Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reverse gene mutation assay: Negative (non-mutagenic); OECD 471; Anon. (2011)

Mammalian cell cytogeneticity assay: Negative (non-clastogenic); OECD 473; Anon. (2017)

Mammalian cell gene mutation assay: Negative (non-mutagenic); OECD 476; Anon. (2017)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 June - 22 July 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ca. 4 ºC in the dark and under nitrogen
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Fully miscible in DMSO (dimethyl sulfoxide) and assumed stable.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (27.6%) of the test item.
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: Concentration of stock solution = 50 mg/mL.
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: No
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Salmonella strains (presence of S9-mix) - 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate
E. coli (presence of S9-mix) - 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
All bacterial strain (absence of S9-mix) - 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: guideline recommended
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation; in suspension (main test)
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): n/a
- Fixation time (start of exposure up to fixation or harvest of cells): n/a

SELECTION AGENT (mutation assays): n/a

SPINDLE INHIBITOR (cytogenetic assays): n/a

STAIN (for cytogenetic assays): n/a

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: n/a

NUMBER OF CELLS EVALUATED: n/a

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): n/a

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: n/a

DETERMINATION OF CYTOTOXICITY
- Method: presence of bacterial lawn
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:n/a
- Determination of endoreplication: n/a
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): n/a

- OTHER:
Rationale for test conditions:
The study was based on the in vitro technique described by Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence (Green and Muriel (1976)) is used to complement the Salmonella strains.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
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:
not examined
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:
not examined
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:
not examined
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:
not examined
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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not examined
- Effects of osmolality: not examined
- Evaporation from medium: not examined
- Water solubility: not examined
- Precipitation: none
- Definition of acceptable cells for analysis: All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: In the range-finding test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 1500 μg/plate in the absence and presence of S9-mix.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: n/a

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: n/a
- Indication whether binucleate or mononucleate where appropriate: n/a

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No
- Negative (solvent/vehicle) historical control data: Yes (2009 - 2010 data)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: presence of bacterial lawn
- Other observations when applicable: no

Table 1       Main test results

Base-pair substitution and frameshift type

Concentration [µg/plate]

No. of revertants per plate*

— MA

Cytotoxicity

+ MA

Cytotoxicity

TA100

(Base pair substitution)

0

110

N

123

N

0.15

104

N

-

-

0.5

113

N

-

-

1.5

98

N

103

N

5

110

N

109

N

15

95

Y

100

N

50

80

Y

93

N

150

0

Y

78

Y

500

-

-

0

Y

1500

-

-

0

Y

5000

-

-

-

-

Positive control

711

N

647

N

TA1535

(Base pair substitution)

0

18

N

11

N

0.15

17

N

-

-

0.5

16

N

-

-

1.5

17

N

10

N

5

15

N

11

N

15

18

N

10

N

50

12

Y

12

N

150

10

Y

8

N

500

-

-

0

Y

1500

-

-

0

Y

5000

-

-

-

-

Positive control

110

N

289

N

WP2uvrA

(Base pair substitution)

0

19

N

39

N

0.15

26

N

-

-

0.5

27

N

-

-

1.5

23

N

-

-

5

26

N

38

N

15

26

Y

37

N

50

21

Y

33

N

150

24

Y

34

N

500

-

-

32

N

1500

-

-

25

Y

5000

-

-

0

Y

Positive control

627

N

250

N

TA98

(Frameshift)

0

18

N

20

N

0.15

19

N

-

-

0.5

13

N

-

-

1.5

17

N

18

N

5

14

N

16

N

15

12

N

22

N

50

14

Y

22

N

150

12

Y

16

N

500

-

-

6

Y

1500

-

-

0

Y

5000

-

-

-

-

Positive control

112

N

190

N

TA1537

(Frameshift)

0

9

N

13

N

0.15

9

N

-

-

0.5

11

N

-

-

1.5

10

N

13

N

5

13

N

15

N

15

9

N

13

N

50

6

Y

14

N

150

2

Y

9

N

500

-

-

0

Y

1500

-

-

0

Y

5000

-

-

-

-

Positive control

617

N

98

N

- not conducted

MA: metabolic activation (S9 mix)

* mean of 3 replicates

Conclusions:
Te test item was considered to be non-mutagenic under the conditions of the test.
Executive summary:

OECD 471 (2011) - In a reverse gene mutation assay in bacteria, strains TA100, TA1535, TA98 and TA1537 of S. typhimuriumand WP2uvrA of E. coliwere exposed to Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene (limonene) and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene (ether MT) in DMSO at concentrations of 0.15, 0.5, 1.5, 5, 1550 and 150 µg/plate without metabolic activation and 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation, in a pre-incubation test system.

