Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a reverse gene mutation assay in bacteria, performed on alpha-pinene multiconstituent according to OECD guideline 471 and in compliance with GLP, the substance was found as non mutagenic in the presence or absence of metabolic activation.

In a study conducted on alpha-pinene multiconstituent according to OECD guideline 476 in compliance with GLP (in vitro HPRT cell mutation assay) the substance did not demonstrate any mutagenic potential.

In a study performed on alpha-pinene multiconstituent according to OECD guideline 487 in compliance with GLP the substance did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
His+ for S. typhimurium; trp+ for E. coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
10 % (v/v) S9 mix; S9 fraction prepared from liver homogenates of rats induced with Phenobarbital sodium/5,6-benzoflavone.
Test concentrations with justification for top dose:
Experiment 1 (plate incorporation method): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in all strains
Experiment 2 (pre-incubation method): 0,05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in all strains
Vehicle / solvent:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-Aminoanthracene
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: Strains of S. typhimurium and E. coli were obtained from

METHOD OF APPLICATION:
Experiment 1: In agar (direct plate incorporation)
Experiment 2: preincubation method

DURATION
- Preincubation period: Exp. 2, 30 minutes at 37 °C
- Incubation period: Approximately 48-72 h at 37 °C for both direct plate incorporation and preincubation methods

NUMBER OF REPLICATIONS:
-3 plates/dose for treatment, vehicle and positive controls

DETERMINATION OF CYTOTOXICITY
- Method: Any toxic effects of the test substance may be detected by a substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both.

OTHER: After 72 h of incubation at 37 °C, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).
Evaluation criteria:
- If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
- If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
- If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times those of the vehicle controls (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 judgement.
Statistics:
- Statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis.
Species / strain:
S. typhimurium TA 1535
Remarks:
Plate incorporation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Remarks:
Plate incorporation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Remarks:
Plate incorporation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
hinning of background lawn observed at 5000 µg per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Remarks:
Plate incorporation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Remarks:
Plate incorporation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Remarks:
: Pre-incubation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight thinning of background lawn observed at 15 µg per plate, without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Remarks:
Pre-incubation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight thinning of background lawn observed at 15 µg per plate without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Remarks:
Pre-incubation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight thinning of background lawn observed at 15µg per plate without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
not valid
Species / strain:
S. typhimurium TA 100
Remarks:
Pre-incubation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight thinning of background lawn observed at 15 µg per plate without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Remarks:
Pre-incubation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1: Positive control results (Plate incorporation assay, without metabolic activation) :

Strain

Addition

Concentration per plate

Mean revertants per plate

Standard Deviation

Fold increase relative to vehicle

Individual revertant
colony counts (Sorcerer)

TA98

2NF

2 µg

238.3

57.3

11.7

174, 284, 257

TA100

NaN3

2 µg

554.0

19.9

3.1

531, 565, 566

TA1535

NaN3

2 µg

635.7

38.3

38.1

647, 593, 667

TA1537

AAC

50 µg

214.7

186.2

16.5

71, 148, 425

WP2 uvrA (pKM101)

NQO

2 µg

2692.7

110.4

16.2

2682, 2808, 2588

Table 2: Positive control results (Plate incorporation assay, with metabolic activation) :

Strain

Addition

Concentration per plate

Mean revertants per plate

Standard Deviation

Fold increase relative to vehicle

Individual revertant
colony counts (Sorcerer)

TA98

B[a]P

5 µg

173.7

17.2

5.3

177, 155, 189

TA100

AAN

5 µg

3118.0

192.7

16.1

3269, 2901, 3184

TA1535

AAN

5 µg

210.3

24.4

16.6

205, 237, 189

TA1537

B[a]P

5 µg

167.7

34.1

14.4

147, 207, 149

WP2 uvrA (pKM101)

AAN

10 µg

1432.0

66.6

7.0

1435, 1497, 1364

Table 3: Positive control results (Pre-incubation assay, without metabolic activation):

