Reaction mass of (4R)-isopropenyl-1-methylcyclohexene and (4S)-isopropenyl-1-methylcyclohexene and 1-isopropyl-4-methylcyclohexa-1,3-diene and 1-isopropyl-4-methylcyclohexa-1,4-diene and 4-isopropylidene-1-methylcyclohexene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLNENE did not induce micrinuclei.

In an Ames test performed according to OECD Guideline 471 and in compliance with GLP, REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE was not mutagenic.

In an in vitro HPRT test performed according to OECD Guideline 476 and in compliance with GLP, REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA-TERPINENES, TERPINOLENE was not mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January- February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

17 June 2015

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item
(1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

The solvent (vehicle) control used was dimethyl sulphoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM:
All the strains were originally sourced from the University of California, Berkley, Syngenta CTL Ltd and the British Industrial Biological Research Association (BIBRA)

METHOD OF APPLICATION: In agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes at 37 ± 3 °C, with shaking
- Incubation period: Plates were incubated at 37 ± 3 °C for approximately 48 hours.

NUMBER OF REPLICATIONS:
- Vehicle and positive controls were included in triplicate plates.
- Treatment (test item) groups were included in triplicate plates

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Sparse bacterial background lawn at 5000µl/plate and In the pre-incubation plate method Sparse bacterial background lawn at concentration from 500 µk/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the pre-incubation plate method only Sparse bacterial background lawn at concentration from 500 µk/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the pre-incubation plate method only Sparse bacterial background lawn at concentration from 500 µk/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the pre-incubation plate method only Sparse bacterial background lawn at concentration from 500 µk/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE was considered to be non-mutagenic in a Ames test.

Executive summary:

REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE was tested for mutagenicity purpose according to OECD Guideline 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. An interim (confirmatory) experiment was performed between the first and second mutation tests employing one strain only (TA1535) following the observation of statistically significant increases in Experiment 1. The experiment employed a tightened test item dose range to potentially enhance any response. The response was not repeated in the interim experiment; therefore a second mutation test was performed on all of the bacterial strains employing the pre-incubation method using fresh cultures of the bacterial strains and fresh test item formulations. The dose range for the second mutation test was amended slightly and was 5 to 5000 μg/plate. Seven test item concentrations were selected in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test, although weakened bacterial background lawns were noted to TA1535 at 5000 μg/plate in both the absence and presence of S9-mix in Experiment 1 (confirmatory test). These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 μg/plate in the second mutation test. The pre-incubation modification was employed for the second mutation test and this change in methodology caused the test item to exhibit a toxic response in the form of weakened bacterial background lawns to all of the Salmonella strains at and above 500 μg/plate in both the presence and absence of metabolic activation (S9-mix). No significant weakening of the bacterial background lawns was noted for Escherichia coli strain WP2uvrA at any test item dose level. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). In the first mutation test, statistically significant increases in TA1535 revertant colony frequency were noted from 1500 μg/plate in both the presence and absence of S9-mix. These results were investigated in a confirmatory experiment employing a tightened concentration range at the upper dose levels. However, there were no toxicologically significant increases in the frequency of TA1535 revertant colonies recorded after the confirmatory test but weakened bacterial background lawns were noted at 5000 μg/plate. Therefore, the responses noted for TA1535 in the original test were considered due to the fact that the test item exhibited toxicity under certain circumstances and exposure conditions (a much stronger toxic response was noted after performing the pre-incubation modification) and the increases in revertant colony frequency noted may have been an artefact resulting from a modest level of toxicity to the tester strain at the upper test item dose levels. Even though weakened background lawns were not noted in the original test there may have been enough weakening (toxicity) to induce a ‘false’ response. A possible mechanism may be that low level toxicity has caused a selective effect on the number of bacterial cells plated, resulting in an increase in non-revertant histidine-dependent bacteria, which are able to manifest as false ‘colonies’.

Therefore, REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE was considered to be non-mutagenic in a Ames test.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January - March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hemizygous hypoxanthine phosphoribosyl transferase (HPRT) gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Source: Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany
- Type and identity of media:Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS))
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No; karyotype was assumed to be stable.
- Other details: Prior to exposure to test item, spontaneous mutants were eliminated from the stock cultures by culturing in HAT medium for four days. After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and beta-naphthoflavone.

