Gum turpentine oil, consisting of the volatile fraction resulting from the distillation of oleoresin collected by tapping softwoods from pinacea derivatives (genus: pinus). It consists of terpenes, mainly monoterpenes such as alpha-pinene and beta-pinene.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a Ames Test conducted according to OECD Guideline 471, the registered substance was non mutagenic up to cytotoxic concentration in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.
In a micronucleus test in cultured human lymphocytes conducted according to OECD Guideline 487, the substance was non clastogenic.

In a gene mutation assay (HPRT) in CHO cells conducted according to OECD Guideline 476, the substance was non 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:

key study

Study period:

04 - 15 October 2017

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 471 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:

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

15 March 2017

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine dependent auxotrophic mutants of Salmonella typhimurium and tryptophan-dependent mutant of Escherichia coli

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (10% - test 1; 20% v/v – test 2); S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with phenobarbital/5,6-benzoflavone

Test concentrations with justification for top dose:

Mutagenicity tests:
- Test 1: 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with (10% S9) and without S9 mix in all 5 strains (plate incorporation method)
- Test 2: 50, 150, 500, 1500 and 5000 μg/plate, with (20% S9) and without S9 mix in all 5 strains (plate incorporation method)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The solubility of Gum turpentine oil was assessed in Envigo study number JC60GL in water, DMSO and ethanol. It was found to be immiscible in water and DMSO and not suitable for dosing. In ethanol it was found to be miscible. Ethanol (analytical grade) was, therefore, used as the vehicle for this study.
- Test item preparation: The highest concentration of Gum turpentine oil tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 μg/plate. This is the standard limit concentration recommended in the regulatory guidelines. The highest concentration in each test was diluted with ethanol to produce a series of lower concentrations, separated by approximately half-log10 intervals.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

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 of S. typhimurium and E. coli were obtained from Moltox Inc.

METHOD OF APPLICATION: In agar (plate incorporation method)

DURATION
- Exposure duration: Plates were inverted and incubated at approximately 34-39 °C for ca 72 h.

NUMBER OF REPLICATIONS:
- Treatment, vehicle and positive control groups: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Toxic effects of the test item may be detected by a reduction in mean revertant colony numbers to ≤50% of the concurrent vehicle control count, by a sparse or absent background bacterial lawn, or both.

OTHER:
Colony counting: After the incubation period of 72 h, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).

Rationale for test conditions:

The highest concentration of Gum turpentine oil tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 μg/plate. This is the standard limit concentration recommended in the regulatory guidelines.

Evaluation criteria:

Criteria for Assessing Mutagenic Potential:
- If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
- If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
- If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below two or three times those of the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
- Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgment.

Statistics:

The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis.

Key result

Species / strain:

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

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
- Water solubility: None
- Precipitation: No precipitate was observed on any plates up to 5000 μg/plate.

MUTAGENICITY TESTS
First Test:
- Toxicity, observed as thinning of the background lawn of non-revertant colonies and/or together with a reduction in revertant colony numbers, was obtained in Salmonella typhimurium strains following exposure to test item at 5000 μg/plate. No precipitate was observed on any plates up to 5000 μg/plate. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test.
- No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test item at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.
Second Test
- No evidence of toxicity was obtained following exposure to test item. No precipitate was observed on any plates up to 5000 μg/plate. A decrease in the revertant colonies observed in strain TA1537 in the absence of S9 mix at to 1500 μg/plate was considered to be natural variation within the assay.
- No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test item at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data:
Without S9 mix: 82-579 (225 ± 87), 293-3769 (757 ± 388), 180-1232 (677 ± 186), 62-1039 (265 ± 161), 438-3730 (1985 ± 770) for TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA (pKM101), respectively.
With S9 mix: 77-473 (211 ± 63), 338-4500 (2167 ± 955), 61-785 (325 ± 118), 45-411 (121 ± 60), 436-3695 (1128 ± 413) for TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA (pKM101), respectively.
- Negative (solvent/vehicle) historical control data:
Without S9 mix: 17-74 (35 ± 10), 97-227 (169 ± 29), 7-39 (22 ± 7), 5-41 (16 ± 7), 90-259 (190 ± 33) for TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA (pKM101), respectively.
With S9 mix: 18-84 (44 ± 15), 103-248 (174 ± 31), 8-47 (21 ± 7), 6-50 (23 ± 11), 109-300 (205 ± 34) for TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA (pKM101), respectively.

