[3R-(3α,3aβ,7β,8aα)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)ethan-1-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test): the substance Methyl Cedryl Ketone (CAS No. 32388-55-9) does not induce mutagenicity using S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A pKM 101 in the presence or absence of rat S9 metabolic activation (OECD 471/GLP);  

Chromosome aberration (in vitro mammalian cell cytogenicity): the substance Methyl Cedryl Ketone (CAS No. 32388-55-9) does not induce chromosome aberrations in CHO-K1 cells (CCL 61) cells in the presence or absence of Aroclor 1254-induced rat liver S9 metabolic activation (OECD 473/GLP);  

Gene mutation (in vitro mammalian cell gene mutation): the substance Methyl Cedryl Ketone (CAS No. 32388-55-9) does not induce gene mutations in L5178Y mouse lymphoma cells in the presence or absence of S9 rat liver metabolic activation (OECD 476/GLP).

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:

16-10-2012 to 30-10-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline and GLP compliant

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Commercially available S9 fraction (Trinova Biochem GmbH, Germany) supplemented with co-factors.Lot No.: 2907 (Date of preparation: 15 March 2012) – used in test 1; Lot No.: 2943 (Date of preparation:18 May 2012) – used in test 2.

Test concentrations with justification for top dose:

Mutation Test - Experiment 1 (direct plate incorporation): 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Mutation Test - Experiment 2 (pre-incubation): 0, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of Methyl Cedryl Ketone was assessed at 50 mg/mL in dimethyl sulphoxide
(DMSO) and was found to be soluble. DMSO (HPLC grade) was, therefore, used as the vehicle for this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

NaN3: 2 µg/plate TA100 and TA1535; AAC: 50 µg/plate TA1537; NQO: 2 µg/plate WP2 uvrA (pKM101); 2-NF:2 µg/plate for strain TA98.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

AAN: 5 µg/plate TA100 and TA1535, 10 µg/plate WP2 uvrA (pKM101); B[a]P:5 µg/plate for TA98 and TA1537

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Mutation Test - Experiment 1: direct plate incorporation method;
Mutation Test - Experiment 2 : preincubation method

DURATION
- Preincubation period: 30 minutes (Mutation Test - Experiment 2)
- Exposure duration: approximately 72 hrs (both methods)

NUMBER OF REPLICATIONS:3 (Experiments 1 & 2)

DETERMINATION OF CYTOTOXICITY
- Method: Substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both.

Evaluation criteria:

If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.

If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system.

Statistical analysis can be performed if results fail to satisfy the criteria for a clear “positive” or “negative” response; see Statistics below.

It is acceptable to conclude an equivocal response if no clear results can be obtained after statistical analysis.

Statistics:

If the results obtained satisfy the criteria for a clear “positive” or “negative” response, no statistical analysis is performed.

If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis.

Biological importance will be considered along with statistical significance. In general,treatment-associated increases in revertant colony
numbers below two or three times those of the vehicle controls are not considered biologically important.

MAHON, G.A.T., GREEN, M.H.L., MIDDLETON, B., MITCHELL, I. de G., ROBINSON,
W.D. and TWEATS, D.J. (1989) Analysis of data from microbial colony assays in:
KIRKLAND, D.J. (Ed.). UKEMS Sub-committee on Guidelines for Mutagenicity Testing.
Report. Part III. Statistical Evaluation of Mutagenicity Test Data, pp.26-65. Cambridge
University Press, Cambridge.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:The mean revertant colony counts for the vehicle controls were within or close to the current
historical control range for the laboratory (Period: 1 May 2007 to 30 April 2012; Appendix I).

Study report attachments:

Table 1 Results obtained in the absence of metabolic activation - test 1 (MBB0003)

Table 2 Results obtained in the presence of metabolic activation - test 1 (MBB0003)

Table 3 Viability counts obtained in test 1 (MBB0003)

Table 4 Results obtained in the absence of metabolic activation - test 2 (MBB0003)

Table 5 Results obtained in the presence of metabolic activation - test 2 (MBB0003)

Table 6 Viability counts obtained in test 2 (MBB0003)

Appendix 1 Historical Control Data (MBB0003)

Conclusions:

It was concluded that Methyl Cedryl Ketone showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

