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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Dihydromyrcenyl acetate is

- Not mutagenic in bacterial cells in an Ames test according to OECD TG 471 but in absence of strain TA 102. The negative result is supported with Ames information from Dihydromyrcenol.

-       Negative for cytogenicity in an in vitro Micronucleus test (OECD TG 487)

-       Negetive for genemutations in mammalian cells based on read across from Dihydromyrcenol, which was tested in a mouse lymphoma assay (OECD TG 476).

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:
1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
TA102 strain is missing
Justification for type of information:
In addition to the Ames test on Dihydromyrcenyl acetate, Ames information from Dihdyromyrcenol is added to make up for the missing TA102 strain. The read across rationale is presented in the Genetic toxicity Endpoint summary, the accompanying files are also attached there.
Qualifier:
according to guideline
Guideline:
other: Ames test method et al. (1975)
Version / remarks:
Ames test method et al. (1975)
GLP compliance:
no
Remarks:
conduected prior to the GLP guideline
Type of assay:
bacterial forward mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 98
Metabolic activation:
with and without
Test concentrations with justification for top dose:
0, 0.008, 0.04, 0.2, 1.0 and 5.0 microL/plate
Vehicle / solvent:
Appropriate dilutions in DMSO were prepared immediately before use.
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
2 ml molten soft agar were added to 0.1 mL of a fully grown culture of one of the tester strains and 0.1 mL of the appropriate dilution /suspension of the test compound (and 0.5 mL of the S-9 mix if indicated). The ingredients were thoroughly mixed and immediately poured into minimal glucose agar plates. After the top agar had been allowed to harden, the plates were incubated at 37°C for two days. Then the colonies (revertants which are histidine-independant) were counted, and the background lawn of baterial growth examined microscopically. All determination were made in triplicate and appropriate controls were included in each assay.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1.0 and 5.0 microL/plate DHMA was clearly toxic to strains TA1537, TA1538, TA98 and TA100 in the absence of S9
Vehicle controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
Incorporation of 0.008 up to 0.5 - 0.5 microL of the test liquid par plate did not induce an increase in the number of his +revertants with any of the five tester strains either in the presence ot absence of S-9 mix. At 1.0 and 5.0 microL/plate DHMA was clearly toxic to strains TA1537, TA1538, TA98 and TA100 in the basence of S9 mix as revealed by a less dense background lawn of bacterial growth.
Conclusions:
From the present results it can be concluded that the substance up to non- inhibitory levels did not reveal any mutagenic activity in the plate incorporation assay with S.Typhimurium TA1535, TA1537, TA1538, TA98 or TA100 in the presence or absence of the liver microsome activation system under the test condition employed in this evaluation.
Executive summary:

The mutagenic activity of the substance was examined in the Salmonella/microsome mutagenicity test, using a set of five histidine requiring mutants of S. Typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) and liver homogenate of Aroclor induced rats. Incorporation of the test liquid up to non-inhibitory levels i.e. 0.2 - 5.0 microl/plate did not increase the number of his +revertants in any of the five tester strains either in the presence or in the absence of the liver microsome activation system.

It was concluded that the present results did not reveal any mutagenic activity of the test material in the Salmonella/microsome metagenicity test.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to generally valid and/or internationally accepted testing guidelines.
Justification for type of information:
Information is used to support the Ames test conducted with Dihydromyrcenyl acetate.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium:
TA 1535, TA 100: hisG46
TA 1537: hisC3076
TA 98: hisD3052
Escherichia coli (WP2uvrA/pKM101):
contains ochre mutation; deficient in DNA repair system 9uvrA); contains the pKM101 plasmid
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:
Rat liver S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 microg/plate

Experiment 1 (Range-finding)
15, 50, 150, 500, 1500 and 5000 microg/plate

Mutation Test (Main Test) - with preincubation
TA100 (with/without S9), TA1535 and TA1537 (without S9) and E. coli (without S9): 1.5, 5, 15, 50, 150, 500 and 1500 microg/plate

TA98 (with/without S9), TA1535 and TA1537 (with S9) and E. coli (with S9): 15, 50, 150, 500, 1500 and 5000 microg/plate
Vehicle / solvent:
dimethyl sulphoxide

The test material was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Pos. Control for TA98 at 5 microg/plate (+S9)
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
Pos. Control for TA100 at 1 microg/plate (+S9); TA1535 and TA1537, 2 microg/plate (+S9); and E. coli WP2uvrA- at 10 microg/plate (+S9)
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Pos. Control for E. coli WP2uvrA- at 2 microg/plate (-S9); TA100 at 3 microg/plate (-S9); TA1535 at 5 microg/plate (-S9)
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Pos. Control for TA1537 at 80 microg/plate (-S9)
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Pos. Control for TA98 at 0.2 microg/plate (-S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
A standard plate incorporation method was employed.

Preliminary Toxicity Test:
In order to select appropriate dose levels for use in the main test, a preliminary test were conducted in the presence or absence of S9. Ten dose levels and controls were tested up to and including 5,000 microg/plate at approximately half-log intervals. The assay was conducted by mixing 0.1 ml the bacterial culture (TA100 or WP2uvrA-), and 0.1 ml of the vehicle or test chemical mixture, 0.5 mL of S9-mix or phosphate buffer and 2.0 ml of molten agar supplemented with trace histidine or tryptophan and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). After approximately 48 hours incubation at 37 deg C, the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Experiment 1 (Range-finding Test):
Six concentrations of the test material were assayed with or without S9-mix against each tester strain using the above described direct plate incorporation method. An additional dose level and expanded dose range were selected in order to achieve both four non-toxic doses and the toxic limit of the test material.