OTHERS:
- The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test item formulation.
- The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 10^9/mL in all cases, and therefore met the acceptance criteria.
- The mean revertant colony counts for the vehicle controls were within the current historical control range for the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

None

Conclusions:

The test item is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98 and TA100) and E. coli WP2 uvrA (pKM101) strains.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to OECD Guideline 471 and in compliance with GLP, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2uvrA (pKM101), were exposed to Gum turpentine oil diluted in Ethanol at the following concentrations:

 

Test 1: 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with (10% S9) and without S9 mix in all 5 strains (plate incorporation method)

Test 2: 50, 150, 500, 1500 and 5000 μg/plate, with (20% S9) and without S9 mix in all 5 strains (plate incorporation method)

 

Metabolic activation system used in this test, S9 mix (10 and 20% v/v S9 fraction), was prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone. Vehicle and positive control groups were also included in mutagenicity tests.

 

In the first mutation test, toxicity (observed as thinning of the background lawn of non-revertant colonies and/or together with a reduction in revertant colony numbers) was seen in all Salmonella typhimurium strains following exposure to the test item at 5000 μg/plate. No precipitate was observed on any plates containing the test item up to 5000 μg/plate. In the second mutation test, no signs of toxicity towards the tester strains were observed following exposure to the test item. No precipitate was observed on any plates containing the test item up to 5000 μg/plate. No evidence of mutagenic activity was seen at any concentration in either mutation test.

 

The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within the current historical control range for the laboratory.

 

Therefore, the test item is not considered as mutagenic in these bacterial systems.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 October to 10 November 2017

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 476 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:

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)

Remarks:

15 March 2017

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

Hypoxanthine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO-K1) cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: CHO-K1 cells were obtained from the European Collection of Cell Cultures.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
H0 Ham’s Nutrient Mixture F12, supplemented with 1 mM L glutamine and 50 ng/mL amphotericin B / 20 IU/mL penicillin / 20 μg/mL streptomycin.
H10: H0 medium supplemented with 10% heat inactivated fetal calf serum
H10 medium was used for cell culture unless otherwise specified.
The selective medium, in which only HPRT deficient cells will grow, consists of H10 supplemented with 6-TG at a final concentration of 10 μg/mL.
All cell cultures were maintained at 34-39 °C in a humidified atmosphere of 5% CO2 in air.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes; Spontaneous mutants were eliminated from the cultures by 3-day incubation in the presence of methotrexate (0.3 μg/mL), thymidine (4 μg/mL), hypoxanthine (15 μg/mL) two days prior to storage between -196 °C and -150 °C, in heat-inactivated foetal calf serum (HiFCS) containing 10% dimethyl sulphoxide (DMSO).

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix contains: S9 fraction (10% v/v) prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test: 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL, 3 h exposure with out and with metabolic activation
Mutation tests:
3 h exposure without metabolic activation: 25, 30, 35, 37.5, 40, 42.5 and 45 μg/mL
3 h exposure with metabolic activation: 20, 40, 60, 80, 90, 100, 110, 120, 130 and 140 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The solubility of Gum turpentine oil was assessed in Envigo study number JC60GL in water, dimethyl sulfoxide (DMSO) and ethanol. It was found to be immiscible in water and DMSO and not suitable for dosing. In ethanol it was found to be miscible. Ethanol (analytical grade) was, therefore, used as the vehicle for this study.
- Gum turpentine oil was dissolved and formulated in ethanol (analytical grade), shortly before dosing. The final volume of ethanol added to the cultures was 1% v/v.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol 1% v/v

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol 1% v/v

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; H10 medium - H0 medium supplemented with 10% heat inactivated fetal calf serum.
- Cell density at seeding (if applicable): 2 x 10^6 cells from each culture were seeded into 150 cm2 flasks

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
All cell cultures were maintained at 34-39 °C in a humidified atmosphere of 5% CO2 in air.

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

NUMBER OF REPLICATIONS:
Preliminary toxicity test: Single culture for test item and duplicate cultures for vehicle controls
Main test: Quadruplicate cultures for vehicle and duplicate cultures for test item and positive controls

NUMBER OF CELLS EVALUATED:
For each culture, three flasks were seeded with 200 cells each, to determine cloning efficiency and five flasks with 5 x 10^5 cells each in selective medium to determine cloning efficiency.
Mutant frequencies expressed per 10^6 viable cells

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency and Relative survival

- OTHER:
For each culture, three flasks were seeded with 200 cells each, to determine cloning efficiency and five flasks with 5 x 10^5 cells each in selective medium to determine cloning efficiency. The flasks were returned to the incubator for approximately seven days at between 34 and 39 °C in a humidified atmosphere of 5% CO2 in air. At the end of this incubation period, colonies growing in the flasks were fixed and stained in a methanol:Giemsa solution (4:1 v/v) and counted.