In a reverse gene mutation assay in bacteria (MBB0003), strains of S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A (pKM101) were exposed to Methyl Cedryl Ketone in DMSO at concentrations of 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (direct plate incorporation; experiment 1) and 0, 50, 150, 500, 1500 and 5000 µg/plate (pre-incubation; experiment 2) in the presence and absence of mammalian metabolic activation (rat S9). Methyl Cedryl Ketone was tested up to the limit concentration (5000 µg/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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25-02-2003 to 25-04-2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline and GLP compliant study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: CHO-K1 cells (CCL 61)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5A medium with 10% FBS, 100 units penicillin/mL, 100µg streptomycin/mL and 2mM L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: Yes; working cell stocks were not used beyond passage 20.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity tests: 0.25, 0.75, 2.5, 7.4, 24.6, 73.8, 246, 738, 2640 µg/mL +/- S9.
Selection of dose levels for the chromosome aberration assay was based on cell growth inhibition relative to the solvent control. Substantial
toxicity (i.e., at least 50% cell growth inhibition, relative to the solvent control) was observed at dose levels > 24.6 µg/mL in both the non-activated and S9 activated 4 hour exposure groups, and at dose levels > 73.8 µg/mL in the non-activated 20 hour continuous exposure group.


Chromosome aberration assay: 0, 3.125, 6.25, 12.5, 25, 35, 50 µg/mL +/- S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (CAS No. 64-17-5) from Pharmco

- Justification for choice of solvent/vehicle: A solubility test was conducted using DMSO and ethanol. Ethanol was determined to be the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test substance was soluble in ethanol at a concentration of 500 mg/mL, the maximum concentration tested for solubility.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol (CAS No. 64-17-5)

True negative controls:

no

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

mitomycin C

Remarks:

Mitomycin C (CAS 50-07-7) at 0.1 and 0.2 µg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

Cyclophosphamide (CAS 6055-19-2) at 10 and 20 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in cell culture medium

DURATION
- Exposure duration: 4 hours +/-S9; 20 hours - S9.
- Fixation time (start of exposure up to fixation or harvest of cells): All cultures harvested at 20 hours and fixed.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid at 0.1µg/mL
STAIN (for cytogenetic assays): Cells fixed in Carnoys fixative and Giesma stained.

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: % of cells in mitosis per 500 cells scored determined for each dose. Metaphase cells with 20+/-2 centromeres were scored. Minimum of 200 cells per metaphase spread (100 per duplicate) scored for abberrations.

DETERMINATION OF CYTOTOXICITY
- Method: Cell viability

OTHER EXAMINATIONS:
- Determination of polyploidy: Polyploidy was evaluated from each treatment flask per 100 treatment cells evaluated.
- Determination of endoreplication: Endoreplication was evaluated from each treatment flask per 100 treatment cells evaluated.

Evaluation criteria:

The toxic effects of treatment were based upon cell growth inhibition relative to the solvent-treated control.
The number and types of aberrations found, the percentage of structurally and numerically damaged cells (percent aberrant cells) in the total population of cells examined, and the mean aberrations per cell were calculated and reported for each group.

1. The test article was considered to induce a positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant (p<0.05). Test articles not demonstrating a statistically significant increase in aberrations will be concluded to be negative.

2. The frequency of cells with structural chromosome aberrations in the solvent control must be within the range of the historical solvent control.
Values that are statistically significant but do not exceed the range of historic solvent controls may be judged as not biologically significant.


3. The percentage of cells with chromosome aberrations in the positive control must be statistically increased (p<0.05, Fisher's exact test) relative to the solvent control.

Statistics:

Percent aberrant cells: Fisher's exact test.
Positive Fisher's test: Cochran-Armitage test (dose-response).
Significance: p<0.05.

Species / strain:

other: CHO-K1 cells (CCL 61)

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 24.6 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

other: CHO-K1 cells (CCL 61)

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 73.8 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

other: CHO-K1 cells (CCL 61)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

35 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

other: CHO-K1 cells (CCL 61)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

12.5 µg/mL (16%)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

other: CHO-K1 cells (CCL 61)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

25 µg/mL (40%)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the highest concentration of test article in treatment medium was approximately 7.0.

- Effects of osmolality: The osmolality of the test substance concentrations in treatment medium are acceptable because they did not exceed the osmolality of the solvent by more than 20%.
- Precipitation: In the preliminary test - Visible precipitate was observed in treatment medium at dose levels > 246 µg/mL; Visible precipitate was observed after treatment period at concentrations > 738 µg/mL.