Test concentrations with justification for top dose:

Preliminary toxicity test: 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL.
Second premiminary toxicity test: 0.0125, 0.25, 0.5, 1, 2, 4, 8, 16 and 32 μg/mL
Mutation tests: The upper concentration levels were selected based on cytotoxicity.
-S9 mix Test 1 (4 hours) 2, 4, 8, 10, 12, 14, 16 and 20 μg/mL.
+S9 mix Test 1 (4 hours) 7.5, 15, 30, 45, 60 and 75 μg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

500 and 750 µg/mL for 4-hour exposure, in the absence of S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: dimethyl benzanthracene

Remarks:

1 and 2 µg/mL for 4-hour exposure in the presence of S9

Details on test system and experimental conditions:

DURATION
- Exposure duration: 4 h (in serum free media (MEM))
- Expression time (cells in growth medium): 7 days
- All incubations were performed at 37 °C in a humidified atmosphere of 5 % CO2 in air.

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture/dose for test item
- Main test: 2 cultures/dose
NUMBER OF CELLS EVALUATED: 200 cells/plate were seeded for cloning efficiency and 10^6 cells were analyzed for mutant frequencies.

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency,
% Cloning efficiency (CE): ( CE counts/200) x100
% control : (CE% of Dose IDx/CE% of Dose ID0) x100
where
Dose ID0 =Vehicle control values
Dose IDx = Dose level values


OTHER:
Mutant Frequency (MF) per 10^6 for each dose was calculated as: Total mutant plate counts/2
mutant frequency per 10^6 survival rate (MFSV) for each dose was calculated as: (MF10-6/CE) x100

Rationale for test conditions:

Mutation tests: The upper concentration levels were selected based on cytotoxicity.

Evaluation criteria:

The criteria for a positive response will be:
- At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
- Evidence of a dose-response relationship, over at least two dose levels, when any single dose level shows a significant increase in mutant frequency;
- The increase is considered to be concentration-related.
- The results are outside the range of the historical negative control data for the test item concentrations.

The criteria for a negative response will be:
- None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
- There is no concentration related increase.
- The results for the test item concentrations are within the range of the historical negative control data.

Statistics:

When there is no indication of any increases in mutant frequency at any dose level then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual dose level, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuations in pH when the test item was dosed into media.
- Effects of osmolality: No fluctuations in osmolality of more than 50 mOsm at the dose levels investigated
- Precipitation: Precipitate was seen at 9.77 µg/mL and greasy/oily precipitate was observed at and above 39.06 µg/mL in the 4-hour exposure group in the absence of S9. In the presence of S9, precipitate (cloudy and greasy/oily) was observed at the end of exposure at and above 78.13 µg/mL.

PRELIMINARY TOXICITY TEST:
- A dose range of 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL was used initially in the preliminary cytotoxicity test. Although the test item is a multiconstituent substance, the maximum dose concentration was set at 5000 µg/mL. Due to excessive toxicity demonstrated in the absence of S9, with no surviving dose levels remaining at the end of the exposure period, this exposure group was repeated with a dose range of 0.0125, 0.25, 0.5, 1, 2, 4, 8, 16 and 32 µg/mL. A greasy/oily precipitate of the test item was observed at the end of exposure at and above 312.5 µg/mL in the 4-hour exposure group in the absence of S9 and at and above 156.25 µg/mL in the presence of S9. There was no precipitate observed at the end of the exposure period in the repeated 4-hour exposure in the absence of S9. A dose-related reduction in the cloning efficiency (CE) was demonstrated in both the presence and absence of S9. The 4-hour exposure in the absence of S9 demonstrated much greater toxicity than in the presence of S9 and 85% reduction in cloning efficiency was seen at 16 µg/mL. In the presence of S9 there was a steep toxicity curve between 39.06 µg/mL which demonstrated a 24% reduction in cloning efficiency and 78.13 µg/mL which demonstrated a 95% reduction in cloning efficiency. The maximum dose level selected for the main mutagenicity experiment was based on the toxicity seen in the preliminary cytotoxicity test and was 20 µg/mL in the absence of S9 and 75 µg/mL in the presence of S9.
Concentrations for the main test were based upon these data and toxicity was the primary determinant for the dose selection.