Rationale for test conditions:

The highest final concentration used in the preliminary toxicity test was 5000 μg/mL. This is the standard limit concentration within this test system as recommended in the regulatory guidelines.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
b) the increase is concentration-related when evaluated with an appropriate trend test
c) any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
b) there is no concentration-related increase when evaluated with an appropriate trend test
c) all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly positive or negative response.
In cases when the response is neither clearly negative nor clearly positive as described above, or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions [i.e. S9 concentration or S9 origin]) could be useful.

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 item, for non-linearity and for the comparison of positive control and treated groups to solvent control. The data was analyzed using SAS (SAS Institute Inc., 2002).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

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 were observed at 5000 μg/mL of more than 1.0 unit compared with the vehicle control.
- Effects of osmolality: The osmolality of the test item in medium was tested at 5000 μg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.
- Precipitation: Precipitate was observed by eye at the end of treatment.

PRELIMINARY TOXICITY TEST:
- Precipitate was observed by eye at the end of treatment at concentrations of 78.1 μg/mL and above in the absence of S9 mix and 156.31 μg/mL and above in the presence of S9 mix and these were, therefore, the highest concentrations plated for determination of relative survival (RS).
- After exposure to test item at concentrations from 39.1 to 78.1 μg/mL in the absence of S9 mix and from 39.1 to 156.3 μg/mL in the presence of S9 mix, RS values ranged from 92 to 1% and from 75 to 0%, in the absence and presence of S9 mix respectively. Concentrations for the main test were based upon these data.

MAIN TEST:
3 h treatment in the absence of S9 mix:
- No precipitate was seen by eye at the end of treatment.
- Exposure to test item resulted in mean RS values from 93 to 6%.
- All cultures were plated out for determination of cloning efficiency and mutant frequency. Cultures treated at 40 and 42.5 μg/mL were not analysed for mutant frequency as mean RS was <10% at these concentrations.
- No significant increases in mean mutant frequency were observed after exposure to test item at any concentration analysed. Tests for both a linear trend and non-linearity were applied across all treatment groups, neither of which was statistically significant. All of the test item treated cultures were within the laboratory historical control limits.

3 h treatment in the presence of S9 mix:
- No precipitate was seen by eye at the end of treatment.
- Exposure to test item resulted in mean RS values from 98 to 6%.
- All cultures except those treated at 80 μg/mL were plated out for determination of cloning efficiency and mutant frequency. Cultures treated at 80 μg/mL were found to be contaminated and therefore were discarded. Cultures treated at 140 μg/mL were not analysed for mutant frequency as mean RS was <10% at this concentration.
- No significant increases in mean mutant frequency were observed after exposure to test item at any concentration analysed. Tests for both a linear trend and non-linearity were applied across all treatment groups. The test for a linear trend was statistically significant for a non-relevant inverse trend and the test for non-linearity was not significant. All of the test item treated cultures were within the laboratory historical control limits.

HISTORICAL CONTROL DATA (mean and standard deviation)
- Positive historical control data:
Mean Mutant Frequency (x 10^-6): 188.4 ± 149 (Ethyl methanesulphonate, -S9); 248.0 ± 216 (3-Methylcholanthrene, +S9)
- Negative (solvent/vehicle) historical control data:
Mean Mutant Frequency (x 10^-6): 2.5 ± 1.1 (-S9); 2.9 ± 1.9 (+S9);
Mean Day 1 CE (%): 72 ± 0.1 (-S9); 73 ± 0.1 (+S9)

Table 7.6.1/1: HPRT summary table

 

Groups	Concentration (μg/mL)	3 h treatment -S9 mix		3 h treatment +S9 mix
		Mean RS (%)	Mean MF^a	Mean RS (%)	Mean MF^a
Vehicle Ethanol	0	100	1.72	100	1.83
Test item	20	NT	NT	98	2.01
	25	91	1.56	NT	NT
	30	93	2.30	NT	NT
	35	56	2.25	NT	NT
	37.5	39	2.36	NT	NT
	40	6	NA	74	1.53
	42.5	6	NA	NT	NT
	45	11	0.96	NT	NT
	60	NT	NT	69	0.92
	80	NT	NT	69	NA
	90	NT	NT	55	1.57
	100	NT	NT	53	2.78
	110	NT	NT	53	1.95
	120	NT	NT	25	1.41
	130	NT	NT	17	2.33
	140	NT	NT	6	NA
Ethyl methanesulphonate	250	67	92.99***	NT	NT
3-methylcholanthrene	5	NT	NT	71	83.05***

 

^a Mutant frequencies expressed per 10⁶ viable cells

RS: Relative Survival

MF: Mutant Frequency

NT: Not tested

NA: Not analysed

***p<0.001; all other culturesp≥0.05

Conclusions:

The test item did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay.