 

Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene (limonene) and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene (ether MT)was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains.

 

There was no evidence of induced mutant colonies over background in the test item treated colonies.

 

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:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 September 2016 - 06 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell transformation assay
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ca. 4 ºC in the dark and under nitrogen
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Miscible at 400 mg/mL in acetone
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Due to the sensitivity of human lymphocytes to acetone, the formulations were prepared at twice the concentration required in culture and dosed in 50 μl aliquots.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dissolved in acetone.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: Due to the sensitivity of human lymphocytes to acetone, the formulations were prepared at twice the concentration required in culture and dosed in 50 μl aliquots.
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): applied as a liquid

OTHER SPECIFICS: no
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: 18-35 year old non-smoking volunteers (human)
- Suitability of cells: previously screened for suitability
- Cell cycle length, doubling time or proliferation index: doubling time = approximately 16 hours.
- Sex, age and number of blood donors if applicable: 18-35 year old non-smokers- prelim test (female, 28 y/o), 4-h main test (male, 26 y/o) and 24-h main test (female, 25 y/o)
- Whether whole blood or separated lymphocytes were used if applicable: The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Number of passages if applicable: 1
- Methods for maintenance in cell culture if applicable: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % fetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Modal number of chromosomes: 46
- Normal (negative control) cell cycle time: 16 h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagles Minimal Essential Media
- Properly maintained: n/a - supplied by GIBCO BRL
- Periodically checked for Mycoplasma contamination: n/a - supplied by GIBCO BRL
- Periodically checked for karyotype stability: n/a - supplied by GIBCO BRL
- Periodically 'cleansed' against high spontaneous background: n/a - supplied by GIBCO BRL
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
4 h without S9 mix: 0, 3.75, 7.5, 15, 30, 45, 60 and 120 µg/mL
4 h with S9 mix: 0, 15, 30, 45, 60, 75, 90 and 120 µg/mL
24 h without S9 mix: 0, 15, 30, 45, 60, 75, 90 and 120 µg/mL

The dose levels used in the Main Experiment were selected using data from the Cell Growth Inhibition Test (Preliminary Toxicity Test) where the results indicated that the maximum concentration should be limited on toxicity.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: untreated solvent control preppared to demonstrate that no deleterious or clastogenic effects were induced by the chosen solvent
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension
- Cell density at seeding (if applicable): 0.75 mL blood

DURATION
- Preincubation period: 48 h
- Exposure duration: 4 h (short-term) or 24 h (continuous)
- Expression time (cells in growth medium): 20 h (short-term) and 0 h (continuous), respectively.
- Selection time (if incubation with a selection agent): Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours and 2.5 hours in the Cell Growth Inhibition Test and Main Test, respectively, before the required harvest time.
- Fixation time (start of exposure up to fixation or harvest of cells): After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

SELECTION AGENT (mutation assays): n/a

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (Colcemid 0.1 μg/mL)

STAIN (for cytogenetic assays): 5 % Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: When the slides were dry they were stained in 5 % Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate)

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

DETERMINATION OF CYTOTOXICITY
- Method: Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
- Any supplementary information relevant to cytotoxicity: no

OTHER EXAMINATIONS:
- Determination of polyploidy: In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.
- Determination of endoreplication: If the chromosomes were arranged in closely apposed pairs, i.e. 4 chromatids instead of 2, the cell was scored as endoreduplicated (E).
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): no

- OTHER:

Gaps (g)
Gaps are small areas of the chromosome that are unstained. The chromatids remain aligned as normal and the gap does not extend along the chromatid for a distance greater than the width of a chromatid. If the gap occurs on one chromatid only it is a chromatid gap (g). If a gap appears in both chromatids at the same position it is a chromosome gap (G).

Chromatid Breaks (ctb)
Chromatid breaks (ct) vary in appearance. The chromatid may remain aligned but show a gap which is too large to classify as a gap. Alternatively, the chromatid may be broken so that the broken fragment is displaced. In some cases, the fragment is not seen at all. A chromatid fragment (f) should be scored if the chromosome of origin cannot be identified. Very small fragments are scored as minutes (m).

