Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Vinylcyclohexane was tested in a standard in vitro genotoxicity battery as required by Annex VIII of the REACH regulation 1907/2006. In a bacterial reverse gene mutation test conducted according to OECD 471, the target substance did not induce mutagenicity. The substance was also tested negative in an in vitro mammalian cell HPRT mutation assay conducted according to OECD 476, and an in vitro cytogenicity study in Chinese hamster V79 cells conducted according to OECD 487. Based on the lack of mutagenicity/cytogenicity in all three in vitro assays, Vinylcyclohexane is considered to be non-genotoxic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-10-17 to 2018-01-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr. J. Thacker, MRC Radiobiology Unit, Harwell, UK

MEDIA USED
- Type and identity of media: EMEM Minimal medium and EMEM Complete medium (10 %)
Metabolic activation:
with and without
Metabolic activation system:
S9 liver tissue fraction
Test concentrations with justification for top dose:
Preliminary cytotoxicity test: 550, 275, 138, 68.8, 34.4, 17.2, 8.59 and 4.30 µg/mL
Main test, -S9: 275, 212, 163, 125, 96.3 and 74.1 µg/mL
Main test, +S9: 550, 458, 382, 318, 265 and 221 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- justification for choice of solvent: Solubility test and pre-test for toxicity
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
-S9
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
+S9
Details on test system and experimental conditions:
Preliminary Cytotoxicity Test:
The test item was assayed at a maximum dose level of 1100 µg/mL and at a wide range of lower dose levels: 550, 275, 138, 68.8, 34.4, 17.2, 8.59 and 4.30 µg/mL. Treatments were performed both in the absence and presence of S9 metabolism; a single culture was used at each test point and positive controls were not included. In order to evaluate baseline count, at the beginning of treatment two additional control cultures were included in the experimental scheme. These two cultures were trypsinized and cell counts were performed approximately at time 0. The baseline count was the average of the total number of cells from the two flasks. At the end of treatment, cell monolayers were washed with PBS, the cultures were trypsinised, counted, diluted and plated. After incubation for eight days, the colonies were stained with Giemsa solution and counted.

MAIN ASSAY:
A main assay was performed including negative and positive controls, in the absence and presence of S9 metabolising system. The two treatment series were assayed in separate runs. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. For each run, two additional control cultures were included in the experimental scheme, in order to evaluate baseline count. Two days before the experiment, sufficient numbers of 175 cm2 flasks were inoculated with 5 million freshly trypsinised V79 cells from a common pool, in order to have at least 20 million of cells for treatment. At the treatment time, the growth medium was removed from the flasks and replaced with treatment medium; cultures were incubated at 37 °C for three hours.

Determination of survival (Day 0):
At the end of treatment, the medium was removed and the cell monolayers were washed with PBS. The cultures were trypsinised, counted and an aliquot from each culture was diluted and plated to estimate the viability of the cells. Each cell suspension was re-plated (2 x 106 cells/F175) in order to maintain the treated cell populations. Fresh complete medium was added to the flasks which were then returned to the incubator at 37 °C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.

Subculturing (Day 2 and Day 5):
On Days 2 and 5, the cell populations were subcultured in order to maintain them in exponential growth. The cultures were trypsinised, counted and the number of cells taken forward was adjusted to give two million viable cells seeded in 225 cm2 flasks.

Determination of mutant frequency (Day 8):
At the expression time (Day 8), each culture was trypsinised, resuspended in complete medium and counted by microscope. After dilution, an estimated 1 x 10^5 cells were plated in each of twenty 100 mm tissue culture petri dishes containing medium supplemented with 6-thioguanine (at 7.5 µg/mL). These plates were subsequently stained with Giemsa solutions and scored for the presence of mutants. After dilution, an estimated 200 cells were plated in each of three 60mm tissue culture petri dishes. These plates were used to estimate Cloning Efficiency (CE).
Evaluation criteria:
A test item is considered to be clearly positive if:
- At least one of the test concentrations exhibits a statistically significant increase, compared with the concurrent solvent/vehicle control.
- The increase is concentration-related.
- Any of the results are outside the distribution of the historical negative control data (95% confidence limits).