COMPARISON WITH HISTORICAL CONTROL DATA: The percentage of cells with structural aberrations in the test article-treated group (2.5%) was within the historical solvent control range of 0.0% to 6.5%. Therefore, it is not considered to be biologically significant.

Table 1: Summary table of activity of Acetyl cedrene in the induction of chromosome aberrations

							Cells With Aberrations
Treatment µg/mL	S9 Activation	Treatment Time (h)	Mean Mitotic Index	Cells scored	Aberrations per cell (Mean +/- SD)		Numerical (%)	Structural (%)
Ethanol	-	4	10.2	200	0	±0.000	1.5	0
Acetyl cedrene
6.25	-	4	10,2	200	0	±0.000	1.5	0
12.5	-	4	10	200	0	±0.000	2	0
35	-	4	5.9	200	0.005	±0.071	2	0.5
MMC
0.2		4	10.4	150	0.18	±0.479	2.5¶	14.7**
Ethanol	-	4	13.2	200	0	±0.000	1.5	0
Acetyl cedrene
3.125	+	4	12.5	200	0	±0.000	1	0
6.25	+	4	12.1	200	0	±0.000	2	0
12.5	+	4	6.4	200	0.025	±0.157	3.5	2.5*
CP
10	+	4	9.4	200	0.105	±0.338	2	9.5**
Ethanol	-	20	13.3	200	0	±0.000	1.5
Acetyl cedrene
6.25	-	20	12.3	200	0	±0.000	2	0
12.5	-	20	12.5	200	0.015	±0.122	1.5	1.5
25	-	20	6.5	200	0	±0.000	2	0
MMC
0.1	-	20	10.8	150	0.193	±0.459	2.5¶	16.7**

¶ Numerical aberrations are out of 200 cells scored

Treatment: Cells from all treatment conditions were iiarvested 20 hours after the initiation of the treatments

Aberrations per Cell: Severely damaged cells were counted as 10 aberrations.

Percent Aberrant Cells: *p ≤ 0.05; **p ≤ 0.01; using Fisher's exact test

Table 2: Overall assay results

Treatment time	Recovery time	Harvest time	S9	Toxicity* at highest dose scored (µg/mL)	Mitotic index reduction **	LED (1) for structural aberrations	LED (2) for numerical aberrations
4 hr	16 hr	20 hr	-	62% at 35	42%	None	None
20 hr	O hr	20 hr	-	40% at 25	51%	None	None
4 hr	16 hr	20 hr	+	16% at 12.5	52%	None	None

*cell growth inhibition

**relative to solvent control at high dose evaluated for chromosome aberrations

(1) LED = lowest effective dose

(2) µg/mL

Table 3: Historical Control Values - CHO cells (Structural aberrations)

A. NON-ACTIVATED TEST SYSTEM

Historical Values	Solvent (%)	Positive Control (2)
Mean	1.3	21.2
±SD (1)	1.4	12.1
Range	0.0-5.5	6.5-87.0

B. S9-ACTIVATED TEST SYSTEM

Historical Values	Solvent (%)	Positive Control (2)
Mean	1.6	33
±SD (1)	1.5	18.1
Range	0.0-6.5	7.0-84.0

(1) SD = standard deviation

(2) Positive control for non-activated studies, Mitomycin C (MMC, 0.08-0.2, µg/mL).

Positive control for S9-activated studies, cyclophosphamide (CP, 10-60, µg/mL).

Table 4: Historical Control Values - CHO cells (Combined numerical aberrations)

A. NON-ACTIVATED TEST SYSTEM

Historical Values	Solvent (%)	Positive Control (2)
Mean	2.1	2.8
±SD (1)	1.4	1.6
Range	0.0-7.5	0.0-8.0

B. S9-ACTIVATED TEST SYSTEM

Historical Values	Solvent (%)	Positive Control (2)
Mean	2.8	2.6
±SD (1)	1.9	1.5
Range	0.0-11.0	0.0-6.0

(1) SD = standard deviation

(2) Positive control for non-activated studies, Mitomycin C (MMC, 0.08-0.2, µg/mL).

Positive control for S9-activated studies, cyclophosphamide (CP, 10-60, µg/mL).

Conclusions:

Based on the findings of this study, acetyl cedrene was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO-K1 cells.

Executive summary:

In a mammalian cell cytogenetics assay [Chromosome aberration] (AA71KY.331.BTL), CHO-K1 cells (CCL 61) were exposed to Methy Cedryl Ketone in ethanol at concentrations of 0, 3.125, 6.25, 12.5, 25, 35, 50 µg/mL with and without metabolic activation.