Experiment 2 (Main Test):
A second experiment was performed with fresh bacterial cultures, test material and control solutions. The dose ranges selected were based on the range-finding test and an aborted main test (data not shown). Preincubation was employed. Thus, measured aliquots (0.1 mL) of each bacterial culture were dispensed into sets of test tubes followed by 0.5 mL of S9-mix or phosphate buffer and 0.1 mL of vehicle or test material formulation and incubated for 20 minutes at 37 deg C with shaking at approximately 130 rpm prior to addition of 2 mL of molten trace histidine or tryptophan supplemented top agar. The contents of the tubes were mixed and poured onto the surface of Vogel-Bonner Minimal agar plates. This procedure was repeated for each bacterial strain either with or without S9.

NUMBER OF REPLICATIONS:
3 replicates/strain

DETERMINATION OF CYTOTOXICITY
Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony counts or by the absence of a complete bacterial lawn.
Evaluation criteria:
Acceptance Criteria:

The following criteria must be met for acceptance:
- All tester strain cultures must exhibit a characteristic number of spontaneous revertants per plate in vehicle and untreated controls.
- The appropriate characteristics of each tester strain must be confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor.
- All tester strain cultures should be in the range of 1 to 9.9 x 10^9 bacteria per mL.
- Each mean positive control value should be at least 2x the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains and the integrity of the S9-mix
- The test should include a minimum of four non-toxic dose levels.

Evaluation Criteria:

A dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain either with or without metabolic activation. Biological relevance of the response is to be considered first, as recommended by the UKEMS sub-committee on Guidelines for Mutagenicity Testing (1989). Statistical methods can be used as an aid to evaluation but may not be the only determining factor for a positive response.

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Mean values with standard deviations were reported.
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Please refer to Tables 1 to 5 for details of results.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Preliminary Toxicity Test

The test material was toxic at 5000 microg/plate to tester strains TA100 and WP2uvrA-. The results are summarized in Table 1.

Table 1: Numbers of revertant colonies in the preliminary toxicity test

With (+) or without (-) S9-mix

Strain

Dose (mg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

93

100

120

101

123

133

132

106

128

152

113*

+

TA100

62

107

77

82

81

88

94

84

84

74

0*

-

WP2uvrA-

18

15

16

18

22

33

24

14

19

20

17*

+

WP2uvrA-

28

32

22

27

22

20

24

25

25

31

15*

* Partial absence of bacterial background lawn

Table 2: Range-finding test without metabolic activation

Revertant colony counts (mean 3 replicates)

Addition (mg)

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

101

24

18

17

11

15

81

30

19

15

10

50

86

28

19

19

10

150

91

23

17

20

9

500

99

27

18

18

11

1500

92

27

20

14

10

5000

82*

15*

13

12*

2*

ENNG (3)

421

ENNG (5)

907

ENNG (2)

596

4NQO (0.2)

167

9AA (80)

354

* Partial absence of background lawn in all replicate plates.

Abreviations: ENNG, N-ethyl-N’-nitro-N-nitrosoguanidine; 9AA, 2-aminoacridine; BP, benzo(a)pyrene; 2AA, 2-aminoanthracene; 4NQO, 4-nitroquinoline-1-oxide

Table 3: Range-finding test with metabolic activation

Revertant colony counts (mean 3 replicates)

Addition (mg)

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

88

14

22

19

14

15

90

15

22

21

14

50

87

14

21

15

14

150

88

10

19

21

10

500

74

11

23

19

10

1500

84

9

17

16

9

5000

72*

2*

17

12*

3*

2AA (1)

760

2AA (2)

212

195

2AA (10)

244

BP (5)

119

* Partial absence of background lawn in all replicate plates.

Table 4: Main Test without metabolic activation

Revertant colony counts (mean 3 replicates)

Addition (mg)

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

106

28

26

11

12

1.5

107

24

26

N/T

14

5

103

27

17

N/T

12

15

108

29

20

15

9

50

100

25

21

10

8

150

107*

28

26

15

9

500

76*

27*

25

10

8*

1500

0*

0*

0*

0*

0*

5000

N/T

N/T

N/T

0*

N/T

ENNG (3)

406

ENNG (5)

291

ENNG (2)

498

4NQO (0.2)

100

9AA (80)

937

* Partial absence of background lawn in all replicate plates.

N/T - not tested at this dose level.

Table 5: Main Test with metabolic activation

Revertant colony counts (mean 3 replicates)

Addition (mg)

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

114

10

18

19

9

1.5

102

N/T

N/T

N/T

N/T

5

115

N/T

N/T

N/T

N/T

15

90

11

17

22

11

50

102

11

21

25

8

150

103

14

19

20

10

500

95*

11

15

18

7

1500

0*

0*

0*

0*

0*

5000

N/T

0*

0*

0*

0*

2AA (1)

813

2AA (2)

230

199

2AA (10)

212

BP (5)

248

* Partial absence of background lawn in all replicate plates.

N/T - not tested at this dose level.

Conclusions:
Dihydromyrcenol is negative in the Ames test (OECDTG 471, Rel. 1), with and without S9.
Executive summary:

In the first experiment, the test material caused a visible reduction in the growth f the bacterial background lawns of all the Salmonella stains at 5000 microg/plate in both the presence and absence of S9. No toxic response to Escherichia coli strain WP2uvrA- was noted. In the second experiment (with pre-incubation) the test material reduced the bacterial background lawns of all the tester strains in both the presence and absence of S9. The sensitivity of the various stains varied with the presence or absence of S9 and the strain involved; therefore, the test material was tested up to the maximum recommended dose level. No test material precipitate was observed on the plates at any of the doses tested either with or without S9.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9 -mix and the sensitivity of the bacterial strains.