A test item is considered to be clearly negative if:
- None of the test concentrations exhibits a statistically significant increase, compared with the concurrent solvent/vehicle control.
- There is no concentration-related increase.
- All results are inside the distribution of the historical negative control data (95% confidence limits).
Statistics:
Mean of the experimental values was calculated.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No statistically significant increase over the spontaneous mutation frequency was observed at any treatment level, in the absence or presence of S9 metabolic activation. All results were inside the distribution of the historical negative control data, both in the absence and presence of S9 metabolism.
Conclusions:
In this study, the test item did not induce any mutation in the Chinese hamster V79 cells after in vitro treatment in the presence or absence of metabolic activation.
Executive summary:

In a mammalian cell HPRT gene mutation assay conducted according to OECD guideline 476, V79 cells cultured in vitro were exposed for 3 hours to the test item (99.8 % purity) in DMSO at concentrations of 275, 212, 163, 125, 96.3 and 74.1 µg/mL in the absence and 550, 458, 382, 318, 265 and 221 µg/mL in the presence of mammalian metabolic activation. The treated cells were maintained in growth medium for 9 days to allow phenotypic expression of induced mutation. For all tested treatment groups no dose-response relationship could be observed. The positive and negative controls did induce the appropriate response. There was no evidence of induced mutant colonies over background. 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data. 

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-09-26 to 2018-03-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Storage conditions: Room temperature in tightly closed container and in a well-ventilated place
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr. J. Thacker, MRC Radiobiology Unit, Harwell, UK

MEDIA USED
- Type and identity of media:
Minimal Medium:
- Eagle´s Minimal Essential Medium (10x)
- L-glutamine (200 mM)
- Sodium bicarbonate (7.5%)
- Non-essential amino acids (100x)
- Antibiotic solution
- Sterile distilled water

EMEM complete medium:
- Minimal medium (900 mL) and Foetal Calf Serum (100 mL)
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B (3 µg/mL)
Metabolic activation:
with and without
Metabolic activation system:
Microsomal liver enzyme (S9)
Test concentrations with justification for top dose:
Top dose was selected as per the guideline 1100 µg/mL corresponds to 10 mM)
Main Experiment 1, short-term (3 hours, -S9, +S9): 1100, 733, 489, 326, 217, 145, 96.6, 64.4 and 42.9 µg/mL
On the basis of the results from Main Experiment 1 the following concentrations were chosen:
Main Experiment 2, short term (3 hours, - S9): 100, 87.0, 75.6, 65.8, 57.2, 49.7, 43.2, 37.6 and 32.7 µg/mL
Main Experiment 2, long-term (24 hours, -S9): 100, 87.0, 75.6, 65.8, 57.2, 49.7, 43.2, 37.6, 32.7 amd 28.4 µg/mL
Main Experiment 3, short-term (3 hours, - S9): 88.4, 83.0, 79.6, 73.7, 66.3, 61.4, 55.3, 51.2 and 46.0 µg/mL
Main Experiment 3, short-term (3 hours, +S9): 220, 198, 183, 165, 153, 138, 127 and 115 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO was used as vehicle (1%)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1% DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: Colchicine & Mitomycin-C without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable): Each 25 cm² flask was seeded with 5x10^5 cells in EMEM complete medium

DURATION
- Preincubation period: 20
- Exposure duration: short-term: 3 hours; long-term: 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24-25 hours

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin-B (3 µg/ mL) was added 3 hours after start of the treatment

STAIN (for cytogenetic assays): Acridine Organge

NUMBER OF REPLICATIONS: 2 cell cultures per concentration

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The medium was removed from the flasks and the cells were brought into suspension by trypsinization. For each culture, the number of cells was adjusted to approximately 5 x10^4 cells/mL. A suitable volume of each cell suspension was added to the cytofunnels in order to prepare slides using a CytospinTM. The cells were fixed with 90% methanol (-20 °C). The slides were allowed to dry and were kept at room temperature prio to staining. The slides were stained with Acridine Orange in PBS (12.5 mg/100 mL PBS) and rinsed in PBS.

NUMBER OF CELLS EVALUATED: 1000 binucleated cells/culture were scored for micronuclei.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
- the micronucleus diameter was less than 1/3 of the nucleus diameter
- the micronucleus diameter was greater than 1/16 of the nucleus diameter
- No overlapping with the nucleus was observed
- the aspect was the same as the chromatin

DETERMINATION OF CYTOTOXICITY
- Method: the percentage cytotoxicity was evaluated according to the following formula:
% cytotoxicity: 100 - 100 [(CBPI(treatment)-1)/(CBPI(solvent control)-1)], wheras the cytokinesis-block proliferation index (CBPI) was calculated as follows: CBPI= [(mononucleated+ 2 x binucleated + 3 x multinucleated)/ total number of cells counted]. Mononucleated, binucleated and multinucleated are the number of mononucleated, binucleated and multinucleated cells. 500 cells per culture were analysed. At least five dose levels were analysed.
Evaluation criteria:
In this assay, the test item is considered as clearly positive if the following criteria are met:
- Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations,
- The proportion of micronucleated cells at such data points exceeds the normal range. If the increases fall within the range of values normally observed in the negative control cultures, the test item can not be classified as positive. Any significant increases over the concurrent negative controls are therefore compared with historical control values derived from recent studies,
- There is a significant dose effect relationship.