Strain

Addition

Concentration per plate

Mean revertants per plate

Standard Deviation

Fold increase relative to vehicle

Individual revertant
colony counts (Sorcerer)

TA98

2NF

2 µg

295.7

40.0

12.5

308, 328, 251

TA100

NaN3

2 µg

624.3

9.3

4.1

614, 627, 632

TA1535

NaN3

2 µg

632.3

49.2

41.2

577, 649, 671

TA1537

AAC

50 µg

226.3

66.3

33.9

282, 244, 153

WP2 uvrA (pKM101)

NQO

2 µg

2622.3

12.9

16.7

2613, 2617, 2637

Table 4: Positive control results (Pre-incubation assay, with metabolic activation):

Strain

Addition

Concentration per plate

Mean revertants per plate

Standard Deviation

Fold increase relative to vehicle

Individual revertant
colony counts (Sorcerer)

TA98

B[a]P

5 µg

236.0

22.1

8.6

244, 253, 211

TA100

AAN

5 µg

912.0

327.7

5.9

1286, 775, 675

TA1535

AAN

5 µg

314.3

22.1

24.8

335, 291, 317

TA1537

B[a]P

5 µg

157.3

3.2

9.4

155, 156, 161

WP2 uvrA (pKM101)

AAN

10 µg

1661.0

48.1

9.1

1716, 1627, 1640

Conclusions:
alpha-Pinene multiconstituent is not considered as mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2uvrA) strains.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD 471 Guideline and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli (WP2uvrA) were exposed to alpha-pinene multiconstituent at the following concentrations:

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

Experiment 2 (pre-incubation method) 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in all strains.

 

Metabolic activation system used in this test was10 % (v/v) S9 mix;S9 fraction prepared from liver homogenates of rats induced with Phenobarbital sodium/5,6-benzoflavone.Vehicle and positive control groups were also included in mutagenicity tests.

 

No signs of toxicity towards the tester strains were observed in the first experiment following exposure to test item. Toxicity, observed as a reduction in revertant colony numbers, ans slight to severe thinning of the background lawn, was obtained in all strains in the second experiment following exposure to test item at 15 µg/plate in the absence of S9 mix. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to alpha-pinene multiconstituent, at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix. The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study.

 

Therefore, alpha-pinene multiconsituent is not considered as mutagenic in these bacterial systems

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
September-October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: Human
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM).
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 3,91, 7.92, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL; 3 h exposure with and without S9-mix; 20 h continuous exposure without S9-mix

3 h exposure to the test item formulations without S9-mix, followed by a 20 h incubation period in treatment-free media: 62.5, 125, 150, 175, 200, 225 and 250 µg/mL
3 h exposure to the test item formulations with S9-mix (2%) , followed by a 20 h incubation period in treatment-free media: 125, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 µg/mL
20 h continuous exposure to the test item without S9-mix, followed by a 20 h incubation period in treatment-free media: 62.5, 125, 150, 175, 200, 225 and 250 µg/mL

Fisrt additional main tests:
3 h exposure to the test item formulations without S9-mix, followed by a 20 h incubation period in treatment-free media: 1, 10, 20, 40, 45, 50, 55, 60, 80, 100, 150, 200 and 250 µg/mL
3 h exposure to the test item formulations with S9-mix (2%) , followed by a 20 h incubation period in treatment-free media: 50, 100, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 and 1000 µg/mL

Second additional main tests:
3 h exposure to the test item formulations without S9-mix, followed by a 20 h incubation period in treatment-free media: 1, 10, 20, 25, 30, 35, 40, 45, 50, 55 and 60 µg/mL
3 h exposure to the test item formulations with S9-mix (2%) , followed by a 20 h incubation period in treatment-free media: 0.1, 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31 µg/mL