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 Gum turpentine oil for 3 h, with and without metabolic activation (10 % v/v S9).

The following concentrations were tested. 

Preliminary toxicity test: 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL, 3 h exposure with out and with metabolic activation

Mutation tests:

3 h exposure without metabolic activation: 25, 30, 35, 37.5, 40, 42.5 and 45 μg/mL

3 h exposure with metabolic activation: 20, 40, 60, 80, 90, 100, 110, 120, 130 and 140 μg/mL

 

In a preliminary toxicity test, precipitate was observed by eye at the end of treatment at concentration of 78.1 μg/mL and above in the absence of S9 mix and at concentration of 156.31 μg/mL in the presence of S9 mix and these were, therefore, the highest concentrations plated for determination of toxicity. Cytotoxicity was measured as Day 1 relative survival (RS). After exposure to the test item at concentrations from 39.1 to 78.1 μg/mL in the absence of S9 mix and from 39.1 to 156.3 μg/mL in the presence of S9 mix, RS values ranged from 92 to 1% and from 75 to 0%, in the absence and presence of S9 mix respectively.

 

In the main mutation test in the absence of S9 mix, cells were exposed to the test item at concentrations from 25 to 45 μg/mL. No precipitate was observed by eye at the end of treatment. Mean RS values ranged from 93 to 6% relative to the vehicle control. The test item did not induce a statistically significant increase in mutant frequency.

 

In the main mutation test in the presence of S9 mix, cells were exposed to the test item at concentrations from 20 to 140 μg/mL. No precipitate was observed by eye at the end of treatment. Mean RS values ranged from 98 to 6% relative to the vehicle control. The test item did not induce a statistically significant increase in mean mutant frequency.

 

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.

 

Therefore, the test item did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 September to 27 November 2017

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:

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:

15 March 2017

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Following treatment (3 h exposure) or during treatment (20 h exposure), cytokinesis was blocked using the inhibitor Cytochalasin B at 6 μg/mL.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (10% v/v S9 fraction): S9 fraction, prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 8.3, 16.6, 33.1, 66.3, 132.5, 265.1, 530.2, 1060.4, 2120.8 and 4241.5 μg/mL; 3 h exposure with and without S9-mix; 20 h continuous exposure without S9-mix

Main Test
3 h exposure: 5, 15, 20, 25, 30, 35, 40 and 50 μg/mL (without S9-mix); 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg/mL (with S9-mix)
Additional 3 h exposure with S9-mix: 20, 30, 40, 45, 50, 55 and 60 μg/mL
Second additional 3 h exposure with S9-mix: 20, 40, 45, 50, 55, 60, 65 and 70 μg/mL
20 h continuous exposure to the test item without S9-mix: 1, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 and 25 μg/mL
Additional 20 h continuous exposure to the test item without S9-mix: 1, 5, 10, 15, 20, 22.5, 25, 27.5, 30, 32.5 and 35 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The solubility of Gum turpentine oil was assessed in water, dimethyl sulfoxide (DMSO) and ethanol. It was found to be immiscible in water and DMSO and not suitable for dosing. In ethanol it was found to be miscible. Ethanol was, therefore, used as the vehicle for this study.
- An accurately measured sample was weighed and the maximum concentration achieved in the preliminary toxicity test was a final concentration of 4241.5 μg/mL when dosed at 0.5% v/v.
- Test item preparation: Gum turpentine oil was dissolved and diluted in ethanol (analytical grade), shortly before dosing. The final volume of ethanol added to the cultures was 0.5% v/v. All concentrations are expressed in terms of Gum turpentine oil as received and containers of the neat test item were used within 7 days of opening for the first time.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol (0.5% v/v)

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: Colchicine

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol (0.5% v/v)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

CULTURE OF LYMPHOCYTES: Human blood was collected aseptically from two healthy, non-smoking, adult (between 18-35 years of age) donors, pooled (in equal volumes from each donor) and diluted with HML media. 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 and dispensed as 5 mL aliquots (in sterile universal containers) so that each culture contained blood (0.4 mL), HML media (4.5 mL) and PHA solution (0.1 mL). All cultures were then incubated at 34-39 °C, and the cells were re-suspended (twice daily) by gentle inversion.

METHOD OF APPLICATION: in medium
HML Media RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM L-glutamine.