Chromosome Breaks (csb)
Chromosome breaks (CS) are breaks in both chromatids of the chromosome. A fragment with two chromatids is formed and may be displaced by varying degrees. Breaks are distinguished from gaps by the size of the unstained region. A chromosome break is scored if the fragment is associated with a chromosome from which it was probably derived. However, fragments are often seen in isolation and are then scored as chromosome fragments (F). Very small fragments are scored as minutes (M).

Exchanges (cte and cse)
Exchanges are formed by faulty rejoining of broken chromosomes and may be of the chromosome or chromatid type. Chromatid exchanges (c/c,r) have numerous different forms but are generally not further classified. Where multiple exchanges have occurred each exchange point is counted as one chromatid exchange. Chromosome exchanges generally appear as either a dicentric (D) or a ring (R) form, either of which can be associated with a fragment, which if possible should be scored as part of the exchange.

Multiple Aberrations
If many aberrations are present in one metaphase, the exact details may not be scorable. This is particularly the case when chromosome pulverisation occurs. If the number of aberrations is 10 or more then the cell is classified as X.

Evaluation criteria:
The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range. The level of spontaneous background aberrations was slightly elevated above the normal range and the experiment still considered valid.
• All the positive control chemicals induced a positive response (p ≤ 0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
hemolysis was observed at and above 30 μg/mL which was accompanied by a reduced cell pellet from 60 μg/mL. This indicates that maximum exposure was reached at and above 60 μg/mL.
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: not affected
- Effects of osmolality: not affected
- Evaporation from medium: not affected
- Water solubility: not affected
- Precipitation: precipitation present in prelim test at and above 125 µg/mL. Main test concentrations were tested below this level.
- Definition of acceptable cells for analysis:
- Other confounding effects: hemolysis (cytotoxic response) - see below.

RANGE-FINDING/SCREENING STUDIES:

The dose range for the Cell Growth Inhibition Test was 7.81 to 2000 μg/mL. The maximum dose was the maximum recommended dose level.

A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 125 μg/mL in the 4(20)-hour exposure group in the absence of metabolic activation (S9) and at and above 250 μg/mL in the 4(20)-hour exposure group in the presence of metabolic activation and the 24-hour continuous exposure groups.

Hemolysis was observed following exposure to the test item at and above 7.81 μg/mL in the 4(20)-hour exposure groups and at and above 31.25 μg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. In addition to the hemolysis, a reduced cell pellet was observed at and above 125 μg/mL in all three exposure groups and is indicative of toxicity to the cell population present and can indicate that maximum exposure has been reached.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 62.5 μg/mL in all three exposure groups. Above this dose level there were no metaphases present for assessment. The test item induced evidence of severe and steep toxicity in all of the exposure groups.

The selection of the maximum dose level was based on toxicity for all three exposure groups used in the Main Experiment.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: n/a

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Hemolysis was observed following exposure to the test item at and above 7.81 μg/mL in the 4(20)-hour exposure groups and at and above 31.25 μg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. In addition to the hemolysis, a reduced cell pellet was observed at and above 125 μg/mL in all three exposure groups and is indicative of toxicity to the cell population present and can indicate that maximum exposure has been reached.

Table 1       Short term exposure: 4(20) hours without metabolic ativation (S9 mix)

Treatment period

(h)

S9 mix

Conc.

µg/mL

 

 

g

Cell growth index

Number and % of cells showing numerical aberrations (%)

Observed

ctb

cte

csb

cse

Other

Total

Mitotic index

%

Observed

Polyploids

Others

Total

4

-

Solvent control

0

A

150

0

0

0

0

0

0

0

7.00

150

0

0

0

B

150

2

0

0

0

0

2

4

5.40

150

0

0

0

Total (%)

300

(100)

2

(0.7)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

2

(0.7)

4

(1.3)

(100)

300

0

0

0

(0.0)

-

15

A

150

1

0

0

0

0

1

0

5.75

150

0

0

0

B

150

2

0

0

0

0

2

2

3.85

150

0

0

0

Total (%)

300

(100)

3

(1.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

3

(1.0)

2

(0.7)

(77)

300

0

0

0

(0.0)

-

30

A

150

0

0

0

0

0

0

0

5.55

150

0

0

0

B

150

1

0

2

0

0

2

2

4.85

150

0

0

0

Total (%)