The test material was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across information.
Justification for type of information:
The gene matation in mammalian cells is derived from read across with Dihydromyrcenol. The read across rationale is presented in the Genetic toxicity Endpoint summary and the accompanying files are also attached there.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at highest conc. of 200 μg/ml each
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
It is concluded that the substance is not a mutagenic substance in the HPRT locus assay using CHO cells, based on the results of the source substance.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 November 2009 to 07 December 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This information is used for read across to Dihydromyrcenyl acetate.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
No. 440/2008 of 30 May 2008
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase heterozygous system, TK +/- to TK -/- conversion
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y TK +/- 3.7.2c mouse lymphoma cell line, obtained from Dr. J. Cole of the MRC Cell Mutation Unit, University of Sussex, Brighton, UK
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test:
0, 6.09, 12.19, 24.38, 48.75, 97.5, 195, 390, 780, 1560 micrograms/mL

Main Experiment, 4-Hour, Without S9:
0, 12.5, 25, 50, 100, 125, 150, 175, 200 micrograms/mL

Main Experiment, 4-Hour, With S9:
0, 25, 50, 100, 125, 150, 175, 200, 250 micrograms/mL

Main Experiment, 24-Hours, Without S9:
0, 6.25, 12.5, 25, 50, 75, 100, 125, 150 micrograms/mL
Vehicle / solvent:
Dimethyl sulfoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without S9 activation Migrated to IUCLID6: Sigma batch 1419706 15108051 at 400 micrograms/mL (4 hour) and 150 micrograms/mL (24-hour)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9 activation Migrated to IUCLID6: Acros batch A0164185 at 2 micrograms/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in suspension;
- Cell density at seeding (if applicable): 4 hour exposures: 1 x 10^6 cells/ml in 10 ml aliquots, 24 hours exposures: .3 x 10^6 cells/ml in 10 ml

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): expression period of two days
- Selection time (if incubation with a selection agent): ten to fourteen days of incubation
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): 4 μg/ml 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicate cultures

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: To assist the scoring of the TFT mutant colonies 0.025 ml of MTT solution (2.5 mg/ml in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours. the MTT vital stain was taken up by viable cells to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

NUMBER OF CELLS EVALUATED:The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

- OTHER:
Cell Culture
Cells were stored in liquid nitrogen at -196°C. Cells were cultured at 37°C with 5% CO2 in air in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 μg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/ml) and 10% donor horse serum (giving R10 media). Cells had a generation time of approximately 12 hours and were sub-cultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) were used during the course of the study. Master cultures of stock cells were checked and found to be mycoplasma free.

Preparation of test and control materials
The test material was dissolved in dimethyl sulfoxide (DMSO) and the appropriate dilutions were made. The maximum dose level investigated in the preliminary toxicity test was 1560 μg/ml which was equivalent to approximately 10 mM.. Analysis for concentration, homogeneity and stability of the test material preparations were not a requirement of the test method and were therefore not performed.
Vehicle and positive controls were used in parallel with the test material. Solvent (DMSO) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) at 400 μg/ml and 150 μg/ml for the 4-hour and 24-hour exposures respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) 2 μg/ml was used as the positive control in the presence of metabolic activation.

Preliminary toxicity test
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/ml, using a 4-hour exposure time both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/ml using a 24-hour exposure without S9. The dose range used in the preliminary toxicity test was 6.09 to 1560 μg/ml for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a coulter counter and then serially diluted to 2 x 10^5 cells/ml.
The cultures were incubated and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/ml. After a further 24 hours, the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth values (SG). The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a % Relative Suspension Growth Value (%RSG).
Results from the preliminary toxicity test were used to set the test material dose levels for the mutagenicity experiment. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 μg/ml or 10 mM.
ii) The presence of excessive precipitate where no test material-induced toxicity was observed.
iii) Test material-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required).

Mutagenicity test (main test)
4-Hour exposures, with and without metabolic activation
An exponentially growing stock culture of cells was set up to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/ml in 10 ml aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at eight dose levels of the test material (12.5 to 200 μg/ml in the absence of metabolic activation, and 25 to 250 μg/ml in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 ml of S9-mix if required, 0.2 ml of the treatment dilutions, (0.2 ml for the positive control) and sufficient R0 medium to bring the total volume to 20 ml.
The treatment vessels were incubated at 37°C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

24-Hour exposures, without metabolic activation
Cells were counted and processed (as above) to give 0.3 x 10^6 cells/ml in 10 ml duplicate cultures established in 25 cm2 tissue culture flasks. To each culture was added 2 ml of the treatment dilutions (0.2 ml for the positive control) and sufficient R10 medium to give a final volume of 20 ml. The dose range of the test material was 6.25 to 150 μg/ml. The treatment vessels were incubated at 37°C with continuous shaking using an orbital shaker for 24 hours.
Cell processing following treatment periods
At the end of the treatment periods for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/ml. The cultures were incubated and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10^5 cells/ml.
On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium. The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Plate scoring
Microtitre plates were scored using a magnifying mirror box after ten to fourteen days incubation. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies were scored manually by eye using qualitative judgment. Large colonies were defined as those covering approximately ¼ to ¾ of the surface of the well and were generally no more than one or two cells thick. As a rule of thumb, all colonies less than 25% of the average area of the large colonies were scored as small colonies. Small colonies were normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 ml of MTT solution (2.5 mg/ml in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours. the MTT vital stain was taken up by viable cells to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.
Evaluation criteria:
The normal range for mutant frequency per survivor is 50-200 x 10^-6 for the TK+/- locus in L5178Y cells at this testing laboratory. Vehicle control results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 250 x 10^-6 mutant frequency per survivor are not normally acceptable and will be repeated. Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. A Global Evaluation Factor (GEF) value was set following International Workshop (Moore et al., 2003; Moore et al, 2006) at 126 x 10^-6 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10^-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgment will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Statistics:
Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.