DURATION
- Exposure duration: 3 h (± S9) and 20 h continuous exposure (-S9) in preliminary toxicity test; 3 h (± S9) and 20 h continuous exposure (-S9) in main tests
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

SPINDLE INHIBITOR (cytogenetic assays): Following treatment (3 h exposure) or during treatment (20 h exposure), cytokinesis was blocked using the inhibitor Cytochalasin B at a final concentration of 6 μg/mL.

STAIN (for cytogenetic assays): Slides were stained with Acridine orange solution (0.0125 mg/mL using purified water)

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single cultures were prepared for each treatment level and duplicate cultures were prepared for vehicle controls.
- Main test: Duplicate cultures were prepared for each treatment level and positive control cultures; quadruplicate cultures were prepared for vehicle controls; two slides were prepared from each culture.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Harvesting and Fixation: The cells were harvested by centrifugation at 500 g for 5 minutes. The supernatant was removed and the cell pellet re-suspended and treated with a 4 mL hypotonic solution (0.075M KCl) at 34 to 39°C, cultures were then incubated for 3 minutes at 34-39 °C to cause swelling. Cultures were agitated, 4 mL of ice-cold fixative (3:1 v/v methanol: acetic acid) was added slowly onto the culture surface and the cultures were slowly inverted to mix. The cultures were centrifuged at 500 g for five minutes. The supernatant was removed, and the cell pellet re-suspended. A further 4 mL of fresh fixative was then added and the cells stored at 2 to 8°C until slide preparation.
Slide Preparation: The cultures were centrifuged at 500 g for 5 minutes and the supernatant removed. A homogeneous cell suspension was prepared. Pre-cleaned microscope slides were prepared for each culture by aliquoting the re-suspended cells onto the slides, and allowing the slides to air-dry. One slide was prepared from each culture. The slides were then stained using an acridine orange solution at 0.0125 mg/mL in purified water. The remaining cell cultures were stored at 2-8 °C until slide analysis was complete.
Microscopic Examination: Immediately prior to scoring, slides were wet mounted with glass coverslips using purified water. The prepared slides were examined by fluorescence microscopy. The incidences of mononucleate, binucleate and polynucleate cells were assessed per culture. The presence of an unusual number of, for example, cells undergoing mitosis, polyploid cells, necrotic cells and debris, if any, was also noted.

NUMBER OF CELLS EVALUATED:
- Scoring of Micronuclei: Interphase cells were examined by fluorescence microscopy and the incidence of micronucleated cells per 1000 binucleate cells per culture were scored where possible.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The analysis for micronucleated cells was based on the following criteria (Fenech and Morley 1985 and Fenech, 1993):
Cells were included in the analysis provided the cytoplasm remained essentially intact and any micronuclei present were separate in the cytoplasm or only just touching the main nucleus (not connected to the nucleus by a nucleoplasmic bridge). Micronuclei should lie in the same focal plane as the cell, and should possess a generally rounded shape with a clearly defined outline. The main nuclei of the binucleate cells scored for micronuclei should be of approximately equal size. The diameter of the micronucleus should be between 1/16 and 1/3 that of the main nucleus. The color of the micronuclei should be the same or lighter than the main nucleus. There should be no micronucleus-like debris in the surrounding area.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of test item in the lymphocyte cultures was determined using the cytokinesisblock proliferation index (CBPI index).
% Cytostasis = 100-100{(CBPIT – 1)/(CBPIC –1)}
CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / [Total number of cells]
T = test item treatment culture
C = solvent control culture
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.
To calculate the CBPI at least 500 cells were assessed per culture.

OTHER:
At least three concentrations were selected for micronucleus analysis. The highest concentration was intended to be that which caused a depression in the cytokinesis-block proliferative index (CBPI) equivalent to 55 ± 5 % cytotoxicity (approximately) when compared with the concurrent vehicle control or, where no cytotoxicity was observed, the maximum concentration as recommended in the test guidelines or the limit of solubility.

Evaluation criteria:

Providing that all of the acceptance criteria have been met, the test item was considered to be clearly positive if, in any of the experimental conditions examined:
At least one of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control.
The increase in the frequency of micronucleated cells is dose-related when evaluated with an appropriate trend test.
Any of the results are outside the distribution of the historical vehicle control data (above the upper control limit).
If all of these criteria are met, the test item was considered able to induce chromosome breaks and/or gain or loss in the test system.

Providing that all of the acceptance criteria have been met, a negative response will be claimed if, in all of the experimental conditions examined:
None of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells 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 (below the upper control limit).
If all of these criteria are met, the test item was considered unable to induce chromosome breaks and/or gain or loss in the test system.

