper degree of cytotoxicity, RTG value was 15%), but in Assay 3 the Dn2/var(Dn) value of the highest evaluated concentration (Dn2/var(Dn)=5.703) was close to the limit value (significant if Dn2/var(Dn) > 5.48). No statistically significant increase was observed at further concentrations. Concentration related increase was indicated by the linear trend analysis, however the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF (thus showing no biological relevance) in each case. Reproducibility was seen between replicates. This experiment was considered to be negative.

 

Assay 3 (Absence of S9-mix (24-hour treatment)):

In Assay 3, following a 24-hour treatment without metabolic activation, aconcentration related increase was indicated by the linear trend analysis, however no statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. Reproducibility was seen between replicates. Therefore, this experiment was considered to be negative.

 

VALIDITY OF THE MUTATION ASSAYS

Untreated, negative (vehicle/solvent) and positive controls were run concurrently in the study. The spontaneous mutation frequency of the negative (vehicle/solvent) and untreated controls were in the recommended range (50-170 x 10^-6) in all cases.

 

The positive controls (Cyclophosphamide in the presence of metabolic activation and

4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all. All of the positive control samples in the performed experiments fulfilled at least one of the relevant OECD No. 490 criteria.

 

The plating efficiencies for the negative (vehicle/solvent) control of the test item and the untreated control samples at the end of the expression period (PE_viability) were within the acceptable range (65-120%) in all assays.

 

The number of test concentrations evaluated was at least five in Assay 1 and Assay 3, which met the acceptance criteria about the minimum number of evaluated concentrations.

 

The tested concentration range in the study was considered to be adequate as the selected range properly covered concentrations from cytotoxicity to no or little cytotoxicity. More closely spaced test item concentrations were used in the expected cytotoxic range. Furthermore, the highest evaluated concentration showed proper degree of cytotoxicity (approximately 80-90%, i.e. approximately 10-20 relative total growth)*.

 

*Notes:

In Assay 1 the experiment of 3-hour with metabolic activation, the relative total growth of the highest evaluated concentration (80 µg/mL) was 33%, thus a lower degree of cytotoxicity was observed than the recommendation of the relevant OECD guideline. However, in Assay 3 the experiment of 3-hour with metabolic activation, the relative total growth of the highest evaluated concentration (85 µg/mL) was 15%, therefore the RTG value of this concentration was within the target range of 10-20%. Thus, the results were considered to cover appropriate concentrations, being acceptable to justify the study for the exposures with metabolic activation.

 

In Assay 1 the experiment of 3-hour without metabolic activation there were no concentrations with significant cytotoxicity, but in Assay 3 the RTG value of the highest tested concentration (at 30 µg/mL, RTG value of 18%) was within the target cytotoxicity range (approximately 10-20%). It is considered that the degree of cytotoxicity without S9 was in accordance with the recommendation of the OECD No. 490 guideline, although only at the 24-hour exposure, when same concentration was used. Closely spaced concentration range was used to properly cover concentrations from cytotoxicity, to no or little cytotoxicity. Thus, the results were considered to cover appropriate concentrations, being acceptable to justify the study for the exposures without metabolic activation.

 

Suspension growth value of the untreated and negative (vehicle/solvent) control samples were in line with the recommended range in all cases (i.e. 8-32 fold in case of short treatments and 32-180 fold in case of long treatment).

 

The overall study was considered to be valid.

Conclusions:

No mutagenic effect of the test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile) was observed in the presence or absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Executive summary:

A Key OECD Guideline 490 in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to evaluate the potential of the test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile) to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).

 

DMSO was used as vehicle in this study. Based on the results of the preliminary experiment the following test material concentrations were examined in the mutation assays:

 

Assay 1 (3-hour treatment with metabolic activation): 100, 80, 60, 40, 20, 10 and 5 µg/mL,

Assay 1 (3-hour treatment without metabolic activation): 80, 70, 60, 50, 40, 20, 10 and 5 µg/mL,

Assay 3 (3-hour treatment with metabolic activation): 90, 85, 80, 60, 40, 20, 10 and 5 µg/mL,

Assay 3 (24-hour treatment without metabolic activation): 55, 50, 40, 30, 20, 10, 5 and 2.5 µg/mL.