MAIN MUTATION TEST

There was a dose related reduction in in the Day 0 cloning efficiency demonstrated in both the absence and presence of S9. The 4-hour exposure group in the absence of S9 demonstrated optimum toxicity with a reduction in cloning efficiency of 83% and 82% at 16 µg/mL and 20 µg/mL, respectively. In the presence of S9, optimum toxicity was also achieved at 75 µg/mL with a reduction in cloning efficiency of 86% compared to the vehicle control. There was no marked reduction in the Day 7 cloning efficiencies of either exposure group indicating full recovery from the toxicity observed at the end of exposure to the test item.
There were no dose related increases in mutation frequency per survivor or any dose level exceeded the current historical control range for a vehicle control.
The vehicle control values were all considered to be within an acceptable range, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

The test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation. Substance Reaction mass of DL-Limonene, Alpha- Gamma- Terpinenes, Terpinolene: was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Table Summary results

Main Experiment – 4-Hour Exposure without Metabolic Activation (S9)

		Day 0 viability						Day 7 viability						Day 7 mutant
Dose (µg/mL)		Colonies/flask (200 cells plated/flask)			%CE	% control	Mean % control	Colonies/flask (200 cells plated/flask)			%CE	% control	Mean % control	Colonies/flask (2 x 10^5 cells plated/flask)										MF	MFS 10^-6	SD	Group MFS 10^-6
0	A	140	167	150	76.2	100	100	179	161	183	87.2	100	100	0	2	1	0	0	1	0	1	1	1	3.5	4.0	0.91	5
	B	148	142	150	73.3	100		182	186	179	91.2	100		1	2	2	0	3	2	0	1	0	0	5.5	6.0
2	A	143	139	137	69.8	91.7	91	185	175	178	89.7	102.9	102	0	0	0	0	0	0	0	0	1	1	1.0	1.1	1.29	4
	B	139	124	136	66.5	90.7		185	188	179	92.0	100.9		5	3	2	0	0	1	0	1	0	1	6.5	7.1
4	A	160	140	144	74.0	97.2	93	172	180	177	88.2	101.1	97	2	0	0	1	0	0	0	0	0	1	2.0	2.3	1.67	8
	B	150	4269	118	65.7	89.5		182	160	171	85.5	93.8		2	2	0	6	2	2	2	2	5	2	12.5	14.6
8	A	132	128	127	64.5	84.7	90	198	201	186	97.5	111.9	107	2	2	0	1	1	1	0	1	1	2	5.5	5.6	0.81	6
	B	124	149	145	69.7	95.0		187	187	186	93.3	102.4		2	1	2	2	2	0	0	2	0	1	6.0	6.4
10	A	135	149	117	66.8	87.7	86	192	193	196	96.8	111.1	104	1	0	0	0	1	0	0	0	0	0	1.0	1.0	1.17	5
	B	117	123	127	61.2	83.4		179	181	174	89.0	97.6		3	1	1	0	2	1	0	2	4	2	8.0	9.0
12	A	156	151	142	74.8	98.2	87	189	168	178	89.2	102.3	100	0	1	1	1	0	1	1	0	0	1	3.0	3.4	0.97	6
	B	111	112	106	54.8	74.8		165	177	187	88.2	96.7		0	2	1	0	3	0	1	2	3	2	7.0	7.9
14	A	70	70	71	35.2	46.2	41	182	172	183	89.5	102.7	103	1	2	0	0	1	1	1	0	1	0	3.5	3.9	1.36	8
	B	57	50	52	26.5	36.1		198	184	187	94.8	104.0		2	2	3	1	2	5	4	3	1	1	12.0	12.7
16	A	30	30	26	14.3	18.8	17	164	173	174	86.8	99.6	100	0	1	1	0	1	0	2	1	0	0	3.0	3.5	0.89	5
	B	25	18	21	10.7	14.5		182	182	190	92.3	101.3		2	2	1	0	2	2	0	2	2	0	6.5	7.0
20	A	30	27	29	14.3	18.8	18	149	148	162	76.5	87.8	93	1	0	0	1	0	0	1	0	0	0	1.5	2.0	0.57	2
	B	24	27	26	12.8	17.5		167	184	181	88.7	91.3		0	2	0	0	0	0	1	0	0	0	1.5	1.7
EMS 500	A	106	125	125	59.3	77.9	72	201	209	197	101.2	116.1	110	13	28	34	28	22	16	31	32	51	PL	140.7	140.1	9.24	171
	B	96	104	92	48.7	66.4		189	187	188	94.01	103.1		39	34	42	39	32	35	41	42	39	36	189.5	201.6
EMS 700	A	81	74	74	38.2	50.1	52	148	156	160	77.3	88.7	88	39	54	46	56	55	53	49	43	55	58	254.0	328.4	6.45	340