Statistics:

The analysis assumed that the replicate was the experimental unit. An arcsine transformation was used to transform the data. Gum turpentine oil treated groups were then compared to control using Williams’ tests (Williams 1971, 1972), unless there was evidence against a monotonic dose-response relationship in which case Dunnett’s test was used instead (Dunnett 1955, 1964). Positive controls were compared to control using t-tests. Trend tests have also been carried out using linear contrasts by group number. These were repeated, removing the top dose group, until there were only 3 groups.
Statistical significance was declared at the 5% level for all tests.
Data were analyzed using SAS (SAS Institute 2002) and Quasar (Quasar 1.5 2016).

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
- Effects of pH: No fluctuations in pH of the medium were observed at 100 μg/mL of more than 1.0 unit compared with the vehicle control.
- Effects of osmolality: The osmolality of the test item in medium was tested at 100 μg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.
- Precipitation: Yes

PRELIMINARY TOXICITY TEST:
- In all exposure conditions the highest concentration tested was 4241.5 μg/mL and precipitate was observed by eye at the end of treatment at 2120.8 μg/mL and above.
- After 3 h treatment in the absence of S9-mix, a reduction in CBPI compared with vehicle control values, equivalent to 46.8% cytotoxicity, was obtained with test item at 33.1 μg/mL. At higher tested concentrations, overt toxicity was observed.
- After 3 h treatment in the presence of S9-mix, a reduction in CBPI compared with vehicle control values, equivalent to 45.4% cytotoxicity, was obtained with test item at 66.3 μg/mL. At higher tested concentrations, overt toxicity was observed. The result seen following exposure at 2120.80 μg/mL is considered to be caused by the effect of precipitate in the culture and is not considered to be a true measurement of cytostasis.
- After 20 h treatment in the absence of S9-mix, a reduction in CBPI compared with vehicle control values, equivalent to 46.2% cytotoxicity, was obtained with test item at 16.6 μg/mL. At higher tested concentrations, overt toxicity was observed.

MAIN TEST
3 h treatment in the absence of S9 mix:
Cytotoxicity: A reduction in CBPI compared to vehicle control values equivalent to 60.3% cytotoxicity, was obtained with test item at 30 μg/mL. Concentrations of test item selected for micronucleus analysis were 5, 20 and 30 μg/mL.
3 h treatment in the presence of S9 mix / Additional 3 h treatment in the presence of S9 mix: As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels.
Second additional 3 h treatment in the presence of S9 mix: A reduction in CBPI compared with vehicle control values, equivalent to 57.0% cytotoxicity, was obtained with test item at 55 μg/mL. Concentrations of test item selected for micronucleus analysis were 20, 45 and 55 μg/mL.
20 h treatment in the absence of S9 mix: As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels.
Additional 20 h treatment in the absence of S9 mix: A reduction in CBPI compared with vehicle control values, equivalent to 58.5% cytotoxicity, was obtained with test item at 30 μg/mL. Concentrations of test item selected for micronucleus analysis were 1, 25 and 30 μg/mL.

MICRONUCLEUS ANALYSIS
In both the absence and presence of S9 mix, following 3 h treatment, and in the absence of S9 mix, following 20 h treatment, test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls. There was no evidence of a positive linear dose-concentration relationship and all of the mean micronucleus frequencies for the test item treated cultures and the vehicle were within the laboratory historical 95% confidence limits.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Binucleate Individual MN/1000 cells (3 h treatment in the absence of S9 mix): Mitomycin C: 16-67 (34.9 ± 11.5); Colchicine: 15-43 (24.7 ± 6.0)
Binucleate Group MN (3 h treatment in the absence of S9 mix): Mitomycin C: 18-64.5 (34.9 ± 11.4), 95% Cl: 21.5-57.6; Colchicine: 17.0-38 (24.7± 5.2), 95% Cl: 14.4-35.1
Binucleate Individual MN/1000 cells (20 h treatment in the absence of S9 mix): Mitomycin C: 15-41 (27 ± 7.3); Colchicine: 13-25 (17.3 ± 2.3)
Binucleate Group MN (20 h treatment in the absence of S9 mix): Mitomycin C: 16-37.5 (27 ± 6.7), 95% Cl: 18.2-40.5; Colchicine: 14-23 (17.3 ± 1.9), 95% Cl: 13.6-21.0
Binucleate Individual MN/1000 cells (3 h treatment in the presence of S9 mix): Cyclophosphamide: 14-38 (22.5 ± 5.3)
Binucleate Group MN (3 h treatment in the presence of S9 mix): Cyclophosphamide: 14.5-36.5 (22.5 ± 4.8), 95% Cl: 12.9-32.0
- Negative (solvent/vehicle) historical control data:
Binucleate Individual MN/1000 cells (3 h treatment in the absence of S9 mix): 2-14 (6.5 ± 2.4)
Binucleate Group MN (3 h treatment in the absence of S9 mix): 3.3-10.5 (6.5 ± 1.7), 95% Cl: 3.2-9.8
Binucleate Individual MN/1000 cells (20 h treatment in the absence of S9 mix): 0-13 (6.9 ± 2.5)
Binucleate Group MN (20 h treatment in the absence of S9 mix): 2-11.5 (6.9 ± 1.9), 95% Cl: 3.2-10.6
Binucleate Individual MN/1000 cells (3 h treatment in the presence of S9 mix): 0-15 (6.5 ± 2.7)
Binucleate Group MN (3 h treatment in the presence of S9 mix): 2.5-12.5 (6.5 ± 2.0), 95% Cl: 2.4-10.6