Methyl Cedryl Ketone was tested up to cytotoxic concentrations in each exposure group (selection of doses for microscopic analysis measured by at least 50% cell growth inhibition). Positive controls induced the appropriate response and were statistically significant. There was no evidence of chromosome aberration induced over background in non-activated 4h and 20h exposure groups. There was evidence of chromosome aberration (structural) in S9-activated 4h exposure groups; however the increase was within laboratory historical controls.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 (In vitro mammalian cytogenetics - chromosome aberration).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09-10-12 to 13-11-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline and GLP compliant study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase

Species / strain / cell type:

other: L5178Y mouse lymphoma (3.7.2c) cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
Cell culture: R10p: R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v;
Cloning efficiency plating: Equal volumes of R10p and R30p (r30p:R0, supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v.)
Selective medium: R10p containing 4µg/mL trifluorothymidine (TFT).

- Properly maintained: Yes; cultures were used within ten days of recovery from frozen stock
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: Yes - Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24 hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196°C, in heat-inactivated donor horse serum (HiDHS) containing 10% DMSO.

Metabolic activation:

with and without

Metabolic activation system:

S9 from rat liver dosed with phenobarbital and 5,6 benzoflavone (commercial source; Lot No: 2907 (Date of preparation: 15-03-12)). S9 mix: S9 fraction (5% v/v), glucose-6-phosphate (6.9 mM), NADP (1.4 mM) in R0. Final concentration in experiments: 2%

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 4.81, 9.63, 19.25, 38.5, 77, 154, 308, 616, 1232 and 2464 µg/mL
The RSG was used to determine the concentrations of test substance used in the main test; ideally the maximum concentration should reduce RTG to approximately 10 to 20% of the concurrent vehicle control value.

Mutation test (3 hrs, without S9): 0, 10, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5 and 40 µg/mL

Mutation test (3 hrs, with S9): 0, 10, 40, 50, 55, 60, 65, 70, 75, 80, and 90 µg/mL

Mutation test (24 hrs, without S9): 0, 10, 20, 25, 30, 32.5, 35, 37.5, 40 and 45 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Prior to commencing testing, the solubility of the test substance in vehicle compatible with this test system was assessed. Methyl Cedryl Ketone was found to be soluble at 246.4 mg/mL in acetone. A solution of 246.4 mg/mL, dosed at 1% in medium, showed no precipitate in the culture medium.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

methylmethanesulfonate

Remarks:

10 µg/mL (3 hour exposure) 5 µg/mL (24 hour exposure) in DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

benzo(a)pyrene

Remarks:

1 µg/mL (3 hour exposure) in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 3 hrs and 24 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): R10p containing 4 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
Preliminary toxicity test: Control (2); Test (1)
Mutation test: Control (4); Test (2)

DETERMINATION OF CYTOTOXICITY
- Method:
Preliminary toxicity test:relative suspension growth (RSG)
Mutation test: relative total growth (RTG).

Evaluation criteria:

The assay was considered valid in accordance with the assay acceptance criteria for cytotoxicity, mutant frequency, cloning efficiency, suspension growth and small colony mutants.

The test agent was regarded as negative if:
The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the GEF.

If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend testwas applied:
If the linear trend test was negative, the result was regarded as negative.
If the linear trend test was positive, this indicated a positive, biologically relevant response.

GEF = Global Evaluation Factor. For microwell assays this is 126 x 10-6

Statistics:

The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989) using a one-sided F-test, where p<0.001.

ROBINSON, W.D., GREEN, M.H.L., COLE, J., HEALY, M.J.R., GARNER, R.C., and GATEHOUSE, D. (1989). Statistical evaluation of bacterial/mammalian fluctuation tests. In: KIRKLAND, D. J. (Ed). UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part 111. Statistical Evaluation of Mutagenicity Test Data, p.102-140. Cambridge University Press, Cambridge.

Species / strain:

other: L5178Y mouse lymphoma (3.7.2c) cells

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

38.5 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

other: L5178Y mouse lymphoma (3.7.2c) cells

Metabolic activation:

with

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

77 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

other: L5178Y mouse lymphoma (3.7.2c) cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

35 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

other: L5178Y mouse lymphoma (3.7.2c) cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

70 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

other: L5178Y mouse lymphoma (3.7.2c) cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

30 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuations in pH of the medium were observed at 90 µg/mL of more than 1.0 unit compared with the vehicle control.