The test item is considered clearly negative if the following criteria are met:
- None of the dose level shows a statistically significant increase in the incidence of micronucleated cells,
- There is no concentration related increase when evaluated with an appropriate trend test,
- All the results are inside the distribution of the historical control data.
Statistics:
For the statistical analysis, a modified x2 test was used to compare the number of cells with micronuclei in control and treated cultures. Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: Following treatment with the test item, no remarkable variation of pH or osmolality was observed at any dose level, in the absence or presence of S9 metabolism.

RANGE-FINDING/SCREENING STUDIES:
Solubility Test:
Solubility of the test item was evaluated in a preliminary trial using DMSO. An opaque solution was obtained at 110 mg/mL, while a clear solution was noted at 73.3 mg/mL. The addition of the stock solution at 73.3 mg/mL to the culture medium in the ratio 1:100 generated a clear solution. Based on these results, the test item was assayed at the maximum dose level of 1100 µg/mL, which corresponds to 10 mM, the upper limit to testing indicated in the Study Protocol. Following treatment with the test item, a slight opacity of the medium was only observed at the beginning of treatment at the dose level of 1100 µg/mL, both in the absence and presence of S9.

CYTOTOXICITY RESULTS:
First main experiment:
Both in the absence and presence of S9 metabolism, severe toxicity was observed at all dose levels with the exception of the lowest dose levels, where no toxicity was noted. In the first instance no concentration was considered adequate for scoring.
Second main experiment:
Using the short treatment time in the absence of S9 metabolism, severe toxicity was observed at the two highest dose levels tested, while a mild to no remarkable toxicity was observed over the remaining dose range. Due to the steep decline of toxicity, once again no dose level could be selected for cytogenetic effect analysis. Using the continuous treatment, severe toxicity was observed at the three highest dose levels tested, moderate cytotoxicity was noted at the next lower dose level (60%), while no remarkable cytotoxicity was observed over the remaining dose range. Based on these results, the dose levels selected for scoring were 65.8, 57.2 and 49.7 µg/mL.
Third main experiment:
Using the short treatment in the absence of S9, marked to moderate cytotoxicity was observed from the dose level of 88.4 to 66.3 µg/mL. Mild toxicity was observed at the next lower dose level of 61.4 µg/mL and no toxicity was observed at the two lowest dose levels analysed. An unforeseen cytotoxicity result (29%), probably due to a technical problem, was noticed at 83.0 µg/mL. Based on the results obtained, the dose levels selected for scoring were: 66.3, 61.4 and 55.3 µg/mL. In the presence of S9 metabolic activation, no cells survived treatment from 153 µg/mL onwards. At the three lowest concentrations, the observed toxicity was not dose-related and a high variability was observed btween replicate cultures at 127 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
Results show that the incidences of micronucleated cells of the negative controls were within the distribution range of our historical control values. Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells were analysed. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system. The study was accepted as valid.

ANALYSIS OF MICRONUCLEI IN TREATMENT GROUPS:
Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrent negative control value was observed in the presence or absence of S9 metabolism, at any concentration, nor concentration related increase of cells bearing micronuclei was seen in any treatment series. The incidences were within the normal distribution range of historical values for negative controls except for the incidence observed at the lowest dose level in the presence of S9 (Main Experiment 3), which slightly exceeded the distribution range. This result was a chance event not related to the action of the test item and of no biological significance.

Table 1: Main experiment III, without S9 metabolism

Treatment

Dose Level

(µg/mL)

Absence of S9 metabolism

% Mn cells

Sig.

% Cytotoxicity

Solvent

1 %

1.10

 

0

Test item

55.3

1.00

N.S.

7

Test item

61.4

0.75

N.S.

31

Test item

66.3

0.65

N.S.

58

Solvent: DMSO

Treatment Time: 3 hours

Sampling Time: 24 hours

Table 2: Main experiment I and III, with S9 metabolism

Presence of S9 metabolism

Treatment

Dose level

(µg / mL)

Main Experiment I

Dose level

(µg / mL)

Main Experiment III

 

% Mn cells

Sig.