300

(100)

1

(0.3)

0

(0.0)

2

(0.7)

0

(0.0)

0

(0.0)

2

(0.7)

2

(0.7)

(84)

300

0

0

0

(0.0)

-

45

A

150

4

0

0

0

0

4

4

1.90

150

0

0

0

B

150

0

0

0

0

0

0

0

3.75

150

0

0

0

Total (%)

300

(100)

4

(1.3)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

4

(1.3)

4

(1.3)

(46)

300

0

0

0

(0.0)

-

Positive control

0.2

A

150

7

3

1

0

0

11

2

3.10

150

0

0

0

B

57a

14

2

0

0

0

16

0

3.15

57

0

0

0

Total (%)

207

(100)

21

(10.1)

5

(2.4)

1

(0.5)

0

(0.0)

0

(0.0)

27***

(13.0)

2

(1.0)

(50)

207

0

0

0

(0.0)

ctb: chromatid breaks

cte and cse: exchanges

csb: chromosome breaks

g: gaps

Solvent control: acetone

Positive control: mitomycin C

*** p < 0.001

a: slide evaluation terminated when at least 15 cells with aberrations (excluding gaps) had been observed

 

Table 2       Short term exposure: 4(20) hours with metabolic activation (S9 mix)

Treatment period

(h)

S9 mix

Conc.

µg/mL

 

 

g

Cell growth index

Number and % of cells showing numerical aberrations (%)

Observed

ctb

cte

csb

cse

Other

Total

Mitotic index

%

Observed

Polyploids

Others

Total

4

+

Solvent control

0

A

150

2

0

1

0

0

3

0

6.20

150

0

0

0

B

150

1

0

1

0

0

2

4

4.30

150

0

0

0

Total (%)

300

(100)

3

(1.0)

0

(0.0)

2

(0.7)

0

(0.0)

0

(0.0)

5

(1.7)

4

(1.3)

(100)

300

(100)

0

0

0

(0.0)

+

45

A

150

1

0

0

0

0

1

4

4.10

150

0

0

0

B

150

2

0

0

0

0

2

3

3.15

150

0

0

0

Total (%)

300

(100)

3

(1.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

3

(1.0)

7

(2.3)

(69)

300

(100)

0

0

(0.0)

+

60

A

150

0

0

0

0

0

2

2

3.25

150

1

0

1

B

150

1

0

0

0

0

1

1

5.55

150

1

0

1

Total (%)

300

(100)

1

(0.3)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

3

(1.0)

3

(1.0)

(84)

300

(100)

2

0

2

(0.7)

+

90

A

150

2

0

0

0

0

4

4

2.05

150

1

0

1

B

150

0

0

0

0

0

1

1

2.40

150

0

0

0

Total (%)

300

(100)

2

(0.7)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

5

(1.7)

5

(1.7)

(42)

300

(100)

1

0

1

(0.3)

+

Positive control

2.0

A

69a

10

3

3

0

0

15

1

2.15

69

0

0

0

B

108a

8

5

3

0

0

15

2

3.80

108

0

0

0

Total (%)

177

(100)

18

(10.2)

8

(4.5)

6

(3.4)

0

(0.0)

0

(0.0)

30***

(16.9)

3

(1.7)

(57)

177

(100)

0

0

0

(0.0)

ctb: chromatid breaks

cte and cse: exchanges

csb: chromosome breaks

g: gaps

Solvent control: acetone

Positive control: cyclophosphamide

*** p < 0.001

a: slide evaluation terminated when at least 15 cells with aberrations (excluding gaps) had been observed

Table 3       Contiuous exposure: 24 hours without metabolic activation (S9 mix)

Treatment period

(h)

S9 mix

Conc.

µg/mL

 

 

g

Cell growth index

Number and % of cells showing numerical aberrations (%)

Observed

ctb

cte

csb

cse

Other

Total

Mitotic index

%

Observed

Polyploids

Others

Total

24

-

Solvent control

0

A

150

0

0

0

0

0

0

4

12.05

150

0

0

0

B

150

0

0

0

0

0

0

0

10.60

150

0

0

0

Total (%)

300

(100)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

4

(1.3)

(100)

300

(100)

0

0

0

(0.0)

-

60

A

150

0

0

0

0

0

0

2

8.65

150

0

0

0

B

150

2

0

1

0

0

3

1

10.45

150

0

0

0

Total (%)