Table 7.6.1/1: Main test results

 

Treatment/ Concentration	Mean Cytostasis (%)	Binucleated cells containing micronuclei
		per 1000 cells (Mean)	p-value^b	Trend test p-value^c
3 h treatment in the absence of S9 mix
Vehicle^a	0.0	8.5
Test item 5 μg/mL	4.5	9.5	0.949
Test item 20 μg/mL	21.2	8.5	0.949	0.949
Test item 30 μg/mL	60.3	9.0	0.949	0.868
Mitomycin C 0.3 μg/mL	32.8	32.0	<0.001***
Colchicine 0.06 μg/mL	43.0	23.5	<0.001***
3 h treatment in the presence of S9 mix
Vehicle^a	0	5.3
Test item 20 μg/mL	3.4	6.5	0.398
Test item 45 μg/mL	38.5	7.0	0.335	0.281
Test item 55 μg/mL	57.0	8.5	0.107	0.096
Cyclophosphamide 10 μg/mL	39.8	22.5	<0.001***
20 h treatment in the absence of S9 mix
Vehicle^a	0	4.3
Test item 1 μg/mL	4.4	4.5	1.000
Test item 25 μg/mL	34.1	4.0	1.000	0.869
Test item 30 μg/mL	58.5	7.5	0.070	0.087
Mitomycin C 0.1 μg/mL	19.8	20.0	<0.001***
Colchicine 0.015 μg/mL	36.5	15.5	<0.001***

 

^a Vehicle control = Ethanol (0.5% v/v)

^b p-values are for comparisons to control using Williams' test for test item and the t-test otherwise

^c Trend test p-values are for the linear contrast including the control group and lower concentrations of the same test item

***p<0.001

Conclusions:

The test item did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system.

Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to Gum turpentine oil in the presence and absence of a metabolic activation system. Metabolic activation system used in this test was 10% (v/v) S9 fraction; S9 fraction was obtained from the liver homogenates of male Sprague-Dawley derived rats induced with phenobarbital and 5,6-benzoflavone.

The tested concentrations were the following. 

Preliminary Toxicity Test: 8.3, 16.6, 33.1, 66.3, 132.5, 265.1, 530.2, 1060.4, 2120.8 and 4241.5 μg/mL; 3 h exposure with and without S9-mix; 20 h continuous exposure without S9-mix 

Main Test

3 h exposure: 5, 15, 20, 25, 30, 35, 40 and 50 μg/mL (without S9-mix); 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg/mL (with S9-mix)

Additional 3 h exposure with S9-mix: 20, 30, 40, 45, 50, 55 and 60 μg/mL

Second additional 3 h exposure with S9-mix: 20, 40, 45, 50, 55, 60, 65 and 70 μg/mL

20 h continuous exposure to the test item without S9-mix: 1, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 and 25 μg/mL

Additional 20 h continuous exposure to the test item without S9-mix: 1, 5, 10, 15, 20, 22.5, 25, 27.5, 30, 32.5 and 35 μg/mL

 

Cytokinesis was blocked following mitosis using Cytochalasin B. Then the cells were harvested and slides prepared, so that binucleate cells could be examined for micronucleus induction. Vehicle (ethanol) and positive control cultures were included in all appropriate test conditions.

 

Three concentrations were assessed for determination of induction of micronuclei. The highest concentrations selected for micronucleus analysis were those which caused a reduction in cytokinesis-block proliferative index (CBPI) equivalent to 55 ± 5 % cytotoxicity. Following 3 h of treatment in the absence of S9 mix, a reduction in CBPI equivalent to 60.3% cytotoxicity was obtained with the test item at 30 μg/mL. Concentrations of the test item selected for micronucleus analysis were 5, 20 and 30 μg/mL. Following 3 h of treatment in the presence of S9 mix, a reduction in CBPI equivalent to 57% cytotoxicity was obtained with the test item at 55 μg/mL. Concentrations of the test item selected for micronucleus analysis were 20, 45 and 55 μg/mL. In the absence of S9 mix following 20 h treatment, a reduction in CBPI equivalent to 58.5% cytotoxicity was obtained with the test item at 30 μg/mL. Concentrations of the test item selected for micronucleus analysis were 1, 25 and 30 μg/mL.