Third additionnal main test:
3 h exposure to the test item formulations without S9-mix, followed by a 20 h incubation period in treatment-free media: 1, 11, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 and 45 µg/mL
Vehicle / solvent:
The vehicle was DMSO.
alpha-Pinene multiconstituent was found to be miscible at 400 mg/mL in dimethyl sulphoxide (DMSO). This gave a final concentration of 2000 µg/mL when dosed at 0.5% v/v.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Colchicine
Details on test system and experimental conditions:
TEST SYSTEM: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (18-35) who had been previously screened for suitability.
CELL CULTURE: Cells (whole blood cultures) were grown in 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 experiment

CYTOKINESIS INHIBITOR (cytogenetic assays): Prior to the mitosis (after exposure of the test substance) the chemical cytochalasin B was added to the cultures.

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture for test item and vehicle control
- Main test: Duplicate cultures per dose for test item, vehicle and positive controls

NUMBER OF CELLS EVALUATED:
- Cytotoxicity: A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B.
- Scoring of Micronuclei: The micronucleus frequency in 2000 binucleated cells was analyzsed per concentration (1000 binucleated cells per culture, two cultures per concentration), except for vehicle control (4000 cells).

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of test item in the lymphocyte cultures was determined using the cytokinesis-block 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 substance treatment culture
C = vehicle control culture
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 negative 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 negative control data.
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 negative control.
- There is no concentration-related increase when evaluated with an appropriate trend test.
- All results are inside the distribution of the historical negative control data.
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 square-root transformation was used to transform the data. Alpha-pinene multiconstituent treated groups were then compared to control using Williams’ tests (Williams 1971, 1972). 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 9.1.3 (SAS Institute 2002) and Quasar 1.5 (Quasar 1.5 2016).
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no fluctuations in pH when the test item was dosed into media.
- Effects of osmolality: no fluctuations in osmolality of more than 50 mOsm at the dose levels invest
igated

- Cytotoxicity: concentrations of alpha-pinene multiconstituent used for the main micronucleus test were 0.1, 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31 µg/mL.
No precipitate was observed by eye at the end of treatment. A reduction in CBPI compared to vehicle control values, equivalent to 54.5% cytotoxicity, was obtained with Alpha-pinene multiconstituent at 27 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 1, 13 and 27 µg/mL.

Preliminary Toxicity Test:

In all exposure conditions the highest concentration tested was 2000 µg/mL and no precipitate was observed by eye at the end of treatment.

After 3-hour treatment in the absence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 68.8% cytotoxicity, was obtained with alpha-pinene multiconstituent at 500 µg/mL. At higher tested concentrations, overt toxicity was observed.

After 3-hour treatment in the presence of S9-mix, no significant reduction in CBPI compared to vehicle control values was observed with alpha-pinene multiconstituent up to 250 µg/mL. At higher tested concentrations, overt toxicity was observed.

After 20-hour treatment in the absence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 31% cytotoxicity, was obtained with alpha-pinene multiconstituent at 125 µg/mL. At higher tested concentrations, overt toxicity was observed.

These results were used to select concentrations for the main test. 

Main Test:

3-hour treatment in the absence of S9 Mix:

Concentrations of alpha-pinene multiconstituent used for the main micronucleus test were 62.5, 125, 150, 175, 200, 225 and 250 µg/mL. No precipitate was observed by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels.

Additional 3-hour treatment in the absence of S9 Mix:

Concentrations of alpha-pinene multiconstituent used for the additional main micronucleus test were 1, 10, 20, 40, 45, 50, 55, 60, 80, 100, 150, 200 and 250 µg/mL. No precipitate was observed by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels.

Second additional 3 -hour treatment in the absence of S9 Mix:

Concentrations of alpha-pinene multiconstituent used for the second additional main micronucleus test were 1, 10, 20, 25, 30, 35, 40, 45, 50, 55 and 60 µg/mL. No precipitate was observed by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels.

