Reaction mass of 2-(cis-4-methylcyclohexyl)propan-2-ol and 2-(trans-4-methylcyclohexyl)propan-2-ol and cis-1-methyl-4-(propan-2-yl)cyclohexanol and trans-1-methyl-4-(propan-2-yl)cyclohexanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Dihydroterpineol multiconstituent is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98, TA100 and TA102) strains in a study conducted according to OECD Guideline 471.

Dihydroterpineol multiconstituent did not induce structural chromosome aberrations in cultured human peripheral blood lymphocytes, when tested to the limit of cytotoxicity in both the absence and presence of an Aroclor 1254-induced rat liver metabolic activation system (S-9).

Dihydroterpineol multiconstituent did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay, in which adequate toxicity was achieved in all tests.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 February - 07 April 2014

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

None

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1 (plate-incorporation method):
- TA1535, TA1537, TA98, TA100 and TA102: 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, with and without S9-mix

Experiment 2 (plate-incorporation method without S9 mix; preincubation method with S9 mix):
- TA1535, TA1537, TA98, TA100 and TA102: 8.192, 20.48, 51.20, 128, 320, 800 and 2000 μg/plate, without S9-mix
- TA100, TA1537 and TA102: 8.192, 20.48, 51.20, 128, 320, 800 and 2000 μg/plate, with S9-mix
- TA98 and TA1535: 20.48, 51.20, 128, 320, 800, 2000 and 5000 μg/plate, with S9-mix

Experiment 3 (preincubation method):
- TA98 and TA1535: 3.277, 8.192, 20.48, 51.2, 128, 320 and 800 μg/plate, with S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Dihydroterpineol multiconstituent was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to at least 100 mg/mL.Therefore, DMSO was selected as vehicle.
- Test substance preparation: Test substance stock solutions were prepared by formulating Dihydroterpineol multiconstituent under subdued lighting in DMSO, to give the maximum required treatment concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 3.5 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:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM:
Strains TA98, TA1535 and TA1537 were originally obtained from the UK NCTC. Strains TA100 and TA102 were derived from cultures originally obtained from Covance Laboratories Inc., USA.

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

DURATION
- Preincubation period: 20 minutes at 37 ± 1 °C, with shaking
- Incubation period: Plates were inverted and incubated at 37 ± 1 °C in the dark for 3 days in both direct plate and preincubation methods.

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

DETERMINATION OF CYTOTOXICITY
- Method: The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response. Where mutation data from fewer than five treatment concentrations was obtained, an evaluation of the mutation data for the study as a whole was made. If the mutation data for any strain treatment was considered insufficient to provide a thorough and robust assessment of mutagenicity then additional testing was conducted.

OTHER:
- Strain characteristics: The inocula were taken from master plates or vials of frozen cultures, which had been checked for strain characteristics (histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline). Checks were carried out according to Maron and Ames, 1983 and De Serres and Shelby, 1979.
- Colony counting: Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments). The background lawn was inspected for signs of toxicity.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control values.
The test article was considered positive in this assay if the above criterion was met.
The test article was considered negative in this assay if the above criterion was not met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and between experiments.

Statistics:

The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate). However, adequate interpretation of biological relevance was of critical importance (OECD, 1997; ICH S2(R1), 2011).

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

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
- Precipitation: None
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA: Mean vehicle control counts fell within the laboratory’s historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment 1: Following the treatment, evidence of toxicity ranging from a diminution of the background bacterial lawn, with or without a concurrent marked reduction in revertant numbers, to a complete killing of the test bacteria was observed at 500 μg/plate and/or 1600 μg/plate and above in all strains in the absence and presence of S-9.
- Experiment 2: Following the treatment, evidence of toxicity ranging from a slight thinning of the background bacterial lawn, to a complete killing of the test bacteria was observed at 800 μg/plate and/or at 2000 μg/plate and/or 5000 μg/plate in the absence and presence of S-9 in all strains. In addition, complete toxicity was observed at 320 μg/plate on a single plate for strain TA1537 in the presence of S-9 only. Since mutation data were only available for four concentrations in the presence of S-9 for strains TA98 and TA1535 due to toxicity, a further Experiment (Experiment 3) was performed.
- Experiment 3: Following the treatment, evidence of toxicity ranging from a slight thinning of the background bacterial lawn to a complete killing of the test bacteria was observed at 320 μg/plate and above in both strains.

None

Conclusions:

The test item is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98, TA100 and TA102) strains in a study conducted according to OECD Guideline 471.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102) were exposed to test item dihydroterpineol multiconstituent at the concentrations below. 

Experiment 1 (plate-incorporation method)

- TA1535, TA1537, TA98, TA100 and TA102: 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, with and without S9-mix 

Experiment 2 (plate-incorporation method without S9 mix; preincubation method with S9 mix)

- TA1535, TA1537, TA98, TA100 and TA102: 8.192, 20.48, 51.20, 128, 320, 800 and 2000 μg/plate, without S9-mix

- TA100, TA1537 and TA102: 8.192, 20.48, 51.20, 128, 320, 800 and 2000 μg/plate, with S9-mix

- TA98 and TA1535: 20.48, 51.20, 128, 320, 800, 2000 and 5000 μg/plate, with S9-mix 

Experiment 3 (preincubation method)

- TA98 and TA1535: 3.277, 8.192, 20.48, 51.2, 128, 320 and 800 μg/plate, with S9-mix 

Metabolic activation system used in this test is 10% S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254. Vehicle and positive control groups were also included in mutagenicity tests.