Robinson W D et al (1989) Statistical evaluation of bacterial/mammalian fluctuation tests. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing (Kirkland D J Ed.), Cambridge University Press Report part III, pp102-140.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The maximum dose level used was limited by test material toxicity.
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity test:The results for Relative Suspension Growth (%RSG) are as shown in Table 1:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A precipitate of the test material was observed at and above 195 μg/ml in the 4-hour exposure group in the absence of metabolic activation, and at and above 390 μg/ml in both the 4-hour exposure group in the presence of metabolic activation and the 24-hour exposure group in the absence of metabolic activation. The precipitate was observed to become greasy and oily in appearance at 1560 μg/ml in both of the 4-hour exposure groups.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Mutagenicity test results
4-Hour exposures with and without metabolic activation (Table 2):
There was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of any significant reductions in (%V) viability in either the absence or presence of metabolic activation, therefore indicating that no residual toxicity had occurred. Near optimum levels of toxicity were achieved in the absence of metabolic activation. Optimum levels of toxicity were not achieved in either the absence or presence of metabolic activation due to the sharp onset of toxicity, despite using a very narrow dose interval. However, a dose level that exceeded the usual acceptable upper limit of toxicity was plated for viability and TFT resistance for each of the exposure groups. It was therefore considered that with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels, including the dose levels that exceeded the usual upper limit of acceptable toxicity, or in the 24-hour exposure group where optimum levels of toxicity were achieved, the test material had been adequately tested. The excessive toxicity observed at 200 μg/ml in the absence of metabolic activation, and at 250 μg/ml in the presence of metabolic activation, resulted in these doses not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.

24-Hour exposure without metabolic activation (Table 3):
As was seen in the Preliminary Toxicity Test, there was evidence of a marked reduction in % RSG and RTG values in cultures dosed with the test material. There was also evidence of a modest reduction in (%V) viability, therefore indicating that residual toxicity had occurred. Optimum levels of test material-induced toxicity were achieved. The positive control induced acceptable levels of toxicity.
The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had an effect on the toxicity of the test material. The vehicle control mutant frequency value was within the acceptable range of 50 to 200 x 10^-6 viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily.
The test material induced a small statistically significant dose related (linear-trend) in the mutant frequency x 10^-6 per viable cell (Table 9). However, statistically significant increases in mutant frequency were not observed at any of the individual dose levels, the GEF was not exceeded at any of the individual dose levels, and the mutant frequency values observed were within the acceptable range for vehicle controls. Therefore, the response was considered to be spurious and of no toxicological significance. Precipitate of the test material was not observed at any of the dose levels.
Remarks on result:
other: all strains/cell types tested

Table 1: Preliminary Toxicity Test Results

Dose

(mg/mL)

% RSG (-S9)

4-Hour Exposure

%RSG (+ S9)

4-Hour Exposure

%RSG (-S9)

24-Hour Exposure

0

100

100

100

6.09

93

103

94

12.19

87

116

81

24.38

87

111

80

48.75

90

103

62

97.5

84

101

34

195

0

28

0

390

0

0

0

780

0

0

0

1560

0

0

0

Table 2: Experimental Results

4-Hour Test Without S9

Treatment

(mg/mL)

%RSG

RTG

MF

0

100

1.00

118.34

12.5*

95

25

92

1.16

91.83

50

88

1.05

101.08

100

84

0.99

104.88

125

73

0.81

120.90

150

37

0.38

122.92

175**

1

0.03

143.07

200***

0

-

-

Linear trend

NS

EMS

400

74

0.60

684.79

4-Hour Test With S9

0

100

1.00

149.96

12.5*

92

25

90

0.98

91.37

50

83

0.88

147.57

100

83

0.97

150.12

125

77

0.81

106.33

150

50

0.50

116.61

175**

9

0.12

111.16

200*

0

-

-

Linear trend

NS

CP

2

48

0.24

781.04

* Not plated for viability or 5-TFT resistence.

** Treatment excluded from statistics due to toxicity.

Table 3: Experimental Results

24-Hour Test Without S9

Treatment

(mg/mL)

%RSG

RTG

MF

0

100

1.00

115.95

6.25*

104

12.5

108

0.92

91.35

25

99

0.96

98.55

50

71

0.66

121.85

75

46

0.43

101.71

100

30

0.16

149.79

125

11

0.10

158.82

150*

2

-

-

Linear trend

p < 0.05

EMS

150

66

0.45

1007.15

* Not plated for viability or 5-TFT resistence.

Conclusions:
The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and was not considered to be mutagenic under the conditions of the test. Therefore, Dihydromyrcenol does not need to be classified as mutagenic according to Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The objective of this in vitro assay (OECD TG 476) was to evaluate the ability of Dihydromyrcenol to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5178Y cell line, using Trifluorothymidine (TFT) as selection agent. The test material was dissolved in Dimethylsulfoxide (DMSO). A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/ml, using a 4-hour exposure time both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/ml using a 24-hour exposure without S9. The dose range used in the preliminary toxicity test was 6.09 to 1560 μg/ml for all three of the exposure groups. The test material produced dose-related toxicity, starting at 195 ug/ml with and without activation. A precipitate of the test material was observed at and above 195 μg/ml in the 4-hour exposure group in the absence of metabolic activation, and at and above 390 μg/ml in both the 4-hour exposure group in the presence of metabolic activation and the 24-hour exposure group in the absence of metabolic activation.