Third additional 3-hour treatment in the absence of S9 Mix:

Concentrations of alpha-pinene multiconstituent used for the third additional main micronucleus test were 1, 11, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 and 45 µg/mL. No precipitate was observed by eye at the end of treatment. A reduction in CBPI compared to vehicle control values equivalent to 56.5% cytotoxicity, was obtained with Alpha-pinene multiconstituent at 33 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 1, 27 and 33 µg/mL.

alpha-Pinene multiconstituent did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle control.

Mean micronucleus induction in the vehicle control was close to the laboratory historical control limit and the data were considered acceptable for addition to the laboratories laboratory historical negative control database.

The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, within the laboratory historical positive control data, demonstrating the sensitivity of the test system

3-hour treatment in the presence of S9 Mix:

Concentrations of alpha-pinene multiconstituent used for the main micronucleus test were 125,250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 µg/mL. No precipitate was observed by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels.

Additional 3 -hour treatment in the presence of S9 Mix:

Concentrations of alpha-pinene multiconstituent used for the additional main micronucleus test were 50, 100, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 and 1000 µg/mL. No precipitate was observed by eye at the end of treatment. A reduction in CBPI compared to vehicle control values, equivalent to 50.6% cytotoxicity, was obtained with Alpha-pinene multiconstituent at 250 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 50, 100 and 250 µg/mL.

alpha-Pinene multiconstituent did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle control.

Mean micronucleus induction in the vehicle control was within the laboratory historical control limit.

The positive control compound (cyclophosphamide) caused a statistically significant increase in the number of binucleate cells containing micronuclei, within the laboratory historical positive control data, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

20 -hour treatment in the absence of S9 Mix:

Concentrations of alpha-pinene multiconstituent used for the main micronucleus test were 62.5, 125, 150, 175, 200, 225 and 250 µg/mL. No precipitate was observed by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels.

Additional 20-Hour Treatment in the Absence of S9 Mix:

Concentrations of alpha-pinene multiconstituent used for the main micronucleus test were 0.1, 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31 µg/mL. No precipitate was observed by eye at the end of treatment. A reduction in CBPI copared to vehicle control values, equivalent to 54.5% cytotoxicity, was obtained with alpha-pinene multiconstituent at 27 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 1, 13 and 27 µg/mL

alpha-Pinene multiconstituent did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle control.

Mean micronucleus induction in the vehicle control was within the laboratory historical control limit.

The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, within the laboratory historical positive control data, demonstrating the sensitivity of the test system.

Conclusions:
alpha-Pinene multiconstituent 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 human lymphocytes were exposed to test item, alpha-pinene multiconstituent.

Three alpha-pinene multiconstituent 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-hour treatment in the absence of S9 mix, reductions in CBPI equivalent to 56.5% cytotoxicity were obtained with alpha-pinene multiconstituent at 33 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 1, 27 and 33 µg/mL. Following 3‑hour treatment in the presence of S9 mix, reductions in CBPI equivalent to 50.6% cytotoxicity were obtained with alpha-pinene multiconstituent at 250 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 50, 100 and 250 µg/mL. In the absence of S9 mix following 20‑hour treatment, a reduction in CBPI equivalent to 54.5% cytotoxicity was obtained with alpha-pinene multiconstituent at 27 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 1, 13 and 27 µg/mL.

In both the absence and presence of S9 mix, following 3-hour treatment, and in the absence of S9 mix, following 20-hour treatment, alpha-pinene multiconstituent did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared to 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.