 

In Experiment 1, following the treatment, evidence of toxicity was observed at 500 μg/plate and/or 1600 μg/plate and above in all strains in the absence and presence of S-9. In Experiment 2, evidence of toxicity was observed at 800 μg/plate and/or at 2000 μg/plate and/or at 5000 μg/plate in the absence and presence of S-9 in all strains. In addition, complete toxicity was observed at 320 μg/plate on a single plate for strain TA1537 in the presence of S-9 only. Since mutation data were only available for four concentrations for strains TA98 and TA1535 in the presence of S-9 due to toxicity, a further experiment (Experiment 3) was performed. In Experiment 3, evidence of toxicity was observed at 320 μg/plate and above in both strains.

 

The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

 

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

 

Therefore, the test item is not considered as mutagenic in this bacterial system.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

5 February 2014 - 20 May 2014

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 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

17-19 July 2013

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

None

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (10% v/v); S9 fraction, prepared from male Sprague-Dawley derived rats,

Test concentrations with justification for top dose:

Preliminary toxicity test: 18.14; 30.23; 50.39; 83.98; 140; 233.3; 388.8; 648; 1080; 1800;3000 and 5000 µg/mL

Main tests:
-S9 mix (3 hours): 50, 100, 125, 150,175, 200, 225, 250, 275, 300, 325, 350, 375, 400 and 500 µg/mL
+S9 mix (3 hours): 75, 750, 200, 250, 275, 300, 315, 330, 345, 360, 375, 390, 410, 450 and 500 µg/mL
-S9 mix (20 hours): 10, 25, 50, 65, 80, 95, 100, 110, 125, 140, 155, 170, 185, 200, 240, 250, 2280, 295, 305, 340, 355, 370, 385, 395, 405, 415, 425 and 505 µg/mL
-S9 mix (20 hours) (additional test): 10, 25, 50, 60, 80, 90, 100, 110, 120, 130, 140, 160, 180, 240, 350, 365, 370, 375, 380, 385, 390, 395, 400, 410 and 420 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl suphoxide (DMSO)
- Test item was dissolved and diluted in DMSO, shortly before dosing.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix: 0.3 and 0.4 μg/mL (3-hour treatment); 0.1 and 0.05 μg/mL (20-hour continuous treatment)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix: 1 and 2 μg/mL

Details on test system and experimental conditions:

PREPARATION OF CULTURES:
- Human blood was collected from three healthy, non-smoking, adult female donors, pooled (in equal volumes from each donor.
As lymphocytes do not normally undergo cell division, they were stimulated to do so by the addition of phytohaemagglutinin (PHA), a naturally occurring mitogen. Cultures were established from the prepared (pooled) sample. All cultures were then incubated at 37 °C, for approximately 48 hours and rocked continuously.

METHOD OF APPLICATION: in medium
HEPES-buffered RPMI media, supplemented with 10% inactivated foetal calf serum, 0.52% penicillin/streptomycin and PHA solution .

DURATION
- Exposure duration: Preliminary toxicity test: 3 h (±S9); 20 h continuous treatment (-S9); Main test: 3 h (±S9); 20 h continuous treatment (-S9)
- Fixation time (start of exposure up to harvest of cells): 20 h, with and without S9 mix in preliminary toxicity and main tests

SPINDLE INHIBITOR (cytogenetic assays): Two hours before the cells were harvested, mitotic activity was arrested by addition of Colchicine to each culture at a final concentration of 1 µg/mL.

STAIN (for cytogenetic assays): Giemsa staining (4% (pH 6.8))

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Duplicate cultures were used for treatment with the vehicle, and single cultures for treatment with test substance for each test condition.
- Main test: Duplicate cultures were used for treatment with the vehicle, test substance and positive controls.

NUMBER OF CELLS EVALUATED:
- The proportion of mitotic cells per 1000 cells in each culture was recorded (except for when clear evidence of overt toxicity was observed, or in cultures where there were no signs of cytotoxicity).
- One hundred metaphase figures were examined from each culture, however, this number was reduced in cultures showing a high level of aberrant cells, where 10 metaphases with structural aberrations were observed. In this study scoring was truncated only for the positive control cultures. Chromosome aberrations were scored according to the classification of the ISCN (2009). Only cells with 44 - 48 chromosomes were analysed.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

OTHER EXAMINATIONS:
- The incidence of polyploid and endoreduplicated cells (i.e. the ploidy status) were each recorded, independently from the analysis for chromosome aberrations.

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic events if:
- A proportion of cells with structural aberrations at one or more concentrations thatexceeded the normal range was observed in both replicate cultures
- A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p≤0.05)
- There was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).

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 to be dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result

Statistics:

The number of aberrant metaphase cells in each test substance group was compared with the vehicle control value using the one-tailed Fisher exact test (Fisher 1973). Probability values of p≤0.05 were accepted as significant.

A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.

D20’s (the minimum concentration (mg/mL) at which aberrations were found in 20% of metaphases) were estimated (where possible) using logistic regression on a log(concentration) scale, allowing the number of control aberrations to be non-zero (Armitage et al., 2002).

The data was analysed using the SAFEStat (SAS statistical applications for end users, version 1.1) Chromosome Aberrations application (version 1.1) which was developed in SAS (SAS INSTITUTE 2002).

Key result

Species / strain:

lymphocytes: human

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 pH or osmolality at the highest concentration tested for each culture, as compared to the concurrent vehicle controls (individual data not reported).

PRELIMINARY TOXICITY TEST:
- In the absence of S9 mix following 3-hour treatment, test item caused a reduction in the mitotic index to 82% of the vehicle control value at 388.8 μg/mL. At higher tested concentrations excessive toxicity was observed.
- In the presence of S9 mix following 3-hour treatment, test item caused a reduction in the mitotic index to 73% of the vehicle control value at 388.8 μg/mL. At higher tested concentrations excessive toxicity was observed. N
- In the absence of S9 mix following 20-hour continuous treatment, test item caused a reduction in the mitotic index to 96% of the vehicle control value at 388.8μg/mL. At higher tested concentrations excessive toxicity was observed.