300

(100)

2

(0.7)

0

(0.0)

1

(0.3)

0

(0.0)

0

(0.0)

3

(1.0)

3

(1.0)

(84)

300

(100)

0

0

0

(0.0)

-

75

A

150

0

0

0

0

0

0

0

7.15

150

0

0

0

B

150

0

0

0

0

0

0

0

8.65

150

0

0

0

Total (%)

300

(100)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

(70)

300

(100)

0

0

0

(0.0)

-

90

A

150

0

1

0

0

0

1

0

5.45

150

0

0

0

B

150

0

0

1

0

0

1

0

10.00

150

0

0

0

Total (%)

300

(100)

0

(0.0)

1

(0.3)

1

(0.3)

0

(0.0)

0

(0.0)

2

(0.7)

0

(0.0)

(68)

300

(100)

0

0

0

(0.0)

-

Positive control

0.1

A

150

8

3

2

0

0

10

2

4.70

150

0

0

0

B

134

9

8

0

0

0

17

5

4.05

134

0

0

0

Total (%)

284

(100)

17

(6.0)

11

(3.9)

2

(0.7)

0

(0.0)

0

(0.0)

27

(9.5)

7

(2.5)

(39)

284

(100)

0

0

0

(0.0)

ctb: chromatid breaks

cte and cse: exchanges

csb: chromosome breaks

g: gaps

Solvent control: acetone

Positive control: mitomycin C

*** p < 0.001

a: slide evaluation terminated when at least 15 cells with aberrations (excluding gaps) had been observed

Conclusions:
Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

OECD 473 (2017) - In a mammalian cell cytogenetics assay (in vitro chromosome aberration, OECD 473), primary lymphocyte cultures were exposed to Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene at concentrations of 0, 3.75, 7.5, 15, 30, 45, 60 and 120 µg/mL for 4 h without metabolic activation or 0, 15, 30, 45, 60, 75, 90 and 120 µg/mL for 4 h with metabolic. Additionally, a continuous exposure of 24 h was tested without metabolic activation at test item concentrations of 0, 15, 30, 45, 60, 75, 90 and 120 µg/mL.

 

Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene was tested up to precipitating and cytotoxic concentrations of 120 µg/mL. In the range-finding test, concentrations above 125 μg/mL in all three exposure groups had a reduced pellet which is indicative of toxicity to the cell population present, indicating that maximum exposure has been reached.  Positive controls induced the appropriate response.

 

There was no evidence (or a concentration related positive response) of chromosome aberration induction over background values at the highest tested concentration.

 

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline In vitro Mammalian Chromosome Aberration Test (OECD 473) in human lymphocyte cells.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 March - 16 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with International guidleines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell transformation assay
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ca. 4 ºC in the dark and under nitrogen
- Stability under test conditions: Asumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Soluble at 400 mg/mL in acetone
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item formulated in acetone at 400 mg/mL. Serial dilutions prepared from stock did not contain more than 0.5 % acetone v/v.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: Stock prepared at 400 mg/mL
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): No, applied as a liquid.

OTHER SPECIFICS: n/a
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type.
- Cell cycle length, doubling time or proliferation index: Doubling time 12 - 16 h in stock cultures.
- Sex, age and number of blood donors if applicable: n/a
- Whether whole blood or separated lymphocytes were used if applicable: n/a
- Number of passages if applicable: n/a
- Methods for maintenance in cell culture if applicable: Laboratory stock cell cultures will be periodically checked for stability and absence of mycoplasma contamination. The stock of cells is stored in liquid nitrogen. Cell stocks spontaneously mutate at a low but significant rate. Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 μg/mL, 100 μM). Aminopterin (0.0178 μg/mL, 0.4 μM) and Thymidine (3.85 μg/mL, 16 μM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.
- Modal number of chromosomes: The cells have a stable karyotype with a modal chromosome number of 22 (Howard-Flanders, 1981).
- Normal (negative control) cell cycle time: The average absolute cloning efficiency of the Day 7 negative controls should exceed 50%.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: For use, a sample of cells was removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10 % fetal bovine serum (FBS)) at approximately 37 °C with 5 % CO2 in humidified air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix. Prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G-6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration.
Test concentrations with justification for top dose:
Prelim Test - 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 and 200 μg/mL (limited by cytotoxicity observed in concurrent Chromosome Aberration Test).
Main Test (with S9-mix) - 3.13, 6.25, 12.5, 25, 30, 35, 40*, 45* and 50* µg/mL (based on results of prelim). **not plated for cloning efficiency and mutant frequency due to high levels of cytotoxicity.
Main Test (without S9-mix) - 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µg/mL (based on results of prelim).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Test item was readily soluble and not toxic to the test system below 0.5 % v/v.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: Pre-incubation with test item
- Cell density at seeding (if applicable): For mutation expression = 2,000,000 cells per 225 cm2 flask; for cloning efficiency = 200 cells per 25 cm2 flask.