 

In Assay 1 and Assay 3, there were no large changes in pH or osmolality after treatment. No insolubility was observed in the final treatment medium at the end of the treatment in Assay 1 and 3 with and without metabolic activation.

 

In Assay 2, with and without metabolic activation, an unexpected infection was observed on the plates / cultures. The observed effect caused major decrease in the cell numbers (even for untreated and negative controls), therefore the Assay 2 was stopped and it was considered to be invalid. An additional experiment (Assay 3) was performed to provide fully valid, interpretable data.

 

In Assay 1 and Assay 3, following a 3-hour treatment with metabolic activation marked or excessive cytotoxicity was seen at higher concentrations. In Assay 1 with metabolic activation no cells survived the expression period in the samples of 100 µg/mL concentration. An evaluation was made using data of six concentrations (concentration range of 80-5 µg/mL) in Assay 1 and seven concentrations (concentration range of 85-5 µg/mL) in Assay 3.

 

There was a statistically significant increase in the mutation frequency values in the highest evaluated concentration in Assay 1 (relative total growth of the highest evaluated concentration of 80 µg/mL was 33%) and Assay 3 (at proper degree of cytotoxicity, RTG was 15%), but in Assay 3 the Dn2/var(Dn) value of the highest evaluated concentration was close to the limit value (significant if Dn2/var(Dn) > 5.48).

 

No statistically significant increase was observed at further concentrations. Concentration related increase was indicated by the linear trend analysis, however the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF (thus showing no biological relevance) in each case. Reproducibility was seen between replicates. These experiments were considered to be negative.

 

In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at the higher concentration range. No cells survived the expression period in the samples of 80, 70 and 60 µg/mL concentration. Lower degree of cytotoxicity was observed at the next concentration (50 µg/mL).

 

No significant cytotoxicity was observed at lower concentrations.

 

An evaluation was made using data of five concentrations (concentration range of 50-5 µg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. Reproducibility was seen between replicates. No concentration related increase was indicated by the linear trend analysis. This experiment was considered to be negative.

 

In Assay 3, following a 24-hour treatment without metabolic activation, excessive or marked cytotoxicity of the test item was observed at higher concentrations. No cells survived the expression period in the samples of 55 µg/mL concentration. Excessive cytotoxicity was observed at 50 and 40 µg/mL concentrations.

 

An evaluation was made using data of five concentrations (concentration range of 30-2.5 µg/mL). Concentration related increase was indicated by the linear trend analysis, however no statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. Reproducibility was seen between replicates. Therefore, this experiment was considered to be negative.

 

The experiments were performed using appropriate untreated, negative (vehicle/solvent) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle/solvent) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were acceptable in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.

 

In conclusion, no mutagenic effect of the test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile) was observed in the presence or absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Reference 4

Endpoint:

genetic toxicity in vitro, other

Remarks:

BlueScreen assay

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Principles of method if other than guideline:

The aim was to assess the genotoxicity and cytotoxicity of Azuril with the BlueScreen HC genotoxicity screening assays, using a protocol with and without metabolic activation in a 96-well microplate format.

GLP compliance:

not specified

Type of assay:

other: Mammalian cell based genotoxicity screening assay (BlueScreen)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Compound name: 3-Cyclohexene-1-carbonitrile, 4-(4-methyl-3-pentenyl)-
- CAS number: 21690-43-7
- Compound ID: GTX 381
- State: supplied as a colourless liquid
- Expiration date of the lot/batch: not specified
- Purity: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was combined with 100% DMSO to give a 1250 mM stock solution. The compound did not precipitate when added to water therefore no further dilution of the DMSO stock solution was required.

Target gene:

GADD45a gene

Species / strain / cell type:

mammalian cell line, other:

Details on mammalian cell type (if applicable):

A genetically modified strain of cultured human lymphoblastoid TK6 cells is used (GLuc-T01), which has a patented Gaussia luciferase (GLuc) reporter system that exploits the proper regulation of the GADD45a gene, which mediates the adaptive response to genotoxic stress.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Highest concentration tested: 1893 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: 96-well plate

(1) BlueScreen HC Assay - WITHOUT METABOLIC ACTIVATION:

>Overview
- A dilution series of the test material is generated in a 96-well, black microplate with an optically clear base.
- A standard genotoxic compound (4-nitroquinoline 1-oxide, 4-NQO) is added as an intra-plate quality control check.
- A genetically modified strain of cultured human lymphoblastoid TK6 cells is used (GLuc-T01), which contains a patented Gaussia luciferase (GLuc) reporter system that exploits the proper regulation of the GADD45a gene, which mediates the adaptive response to genotoxic stress.
- Exposure to a genotoxic compound increases expression of GLuc, which is quantified at the assay endpoint by the detection of luminescence generated from the reaction of GLuc with a coelenterazine substrate, added to the microplate wells just before measurement.
- Each dilution of the test compound is combined with an equal volume of a specialised growth medium containing BlueScreen HC cells. Duplicate dilution sreies of each compound are performed with a single microplate assay.

> Duration - The microplates were covered with a breathable membrane and incubated at 37°C with 5% CO2 and 95% humidity for 48 hours.

> Measurements
- The assay plates are then analysed using a microplate reader that provides measurements of fluorescence and flash luminescence for cells and solutions in the microplate wells.
- A recent protocol enhancement has been added, that measures cytotoxicity by lysis of the cells and addition of a fluorescent DNA binding stain, followed by assessment of the resulting fluorescence.
- This estimation of relative cell density replaces the previous optical absorbance measure.
- Fluorescence is proportional to cell proliferation, which is lowered by toxic analytes, and luminiscence intensity is proportional to the activity of the cell's DNA repair system, which is increased by genotoxic analytes.
- Luminescence is normalised to the fluorescence signal to correct for variation in cell yield caused by cytotoxicity.

> Results
- Raw data collected from BlueScreen HC assay plates are saved to an MS Excel file.
- The luminescence and fluorescence data are automatically analysed using the BlueScreen HC software template to produce a result summary, with data presented both in tabulated and graphical formats, which provides a semi-quantitative assessment of cytotoxicity and genotoxicity.
- The overall assay outcome presented for a compound results from the average of the duplicate test series performed for the compound.


(2) BlueScreen HC S9 Metabolic Activation Assay - WITH METABOLIC ACTIVATION

> Overview: - Exposure to a genotoxic compound increases GLuc expression, which is quantified at the assay endpoint by the detection of luminescence generated from the reaction of GLuc with a coelenterazine substrate, added to the microplate wells just prior to measurement.
- Cell density is determined by subsequent lysis of cells and addition of a fluorescent DNA binding stain, followed by assessment of the resulting fluorescence.

> Cells: - This protocol uses the same cultured TK6 cell strain (GLuc-T01) as the assay without metabolic activation and is also performed in 96-well plates.

> Duration: - The test substance is incubated with test cells n the presence of 1% (v/v) Aroclor-1254 induced rat liver S9 fraction mix in Exposure Medium at 37°C (5% CO2, 95% humidity) for 3 hours.

> Washing and recovery: - Cells were washed in phosphate buffered saline solution, harvested by centrifugation and allowed to recover in Recovery medium for 45 hours at 37ºC with 5% CO2 and 95% humidity.

> Positive control: Cyclophosphamide - a commonly used standard compound in genotoxicity studies using metabolic activation.

Evaluation criteria:

GENOTOXICITY
1) Without metabolic activation:
- The statistically defined threshold for a positive genotoxicity result is 1.8, i.e. 80% induction over and above the baseline for vehilce-treated cells.

2) With metabolic activation:
- The statistically defined threshold for a positive result is 1.5, i.e 50% induction over and above the baseline for vehicle-treated control cells.


CYTOTOXICITY
- The defined condition for a positive cytotoxicity result in the BlueScreen HC assay without S9 metabolic activation is a reduced relative cell density to less than 80%, compared to the vehicle-treated control.

Statistics:

Not specified

Key result

Species / strain:

mammalian cell line, other: Human lymphoblastoid TK6 cells (GLuc-T01)

Remarks:

Screening test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Positive controls validity:

valid

Key result

Species / strain:

mammalian cell line, other: Human lymphoblastoid TK6 cells (GLuc-T01)

Remarks:

Screening test

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

The positive control substances (4-NQO and CPA) passed the test criteria in each case following the standard procedure for the BlueScreen HC and BlueScreen HC S9 metabolic activation assays, respectively.