MAIN TEST:

Cytotoxicity:
Experiment 1:
- In the absence of S9 mix, 3-hour treatment: Test item caused a reduction in the mitotic index to 87% of the vehicle control value at 350 μg/mL. The concentrations selected for metaphase analysis were 100, 225 and 275 μg/mL Precipitate have been detected at 225 µg/ml and above.
- In the presence of S9 mix, 3-hour treatment: Test item caused a reduction in the mitotic index to 73% of the vehicle control value at 400 μg/mL. Since the required toxicity was not obtained a further experiment was conducted. Test item caused a reduction in the mitotic index to 93% of the vehicle control value at 450 μg/mL. The concentrations selected for metaphase analysis were 200, 330, 375 and 410 μg/mL Precipitate have been detected at 275 µg/ml and above.

Experiment 2
- In the absence of S9 mix, 20-hour continuous treatment: Test item caused a reduction in the mitotic index to 80% of the vehicle control value at 250 μg/mL
Since the required toxicity was not obtained further experiment was conducted.
- Additional main test: 20-hour continuous treatment in the absence of S9 mix: Test item caused a reduction in the mitotic index to 84% of the vehicle control value at 180 μg/mL. The concentrations selected for metaphase analysis were 25,50,60 and 110 μg/mL.

STRUCTURAL ABBERATIONS:
Treatment of cells with Dihydroterpineol multiconstituent in the absence and presence of S-9 resulted in frequencies of cells with structural chromosome aberrations which were similar to those observed in concurrent vehicle control cultures for all concentrations analysed in all treatments. Numbers of aberrant cells (excluding gaps) in all treated cultures fell within the normal ranges.

NUMERICAL ABERRATIONS:
Following Experiment 1 treatments, there was an increase in the frequency of cells with numerical aberrations, which exceeded the normal range in a single
culture at 275 μg/mL in the absence of S-9. There were also increases in the frequency of cells with numerical aberrations, which exceeded the normal range in a single culture at 330 μg/mL and in both replicate cultures at 375 and 410 μg/mL in the presence of S-9. These showed some evidence of being concentration related. These increases were attributable to polyploidy. Although increases in polyploidy can indicate mitotic spindle effects, the biological relevance of these observations in vitro is questionable. In addition, this study is not specifically designed to evaluate the inductions of these types of aberrations.

Following Experiment 2 there were increases in the frequency of cells with numerical aberrations, which exceeded the normal range following 3+17 hour treatment in the presence of S-9, in both replicate cultures at 350 μg/mL. These increases were attributable to polyploidy. No other increases in the frequency of cells with numerical aberrations, which exceeded the normal range, were observed in cultures treated with Dihydroterpineol multiconstituent in the absence or presence of S-9 in either Experiment 1 or Experiment 2.

Table of Results:

1.Mitotic Index Determinations – Experiment 1, Trial 1

Treatment (mg/mL)	Mitotic index (%)
	3 hours, -S-9			3 hours, +S-9
	A/C	B/D	MIH*	A/C	B/D	MIH*
Vehicle (DMSO)	9.4/9.2	10.4/9.6	-	7.9/7.0	8.4/8.2	-
50.00	8.7	9.4	6	9.2	9.5	0
100.0	8.3	8.2	15 #	9.2	9.1	0
125.0	10.2	7.6	8	NT	NT	-
150.0	7.5	6.6	27	8.7	8.2	0
175.0	5.9	7.9	28	NT	NT	-
200.0	7.1	5.9	33	7.4	7.9	3
225.0	6.7	7.7	25 # P	7.5	5.7	16 P
250.0	5.5	6.9	36 P	8.2	7.8	0 P
275.0	4.5	5.2	50 # P	8.1	8.5	0 P
300.0	4.8	4.6	51 P	5.7	6.0	26 P
325.0	2.4	3.2	71 P	4.7	6.2	31 P
350.0	1.3	1.2	87 P	6.0	4.5	33 P
375.0	NT	NT	-	5.3	4.3	39 P
400.0	0.0	0.0	100 P	2.2	2.1	73 P
500.0	0.0	0.0	100 P	0.0	0.0	100 P

NT = Not tested

P = Precipitation observed at treatment

*Mitotic inhibition (%) = [1 – (mean MIT/mean MIC)] x 100% (where T = treatment and C = vehicle control)

# Highlighted concentrations selected for analysis.

2. Mitotic Index Determinations – Experiment 1, Trial 2

Treatment (mg/mL)	Mitotic index (%)
	3 hours, +S-9
	A/C	B/D	MIH*
Vehicle	10.2/9.9	10.8/10.8	-
75.00	10.9	10.5	0
150.0	10.6	10.6	0
200.0	8.8	9.0	15 #
250.0	7.9	8.8	20
275.0	8.2	8.7	19 P
300.0	8.2	8.7	19 P
315.0	10.3	7.1	17 P
330.0	7.2	7.1	31 # P
345.0	6.9	6.9	34 P
360.0	6.6	5.6	41 P
375.0	5.7	5.9	44 # P
390.0	3.6	5.1	58 P
410.0	4.2	4.4	59 # P
450.0	0.6	0.8	93 P
500.0	0.0	0.0	100 P

P = Precipitation observed at treatment

*Mitotic inhibition (%) = [1 – (mean MIT/mean MIC)] x 100% (where T = treatment and C = vehicle control)