DURATION
- Preincubation period: Cells were seeded at 1 x 107 cells/225 cm2 flask approximately 24 hours being exposed to the test or control items.
- Exposure duration: Treatment was for 4 hours in serum free media (MEM) at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): n/a
- Fixation time (start of exposure up to fixation or harvest of cells): Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

SELECTION AGENT (mutation assays): n/a

SPINDLE INHIBITOR (cytogenetic assays): n/a

STAIN (for cytogenetic assays): Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

NUMBER OF REPLICATIONS: 1 for growth and mutation expression and 3 for cloning efficiency.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: n/a

NUMBER OF CELLS EVALUATED: n/a

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): n/a

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: n/a

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: n/a

OTHER EXAMINATIONS:
- Determination of polyploidy: n/a
- Determination of endoreplication: n/a
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): n/a

- OTHER: n/a
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical negative control data for the test item concentrations.

When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical negative control data.

The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Statistics:
Comparisons made between the appropriate vehicle control value and each individual concentration, using Student’s t-test.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Tested up to and beyond cytotoxic limit
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: No effect
- Effects of osmolality: No effect
- Evaporation from medium: Not observed
- Water solubility: Formulated in acetone, therefore no effect
- Precipitation: Not plated when observed (see Table 1)
- Definition of acceptable cells for analysis: Reported and within specification. Fresh cultures used for each test.
- Other confounding effects: None.

Table 2 Main Experiment – 4 hour exposure without S9 mix

 

Dose

(µg/mL)

Rep

Day 0 viability

Day 7 viability

Day 7 Mutant

Colonies/ flask

(200 cells plated/flask)

% CE

% control

Mean % control

Colonies/ flask

(200 cells plated/flask)

% CE

% control

Mean % control

Colonies/flask (2x105cells plated/flask)