 

EMS     = Ethyl methane sulphonate

CE       = Cloning efficiency

MF      = Mutant frequency

MFS     = Mutant frequency per survivor

SD       = Standard deviation

Main Experiment – 4-Hour Exposure with Metabolic Activation (2% S9)

		Day 0 viability						Day 7 viability						Day 7 mutant
Dose (µg/mL)		Colonies/flask (200 cells plated/flask)			%CE	% control	Mean % control	Colonies/flask (200 cells plated/flask)			%CE	% control	Mean % control	Colonies/flask (2 x 10^5 cells plated/flask)										MF	MFS 10^-6	SD	Group MFS 10^-6
0	A	183	180	169	88.7	100	100	162	177	170	84.8	100	100	1	0	1	3	1	1	0	0	0	2	4.5	5.3	0.91	5
	B	151	161	153	77.5	100		166	157	184	84.5	100		0	2	1	0	0	2	2	1	0	1	4.5	5.3
7.5	A	152	160	163	79.2	89.3	95	174	194	185	92.2	108.6	109	0	1	0	0	2	1	1	0	0	1	3.0	3.3	0.68	2
	B	170	148	149	77.8	100.4		172	178	204	92.3	109.3		0	0	0	0	2	0	0	0	0	0	0	1.0
15	A	170	164	147	80.2	90.4	96	182	186	179	91.2	107.5	111	2	2	1	0	1	1	0	3	1	0	5.5	6.0	1.14	7
	B	164	151	162	79.5	102.6		190	198	194	97.0	114.8		2	1	2	2	3	4	1	0	2	0	8.5	8.8
30	A	153	179	179	85.2	96.1	95	161	172	178	85.2	100.4	107	0	1	1	1	3	0	2	0	0	PL	4.4	5.2	0.85	4
	B	139	148	152	73.2	94.4		202	197	176	95.8	113.4		2	1	1	1	0	0	0	1	0	1	3.5	3.7
45	A	175	168	153	82.7	93.2	95	184	190	187	93.5	110.2	106	2	1	2	3	0	4	1	1	3	2	9.5	10.5	1.26	5
	B	139	158	150	74.5	96.1		172	170	174	86.0	101.8		0	1	0	0	0	0	0	0	0	0	0.5	0.6
50	A	143	148	128	69.8	78.8	83	177	166	182	87.5	103.1	106	2	1	0	1	3	2	2	1	4	3	9.5	10.9	1.26	7
	B	144	126	134	67.3	86.9		186	182	184	92.0	108.9		1	2	0	0	0	0	1	3	0	0	3.5	3.8
60	A	96	83	92	45.2	50.9	53	157	161	161	79.8	94.1	100	1	0	0	1	0	2	0	1	1	0	3.0	3.8	0.61	3
	B	81	82	91	42.3	54.6		173	183	176	88.7	104.9		0	0	1	0	0	0	1	1	0	1	2.0	2.3
75	A	30	27	24	13.5	15.2	14	153	149	145	74.5	87.8	97	0	0	1	0	0	0	0	0	1	0	1.0	1.3	0.75	2
	B	21	22	19	10.3	13.3		168	179	190	89.5	105.9		0	1	1	0	0	3	0	0	0	0	2.5	2.8
DMBA 2	A	167	141	148	76.0	85.7	86	159	156	151	77.7	91.6	97	45	44	37	43	41	39	33	40	30	36	194.0	249.8	4.82	231
	B	133	132	135	66.7	86.0		177	167	179	87.2	103.2		36	37	43	37	40	28	30	42	40	36	184.5	211.7
DMBA 1	A	157	167	140	77.3	87.2	93	182	172	176	88.3	104.1	98	33	32	39	42	33	38	37	33	37	48	186.0	210.6	5.09	215
	B	149	146	164	76.5	98.7		162	145	161	78.0	92.3		37	36	29	33	26	39	36	36	28	41	170.5	218.6

DMBA     = Dimethyl benzanthracene

CE          = Cloning efficiency

MF         = Mutant frequency

MFS        = Mutant frequency per survivor

SD          = Standard deviation

PL                = Plate lost due to technical error

Conclusions:

The test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation. Therefore, the test item was considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster (V79) cells were exposed to the test item for 4 hours, with and without metabolic activation, at the concentrations below. 