- Effects of osmolality: The osmolality of the test substance in medium was tested at concentrations of 90 µg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.

- Precipitation:
Preliminary toxicity test: Precipitate (observed by eye at the end of treatment) was observed at concentrations of 154 µg/mL and greater in both the absence and presence of S9 mix, following a 3 hour exposure and at concentrations of 1232 µg/mLand greater following 24 hr exposure.
Main mutation tests - No precipitate was observed by eye at the end of treatment.

COMPARISON WITH HISTORICAL CONTROL DATA:The current Historical Vehicle and Positive Control Mutation Frequencies from the test laboratory are attached in Appendix 1 (time period 13-Dec-10 29-Oct-12).

Study report attachments:

Table 1 Preliminary toxicity test - all groups (MBB0002)

Tables 2 & 3 Main mutation test - 3hr Without S9 (MBB0002)

Tables 4 & 5 Main mutation test - 3hr With S9 (MBB0002)

Tables 6 & 7 Main mutation test - 24hr Without S9 (MBB0002)

Appendix 1 Historical Control Data (MBB0002)

Conclusions:

It was concluded that Methyl Cedryl Ketone did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Executive summary:

In a mammalian cell gene mutation assay [TK] (MBB0002), L5178Y mouse lymphoma (3.7.2c) cells cultured in vitro were exposed to Methyl Cedryl Ketone in acetone at concentrations of 0, 10, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5 and 40 µg/mL (3 hr, without S9); 0, 10, 40, 50, 55, 60, 65, 70, 75, 80, and 90 µg/mL (3 hr, with S9) and 0, 10, 20, 25, 30, 32.5, 35, 37.5, 40 and 45 µg/mL (24hr without S9).

Methyl Cedryl Ketone was tested up to cytotoxic concentrations. The positive controls induced 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 (in vitro mammalian cell gene mutation test) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro:

Gene mutation (Bacterial Reverse Mutation Assay/Ames test):

There is one in vitro bacterial reverse mutation assay/Ames test available.

In a reverse gene mutation assay in bacteria (OECD 471/GLP), strains of S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A (pKM101) were exposed to Methyl Cedryl Ketone in DMSO at concentrations of 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (direct plate incorporation; experiment 1) and 0, 50, 150, 500, 1500 and 5000 µg/plate (pre-incubation; experiment 2) in the presence and absence of mammalian metabolic activation (rat S9). Methyl Cedryl Ketone was tested up to the limit concentration (5000 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

Chromosome aberration (in vitro mammalian cell cytogenicity):

There is one in vitro chromosome aberration (in vitro mammalian cell cytogenicity) study available.

In a mammalian cell cytogenetics assay [Chromosome aberration] (OECD 473/GLP), CHO-K1 cells (CCL 61) were exposed to Methy Cedryl Ketone in ethanol at concentrations of 0, 3.125, 6.25, 12.5, 25, 35, 50 µg/mL with and without metabolic activation. Methyl Cedryl Ketone was tested up to cytotoxic concentrations in each exposure group (selection of doses for microscopic analysis measured by at least 50% cell growth inhibition). Positive controls induced the appropriate response and were statistically significant. There was no evidence of chromosome aberration induced over background in non-activated 4h and 20h exposure groups. There was evidence of chromosome aberration (structural) in S9-activated 4h exposure groups; however, the increase was within laboratory historical controls.

Gene mutation (in vitro mammalian cell gene mutation):

There is one in vitro gene mutation (in vitro mammalian cell gene mutation) study available.

In a mammalian cell gene mutation assay [TK] (OECD 476/GLP), L5178Y mouse lymphoma (3.7.2c) cells cultured in vitro were exposed to Methyl Cedryl Ketone in acetone at concentrations of 0, 10, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5 and 40 µg/mL (3 hr, without S9); 0, 10, 40, 50, 55, 60, 65, 70, 75, 80, and 90 µg/mL (3 hr, with S9) and 0, 10, 20, 25, 30, 32.5, 35, 37.5, 40 and 45 µg/mL (24hr without S9). Methyl Cedryl Ketone was tested up to cytotoxic concentrations. The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background.

The studies are suitable to use in the human health risk assessment.

Justification for classification or non-classification

Based on the available information in the dossier, the substance methyl cedryl ketone (CAS No. 32388-55-9) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.