% Cytotoxicity

% Mn cells

Sig.

% Cytotoxicity

Solvent

1 %

0.90

 

 

1 %

1.00

 

 

Test item

64.4

1.00

N.S.

8

115

1.25

N.S.

44

Test item

96.6

0.80

N.S.

6

127

0.45

N.S.

80

Test item

145

1.00

N.S.

10

138

0.85

N.S.

42

Cyclophosphamide

10.0

3.75

***

56

10.0

7.15

***

62

Solvent: DMSO

Treatment Time: 3 hours

Sampling Time: 24 hours

Table 3: Main experiment II, without S9 metabolism

Treatment

Dose Level

(µg / mL)

Absence of S9 metabolism

% Mn cells

Sig.

% Cytotoxicity

Solvent

1 %

0.75

 

0

Test item

49.7

0.45

N.S.

5

Test item

57.2

1.00

N.S.

18

Test item

65.8

0.35

N.S.

60

Colchicine

0.250 µg / mL

10.1

***

87

Solvent: DMSO

Treatment Time: 24 hours

Sampling Time: 24 hours

 

Conclusions:
On the basis of these results, it is concluded that Vinylcyclohexane does not induce micronuclei in Chinese hamster V79 cells after in vitro treatment, under the reported experimental conditions.
Executive summary:

In an in vitro mammalian micronucleus assay (OECD 487), Chinese hamster V79 cell cultures were exposed to Vinylcyclohexane (99.8% purity) in 1% DMSO in short-term (3 hours) and long-term (24 hours) experiments at concentrations of up to 10 mM in the presence and absence of metabolic activation. The test item showed severe cytotoxicity and the short-term experiments were repeated with lower concentrations of the test item. The positive controls did induce relevant increases of the micronucleus frequency. In all conducted experiments no biologically and statistically significant induction of micronuclei was noted after treatment with the test item. This study is classified as acceptable and satisfies the requirements for Test Guideline OECD 487.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-08-02 to 2018-01-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium: Histidine locus
E. coli: Tryptophan locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 liver tissue fraction
Test concentrations with justification for top dose:
The highest dose level for the mutation assays will be selected as a concentration which elicits moderate toxicity.
If there is no evidence of toxicity following treatment with the test item, then the highest dose level will be 5 mg/plate or 5 μL/plate. If the test item is not soluble at 5 mg/plate or 5 μL/plate, one or more dose levels at which precipitation is observed will be included in the main assay.

Plate incorporation:
TA1535 (with and without S9): 0.250, 0.125, 0.0625, 0.0313, 0.0156, 0.00781 µL/plate
TA1537 (with and without S9): 0.500, 0.250, 0.125, 0.0625, 0.0313, 0.0156 µL/plate
WP2 uvrA (without S9): 1.00, 0.500, 0.250, 0.125, 0.0625, 0.0313 µL/plate
WP2 uvrA (with S9): 2.00, 1.00, 0.500, 0.250, 0.125 µL/plate
TA98 (without S9): 0.250, 0.125, 0.0625, 0.0313, 0.0156, 0.00781 µL/plate
TA98 (with S9): 0.500, 0.250, 0.125, 0.0625, 0.0313, 0.0156 µL/plate
TA100 (without S9): 0.500, 0.250, 0.125, 0.0625, 0.0313, 0.0156 µL/plate
TA100 (with S9): 0.250, 0.125, 0.0625, 0.0313, 0.0156, 0.00781 µL/plate

Pre-incubation:
TA1535, TA100 (with and without S9): 0.200, 0.100, 0.0500, 0.0250, 0.0125, 0.00625 µL/plate
TA1537 (with and without S9): 0.400, 0.200, 0.100, 0.0500, 0.0250, 0.0125 µL/plate
WP2 uvrA (without S9): 0.400, 0.200, 0.100, 0.0500, 0.0250, 0.0125 µL/plate
WP2 uvrA (with S9): 1.600, 0.800, 0.400, 0.200, 0.100, 0.0500 µL/plate
TA98 (without S9): 0.200, 0.100, 0.0500, 0.0250, 0.0125, 0.00625 µL/plate
TA98 (with S9): 0.400, 0.200, 0.100, 0.0500, 0.0250, 0.0125 µL/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Remarks:
medium control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535, TA100: without S9, 1 µg/plate
Untreated negative controls:
yes
Remarks:
medium control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537, without S9, 50 µg/plate
Untreated negative controls:
yes
Remarks:
medium control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA98, without S9, 2 µg/plate
Untreated negative controls:
yes
Remarks:
medium control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA, without S9, 500 µg/plate
Untreated negative controls:
yes
Remarks:
medium control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA1535 1 μg/plate, TA100 1 μg/plate (2 μg/plate), TA1537 1 μg/plate, TA98 1 μg/plate (2 μg/plate), WP" uvrA 10 μg/plate (20 μg/plate), with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: Main assay I - in agar (plate incorporation); Main assay II - preincubation
- Cell density at seeding (if applicable): 1-5 x 10^9 cells per mL