- Preliminary toxicity test: 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL.

 

- Mutation tests:

-S9 mix Test 1 (4 hours) 2, 4, 8, 10, 12,14, 16 and 20 μg /mL

+S9 mix Test 1 (4 hours) 7.5, 15, 30, 45, 50, 60 and 75 μg/mL

The vehicle (DMSO) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system.

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation.

 

Therefore, the test item was not mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27 March to 24 May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well conducted and well described study in accordance with GLP and OECD Guideline 487 without any deviation.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

07 September 2012

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

other: human lymphocytes

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

2 % S9 (final concentration); S9 fraction was prepared from liver homogenates of rats treated with Aroclor 1254.

Test concentrations with justification for top dose:

Range-Finder Experiment:
3+21 h treatments with and without S9; 24+0 h treatments without S9: 2.902, 4.837, 8.062, 13.44, 22.39, 37.32, 62.21, 103.7, 172.8, 288, 480 and 800 µg/mL

Micronucleus Experiment:
3+21 h treatments without S9: 10.00, 20.00, 22.50, 25.00, 27.50, 30.00, 32.50, 35.00, 40.00, 45.00, 50.00, 55.00 and 65.00 µg/mL
3+21 h treatments with S9: 20, 40, 60, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 135 and 150 µg/mL
24+0 h treatments without S9: 10.00, 20.00, 30.00, 35.00, 40.00, 42.50, 45.00, 47.50, 50.00, 52.50, 55.00, 60.00 and 75.00 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that test item was miscible with anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 500 mg/mL.
- Formulation preparation: Test article stock solutions were prepared by formulating test item under subdued lighting in DMSO, with the aid of vortex mixing, as required, to give the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 2.5 h of initial formulation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: Vinblastine

Remarks:

Without metabolic activation - Mitomycin C: 0.60 and 0.80 μg/mL; Vinblastine: 0.02, 0.03 and 0.04 μg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation - 6.25 and 12.50 μg/mL

Details on test system and experimental conditions:

PREPARATION OF CULTURES:
- Blood from two healthy, non-smoking male volunteers from a panel of donors was used for each experiment in this study. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within one day of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use. Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 9.0 mL pre-warmed (in an incubator set to 37 ± 1°C) HEPES-buffered RPMI medium containing 10 % (v/v) heat inactivated foetal calf serum and 0.52 % penicillin / streptomycin, so that the final volume following addition of S-9 mix/KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2 % of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37 ± 1 °C for ca 48 h and rocked continuously.

DURATION
- Exposure duration: 3 h (±S9); 24 h (-S9) - no recovery period (24+0 h)
- Fixation time (start of exposure up to harvest of cells): 24 h (±S9)

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B, formulated in DMSO, was added directly (0.1 mL/culture) to all continuous cultures at the time of treatment to give a final concentration of 6 μg/mL per culture.

STAIN (for cytogenetic assays): Acridine Orange (125 μg/mL in phosphate buffered saline, pH 6.8 for approximately 10 seconds)

NUMBER OF REPLICATIONS:

Cytotoxicity Range-Finder Experiment: Two cultures/dose for vehicle control; one culture/dose for test item
Micronucleus Experiment: Two cultures/dose for test item and positive controls; four cultures for vehicle control

NUMBER OF CELLS EVALUATED:
- Selection of concentrations for Micronucleus Experiment: Slides from the cytotoxicity Range-Finder Experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) was determined.
- Selection of concentrations for micronucleus analysis (Micronucleus Experiment only): Slides were examined, uncoded, for RI to a minimum of 500 cells per culture. The highest concentration for micronucleus analysis was one at which approximately 55 ± 5% reduction in RI had occurred. Analysis of slides from highly cytotoxic concentrations was avoided, where possible.
- Where possible, one thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei.