Remarks on result:

other: With metabolic activation (+S9)

Conclusions:

Based on the results of the study and the evaluation criteria, the test substance, 3-Cyclohexene-1-carbonitrile, 4(4-methyl-3-pentenyl)- was cytotoxic and classified as negative for genotoxicity at the concentrations tested with and without metabolic activation in the BlueScreen HC assay.

Executive summary:

The aim of this study was to assess the genotoxicity and cytotoxicity of the test substance, 3 -Cyclohexene-1 -carbonitrile, 4 -(4 -methyl-3 -pentenyl)-, with the BlueScreen HC genotoxicity screening assays, using a protocol with and without metabolic activation.

The test substance was combined with 100% DMSO to give a 1250 mM stock solution. It did not precipitate when added to water and therefore no further dilution of the stock solution was needed. The highest concentration of the test substance tested was at 1893 µg/mL.

The BlueScreen HC assay was performed in a 96 -well microtitre plate without metabolic activation (-S9). A dilution series of the test susbtance was generated in the microtitre plate. A standard genotoxic compound (4 -nitroquinoline 1 -oxide) was employed as a positive control and was added as an intra-plate quality control check to the microtitre plate at concentrations of 0.5 µg/mL and 0.125 µg/mL. The test system used was a genetically modified strain of cultured human lymphoblastoid TK6 cells (GLuc-T01) with a patented reported system, which explots the proper regulation of the GADD45a gene. This target gene mediates the adaptive response to genotoxic stress. The BlueScreen HC S9 assay was performed in the same cultured TK6 cell strain in a 96 -well microtitre plate as the first assay, but instead in the presence of metabolic activation (+S9). The test material was incubated with the test cells in the presence of 1% (v/v) Aroclor-1254 induced rat liver S9 fraction mix in Exposure Medium at 37°C (5% CO2, 95% humidity) for 3 hours. Following this incubation period, the cells were washed in a phosphate buffered saline solution, harvested by centrifugation and allowed to recover in Recovery Medium for 45 hours at 37°C (5% CO2, 95% humidity). Cyclophosphate was employed as positive control, tested at concentrations of 25 µg/mL and 5µg/mL. A coelenterazine substrate was added to the microplate wells in both assays just prior to measurement, which reacts with GLuc resulting in luminescence. Cell exposure to a genotoxic compound increases GLuC expression that can be quantified by luminscence detection. Cytotoxicity was measured by lysis of cells and the addition of a fluorescent DNA binding stain, followed by the assessment of the resulting fluorescence. Fluorescence is proportional to cell proliferation, which is lowered by toxic analytes, and luminescence intensity is proportional to the activity of the cell's DNA repair system, which is increased by genotoxic analytes. The quanity of luminescence is normalised to the fluorescence signal to correct for the variation in cell yield caused by cytotoxicity.

Based on the results of the study and the evaluation criteria, the test substance, 3-Cyclohexene-1-carbonitrile, 4(4-methyl-3-pentenyl)- was cytotoxic and classified as negative for genotoxicity at the concentrations tested with and without metabolic activation in the  BlueScreen HC assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

AMES TEST (OECD 471)

A Key OECD Guideline 471 study (Citoxlab Hungary Ltd., 2018; K = 1) was conducted 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 its 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 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.

MICRONUCLEUS TEST (OECD 487)

A Key OECD Guideline 487 in vitro study (Citoxlab France., 2019; K = 1) was conducted to evaluate the potential of the test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile) to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/-.

 

After a preliminary cytotoxicity test, the test material diluted in dimethylsulfoxide (DMSO), was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

 

Without S9 Mix (-S9): First and Second Experiment: 3 h treatment + 24 h recovery and 24 h treatment + 0 h recovery.

With S9 Mix (+S9): First and Second Experiment: 3 h treatment + 24 h recovery

 

Each treatment was coupled to an assessment of cytotoxicity at the same dose levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.

 

After the final cell counting, the cells were washed and fixed. Then, cells from at least three dose levels of the test item treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

 

Since the test item was found to be cytotoxic in the preliminary test, the highest dose level selected for the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international regulations.

 

The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

An emulsion was observed in the culture medium at the end of the 3-hour treatment periods at dose levels ≥ 0.10 mM in the second experiment.