# Highlighted concentrations selected for analysis.

3.Mitotic Index Determinations – Experiment 2, Trial 1

Treatment (mg/mL)	Mitotic index (%)
	20 hours, -S-9				3 hours, +S-9
	A/C		B/D	MIH*	A/C	B/D	MIH*
Vehicle	8.2/9.0	6.6/11.1		-	11.5/10.5	9.4/8.4	-
10.00	9.5	8.6		0	NT	NT	-
25.00	6.6	7.8		17	NT	NT	-
50.00	7.6	7.1		16	NT	NT	-
65.00	6.7	6.4		25	NT	NT	-
80.00	6.5	7.4		20	NT	NT	-
95.00	3.0	5.4		52	NT	NT	-
100.0	NT	NT		-	12.6	9.7	0
110.0	2.6	5.4		54	NT	NT	-
125.0	3.1	3.7		61	NT	NT	-
140.0	1.6	2.7		75	NT	NT	-
155.0	1.5	2.1		79	NT	NT	-
170.0	2.0	1.7		79	NT	NT	-
185.0	2.4	1.7		77	NT	NT	-
200.0	1.7	2.0		79	NT	NT	-
240.0	NT	NT		-	7.8	8.5	18 P
250.0	1.8	1.7		80 P	NT	NT	-
280.0	NT	NT		-	6.8	8.9	21 P
295.0	NT	NT		-	7.4	7.2	27 P
305.0	NT	NT		-	6.4	9.0	23 P
340.0	NT	NT		-	6.9	5.7	37 P
355.0	NT	NT		-	8.7	6.3	25 P
370.0	NT	NT		-	6.7	4.6	43 P
385.0	NT	NT		-	2.4	4.4	66 P
395.0	NT	NT		-	1.9	3.6	72 P
405.0	NT	NT		-	2.0	1.5	82 P
415.0	NT	NT		-	0.2	0.6	96 P
425.0	NT	NT		-	0.0	0.1	99 P
505.0	NT	NT		-	0.0	0.0	100 P

NT = Not tested

P = Precipitation observed at treatment

*Mitotic inhibition (%) = [1 – (mean MIT/mean MIC)] x 100% (where T = treatment and C = vehicle control)

# Highlighted concentrations selected for analysis.

4. Mitotic Index Determinations – Experiment 2, Trial 2

Treatment (mg/mL)	Mitotic index (%)
	20 hours, -S-9				3 hours, +S-9
	A/C		B/D	MIH*	A/C	B/D	MIH*
Vehicle	4.7/9.1	7.0/6.1		-	7.2/8.0	9.1/8.7	-
10.00	8.4	7.4		0 #	NT	NT	-
25.00	4.4	5.8		24 #	NT	NT	-
50.00	7.5	3.8		16	NT	NT	-
60.00	5.0	4.4		30 #	NT	NT	-
80.00	3.5	4.7		39	NT	NT	-
90.00	4.4	4.7		32	NT	NT	-
100.0	3.2	4.4		43	7.2	7.4	12 #
110.0	3.2	2.9		55 #	NT	NT	-
120.0	2.0	2.6		66	NT	NT	-
130.0	2.3	3.0		61	NT	NT	-
140.0	2.8	2.3		62	NT	NT	-
160.0	2.1	1.5		73	NT	NT	-
180.0	1.3	0.9		84	NT	NT	-
240.0	NT	NT		-	5.0	6.1	33 #
350.0	NT	NT		-	3.6	3.8	55 # P
365.0	NT	NT		-	4.0	2.8	59 P
370.0	NT	NT		-	3.6	2.8	61 P
375.0	NT	NT		-	3.6	3.2	59 P
380.0	NT	NT		-	2.9	2.9	65 P
385.0	NT	NT		-	3.6	2.2	65 P
390.0	NT	NT		-	2.7	1.5	75 P
395.0	NT	NT		-	2.2	2.5	72 P
400.0	NT	NT		-	3.8	0.7	73 P
410.0	NT	NT		-	2.0	1.6	78 P
420.0	NT	NT		-	0.3	0.0	98 P

NT = Not tested

P = Precipitation observed at treatment

*Mitotic inhibition (%) = [1 – (mean MIT/mean MIC)] x 100% (where T = treatment and C = vehicle control)

# Highlighted concentrations selected for analysis

Conclusions:

Dihydroterpineol multiconstituent did not induce structural chromosome aberrations in cultured human peripheral blood lymphocytes, when tested to the limit of cytotoxicity in both the absence and presence of an Aroclor 1254-induced rat liver metabolic activation system (S-9).

Executive summary:

In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured human lymphocytes were exposed to the test item at the concentrations below.

Preliminary toxicity test

18.14; 30.23; 50.39; 83.98; 140; 233.3; 388.8; 648; 1080; 1800;3000 and 5000  µg/mL

Main tests

-S9 mix (3 hours): 50, 100, 125, 150,175, 200, 225, 250, 275, 300, 325, 350, 375, 400 and 500  µg/mL

+S9 mix (3 hours): 75, 750, 200, 250, 275, 300, 315, 330, 345, 360, 375, 390, 410, 450 and 500 µg/mL

-S9 mix (20 hours):  10, 25, 50, 65, 80, 95, 100, 110, 125, 140, 155, 170, 185, 200, 240, 250, 2280, 295, 305, 340, 355, 370, 385, 395, 405, 415, 425 and 505 µg/mL

-S9 mix (20 hours) (additional test): 10, 25, 50, 60, 80, 90, 100, 110, 120, 130, 140, 160, 180, 240, 350, 365, 370, 375, 380, 385, 390, 395, 400, 410 and 420 µg/mL

 

Two hours before the cells were harvested, mitotic activity was arrested by addition of Colchicine to each culture at a final concentration of 1 µg/mL. The cells were then fixed, stained and examined for mitotic indices and chromosomal aberrations. Metabolic activation system used in this test was S9 fraction (10% v/v); S9 fraction, prepared from male Sprague-Dawley derived rats.

A preliminary toxicity test was performed using a dose range of 18.14 to 5000 μg/mL. Cells were exposed for a 3-hour treatment in the absence and presence of S9 mix, and a 20-hour continuous treatment in the absence of S9 mix and mitotic index data were used to determine toxicity. Based on the mitotic index data, concentrations were selected for the main test.

 

In the main test, the mitotic index was assessed for all cultures treated with the test item and the vehicle control, Ethanol. Justification for the highest analysed concentration was determined by cytotoxicity. On the basis of these data, the concentrations below were selected for metaphase analysis.