It was concluded that alpha-pinene multiconstituent 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:
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:
September- November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
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. certificate)
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 mix = S9 fraction (10% v/v), glucose-6-phosphate (6.9 mM), NADP (1.4 mM) in H0. 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:
-S9 mix Test 1 (3 hours) 60, 65, 70, 75, 80, 85, 90 and 100 μg/mL
+S9 mix Test 1 (3 hours) 25, 50, 100, 120, 140, 150, 160, 170, 180, 200 and 220 μg/mL
-S9 mix Test 2 (3 hours) 1, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg/mL
+S9 mix Test 2 (3 hours) 20, 40, 80, 90, 95, 100, 105, 110, 115, 120, 125 and 130 μg/mL
- S9 mix Test 3 (3 hours) 1, 20, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45 and 50 µg/mL
+ S9 mix Test 3 (3 hours) 20, 40, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 80, 90 and 100 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Formulation preparation: Alpha-pinene multiconstituent was dissolved and formulated in DMSO (ACS reagent grade), shortly before dosing. The final volume of DMSO added to the cultures was 1% v/v.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
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: 4 cultures for vehicle control, 2 cultures/dose for test item and positive controls

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 co
ncurrent 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:
Alpha-pinene multiconstituent was dosed at concentrations up to 2000 µg/mL. No precipitate was observed by eye at the end of treatment. Exposure to Alpha-pinene multiconstituent for 3 hours at concentrations from 15.63 to 2000 µg/mL in both the absence and presence of S9 mix resulted in RS values from 98 to 0% and 90 to 0%, respectively. Concentrations for the main test were based upon these data.

MAIN TEST:

3-Hour Treatment in the Absence of S9 Mix
- In a first experiment: cultures were exposed to Alpha-pinene multiconstituent at concentrations from 60 to 100 µg/mL. No precipitate was seen by eye at the end of treatment. The cell counts obtained on Day 1 indicated that an appropriate toxicity profile would not be achieved in this experiment, therefore, the test was abandoned and an additional test was performed using modified dose concentrations.
- In a second experiment : cultures were exposed to Alpha-pinene multiconstituent at concentrations from 1 to 100 µg/mL. No precipitate was seen by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and a second additional test was performed using modified dose levels.
- In a third experiment: cultures were exposed to Alpha-pinene multiconstituent at concentrations from 1 to 50 µg/mL. No precipitate was seen by eye at the end of treatment. Cultures treated at 1, 20, 25, 27.5 and 30 µg/mL where RS values from 111 to 19% were observed; these cultures were plated out for determination of cloning efficiency and mutant frequency. Cultures treated at 32.5 µg/mL and above were not analysed for mutant frequency as RS was <10% at these concentrations. No significant increases in mutant frequency were observed after exposure to Alpha-pinene multiconstituent at any concentration analysed.
EMS, the positive control, induced a significant increase in mutant frequency.

3-Hour Treatment in the Presence of S9 Mix
- In a first experiment: cultures were exposed to Alpha-pinene multiconstituent at concentrations from 25 to 220 µg/mL. No precipitate was seen by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels
- In a second experiment: cultures were exposed to Alpha-pinene multiconstituent at concentrations from 20 to 130 µg/mL. No precipitate was seen by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and a second additional test was performed using modified dose levels.
- In a third experiment: Cultures were exposed to Alpha-pinene multiconstituent at concentrations from 20 to 100 µg/mL. No precipitate was seen by eye at the end of treatment. Cultures treated at 20, 40, 45, 47.5, 50, 52.5, 55, 57.5 and 60 µg/mL where RS values of 92 to 0% were observed; these cultures were plated out for determination of cloning efficiency and mutant frequency. Cultures treated at 62.5 µg/mL and above were not analysed for mutant frequency as RS was <10% at these concentrations. No significant increases in mutant frequency were observed after exposure to Alpha-pinene multiconstituent at any concentration analysed.
3MC, the positive control, induced a significant increase in mutant frequency.