 

The 4-hour exposure to dihydromyrcenol, with and without metabolic activation was performed in duplicate (A + B), at eight dose levels of the test material (12.5 to 200 μg/ml in the absence of metabolic activation, and 25 to 250 μg/ml in the presence of metabolic activation), vehicle and positive controls. For the 24-hour exposure without metabolic activation, the dose range of the test material was 6.25 to 150 μg/ml. The treatment vessels were incubated at 37°C with continuous shaking using an orbital shaker.

 

At 4 hours of exposure, there was evidence of a marked reduction in % RSG and RTG values in cultures dosed with the test material. There was also evidence of a modest reduction in (%V) viability, therefore indicating that residual toxicity had occurred. Optimum levels of test material-induced toxicity were achieved. The positive control induced acceptable levels of toxicity. The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had an effect on the toxicity of the test material. The vehicle control mutant frequency value was within the acceptable range of 50 to 200 x 10^-6 viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily.

 

Dihydromyrcenol was evaluated as negative with and without metabolic activation in the L5178Y mouse lymphoma mutation assay under the conditions used in this assay. It was concluded that Dihydromyrcenol does not need to be classified as mutagenic according to Annex I of the CLP Regulation (1272/2008/EC).

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 March 2014 - 24 June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: cultures prepared from blood of a male donor
Details on mammalian cell type (if applicable):
Peripheral blood lymphocytes were cultured in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37°C with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohemagglutinin (PHA).
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from male rats induced with Phenobarbitione/β-Naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
With and without S9-mix, 4 hr exposure; 28 hr recovery: 0, 7.75, 15.49, 30.98, 61.97, 123.94, 247.88, 495.75, 991.5 and 1983 μg/mL
Without S9-mix, 24 hr exposure; 28 hr recovery: 0, 7.75, 15.49, 30.98, 61.97, 123.94, 247.88, 495.75, 991.5 and 1983 μg/mL
First cytogenetic test:
Without S9-mix, 4 hr exposure; 28 hr recovery: 0, 15, 30, 40, 60, 80 and 120 μg/mL
With S9-mix, 4 hr exposure; 28 hr recovery: 0, 15, 30, 60, 80, 120 and 160 μg/mL
The following dose levels were selected for scoring of micronuclei:
Without S9-mix, 4hr exposure; 28 hr recovery: 40, 60, 80 and 120 μg/mL
With S9-mix, 4 hr exposure; 28 hr recovey: 60, 80, 120 and 160 μg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 28 hr recovery: 0, 15, 30, 60, 80, 120 and 160 μg/mL
The following dose levels were selected for scoring of micronuclei:
Without S9-mix, 24 hr exposure; 28 hr recovey: 30, 60, 80 and 120 μg/mL
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: The test substance was fully miscible in dimethyl sulphoxide at 198.3 mg/mL in solubility checks performed in-house.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcine (DC)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration:
Short-term treatment
Without and with S9-mix: 4 hr treatment, 28 hr recovery/harvest time
Continuous treatment
Without S9-mix: 24 hr treatmen, 28 hr recovery/harvest time

ARREST OF CELL DIVISION: 4.5 μg/mL Cytochalasine B
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375 KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 °C prior to slide making.
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.
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 minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B.
The micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration). For the 4 hour in the absence of S9 exposure group, the positive control 'A' culture (MMC 0.2 μg/mL) was assessed from 2000 cells due to a poor positive control response in the first 1000 cells that were assessed. Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.


DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index)
Evaluation criteria:
The following criteria were used to determine a valid assay:
- The frequency of binucleate cells with micronuclei in the vehicle control cultures will normally be within the range of the laboratory historical control data. The frequency of spontaneous background micronuclei may be slightly elevated above the normal range but the experiment can still be considered valid.
- All the positive control chemicals must induce positive responses (p ≤ 0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix
Statistics:
The frequency of cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of cells with micronuclei which was reproducible.
Key result
Species / strain:
lymphocytes: human peripheral blood primary culture
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH/osmolality:
The osmolality in treatment medium of the highest concentration tested, 1983 μg/mL, was 403 mmol/kg. The osmolality of the vehicle (DMSO) in the treatment medium was 455 mmol/kg. The pH of the highest concentration of test substance in treatment medium was 7.32, the pH of the vehicle (DMSO) in the treatment medium was 7.26.
- Precipitation: No precipitate of test item was noted at any dose level in either exposure group in experiment 1. A precipitate of test item (greasy/oily) was noted at the end of exposure at 160 μg/mL in experiment 2.

RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 7.75 to 1983 μg/mL. The maximum dose was the 10 mM concentration.
A greasy/oily precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 123.94 μg/mL, in the exposure groups in the absence of metabolic activation (S9) and at and above 495.75 μg/mL in the exposure group in the presence of S9. Haemolysis was observed in the blood cultures following exposure to the test item at and above 7.75 μg/mL in the 4-hour exposure group without S9, at and above 15.49 μg/mL in the 4-hour exposure group with S9 and at and above 30.98 μg/mL in the 24-hour continuous exposure group. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 61.97 μg/mL in the exposure groups without S9 and up to 123.94 μg/mL in the exposure group dosed in the presence of S9. The CBPI data confirms that the test item induced evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level was based on toxicity for both the 4-hour exposure groups used in Experiment 1 and the 24-hour exposure group used in Experiment 2.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:
See "Any other information on results incl. tables"

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
See "Any other information on results incl. tables"

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
4-Hour Exposure without S9 4-Hour Exposure withS9 24-Hour Exposure without S9
%Cells with Micronuclei %Cells with Micronuclei %Cells with Micronuclei
Minimum 2.50 1.40 1.60
Maximum 10.90 6.05 7.20
Mean 5.61 3.36 3.98
SD 2.41 1.36 1.34
Number 17 17 17