In the absence of S9 mix, 3-hour treatment: 100, 225 and 275 μg/mL.

In the presence of S9 mix, 3-hour treatment: 200, 300, 375 and 410 μg/mL.

In the absence of S9 mix, 21-hour continuous treatment: 25, 50, 60 and 110 μg/mL.

 

Treatment of cells with dihydroterpineol multiconstituent in the absence and presence of S-9 resulted in frequencies of cells with structural chromosome aberrations which were similar to those observed in concurrent vehicle control cultures for all concentrations analysed in all treatments. Numbers of aberrant cells (excluding gaps) in all treated cultures fell within the normal ranges.

In the absence of S9 mix following a 3-hour treatment with the test item, an increase in the frequency of cells with numerical abberration, exceding the normal range was seen at 275 µg/mL.

In the presence of S9 mix following a 3-hour treatment with the test item, an increase in the frequency of cells with numerical abberration, exceding the normal range was seen at 330 and 375, and 410 µg/mL.

These increases were attributable to polyploidy. Although increases in polyploidy can indicate mitotic spindle effects, the biological relevance of these observations in vitro is questionable. In addition, this study is not specifically designed to evaluate the inductions of these types of aberrations.

 

In the absence of S9 mix following a 20-hour continuous treatment, no increases in the frequency of cells with numerical aberrations, which exceeded the normal range, were observed in cultures.

 

Both positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

Therefore, dihydroterpineol multiconstituent did not induce structural chromosome aberrations in cultured human peripheral blood lymphocytes, when tested to the limit of cytotoxicity in both the absence and presence of an Aroclor 1254-induced rat liver metabolic activation system (S-9).

Reference 3

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:

July -August 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 Ovary (CHO)

Details on mammalian cell type (if applicable):

- Source: European Collection of Cell Cultures
- CHO-KI cells are functionally hemizygous at the HPRT locus.
- Type and identity of media: Ham’s Nutrient Mixture F12 medium
- 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 incubating the cells in H10 containing 15 μg/mL hypoxanthine, 0.3 μg/mL amethopterin and 4 μg/mL thymidine for three days. All cell cultures were maintained at 37 °C in an atmosphere of 5 % CO2 in air.

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 5,6-benzoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test: 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μg/mL.
Mutation tests: The upper concentration levels were selected based on cytotoxicity.
-S9 mix Test (3 hours) 62.5, 125, 250, 300, 350, 400, 450 and 500 μg/mL.
+S9 mix Test (3 hours) 15.63, 31.25, 62.5, 125, 250 and 500 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Dihydroterpineol multiconstituent was found to be soluble at 200 mg/mL in dimethyl sulfoxide (DMSO).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

250 μg/mL; without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

5 μg/mL; with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: Ham’s Nutrient Mixture F12 medium
- Ham’s Nutrient Mixture F12, supplemented with 2 mM L-glutamine and 50 μg/mL gentamicin. The resulting medium is referred to as H0.
- H0 medium supplemented with 10 % HiFCS referred to as H10, is used for general cell culture, e.g. when growing cells up from frozen stocks.

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
- All incubations were performed at 37 °C in a humidified atmosphere of 5 % CO2 in air.

SELECTION AGENT (mutation assays): Selective medium, in which only HPRT deficient cells will grow, consisted of H10 supplemented with 6-thioguanine (6-TG) at a final concentration of 10 μg/mL.

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture/dose for test item and 2 cultures for vehicle control
- Main test: , 2 cultures/dose for test item

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, Survival and Relative Survival
Cloning efficiency: Total no of colonies for each culture / (Number of plates scored for colony formation x 200)
Survival: Cloning efficiency x Cell count Correction Factor
Relative Survival (RS): (Individual survival value x100) / Mean control survival value
Following the expression period, three plates were scored for the presence of colonies from each culture and the CE was calculated.
Relative Cloning Efficiency (RCE): (Individual CE x100) / Mean control CE

OTHER:
Mutant Frequency (MF) per 10^6 viable cells for each set of plates was calculated as: (Total no. of mutant colonies x 5) / (CE x no. of uncontaminated plates)

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 vehicle control
- the increase is concentration-related when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical vehicle control data

The criteria for a negative response will be:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
- there is no concentration-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the historical vehicle control data.

Statistics:

The statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution following the methods described by Arlett et al. (1989). Tests were conducted for a linear concentration-response relationship of the test substance, for non-linearity and for the comparison of positive control and treated groups to solvent control. Data was analysed using SAS (SAS Institute Inc., 2002).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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 of the medium of more than 1.0 unit compared with the vehicle control were observed at 2000 μg/mL.
- Effects of osmolality: No fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed when compared with the vehicle control at 2000 µg/mL.

PRELIMINARY TOXICITY TEST:
- In the preliminary toxicity test, a three-hour exposure at concentrations from 15.63 to 500 µg/mL of test item, in the absence, resulted in Day 1 relative survivals of 86 to 0 % and a three-hour exposure at concentrations from 15.63 to 250 µg/mL of test item, in the presence of S9 mix118 to 66 % respectively. Precipitate was seen at 500 μg/mL at the end of treatment.
Concentrations for the main test were based upon these data and toxicity was the primary determinant for the dose selection.

MAIN MUTATION TEST 1
3-hour treatment in the absence of S9 mix: Precipitate was observed by eye at the end of treatment at concentrations of 400 µg/mL and above and this was, therefore, the highest concentration plated for determination of RS. Exposure to test item resulted in Day 1 relative survivals of between 91 and 0 %. Cultures treated at concentrations from 62.5 to 350 µg/mL were plated out for determination of cloning efficiency and mutant frequency. Cultures treated at 400 µg/mL were not analysed for mutant frequency as RS was <10% at this concentration. No significant increases in mutant frequency were observed after exposure to Dihydroterpineol multiconstituent.
3-hour treatment in the presence of S9 mix: Precipitate was observed by eye at the end of treatment at concentrations of 250 µg/mL and above and this was, therefore, the highest concentration plated for determination of RS.. Exposure to test item resulted in Day 1 relative survivals of between 108 and 71 %. Exposure to Dihydroterpineol multiconstituent resulted in RS values from 108 to 71 %. All remaining cultures were plated out for determination of cloning efficiency and mutant frequency. No significant increases in mutant frequency were observed after exposure to Dihydroterpineol multiconstituent.