MF

MFS 10-6

SD

Group MFS 10-6

160

A

186

171

170

87.8

100

100

138

144

143

70.8

100

100

3

3

3

0

3

4

2

0

2

4

12

16.9

1.23

17

B

176

210

213

99.8

100

134

148

137

69.8

100

1

3

2

3

2

3

1

4

3

1

11.5

16.5

17317.13

A

170

168

180

86.3

98.3

97

142

143

139

70.7

99.8

101

0

4

5

3

2

4

4

2

2

1

13.5

19.1

1.41

16

B

195

200

184

96.5

96.7

146

139

147

72.0

103.1

1

2

4

1

2

3

0

2

2

1

9

12.5

6.25

A

192

201

202

99.2

112.9

105

137

140

136

68.8

97.2

98

2

1

3

2

1

4

4

0

3

1

10.5

15.3

1.46

17

B

194

187

205

97.7

97.8

150

125

138

68.8

98.6

4

0

3

4

2

4

2

3

0

4

13

18.9

12.5

A

198

197

184

96.5

109.9

107

138

148

146

72.0

101.6

101

3

4

0

3

4

0

1

4

3

1

11.5

16.0

1.90

17

B

207

204

211

103.7

103.8

142

141

139

70.3

100.7

3

3

5

0

0

6

1

4

0

4

13

18.5

25

A

167

161

163

81.8

93.2

98

141

145

137

70.5

99.5

100

3

0

4

3

3

0

0

3

5

1

11

15.6

1.50

15

B

204

209

199

102.0

102.2

148

138

139

70.8

101.4

0

4

4

2

2

1

2

2

2

2

10.5

14.8

30

A

68

64

59

31.8

36.2

51

139

143

140

70.3

99.3

100

0

2

1

3

3

2

3

2

2

1

9.5

13.5

1.27

13

B

181

111

104

66.0

66.1

140

142

140

70.3

100.7

3

2

1

0

1

3

0

2

5

1

9

12.8

35

A

3

15

5

3.8

4.4

19

142

148

139

71.5

100.9

102

0

2

0

5

2

2

3

1

3

0

9

12.6

1.41

13

B

66

70

64

33.3

33.4

139

140

153

72.0

103.1

3

2

1

1

3

1

4

2

0

3

10

13.9

40

A

3

3

0

1.0

1.1

5

Not plated due to toxicity

Not plated due to toxicity

B

16

22

19

9.5

9.5

Not plated due to toxicity

Not plated due to toxicity

45

A

2

1

0

0.5

0.6

10

Not plated due to toxicity

Not plated due to toxicity

B

39

37

35

18.5

18.5

Not plated due to toxicity

Not plated due to toxicity

50

A

Not plated due to toxicity

Not plated due to toxicity

Not plated due to toxicity

B

Not plated due to toxicity

Not plated due to toxicity

Not plated due to toxicity

EMS 500

A

137

152

157

74.3

84.6

91

145

147

142

72.3

102.1

99

39

42

35

41

39

30

36

39

37

23

180.5

249.5

6.35

283

B

182

203

196

96.8

97.0

137

146

121

67.3

96.4

46

38

40

44

51

39

33

42

44

49

213

316.3

EMS 750

A

165

169

168

83.7

95.3

91

128

128

122

63.0

88.9

92

50

64

63

69

55

54

57

73

75

64

312

495.2

8.77

469

B

171

163

187

86.8

87.0

133

131

137

66.8

95.7

56

53

61

62

64

79

58

65

47

48

296.5

443.6

EMS: ethyl methane sulphonate

CE: cloning efficiency

MF: mutant frequency

MFS: mutant frequency per survivor

SD: standard deviation

 

Table 3 Main Experiment – 4 hour exposure with S9 mix

 

Dose

(µg/mL)

Rep

Day 0 viability

Day 7 viability

Day 7 Mutant

Colonies/ flask

(200 cells plated/flask)

% CE

% control

Mean % control

Colonies/ flask

(200 cells plated/flask)

% CE

% control

Mean % control

Colonies/flask (2x105cells plated/flask)