Table 7.6.1/1: Summary results

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

Day 1 relative survival

Day 8 cloning efficiency

mutant frequency

Concn.of test item   (µg/mL)

Cell Count Day 1 (x106/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 Frequencya

Mean Mutant Frequencya

Plate 1

Plate 2

Plate 3

Plate 1

Plate 2

Plate 3

Plate 1

Plate 2

Plate 3

Plate 4

Plate 5

0

0.92

121

133

139

393

69

65

100

100

200

168

175

543

91

0

0

1

0

1

2

0.00008

0.88

1.35

0.96

148

148

132

428

187

171

167

525

88

0

0

2

0

1

3

0.00012

1.37

1.25

150

143

142

435

159

108

155

422

70

2

1

0

0

1

4

0.00016

2.27

1.15

127

138

139

404

171

177

199

547

91

0

0

1

0

1

2

0.00008

0.88

1

1.20

105

109

107

321

54

57

87

99

150

161

159

470

78

0

0

0

0

1

1

0.00004

0.51

0.79

1.16

142

130

151

423

71

72

111

150

160

141

451

75

0

1

0

0

1

2

0.00008

1.06

20

1.34

128

137

137

402

67

79

122

111

147

150

168

465

78

2

1

0

1

1

5

0.00020

2.58

1.64

1.31

101

139

103

343

57

66

101

115

116

114

345

58

0

0

0

1

0

1

0.00004

0.70

25

0.98

106

88

110

304

51

44

67

62

121

171

159

451

75

0

0

0

2

0

2

0.00008

1.06

1.38

0.88

99

85

100

284

47

37

56

154

152

121

427

71

1

0

0

2

0

3

0.00012

1.69

27.5

0.86

86

82

87

255

43

32

50

49

155

157

158

470

78

1

0

1

2

0

4

0.00016

2.04

1.06

1.55

0.84

82

82

85

249

42

31

47

154

148

151

453

76

0

1

0

1

0

2

0.00008

30

0.44

77

57

53

187

31

12

19

19

139

127

105

371

62

0

0

0

1

1

2

0.00008

1.29

1.89

1.59

0.42

85

60

65

210

35

13

20

101

161

119

381

64

2

0

1

0

0

3

0.00012

32.5

0.20

Cultures discontinued due to low day 1 cell counts

0.16

35

0.12

0.16

37.5

0.20

0.20

40

0.07

0.06

42.5

0.07

0.06

45

0.07

0.07

50

0.06

0.10

EMS – positive control

250

1.27

150

127

130

407

68

76

117

121

139

150

140

429

72

20

26

29

20

22

117

0.00468

65.45

62.52

1.35

140

131

143

414

69

82

126

158

140

129

427

71

20

20

30

17

19

106

0.00424

59.58

***

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

  EMS: Ethyl methanesulphonate

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

Day 1 relative survival

Day 8 cloning efficiency

mutant frequency

 

Concn.of test item   (µg/mL)

Cell Count Day 1 (x106/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 Frequencya

Mean Mutant Frequencya

Plate 1

Plate 2

Plate 3

Plate 1

Plate 2

Plate 3

Plate 1

Plate 2

Plate 3

Plate 4

Plate 5

 

 