DETERMINATION OF CYTOTOXICITY
- Method: Relative replication index
Replication index (RI), which indicates the relative number of nuclei compared to vehicle controls, was determined using the formulae below:
RI = number binucleate cells + 2 (number multinucleate cells) / total number of cells in treated cultures
Relative RI (expressed in terms of percentage) for each treated culture was calculated as follows:
Relative RI (%) = (RI of treated cultures / RI of vehicle controls) x 100
Cytotoxicity (%) is expressed as (100 – Relative RI).

OTHER:
Binucleate cells were only included in the analysis if all of the following criteria were met:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.
A micronucleus was only recorded if it met the following criteria:
1. The micronucleus has the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed.
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of MNBN cells was observed.
- The test article was considered positive in this assay if all of the above criteria were met.
- The test article was considered negative in this assay if none of the above criteria were met.
- Results which only partially satisfied the above criteria were dealt with on a case-by-case basis.

Statistics:

The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al, 1989).
The proportion of MNBN cells for each treatment condition was compared with the proportion in vehicle controls by using Fisher's exact test (Richardson et al, 1989). Probability values of p ≤ 0.05 were accepted as significant. Additionally, the number of micronuclei per binucleate cell were obtained and recorded.

Key result

Species / strain:

other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- No marked changes in osmolality or pH were observed at the highest concentration tested in the Range-Finder (800 μg/mL), compared to the concurrent vehicle controls.
- Precipitation: The miscibility limit in culture medium was in the range of 156.3 to 312.5 μg/mL, as indicated by precipitation at the higher concentration which persisted for approximately 22 h after test article addition.

RANGE-FINDER EXPERIMENT
Steep concentration-related toxicity was observed under all three treatment conditions, specifically between 37.32 and 62.21 μg/mL for the 3+21 h and 24+0 h –S-9 treatments and between 103.7 and 172.8 μg/mL for the 3+21 h +S-9 treatment. Concentrations selected for the Micronucleus Experiment were targeted within the regions of steep toxicity for each treatment condition.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical data of the laboratory (April 2010 and September 2012).

OTHERS:
CONCENTRATIONS SELECTED FOR MICRONUCLEUS ANALYSIS
- 3+21 h in the absence of S-9: Concentrations of 27.50, 32.50 and 40.00 μg/mL gave 10, 37 and 52 % reductions in RI, respectively and were considered suitable for micronucleus analysis to cover the range of cytotoxicity.
- 3+21 h in the presence of S-9: Concentrations of 70, 80 and 90 μg/mL gave 6, 21 and 49 % reductions in RI, respectively. Although the reduction in RI seen at 90 μg/mL (49 %) was marginally below the desired range of 55 ± 5 %, a 72 % reduction in RI was seen at 100 μg/mL which was too toxic for analysis, therefore 90 μg/mL was considered an appropriate maximum concentration for micronucleus analysis.
- 24+0 h in the absence of S-9: The lowest three concentrations tested (10, 20 and 30 μg/mL) gave 9, 40 and 61 % reductions in RI, respectively, but 92 % reduction in RI was seen at 35 μg/mL. Although the reduction in RI seen at 30 μg/mL (61 %) was marginally above the desired range of 55 ± 5 %, due to the narrow concentration spacing between the concentrations in question, 30 μg/mL was considered an appropriate maximum concentration for micronucleus analysis.

MICRONUCLEUS ANALYSIS
Treatment of cells with test item for 3+21 h in the absence and presence of S-9 and for 24+0 h in the absence of S-9 resulted in frequencies of MNBN cells that were generally similar to (and not significantly higher than) those observed in concurrent vehicle controls at all concentrations analysed. The MNBN cell frequencies in treated cultures fell within the normal ranges with the exception of a single culture at the intermediate concentration of 80 μg/mL for the 3+21 h +S-9 treatment, which was outside this range but within the observed range. This marginal increase was not observed in the replicate culture at 80 μg/mL or at any other concentration analysed under any of the three treatment conditions, therefore it was considered of no biological relevance.