Table 7.6.1/1: Summary results

Main Test: 3-hour treatment in the absence of S9 mix

Conc^n.of test item   (µg/mL)	Cell Count Day 1 (x10^6/mL)	No. of colonies on plate			Total no. of Colonies	Cloning Efficiency (%)	Adjusted Cloning Efficiency (%)	RS (%)	Mean RS (%)	No. of colonies on plate			Total no. of Colonies	Cloning Efficiency in non‑selective medium (%)	No. of colonies on plate					Total no. of Colonies	Cloning Efficiency in selective medium (%)	Mutant Frequency^a	Mean Mutant Frequency^a
		Plate 1	Plate 2	Plate 3						Plate 1	Plate 2	Plate 3			Plate 1	Plate 2	Plate 3	Plate 4	Plate 5
0b	1.54	124	136	135	395	71	81	100	100	107	117	114	338	56	0	1	2	0	1	4	0.00016	2.84	3.78
	1.52	156	141	138	435					103	105	107	315	53	2	2	2	2	1	9	0.00036	6.86
	1.59	153	138	148	439					150	148	141	439	73	1	0	1	2	0	4	0.00016	2.19
	1.54	146	148	150	444					97	102	96	295	49	2	1	0	1	0	4	0.00016	3.25
62.5	1.46	147	131	143	421	70	76	93	91	120	90	102	312	52	0	1	0	3	3	7	0.00028	5.38	3.88
	1.41	135	142	138	415	69	72	88		93	103	108	304	51	0	1	0	0	2	3	0.00012	2.37
125	1.41	108	147	105	360	60	62	77	78	120	131	113	364	61	1	0	2	0	0	3	0.00012	1.98	2.20
	1.47	130	128	102	360	60	65	80		137	105	156	398	66	0	2	1	0	1	4	0.00016	2.41
250	1.32	115	116	120	351	59	57	70	68	136	137	154	427	71	0	0	0	0	0	0	0.00000	0.00	0.68
	1.28	118	102	123	343	57	54	66		118	129	108	355	59	0	0	0	1	1	2	0.00008	1.35
300	1.17	101	102	91	294	49	42	52	50	139	138	139	416	69	2	0	0	0	0	2	0.00008	1.15	2.46
	1.19	90	86	88	264	44	39	47		106	101	112	319	53	0	1	1	2	1	5	0.00020	3.76
350	0.97	31	23	39	93	16	11	14	13	132	128	141	401	67	3	3	2	3	0	11	0.00044	6.58	3.29
	0.82	36	28	34	98	16	10	12		133	141	137	411	69	0	0	0	0	0	0	0.00000	0.00
400^b	0.72	0	0	2	2	0	0	0	0	Culture not analysed as day 1 relative survival <10%					Culture not analysed as day 1 relative survival <10%
	0.67	1	1	1	3	1	0	0
EMS – positive control
250	1.45	131	128	129	388	65	69	85	74	128	120	132	380	63	37	26	28	31	33	155	0.00620	97.89	107.51
	1.63	94	84	83	261	44	52	64		139	123	72	334	56	35	34	28	34	32	163	0.00652	117.13	***

a. Mutant frequencies expressed per 10⁶viable cells

b. Vehicle control = DMSO 1% (v/v)

***p<0.001, statistically significant increase over concurrent vehicle control mutant frequency

test item – Dihydroterpineol multiconstituent

EMS: Ethyl methanesulphonate

Main Test: 3-hour treatment in the presence of S9 mix

Day 1 relative survival										Day 8 cloning efficiency					mutant frequency
Conc^n.of test item   (µg/mL)	Cell Count Day 1 (x10^6/mL)	No. of colonies on plate			Total no. of Colonies	Cloning Efficiency (%)	Adjusted Cloning Efficiency (%)	RS (%)	Mean RS (%)	No. of colonies on plate			Total no. of Colonies	Cloning Efficiency in non‑selective medium (%)	No. of colonies on plate					Total no. of Colonies	Cloning Efficiency in selective medium (%)	Mutant Frequency^a	Mean Mutant Frequency^a
		Plate 1	Plate 2	Plate 3						Plate 1	Plate 2	Plate 3			Plate 1	Plate 2	Plate 3	Plate 4	Plate 5
0b	1.04	134	152	124	410	71	70	100	100	182	131	134	447	75	0	0	0	0	0	0	0.00000	0.00	1.28
	1.06	151	143	129	423					156	127	137	420	70	1	0	2	1	0	4	0.00016	2.29
	1.02	163	139	143	445					143	129	132	404	67	2	0	1	0	0	3	0.00012	1.78
	1.05	136	162	127	425					149	159	150	458	76	0	0	1	1	0	2	0.00008	1.05
15.63	1.04	163	137	148	448	75	73	105	108	141	146	112	399	67	0	1	0	0	0	1	0.00004	0.60	0.30
	1.05	161	153	157	471	79	77	111		150	147	133	430	72	0	0	0	0	0	0	0.00000	0.00
31.25	1.00	122	118	133	373	62	58	84	83	122	146	161	429	72	3	1	2	0	3	9	0.00036	5.03	3.15
	1.00	118	122	127	367	61	58	83		125	144	112	381	64	1	0	1	0	0	2	0.00008	1.26
62.5	0.91	108	99	118	325	54	47	67	71	110	93	97	300	50	0	2	1	0	0	3	0.00012	2.40	3.71
	0.93	117	119	127	363	61	53	76		145	114	123	382	64	2	0	1	4	1	8	0.00032	5.03
125	0.94	123	104	100	327	55	48	70	73	156	142	121	419	70	2	2	2	0	1	7	0.00028	4.01	4.33
	0.95	125	115	117	357	60	53	76		136	113	112	361	60	1	1	2	2	1	7	0.00028	4.65
250	0.96	119	139	107	365	61	55	79	74	120	98	98	316	53	0	1	1	2	1	5	0.00020	3.80	4.68
	0.92	107	104	121	332	55	48	69		122	134	176	432	72	0	2	3	2	3	10	0.00040	5.56
500	Precipitate observed by eye at the end of treatment, therefore culture discarded									Precipitate observed by eye at the end of treatment					Precipitate observed by eye at the end of treatment
3MC – positive control
5	0.88	90	93	104	287	48	40	57	61	119	136	104	359	60	11	21	17	27	14	90	0.00360	60.17	65.23
	0.98	108	103	87	298	50	46	66		91	125	122	338	56	18	18	17	26	20	99	0.00396	70.30	***