- Negative (solvent/vehicle) historical control data:
4-Hour Exposure without S9 4-Hour Exposure with S9 24-Hour Exposure without S9
%Cells with Micronuclei %Cells with Micronuclei %Cells with Micronuclei
Minimum 0 0.05 0.10
Maximum 1.30 0.80 0.70
Mean 0.39 0.36 0.38
SD 0.31 0.21 0.17
Number 17 17 17

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1:
The qualitative assessment of the slides determined that the toxicity was slightly less than that observed in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring at the maximum dose level of test item, 120 μg/mL and 160 μg/mL in the absence and presence of S9, respectively. It should be noted that reductions in cell pellet size at harvest were observed in both exposure groups at and above 40 μg/mL and 60 μg/mL, in the absence and presence of S9, respectively. Haemolysis was observed at the end of exposure at and above 15 μg/mL and 30 μg/mL in the absence and presence of S9, respectively.
The CBPI data confirm the qualitative observations in that no dose-related inhibition of CBPI was observed. Although there was no appreciable inhibition of CBPI, there was a reduced cell pellet, indicating toxicity to the blood cells in culture. The overall reduction in cell numbers was taken as evidence that the test item had been adequately tested and that this limited the upper dose level for scoring. The maximum dose level selected for analysis of binucleate cells was 120 μg/mL and 160 μg/mL in the absence and presence of S9, respectively.
Experiment 2:
The qualitative assessment of the slides determined that there were binucleate cells suitable for scoring at 120 μg/mL. Haemolysis was observed at the end of exposure at and above 80 μg/mL.
The CBPI data confirm the qualitative observations in that a dose-related inhibition of CBPI was observed, and that 41% inhibition of cell proliferation was achieved at 120 μg/mL, this is close to the optimum 50% level. The maximum dose level selected for binucleate cell analysis was, therefore, 120 μg/mL. Above this dose level, there were no binucleates present.

CBPI and Micronucleus Data -Experiment 1 - 4 -Hour Exposure Without Metabolic Activation(S9)

 

DoseLevel (μg/mL)

 

Exposure

Time+/-S9

 

 

Replicate

 

Nucleate cells/500cells

 

 

CBPI

 

Cytotoxcity (% Control CBPI)

Micronuclei(MN)Per

1000 Binucleate cells

 

%Cells with MN

 

Mean% Cells with MN

 

Mono

 

Bi

 

Multi

 

1MN

 

2MN

 

>2MN

 

0

 

 

 

 

 

 

4Hr-S9

A

136

269

95

1.92

 

100

0

0

0

0.00

 

0.10

 

B

 

205

 

245

 

50

 

1.69

 

2

 

0

 

0

 

0.20

 

60

A

232

213

55

1.65

 

81

0

0

0

0.00

 

0.25

 

B

 

223

 

225

 

52

 

1.66

5

 

0

0

0.50

 

80

A

161

275

64

1.81

 

99

0

0

0

0.00

 

 

B

 

166

277

57

1.78

2

0

0

0.20

0.10

 

120

A

200

230

70

1.74

 

91

3

0

0

0.30

 

0.25          

 

B

 

192

251

57

1.73

2

0

0

0.20

 

MMC0.2

A+

217

243

40

1.65

 

81

21

2

1

2.40

 

2.85***

 

B

 

 

198

 

268

 

34

 

1.67

 

31

 

2

 

0

 

3.30

MMC = Mitomycin C

+ = Scored from 2000 cells due to a poor response in the first 1000 cells

***  =P<0.001

CBPI and Micronucleus Data -Experiment 1 - 4 -Hour Exposure With Metabolic Activation(S9)

 

DoseLevel (μg /mL)

 

Exposure

Time+/-S9

 

 

Replicate

 

Nucleate cells/500 cells

 

 

CBPI

 

Cytotoxcity (% Control CBPI)

Micronuclei(MN)Per

1000 Binucleate cells

 

%Cells with MN

 

Mean% Cells with MN

 

Mono

 

Bi

 

Multi

 

1MN

 

2MN

 

>2MN

 

0

 

 

 

 

 

 

4Hr+S9

A

291

157

52

1.52

 

100

1

0

0

0.10

 

0.30

 

B

 

278

 

161

 

61

 

1.57

 

5

 

0

 

0

 

0.50

 

80

A

318

143

39

1.44

 

93

0

0

0

0.00

 

0.00

 

B

 

268

 

173

 

59

 

1.58

0

 

0

0

0.00

 

120

A

284

163

53

1.54

 

95

1

0

0

0.10

 

 

B

 

303

145

52

1.50

1

1

0

0.20

0.15

 

160

A

256

187

57

1.60

 

98

8

0

0

0.80

 

0.55          

 

B

 

307

145

48

1.48

3

0

0

0.30

 

CP5

A

344

135

2l

1.35

 

60

27

2

3

3.20

 

3.80***

 

B

 

 

354

 

135

 

11

 

1.31

 

38

 

6

 

0

 

4.40

CP = Cyclophosphamide

***  =P<0.001

CBPI and Micronucleus Data -Experiment 2 - 24 -Hour Exposure Without Metabolic Activation(S9)

 

DoseLevel (μg /mL)

 

 

Exposure

Time+/-S9

 

 

Replicate

 

Nucleate cells/500 cells

 

 

CBPI

 

Cytotoxicity (%Control CBPI)

Micronuclei(MN)Per

1000 Binucleatecells

 

 

%Cells with MN

 

Mean% Cells with MN

 

Mono

 

Bi

 

Multi

 

1MN

 

2MN

 

>2MN

 

 

0

 

 

 

 

 

24Hr-S9

A

156

314

30

1.75

 