 

Short treatments without S9 mix

With a treatment volume of 0.5% (v/v) in culture medium, the dose levels selected for the treatments were as follows:

- 0.002, 0.005, 0.009, 0.019, 0.038, 0.075, 0.15 and 0.3 mM for the first experiment,

- 0.0025, 0.0050, 0.0100, 0.0125, 0.0188, 0.025, 0.0375, 0.050, 0.075 and 0.10 mM for the second experiment.

 

Cytotoxicity

In the first experiment, a severe cytotoxicity was induced at dose levels ≥ 0.075 mM.

In the second experiment, a moderate to severe cytotoxicity was induced at dose levels ≥ 0.0125 mM.

 

Micronucleus analysis

The dose levels selected for micronucleus analysis were as follows:

- 0.009, 0.019 and 0.038 mM for the first experiment, the latter inducing only a 28% decrease in the PD but higher dose levels being too cytotoxic,

- 0.0050, 0.0100 and 0.0125 mM for the second experiment, the latter inducing only a 49% decrease in the PD but higher dose levels being too cytotoxic.

 

In the first experiment, no statistically significant increase in the frequency of micronucleated cells was observed at any of the analysed dose levels relative to the corresponding vehicle control. No dose response relationship was demonstrated by the linear regression and none of the analysed dose levels showed frequencies of each replicate culture above the vehicle control historical range.

 

Since the recommended level of cytotoxicity was not reached, a second experiment was undertaken using a narrower range of dose levels, and confirmed the results obtained in the first one.

 

Although none of the selected dose levels induced the recommended level of cytotoxicity in either experiment despite the use of a narrower range of dose levels, since both assays met the criteria for a negative response, the results were considered as suitable to allow a reliable interpretation.

 

Continuous treatments without S9 mix

With a treatment volume of 0.5% (v/v) in culture medium, the dose levels selected for the treatments were as follows:

- 0.001, 0.002, 0.005, 0.009, 0.019, 0.038, 0.075 and 0.15 mM for the first experiment,

- 0.0031, 0.0063, 0.0125, 0.02, 0.025, 0.03, 0.0375 and 0.05 mM for the second experiment.

 

Cytotoxicity

In the first experiment, a slight to severe cytotoxicity was induced at dose levels ≥ 0.019 mM.

In the second experiment, a moderate to severe cytotoxicity was induced at dose levels ≥ 0.0125 mM.

 

Micronucleus analysis

The dose levels selected for micronucleus analysis were as follows:

- 0.005, 0.009 and 0.019 mM for the first experiment, the latter inducing only a 34% decrease in the PD but higher dose levels being too cytotoxic,

- 0.0063, 0.0125 and 0.02 mM for the second experiment, the latter inducing the recommended level of cytotoxicity.

 

In the first experiment, no statistically significant increase in the frequency of micronucleated cells was observed at any of the analysed dose levels relative to the corresponding vehicle control, and no dose response relationship was demonstrated by the linear regression.

 

However, the frequencies of both replicate culture at the highest analysed dose level of 0.019 mM were slightly above the vehicle control historical range. These results did not meet either the criteria for a negative or positive response.

 

In order to check the reproducibility of this increase and to reach the recommended level of cytotoxicity, a second experiment was undertaken using a narrower range of dose levels.

 

In the second experiment, no statistically significant increase in the frequency of micronucleated cells was observed in the second experiment, at any of the analysed dose levels relative to the corresponding vehicle control. No dose-response relationship was demonstrated by the linear regression and none of the analysed dose levels showed frequencies of each replicate culture above the vehicle control historical range.

 

Since the increase in the frequency of micronucleated cells observed in the first experiment was not reproduced in the second experiment despite the use of a narrower range of dose levels, this increase is considered not to be biologically relevant, and the overall results are considered to meet the criteria for a negative response.

 

Treatments with S9 mix

With a treatment volume of 0.5% (v/v) in culture medium, the dose levels selected for the treatments were as follows:

- 0.005, 0.009, 0.019, 0.038, 0.075, 0.15, 0.3 and 0.6 mM for the first experiment,

- 0.0063, 0.0125, 0.0188, 0.025, 0.0375, 0.050, 0.075, 0.10, 0.15 and 0.20 mM for the second experiment.