a. Mutant frequencies expressed per 10⁶viable cells

b. Vehicle control = DMSO 1% (v/v)

***p<0.001, statistically significant increase over concurrent vehicle control mutant frequency

test item – Dihydroterpineol multiconstituent

3MC: 3-Methylcholanthrene

Conclusions:

The test item did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay, in which adequate toxicity was achieved in all tests.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster Ovary (CHO-K1) cells were exposed to dihydroterpineol multiconstituent for 3 h, with and without metabolic activation (S9 fraction of male Sprague Dawley rats liver induced with phenobarbital and 5,6-benzoflavone), at the following concentrations: 

Preliminary toxicity test: 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μg/mL

 

Mutation tests:

-S9 mix Test 1 (3 hours) 62.5, 125, 250, 300, 350, 400, 450 and 500 μg/mL

+S9 mix Test 1 (3 hours) 15.63, 31.25, 62.5, 125, 250 and 500 μg/mL

 

Cytotoxicity was measured as Day 1 relative survival; values were from 86% to 0%, and 118% to 66%, after exposure to concentrations from 15.63 to 500/250 μg/mL in the absence and presence of S9 mix respectively.

 

In the main mutation test in the absence of S9 mix, cells were exposed to dihydroterpineol multiconstituent at concentrations from 62.5 to 500 µg/mL. Precipitate was observed by eye at the end of treatment at concentrations of 400 µg/mL and above and this was, therefore, the highest concentration plated for determination of RS. RS values ranged from 91 to 0% compared to the vehicle control. Cultures treated at concentrations of 62.5 to 350 µg/mL were plated out for determination of cloning efficiency and mutant frequency. Dihydroterpineol multiconstituent did not induce a statistically significant increase in mutant frequency. The positive control, ethyl methanesulphonate, induced a significant increase in mutant frequency.

In the main mutation test in the presence of S9 mix, cells were exposed to dihydroterpineol multiconstituent at concentrations from 15.63 to 500 µg/mL. Precipitate was observed by eye at the end of treatment at concentrations of 250 µg/mL and above and this was, therefore, the highest concentration plated for determination of RS. RS values ranged from 108 to 71% compared to the vehicle control. Dihydroterpineol multiconstituent did not induce a statistically significant increase in mutant frequency. The positive control, 3-methylcholanthrene, induced a significant increase in mutant frequency.

Therefore, dihydroterpineol multiconstituent did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a reverse gene mutation assay in bacteria, performed according to OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium(TA1535, TA1537, TA98, TA100 and TA102) were exposed to test item dihydroterpineol multiconstituent at the concentrations below. 

Experiment 1 (plate-incorporation method)

- TA1535, TA1537, TA98, TA100 and TA102: 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, with and without S9-mix 

Experiment 2 (plate-incorporation method without S9 mix; preincubation method with S9 mix)

- TA1535, TA1537, TA98, TA100 and TA102: 8.192, 20.48, 51.20, 128, 320, 800 and 2000 μg/plate, without S9-mix

- TA100, TA1537 and TA102: 8.192, 20.48, 51.20, 128, 320, 800 and 2000 μg/plate, with S9-mix

- TA98 and TA1535: 20.48, 51.20, 128, 320, 800, 2000 and 5000 μg/plate, with S9-mix 

Experiment 3 (preincubation method)

- TA98 and TA1535: 3.277, 8.192, 20.48, 51.2, 128, 320 and 800 μg/plate, with S9-mix 

Metabolic activation system used in this test is 10% S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254. Vehicle and positive control groups were also included in mutagenicity tests.

In Experiment 1, following the treatment, evidence of toxicity was observed at 500 μg/plate and/or 1600 μg/plate and above in all strains in the absence and presence of S-9.In Experiment 2,evidence of toxicity was observed at 800 μg/plate and/or at 2000 μg/plate and/or at 5000 μg/plate in the absence and presence of S-9 in all strains. In addition, complete toxicity was observed at 320 μg/plate on a single plate for strain TA1537 in the presence of S-9 only. Since mutation data were only available for four concentrations for strains TA98 and TA1535 in the presence of S-9 due to toxicity, a further experiment (Experiment 3) was performed. In Experiment 3, evidence of toxicity was observed at 320 μg/plate and above in both strains.

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

In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured human lymphocytes were exposed to the test item at the concentrations below.