Remarks on result:

other: other: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Table 7.6.1/1: Micronucleus Experiment – Results summary

 

Treatment	Concentration (μg/mL)	Cytotoxicity (%)$	Mean MNBN cell frequency (%)	Historical Control Range (%) #	Statistical significance
3+21 h -S-9	Vehicle^a	-	0.50	0.10-1.00	-
	27.50	10	0.30		NS
	32.50	37	0.55		NS
	40.00	52	0.70		NS
	*MMC, 0.80	ND	12.55		p ≤ 0.001
3+21 h +S-9	Vehicle^a	-	0.65	0.00-1.00	-
	70.00	6	0.35		NS
	80.00	21	0.85		NS
	90.00	49	0.40		NS
	*CPA, 12.5	ND	3.05		p ≤ 0.001
24+0 h -S-9	Vehicle^a	-	0.50	0.10-1.10	-
	10.00	9	0.50		NS
	20.00	40	0.50		NS
	30.00	61	0.45		NS
	*VIN, 0.02	ND	3.66		p ≤ 0.001

 

^aVehicle control was DMSO

* Positive control

# 95th percentile of the observed range

$Based on replication index

NS = Not significant

ND = Not determined

Validity of study

1)The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures.

2) The frequency of MNBN cells in vehicle controls fell within the normal ranges.

3) The positive control chemicals induced statistically significant increases in the proportion of cells with micronuclei. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the current historical vehicle control ranges.

4) A minimum of 50 % of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest.

Conclusions:

The test item did not induce micronuclei in cultured human peripheral blood lymphocytes when tested up to cytotoxic concentrations for 3+21 h in the absence and presence of a rat liver metabolic activation system (S-9) and for 24+0 h in the absence of S-9.

Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured human lymphocytes were exposed to test item REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE at the concentrations below.

- Range-Finder Experiment:

3+21 h treatments with and without S9; 24+0 h treatments without S9: 2.902, 4.837, 8.062, 13.44, 22.39, 37.32, 62.21, 103.7, 172.8, 288, 480 and 800 µg/mL

 

- Micronucleus Experiment:

3+21 h treatments without S9: 10.00, 20.00, 22.50, 25.00, 27.50, 30.00, 32.50, 35.00, 40.00, 45.00, 50.00, 55.00 and 65.00 µg/mL

3+21 h treatments with S9: 20, 40, 60, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 135 and 150 µg/mL

24+0 h treatments without S9: 10.00, 20.00, 30.00, 35.00, 40.00, 42.50, 45.00, 47.50, 50.00, 52.50, 55.00, 60.00 and 75.00 µg/mL

 

Cytochalasin B, formulated in DMSO, was added directly (0.1 mL/culture) to all continuous cultures at the time of treatment to give a final concentration of 6 μg/mL per culture. The cells were then treated with a hypotonic solution, fixed, stained and examined for toxicity and micronuclei. Vehicle and positive controls were also included in the study.

 

- Concentrations selected for micronucleus analysis based on cytotoxicity:

3+21 h in the absence of S-9: 27.50, 32.50 and 40.00 μg/mL

3+21 h in the presence of S-9: 70, 80 and 90 μg/mL

24+0 h in the absence of S-9: 10, 20 and 30 μg/mL

 

Treatment of cells with the test item for 3+21 h in the absence and presence of S-9 and for 24+0 h in the absence of S-9 resulted in frequencies of MNBN cells that were generally similar to (and not significantly higher than) those observed in concurrent vehicle controls at all concentrations analysed. The MNBN cell frequencies in treated cultures fell within the normal ranges with the exception of a single culture at the intermediate concentration of 80 μg/mL for the 3+21 h +S-9 treatment, which was outside this range but within the observed range. This marginal increase was not observed in the replicate culture at 80 μg/mL or at any other concentration analysed under any of the three treatment conditions, therefore it was considered of no biological relevance.

 

The positive control substances (Mitomycin-C and Vinblastine; without S-9 &Cyclophosphamide; with S-9) induced statistically significant increases in the proportion of cells with micronuclei, demonstrating the sensitivity of the test system.

 

Therefore, the test item did not induce micronuclei in cultured human peripheral blood lymphocytes when tested up to cytotoxic concentrations for 3+21 h in the absence and presence of a rat liver metabolic activation system (S-9) and for 24+0 h in the absence of S-9.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE did not induce micronuclei.

In a Ames test performed according to OECD Guideline 471 and in compliance with GLP, REACTION MASS OF DL-LIMONENE ALPHA- GAMMA- TERPINENES, TERPINOLENE was not mutagenic.

In an in vitro HPRT test performed according to OECD Guideline 476 and in compliance with GLP, REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE was not mutagenic.

Therefore, according to these results, REACTION MASS OF DL-LIMONENE, ALPHA-GAMMA-TERPINENES, TERPINOLENE can be considered as non genotoxic.