 

Cytotoxicity

In the first experiment, a slight to severe cytotoxicity was induced at dose levels ≥ 0.075 mM.

In the second experiment, a moderate to severe cytotoxicity was induced at dose levels ≥ 0.0188 mM.

 

Micronucleus analysis

The dose levels selected for micronucleus analysis were as follows:

- 0.038, 0.075 and 0.15 mM for the first experiment, the latter inducing only a 45% decrease in the PD but higher dose levels being too cytotoxic,

- 0.0063, 0.0125 and 0.0188 mM for the second experiment, the latter inducing the recommended level of cytotoxicity.

 

In the first experiment, no statistically increase in the frequency of micronucleated cells was observed at any of the analysed dose levels relative to the corresponding vehicle control and no dose-response relationship was demonstrated by the linear regression.

 

However, the frequencies of both replicate culture at the highest analysed dose level of 0.15 mM were slightly above the vehicle control historical range. These results did not meet either the criteria for a negative or positive response.

 

In order to check the reproducibility of this increase and to reach the recommended level of cytotoxicity, a second experiment was undertaken using a narrower range of dose levels. In the second experiment, no statistically significant increase in the frequency of micronucleated cells was observed at any of the analysed dose levels relative to the corresponding vehicle control. No dose response relationship was demonstrated by the linear regression and none of the analysed dose levels showed frequencies of each replicate culture above the vehicle control historical range.

Since the increase in the frequency of micronucleated cells observed in the first experiment was not reproduced in the second experiment despite the use of a narrower range of dose levels, this increase is considered not to be biologically relevant, and the overall results in the presence of metabolic activation are considered to meet the criteria for a negative response.

 

Under the experimental conditions of the study, the test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile), did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

MAMMALIAN GENE MUTATION TEST (OECD 490)

A Key OECD Guideline 490 in vitro mammalian cell assay (Citoxlab Hungary Ltd., 2019; K = 1) was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to evaluate the potential of the test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile) to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).

 

DMSO was used as vehicle in this study. Based on the results of the preliminary experiment the following test material concentrations were examined in the mutation assays:

 

Assay 1 (3-hour treatment with metabolic activation): 100, 80, 60, 40, 20, 10 and 5 µg/mL,

Assay 1 (3-hour treatment without metabolic activation): 80, 70, 60, 50, 40, 20, 10 and 5 µg/mL,

Assay 3 (3-hour treatment with metabolic activation): 90, 85, 80, 60, 40, 20, 10 and 5 µg/mL,

Assay 3 (24-hour treatment without metabolic activation): 55, 50, 40, 30, 20, 10, 5 and 2.5 µg/mL.

 

In Assay 1 and Assay 3, there were no large changes in pH or osmolality after treatment. No insolubility was observed in the final treatment medium at the end of the treatment in Assay 1 and 3 with and without metabolic activation.

 

In Assay 2, with and without metabolic activation, an unexpected infection was observed on the plates / cultures. The observed effect caused major decrease in the cell numbers (even for untreated and negative controls), therefore the Assay 2 was stopped and it was considered to be invalid. An additional experiment (Assay 3) was performed to provide fully valid, interpretable data.

 

In Assay 1 and Assay 3, following a 3-hour treatment with metabolic activation marked or excessive cytotoxicity was seen at higher concentrations. In Assay 1 with metabolic activation no cells survived the expression period in the samples of 100 µg/mL concentration. An evaluation was made using data of six concentrations (concentration range of 80-5 µg/mL) in Assay 1 and seven concentrations (concentration range of 85-5 µg/mL) in Assay 3.

 

There was a statistically significant increase in the mutation frequency values in the highest evaluated concentration in Assay 1 (relative total growth of the highest evaluated concentration of 80 µg/mL was 33%) and Assay 3 (at proper degree of cytotoxicity, RTG was 15%), but in Assay 3 the Dn2/var(Dn) value of the highest evaluated concentration was close to the limit value (significant if Dn2/var(Dn) > 5.48).

 

No statistically significant increase was observed at further concentrations. Concentration related increase was indicated by the linear trend analysis, however the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF (thus showing no biological relevance) in each case. Reproducibility was seen between replicates. These experiments were considered to be negative.

 

In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at the higher concentration range. No cells survived the expression period in the samples of 80, 70 and 60 µg/mL concentration. Lower degree of cytotoxicity was observed at the next concentration (50 µg/mL).