100

5

0

0

0.50

 

0.35           

B

130

324

46

1.83

2

0

0

0.20

 

60

A

137

314

49

1.82

 

101

0

0

0

0.00

 

0.00

B

141

328

31

1.78

0

0

0

0.00

 

80

A

144

324

32

1.78

 

104

2

0

0

0.20

 

0.15

B

117

339

44

1.85

1

0

0

0.10

 

120

A

274

212

14

1.48

 

59

4

0

0

0.40

 

0.30

B

279

219

2

1.45

2

0

0

0.20

 

DC0.075

A

201

235

64

1.73

 

86

25

4

0

2.90

 

4.20***

B

224

240

36

1.62

42

5

8

5.50

DC  =Demecolcin

***  =P<0.001

Conclusions:
An in vitro micronucleus assay with the substance was performed according to OECD 487 guideline and GLP principles, in cultured peripheral human lymphocytes in two experiments. It is concluded that the substance is not clastogenic or aneugenic in human lymphocytes.
Executive summary:

In an in vitro micronucleus assay, cultured peripheral human lymphocytes were exposed to different concentrations of the substance (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 487 guideline and GLP principles. In the first cytogenetic assay, the substance was tested up to and including concentrations of 120 μg/mL and 160 μg/mL for a 4 h exposure time with a 28 h recovery time in the absence and presence of S9-mix, respectively. Although there was no appreciable inhibition of CBPI, there was a reduced cell pellet, indicating toxicity to the blood cells in culture.

In the second cytogenetic assy, the substance was tested up to and including the cytotoxic concentration of 120 μg/mL, an 41% inhibition of cell proliferation was achieved, for a 20 h exposure time with a 28 h recovery time in the absence of S9 -mix. Reliable positive and negative controls were included.

The substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix. It is concluded

that the substance is not clastogenic or aneugenic in human lymphocytes.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The executive summaries of the tests with the target and source chemicals are presented below followed by the read-across rationale.

Dihydromyrcenol acetate Ames test

The mutagenic activity of the substance was examined in the Salmonella/microsome mutagenicity test, using a set of five histidine requiring mutants of S. Typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) and liver homogenate of Aroclor induced rats. Incorporation of the test liquid up to non-inhibitory levels i.e. 0.2 - 5.0 microl/plate did not increase the number of his +revertants in any of the five tester strains either in the presence or in the absence of the liver microsome activation system.

It was concluded that the present results did not reveal any mutagenic activity of the test material in the Salmonella/microsome metagenicity test.

Dihdyromyrcenol Ames test

In the first experiment, the test material caused a visible reduction in the growth f the bacterial background lawns of all the Salmonella stains at 5000 microg/plate in both the presence and absence of S9. No toxic response to Escherichia coli strain WP2uvrA- was noted. In the second experiment (with pre-incubation) the test material reduced the bacterial background lawns of all the tester strains in both the presence and absence of S9. The sensitivity of the various stains varied with the presence or absence of S9 and the strain involved; therefore, the test material was tested up to the maximum recommended dose level. No test material precipitate was observed on the plates at any of the doses tested either with or without S9. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9 -mix and the sensitivity of the bacterial strains. The test material was considered to be non-mutagenic under the conditions of this test.

Dihydromyrcenol gene mutations in mammalian cells (MLA test)

The objective of this in vitro assay (OECD TG 476) was to evaluate the ability of Dihydromyrcenol to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5178Y cell line, using Trifluorothymidine (TFT) as selection agent. The test material was dissolved in Dimethylsulfoxide (DMSO). A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/ml, using a 4-hour exposure time both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/ml using a 24-hour exposure without S9. The dose range used in the preliminary toxicity test was 6.09 to 1560μg/ml for all three of the exposure groups. The test material produced dose-related toxicity, starting at 195 ug/ml with and without activation. A precipitate of the test material was observed at and above 195μg/ml in the 4-hour exposure group in the absence of metabolic activation, and at and above 390μg/ml in both the 4-hour exposure group in the presence of metabolic activation and the 24-hour exposure group in the absence of metabolic activation. 

The 4-hour exposure to dihydromyrcenol, with and without metabolic activation was performed in duplicate (A + B), at eight dose levels of the test material (12.5 to 200μg/ml in the absence of metabolic activation, and 25 to 250μg/ml in the presence of metabolic activation), vehicle and positive controls. For the 24-hour exposure without metabolic activation, the dose range of the test material was 6.25 to 150μg/ml. The treatment vessels were incubated at 37°C with continuous shaking using an orbital shaker.

 At 4 hours of exposure, there was evidence of a marked reduction in % RSG and RTG values in cultures dosed with the test material. There was also evidence of a modest reduction in (%V) viability, therefore indicating that residual toxicity had occurred. Optimum levels of test material-induced toxicity were achieved. The positive control induced acceptable levels of toxicity. The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had an effect on the toxicity of the test material. The vehicle control mutant frequency value was within the acceptable range of 50 to 200 x 10^-6 viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily. Dihydromyrcenol was evaluated as negative with and without metabolic activation in the L5178Y mouse lymphoma mutation assay under the conditions used in this assay.

Dihydromyrcenyl acetate: In vitro micronucleus test

In an in vitro micronucleus assay, cultured peripheral human lymphocytes were exposed to different concentrations of the substance (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 487 guideline and GLP principles. In the first cytogenetic assay, the substance was tested up to and including concentrations of 120 μg/mL and 160 μg/mL for a 4 h exposure time with a 28 h recovery time in the absence and presence of S9-mix, respectively. Although there was no appreciable inhibition of CBPI, there was a reduced cell pellet, indicating toxicity to the blood cells in culture.