 

In both the absence and presence of S9 mix, following 3 h of treatment, and in the absence of S9 mix, following 20 h of treatment, the test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls.

 

The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

 

Therefore, the test item did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system.

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, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2uvrA (pKM101), were exposed to Gum turpentine oil diluted in Ethanol. Metabolic activation system used in this test, S9 mix (10 and 20% v/v S9 fraction), was prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone. Vehicle and positive control groups were also included in mutagenicity tests.

In the first mutation test, toxicity (observed as thinning of the background lawn of non-revertant colonies and/or together with a reduction in revertant colony numbers) was seen in all Salmonella typhimurium strains following exposure to the test item at 5000 μg/plate. No precipitate was observed on any plates containing test item up to 5000 μg/plate. In the second mutation test, no signs of toxicity towards the tester strains were observed following exposure to test item. No precipitate was observed on any plates containing test item up to 5000 μg/plate. No evidence of mutagenic activity was seen at any concentration in either mutation test.

The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within the current historical control range for the laboratory. Therefore, the test item is not considered as mutagenic in these bacterial systems.

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to Gum turpentine oil in the presence and absence of a metabolic activation system. Cytokinesis was blocked following mitosis using Cytochalasin B. Then the cells were harvested and slides prepared, so that binucleate cells could be examined for micronucleus induction. Vehicle (ethanol) and positive control cultures were included in all appropriate test conditions.

Three concentrations were assessed for determination of induction of micronuclei. The highest concentrations selected for micronucleus analysis were those which caused a reduction in cytokinesis-block proliferative index (CBPI) equivalent to 55 ± 5% cytotoxicity. Following 3 h of treatment in the absence of S9 mix, a reduction in CBPI equivalent to 60.3% cytotoxicity was obtained with the test item at 30 μg/mL. Concentrations of the test item selected for micronucleus analysis were 5, 20 and 30 μg/mL. Following 3 h treatment in the presence of S9 mix, a reduction in CBPI equivalent to 57% cytotoxicity was obtained with the test item at 55 μg/mL. Concentrations of the test item selected for micronucleus analysis were 20, 45 and 55 μg/mL. In the absence of S9 mix following 20 h treatment, a reduction in CBPI equivalent to 58.5% cytotoxicity was obtained with test item at 30 μg/mL. Concentrations of test item selected for micronucleus analysis were 1, 25 and 30 μg/mL.

In both the absence and presence of S9 mix, following 3 h treatment, and in the absence of S9 mix, following 20 h treatment, test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls.

The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

Therefore, the test item did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system.

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 Gum turpentine oil for 3 h, with and without metabolic activation (10 % v/v S9).

In a preliminary toxicity test, precipitate was observed by eye at the end of treatment at concentration of 78.1 μg/mL and above in the absence of S9 mix and at concentration of 156.31 μg/mL in the presence of S9 mix and these were, therefore, the highest concentrations plated for the determination of toxicity. Cytotoxicity was measured as Day 1 relative survival (RS). After exposure to the test item at concentrations from 39.1 to 78.1 μg/mL in the absence of S9 mix and from 39.1 to 156.3 μg/mL in the presence of S9 mix, RS values ranged from 92 to 1% and from 75 to 0%, in the absence and presence of S9 mix respectively.

In the main mutation test in the absence of S9 mix, cells were exposed to the test item at concentrations from 25 to 45 μg/mL. No precipitate was observed by eye at the end of treatment. Mean RS values ranged from 93 to 6% relative to the vehicle control. Test item did not induce a statistically significant increase in mutant frequency.

In the main mutation test in the presence of S9 mix, cells were exposed to the test item at concentrations from 20 to 140 μg/mL. No precipitate was observed by eye at the end of treatment. Mean RS values ranged from 98 to 6% relative to the vehicle control. The test item did not induce a statistically significant increase in mean mutant frequency.

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.

Therefore, the test item did not demonstrate mutagenic potential in thisi n vitro HPRT cell mutation assay.

Justification for classification or non-classification

As negative results were found in an Ames test, in an in vitro gene mutation test (HPRT) in CHO cells and in an in vitro micronucleus test in cultured peripheral human lymphocytes, the registered substance has no genotoxic potential and is therefore not classified according to Regulation (EC) No. 1272/2008 (CLP).