Preliminary toxicity test

18.14; 30.23; 50.39; 83.98; 140; 233.3; 388.8; 648; 1080; 1800;3000 and 5000  µg/mL

Main tests

-S9 mix (3 hours): 50, 100, 125, 150,175, 200, 225, 250, 275, 300, 325, 350, 375, 400 and 500  µg/mL

+S9 mix (3 hours): 75, 750, 200, 250, 275, 300, 315, 330, 345, 360, 375, 390, 410, 450 and 500 µg/mL

-S9 mix (20 hours):  10, 25, 50, 65, 80, 95, 100, 110, 125, 140, 155, 170, 185, 200, 240, 250, 2280, 295, 305, 340, 355, 370, 385, 395, 405, 415, 425 and 505 µg/mL

-S9 mix (20 hours) (additional test): 10, 25, 50, 60, 80, 90, 100, 110, 120, 130, 140, 160, 180, 240, 350, 365, 370, 375, 380, 385, 390, 395, 400, 410 and 420 µg/mL

Two hours before the cells were harvested, mitotic activity was arrested by addition of Colchicine to each culture at a final concentration of 1 µg/mL. The cells were then fixed, stained and examined for mitotic indices and chromosomal aberrations. Metabolic activation system used in this test was S9 fraction (10% v/v); S9 fraction, prepared from male Sprague-Dawley derived rats.

A preliminary toxicity test was performed using a dose range of 18.14 to 5000 μg/mL. Cells were exposed for a 3-hour treatment in the absence and presence of S9 mix, and a 20-hour continuous treatment in the absence of S9 mix and mitotic index data were used to determine toxicity. Based on the mitotic index data, concentrations were selected for the main test.

In the main test, the mitotic index was assessed for all cultures treated with the test item and the vehicle control, Ethanol. Justification for the highest analysed concentration was determined by cytotoxicity. On the basis of these data, the concentrations below were selected for metaphase analysis.

In the absence of S9 mix, 3-hour treatment: 100, 225 and 275μg/mL.

In the presence of S9 mix, 3-hour treatment: 200, 300, 375 and 410μg/mL.

In the absence of S9 mix, 21-hour continuous treatment:25, 50, 60 and 110μg/mL.

Treatment of cells with dihydroterpineol multiconstituent in the absence and presence of S-9 resulted in frequencies of cells with structural chromosome aberrations which were similar to those observed in concurrent vehicle control cultures for all concentrations analysed in all treatments. Numbers of aberrant cells (excluding gaps) in all treated cultures fell within the normal ranges.

In the absence of S9 mix following a 3-hour treatment with the test item, an increase in the frequency of cells with numerical abberration, exceding the normal range was seen at 275 µg/mL.

In the presence of S9 mix following a 3-hour treatment with the test item,an increase in the frequency of cells with numerical abberration, exceding the normal range was seen at 330 and 375, and 410 µg/mL.

These increases were attributable to polyploidy. Although increases in polyploidy can indicate mitotic spindle effects, the biological relevance of these observationsin vitrois questionable. In addition, this study is not specifically designed to evaluate the inductions of these types of aberrations.

In the absence of S9 mix following a 20-hour continuous treatment, noincreases in the frequency of cells with numerical aberrations, which exceeded the normal range, were observed in cultures.

Therefore, dihydroterpineol multiconstituent did not induce structural chromosome aberrations in cultured human peripheral blood lymphocytes, when tested to the limit of cytotoxicity in both the absence and presence of an Aroclor 1254-induced rat liver metabolic activation system (S-9).

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster Ovary (CHO-K1) cells were exposed to dihydroterpineol multiconstituent for 3 h, with and without metabolic activation (S9 fraction of male Sprague Dawley rats liver induced with phenobarbital and 5,6-benzoflavone), at the following concentrations: 

Preliminary toxicity test: 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μg/mL

Mutation tests:

-S9 mix Test 1 (3 hours) 62.5, 125, 250, 300, 350, 400, 450 and 500 μg/mL

+S9 mix Test 1 (3 hours) 15.63, 31.25, 62.5, 125, 250 and 500μg/mL

Cytotoxicity was measured as Day 1 relative survival; values were from 86% to 0%, and 118% to 66%, after exposure to concentrations from 15.63 to 500/250 μg/mL in the absence and presence of S9 mix respectively.

In the main mutation test in the absence of S9 mix, cells were exposed to dihydroterpineol multiconstituent at concentrations from 62.5 to 500 µg/mL. Precipitate was observed by eye at the end of treatment at concentrations of 400 µg/mL and above and this was, therefore, the highest concentration plated for determination of RS. RS values ranged from 91 to 0% compared to the vehicle control. Cultures treated at concentrations of 62.5 to 350 µg/mL were plated out for determination of cloning efficiency and mutant frequency. Dihydroterpineol multiconstituent did not induce a statistically significant increase in mutant frequency. The positive control, ethyl methanesulphonate, induced a significant increase in mutant frequency.

In the main mutation test in the presence of S9 mix, cells were exposed to dihydroterpineol multiconstituent at concentrations from 15.63 to 500 µg/mL. Precipitate was observed by eye at the end of treatment at concentrations of 250 µg/mL and above and this was, therefore, the highest concentration plated for determination of RS. RS values ranged from 108 to 71% compared to the vehicle control. Dihydroterpineol multiconstituent did not induce a statistically significant increase in mutant frequency. The positive control, 3-methylcholanthrene, induced a significant increase in mutant frequency.

Therefore, dihydroterpineol multiconstituent did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay.

 

Justification for classification or non-classification

In an Ames test performed according to OECD guideline 471 and in compliance with GLP, dihydroterpineol multiconstituent was considered to be non mutagenic.

In an in vitro chromosome aberration test performed according to OECD guideline 473 and in compliance with GLP, dihydroterpineol multiconstituent did not induce structural chromosome aberrations in cultured human peripheral blood lymphocytes.

In an in vitro HPRT test performed according to OECD guideline 476 and in compliance with GLP, dihydroterpineol multiconstituent was considered to be non mutagenic.

 

According to these results, dihydroterpineol multiconstituent can be considered as non genotoxic according to the CLP Regulation (EC) N° 1272/2008.