MF

MFS 10-6

SD

Group MFS 10-6

0

A

199

204

192

99.2

100

100

172

185

174

88.5

100

100

2

3

0

0

0

1

2

2

1

3

7

7.9

1.08

9

B

187

205

192

97.3

100

207

220

209

106.0

100

0

2

2

2

2

2

1

3

3

3

10

9.4

10

A

191

192

189

95.3

96.1

97

180

179

174

88.8

100.4

98

0

3

1

0

1

0

2

1

1

0

4.5

5.1

1.52

9

B

193

187

191

95.2

97.8

204

211

194

101.5

95.8

0

2

4

2

3

2

1

5

4

3

13

12.8

20

A

190

201

200

98.5

99.3

95

184

171

177

88.7

100.2

97

1

3

2

2

1

1

1

3

2

2

9

10.2

1.15

10

B

163

183

187

88.8

91.3

201

198

192

98.5

92.9

1

1

2

4

5

1

1

2

1

3

10.5

10.7

40

A

175

192

188

92.5

93.3

92

171

177

185

88.8

100.4

98

3

1

1

2

3

1

3

2

1

1

9

10.1

1.20

10

B

191

169

174

89.0

91.4

211

199

195

100.8

95.1

1

3

0

4

0

4

1

2

1

2

9

8.9

50

A

166

170

174

85.0

85.7

88

182

171

179

88.7

100.2

98

0

1

3

3

2

0

2

1

1

2

7.5

8.5

1.03

7

B

179

171

173

87.2

89.6

193

207

204

100.7

95.0

1

2

0

1

1

0

2

1

0

3

5.5

5.5

60

A

146

148

170

77.3

78.0

63

168

173

172

85.5

95.6

97

1

1

0

2

3

2

4

2

2

5

11

12.9

1.47

11

B

103

100

77

46.7

47.9

210

199

206

102.5

96.7

2

0

3

4

4

1

0

3

1

1

9.5

9.3

70

A

34

45

44

20.5

20.7

20

156

147

162

77.5

87.6

95

0

1

1

1

1

2

2

0

2

1

5.5

7.1

0.99

8

B

34

41

33

18.0

18.5

211

228

214

108.8

102.7

1

2

2

4

2

2

1

1

3

3

10.5

9.6

80

A

8

6

17

5.2

5.2

8

Excluded to due toxicity

Excluded to due toxicity

B

25

17

16

9.7

9.9

Excluded to due toxicity

Excluded to due toxicity

90

A

7

17

15

6.5

6.6

6

Excluded to due precipitate

Excluded to due precipitate

B

12

14

8

5.7

5.8

Excluded to due precipitate

Excluded to due precipitate

100

A

15

12

10

6.2

6.2

11

Excluded to due precipitate

Excluded to due precipitate

B

34

29

31

15.7

16.1

Excluded to due precipitate

Excluded to due precipitate

DMBA 1

A

168

154

181

83.8

84.5

86

172

174

181

87.8

99.2

97

44

38

42

31

50

38

41

42

53

41

210

239.1

8.93

250

B

170

164

182

86.0

88.4

205

193

200

99.7

94.0

63

47

45

41

65

50

48

59

45

59

261

261.9

DMBA 2

A

138

155

129

70.3

70.9

71

134

129

139

67.0

75.7

73

80

81

64

81

70

67

68

72

59

60

351

523.9

11.87

560

B

140

150

130

70.0

71.9

145

153

147

74.2

70.0

97

92

81

87

89

89

81

99

78

90

441.5

595.3

DMBA: dimethyl benzanthracene

CE: cloning efficiency

MF: mutant frequency

MFS: mutant frequency per survivor

SD: standard deviation

 

Conclusions:
The test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.
Executive summary:

OECD 476 (2017) - In a mammalian cell gene mutation assay (HPRT locus), Chinese hamster (V79) cells cultured in vitro were exposed to the Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene and 4(R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexeneat at concentrations of 3.13, 6.25, 12.5, 25, 30 and 35 µg/mL and 10, 20, 40, 50, 60, 70, 80 and 90 µg/mL in the absence and presence of mammalian metabolic activation (S9-mix), respectively.

 

The test item was tested up to cytotoxic and solubility limits. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background levels at the concentrations tested.

 

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 conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No information available.

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable as there were no adverse effects observed.

Additional information

OECD 471 (2011) - In a reverse gene mutation assay in bacteria, strains TA100, TA1535, TA98 and TA1537 of S. typhimuriumand WP2uvrA of E. coliwere exposed to Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene (limonene) and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene (ether MT) in DMSO at concentrations of 0.15, 0.5, 1.5, 5, 1550 and 150 µg/plate without metabolic activation and 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation, in a pre-incubation test system.  

Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene (limonene) and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene (ether MT) was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains.   

There was no evidence of induced mutant colonies over background in the test item treated colonies.  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.

OECD 473 (2017) - In a mammalian cell cytogenetics assay (in vitro chromosome aberration, OECD 473), primary lymphocyte cultures were exposed to Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene at concentrations of 0, 3.75, 7.5, 15, 30, 45, 60 and 120 µg/mL for 4 h without metabolic activation or 0, 15, 30, 45, 60, 75, 90 and 120 µg/mL for 4 h with metabolic. Additionally, a continuous exposure of 24 h was tested without metabolic activation at test item concentrations of 0, 15, 30, 45, 60, 75, 90 and 120 µg/mL.  Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene was tested up to precipitating and cytotoxic concentrations of 120 µg/mL. In the range-finding test, concentrations above 125 μg/mL in all three exposure groups had a reduced pellet which is indicative of toxicity to the cell population present, indicating that maximum exposure has been reached.  Positive controls induced the appropriate response.  There was no evidence (or a concentration related positive response) of chromosome aberration induction over background values at the highest tested concentration.  This study is classified as acceptable.  This study satisfies the requirement for Test Guideline In vitro Mammalian Chromosome Aberration Test (OECD 473) in human lymphocyte cells.

OECD 476 (2017) - In a mammalian cell gene mutation assay (HPRT locus), Chinese hamster (V79) cells cultured in vitro were exposed to the Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene and 4(R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexeneat at concentrations of 3.13, 6.25, 12.5, 25, 30 and 35 µg/mL and 10, 20, 40, 50, 60, 70, 80 and 90 µg/mL in the absence and presence of mammalian metabolic activation (S9-mix), respectively.  The test item was tested up to cytotoxic and solubility limits. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background levels at the concentrations tested.  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.

Justification for classification or non-classification

The substance does not meet the criteria for classfication in accordance with GHS or Regulation (EC) No 1272/2008 (CLP)