In the second cytogenetic assy, the substance was tested up to and including the cytotoxic concentration of 120 μg/mL, an 41% inhibition of cell proliferation was achieved, for a 20 h exposure time with a 28 h recovery time in the absence of S9 -mix. Reliable positive and negative controls were included.

The substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix. It is concluded that the substance is not clastogenic or aneugenic in human lymphocytes.

Dihydromyrcenyl acetate (CAS 53767-93-4) its genemutations in bacterial and mammalian cells using read across from Dihydromyrcenol (CAS no. 106-24-1)

 

Introduction and hypothesis for the analogue approach

Dihydromyrcenyl acetate has a C8 alk-1-ene chain with at C2 an acetate ester group and two methyl groups at C3 and C7.For this substance somewhat limited Ames information and no data on in vitro gene mutations in mammalian cells (MLA) are available. In accordance with Article 13 of REACH, lacking information can be generated by other means, i.e. applying alternative methods such as QSARs, grouping and read-across. For assessing the genotoxic properties of Dihydromyrcenyl acetate, the analogue approach is selected because for the analogue Dihydromyrcenol genemutations in bacterial and mammalian cells is available which can be for read across.

Hypothesis: Dihydromyrcenyl acetate has the same result in an Ames as in an MLA assay as Dihydromyrcenol, because Dihydromyrcenol is the key metabolite of Dihydromyrcenyl acetate next to acetic acid.

Available information: For Dihydromyrcenyl acetate a limited Ames test is available because the effects of strain TA 102 or E. coli is missing. For Dihydromyrcenol a negative Ames test is available (OECD TG 471, Rel. 1). For Dihydromyrcenol also a negative MLA test is available (OECD TG 487, Rel. 1). 

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemical are shown in the data matrix, including physico-chemical properties and toxicological information, thought relevant for genotoxic properties.

Purity / Impurities

The purity and impurities of the target chemical do not indicate genotoxic potential other than indicated by the parent substance. The impurities are all below 10%.

Analogue approach justification

According to Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and a common functional group. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation, which is presented below.

Analogue selection: For Dihydromyrcenyl acetate its key metabolite Dihydromyrcenyl acetate is used for which information on genemutations in mammalian cells is available.

Structural similarities and differences:Dihydromyrcenyl acetate and the key metabolite Dihydromyrcenol have the same hydrocarbon backbone. The difference is that Dihydromyrcenyl acetate has an acetic ester as a functional group, while Dihydromyrcenol is its metabolic derivative.

Toxico-kinetic,Absorption: Dihydromyrcenyl acetate has somewhat higher molecular weight and log Kow compared to Dihydromyrcenol, but is expected to have sufficiently similar absorption characteristics. Dihydromyrcenyl acetate, after metabolisaton, has the same absorption and metabolisation characteristics as Dihydromyrcenol.

Metabolism:Dihydromyrcenyl acetate will be metabolised via all routes into Dihydromyrcenol after which they will follow the same metabolic pathway. The other metabolite is acetic acid (presented in the Toxico-kinetic section), which is a normal constituent of the body.

Genotoxic reactivity:Dihydromyrcenyl acetate will have the same genotoxic reactivity as Dihydromyrcenol because the acetic ester bond in the former is not too reactive and will present a similar genotoxicity profile compared to the alcohol as a functional group. Acetic acid being a normal constituent of the body and has no genemutation activity.

Other experimental information supporting the read across: Dihydromyrcenyl acetate and Dihydromyrcenol are both negative for cytogenicity; the first one in an in vitro micronucleus and the second one in a chromosomal aberration assay, supporting the negative genotoxicity.

Uncertainty of the prediction: There are no uncertainties other than those already addressed above.

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics is presented in the Data matrix.

Conclusions on genemutations in mammalian cells

For Dihydromyrcenyl acetate limited Ames and no genemutations in mammalian cells is available. Such information is available for Dihydromyrcenol and this can be used for read across. When using read across the result derived should be applicable for C&L and/or risk assessment and documented with reliable and adequate documentation. This documentation is presented in the current document. Didyromyrcenol shows absence of mutagenic with and without metabolic activation in the Ames and in the Mouse Lymphoma assay. This information can be used for Dihydyromyrcenyl acetate.

Final conclusion on hazard:Dihydromyrcenyl acetate is not mutagenic in bacterial and mammalian cells, without and with metabolic activation.

Data matrix to support the read across to Dihydromyrcenyl acetate from Dihydromyrcenol

Name

Dihydromyrcenyl acetate

Dihydromyrcenol

 

Source

Target

Chemical structure

CAS

53767-93-4        

C10H20O

EINECS

258-751-7

242-362-4

REACH registration

2018

Registered

Chemical formula

C12H22O2             

C10H20O

Molecular weight

198.0

156

Physico-chemical data

 

 

Physical state

Liquid

Liquid

Vapour pressure, Pa

14.4 at 24°C (IFF, 1999)

20 Pa at 25°C (measured)

Water solubility, mg/l

6.1 at 24°C (IFF, 1999)

939 mg/L at 20°C (measured)

Log Kow

4.9 (IFF, 2017)

Log Pow: 3.25 (measured)

Human health-Genotoxicity

 

 

Ames

Negative

(OECD TG 471, missing TA 102)

Negative

OECD TG 471

In vitro cytogenicity

Negative: in vitro micronucleus

(In vitro micronucleus, OECD TG 487)

Negative

(Chrom Aberr. OECD TG 473)

Genemutation in vitro in mammalian cells

Negative

(Read across)

Negative

(OECD TG 476)

 

 

Justification for classification or non-classification

Based on the information available the substance does not need be classified and labelled for genotoxicity, according to EU CLP (EC No. 1272/2008 and its amendments).