Plate-incorporation:
The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) will be added to molten overlay agar and vortexed. The mixture will then be poured on the surface of a minimal medium agar plate, and allowed to solidify prior to incubation.
The overlay mixture will be composed as follows:
(i) Overlay agar (held at 45 °C) 2.0 mL
(ii) Test or control item solution 0.1 mL
(iii) S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 mL
(iv) Bacterial suspension 0.1 mL
The volume of test item solution, as indicated, will usually be 0.1 mL; in the event that it is necessary to alter this volume, the quantities used will be carefully recorded.

Pre-incubation:
The components will be added in turn to an empty test-tube:
(i) Bacterial suspension 0.1 mL
(ii) Test or control item solution 0.05 mL
(iii) S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 mL
The volume of test item solution, as indicated, will usually be 0.05 mL. Where control or test items are dissolved in aqueous solvents, the volume used may be 0.1 mL. In the event that it is necessary to alter this volume, the quantities used will be carefully recorded. The treatment mixture will be vortexed and placed at 37 °C for 30 minutes. Two mL of overlay agar will then be added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.
Incubation and scoring:
The prepared plates will be inverted and incubated for approximately 72 hours at 37 °C. When the test item is a liquid at ambient temperature, the plates will be incubated in separate closed containers for higher and lower dose levels. After this period of incubation, the plates may be held at 4 °C prior to scoring. Scoring is effected by counting the number of revertant colonies on each plate manually. Contaminated plates will be considered on a case-by-case basis. Thinning of the background law and signs of precipitation will be recorded.

NUMBER OF REPLICATIONS: 3
Rationale for test conditions:
Solubility: Solubility of the test item was evaluated in a preliminary trial using DMSO. This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be fully miscible at 100 μL/mL. This result permitted a maximum concentration of 5.0 μL/plate to be used in the toxicity test.
Toxicity test: The test item Vinylcyclohexane was assayed in the toxicity test at a maximum dose level of 5.00 μL/plate and at four lower concentrations spaced at approximately half-log intervals: 1.58, 0.500, 0.158 and 0.0500 μL/plate.

Main assay: The maximum concentration of the test item to be used in the main experiments was selected
as the concentration which elicits a moderate toxicity. The number of lower dose levels included in each treatment series was selected in order to have a sufficient number of analysable concentrations.
Evaluation criteria:
The assay was considered valid if the following criteria were met:
1. Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 million for each strain.
3. No more than 5% of the plates should be lost through contamination or other unforeseen event.

Criteria for outcome of the assays:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
Statistics:
Regression analysis
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
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:
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No precipitation of test item was observed in any tester strain used in main assay 1 and 2 (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment 1 and 2.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Vinylcyclohexane at any concentration level, neither in the presence nor absence of metabolic activation in main assay 1 and 2.
All criteria of validity were met. The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions.
Conclusions:
The test item is not genotoxic in the bacterial reverse gene mutation assay in the presence and absence of mammalian metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471) strains of S. typhimurium (TA98, TA100, TA1535, TA1537, TA102) were exposed to Vinylcyclohexane (≥ 99.8 % purity) in aqua dest. at concentrations of 1.58, 0.500, 0.158 and 0.0500 µL/plate (main assay 1: plate incorporation, main assay 2: pre-incubation) in the presence and absence of mammalian metabolic activation. Vinylcyclohexane was tested up to the limit dose (5.0 µL/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Vinylcyclohexane was tested in a suitable in vitro genotoxicity testing battery as required by Annex VIII of the REACH regulation 1907/2006. Based on the lack of mutagenicity/cytogenicity in all in vitro assays (OECD 471, 476 and 487), the substance is considered to be non-genotoxic and no classification is warranted in accordance with CLP Regulation 1272/2008.