 

No significant cytotoxicity was observed at lower concentrations.

 

An evaluation was made using data of five concentrations (concentration range of 50-5 µg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. Reproducibility was seen between replicates. No concentration related increase was indicated by the linear trend analysis. This experiment was considered to be negative.

 

In Assay 3, following a 24-hour treatment without metabolic activation, excessive or marked cytotoxicity of the test item was observed at higher concentrations. No cells survived the expression period in the samples of 55 µg/mL concentration. Excessive cytotoxicity was observed at 50 and 40 µg/mL concentrations.

 

An evaluation was made using data of five concentrations (concentration range of 30-2.5 µg/mL). Concentration related increase was indicated by the linear trend analysis, however no statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. Reproducibility was seen between replicates. Therefore, this experiment was considered to be negative.

 

The experiments were performed using appropriate untreated, negative (vehicle/solvent) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle/solvent) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were acceptable in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.

 

In conclusion, no mutagenic effect of the test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile) was observed in the presence or absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

 

Note: The final reports of the above OECD 487 and 490 studies are currently awaited from the CRO/s. A spontaneous update of this dossier will be undertaken by the registrant upon receipt of all pending study reports from the CRO/s.

BLUESCREEN TEST (no TG)

A supporting study (Gentronix Limited, 2013; K = 2) was conducted to assess the genotoxicity and cytotoxicity of the test substance, 3-Cyclohexene-1-carbonitrile, 4-(4-methyl-3-pentenyl)-, with the BlueScreen HC genotoxicity screening assays, using a protocol with and without metabolic activation.

The test substance was combined with 100% DMSO to give a 1250 mM stock solution. It did not precipitate when added to water and therefore no further dilution of the stock solution was needed. The highest concentration of the test substance tested was at 1893 µg/mL.

The BlueScreen HC assay was performed in a 96 -well microtitre plate without metabolic activation (-S9). A dilution series of the test susbtance was generated in the microtitre plate. A standard genotoxic compound (4-nitroquinoline 1-oxide) was employed as a positive control and was added as an intra-plate quality control check to the microtitre plate at concentrations of 0.5 µg/mL and 0.125 µg/mL. The test system used was a genetically modified strain of cultured human lymphoblastoid TK6 cells (GLuc-T01) with a patented reported system, which explots the proper regulation of the GADD45a gene. This target gene mediates the adaptive response to genotoxic stress. The BlueScreen HC S9 assay was performed in the same cultured TK6 cell strain in a 96-well microtitre plate as the first assay, but instead in the presence of metabolic activation (+S9). The test material was incubated with the test cells in the presence of 1% (v/v) Aroclor-1254 induced rat liver S9 fraction mix in Exposure Medium at 37°C (5% CO2, 95% humidity) for 3 hours. Following this incubation period, the cells were washed in a phosphate buffered saline solution, harvested by centrifugation and allowed to recover in Recovery Medium for 45 hours at 37°C (5% CO2, 95% humidity). Cyclophosphate was employed as positive control, tested at concentrations of 25 µg/mL and 5µg/mL. A coelenterazine substrate was added to the microplate wells in both assays just prior to measurement, which reacts with GLuc resulting in luminescence. Cell exposure to a genotoxic compound increases GLuC expression that can be quantified by luminscence detection. Cytotoxicity was measured by lysis of cells and the addition of a fluorescent DNA binding stain, followed by the assessment of the resulting fluorescence. Fluorescence is proportional to cell proliferation, which is lowered by toxic analytes, and luminescence intensity is proportional to the activity of the cell's DNA repair system, which is increased by genotoxic analytes. The quanity of luminescence is normalised to the fluorescence signal to correct for the variation in cell yield caused by cytotoxicity.

Based on the results of the study and the evaluation criteria, the test substance, 3-Cyclohexene-1-carbonitrile, 4(4-methyl-3-pentenyl)- was cytotoxic and classified as negative for genotoxicity at the concentrations tested with and without metabolic activation in the  BlueScreen HC assay.

Justification for classification or non-classification

Based on the results observed in the in vitro assays, the test material Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile does not meet the criteria to be classified for mutagenicity under EU Regulation (EC) No 1272/2008 (CLP).