0

1.05

146

170

173

489

76

70

100

100

178

181

173

532

89

1

1

0

2

0

4

0.00016

1.80

1.91

1.02

137

151

132

420

183

171

175

529

88

2

0

1

0

2

5

0.00020

2.27

0.97

153

149

151

453

179

181

172

532

89

2

0

1

1

0

4

0.00016

1.80

0.99

130

152

177

459

174

186

179

539

90

1

1

0

1

1

4

0.00016

1.78

20

0.89

141

138

147

426

71

58

83

92

179

172

181

532

89

1

1

0

1

0

3

0.00012

1.35

1.57

0.97

150

172

153

475

79

70

101

182

178

177

537

90

2

1

0

1

0

4

0.00016

1.79

40

0.89

150

141

151

442

74

60

85

88

169

175

174

518

86

0

0

0

1

0

1

0.00004

0.46

0.70

1.01

116

147

151

414

69

63

91

172

171

174

517

86

1

1

0

0

0

2

0.00008

0.93

45

0.90

109

137

120

366

61

50

72

67

178

183

181

542

90

1

0

2

0

0

3

0.00012

1.33

0.89

0.72

160

127

105

392

65

43

61

188

171

172

531

89

0

0

1

0

0

1

0.00004

0.45

47.5

0.85

132

137

120

389

65

51

72

70

178

176

181

535

89

1

1

1

1

2

6

0.00024

2.69

2.23

2.46

1.04

116

105

80

301

50

48

69

183

174

182

539

90

2

1

0

1

1

5

0.00020

50

0.96

115

130

120

365

61

53

76

64

178

181

174

533

89

0

0

1

0

0

1

0.00004

0.45

1.40

0.92

0.69

110

105

124

339

57

36

51

172

171

173

516

86

0

1

0

2

0

3

0.00012

52.5

0.86

130

145

147

422

70

55

79

82

164

167

174

505

84

1

1

0

1

0

3

0.00012

1.43

1.63

0.84

165

153

151

469

78

60

86

172

174

179

525

88

0

2

1

1

0

4

0.00016

1.83

55

0.89

109

83

102

294

49

40

57

71

181

173

175

529

88

1

1

2

0

0

4

0.00016

1.81

1.60

1.07

140

111

116

367

61

60

85

173

184

161

518

86

1

0

1

0

1

3

0.00012

1.39

57.5

0.69

105

61

82

248

41

26

37

38

169

172

173

514

86

0

0

0

1

0

1

0.00004

0.47

1.16

0.72

90

81

77

248

41

27

39

174

182

162

518

86

1

0

2

1

0

4

0.00016

1.85

60

0.51

50

50

47

147

25

11

16

15

174

161

157

492

82

0

0

1

0

1

2

0.00008

0.98

0.98

0.53

40

48

39

127

21

10

15

163

163

158

484

81

1

0

1

0

0

2

0.00008

0.99

62.5

0.47

11

8

7

26

4

2

3

7

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.53

35

39

30

104

17

8

12

 

 

 

 

 

 

 

 

 

 

 

 

 

65

0.45

6

7

3

16

3

1

2

2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.52

8

3

13

24

4

2

3

 

 

 

 

 

 

 

 

 

 

 

 

 

67.5

0.50

7

8

10

25

4

2

3

7

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.60

19

35

34

88

15

8

11

 

 

 

 

 

 

 

 

 

 

 

 

 

70

0.55

7

7

7

21

4

2

3

3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.62

8

4

13

25

4

2

3

 

 

 

 

 

 

 

 

 

 

 

 

 

72.5

0.56

2

4

4

10

2

1

1

2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.44

13

12

5

30

5

2

3

 

 

 

 

 

 

 

 

 

 

 

 

 

75

0.45

1

1

1

3

1

0

0

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.57

0

1

3

4

1

0

0

 

 

 

 

 

 

 

 

 

 

 

 

 

80

0.51

2

1

1

4

1

0

0

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.41

0

1

2

3

1

0

0

 

 

 

 

 

 

 

 

 

 

 

 

 

90

0.44

Cultures discontinued due to low day 1 cell counts

 

0.20

100

0.27

 

0.18

3MC– positive control

5

1.15

118

105

108

331

55

58

83

89

127

131

122

380

63

23

18

26

23

28

118

0.00472

74.53

77.48

1.16

115

127

137

379

63

67

96

125

124

118

367

61

26

24

22

25

26

123

0.00492

80.44

***

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

 

3MC: 3-Methylcholanthrene

Conclusions:
alpha-Pinene multiconstituent 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 test item for 3 hours, with and without metabolic activation (25% S9 [v/v] 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 μg/mL

 

Mutation tests:

- S9 mix Test 3 (3 hours) 1, 20, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45 and 50  µg/mL

+ S9 mix Test 3 (3 hours) 20, 40, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 80, 90 and 100  µg/mL

 

The vehicle for the test item was dimethyl sulphoxide (DMSO). The highest final concentration used in the preliminary toxicity test was 2000 µg/mL. This is the standard limit concentration within this test system as recommended in the regulatory guidelines for test items of well-known composition. No precipitate was observed by eye at the end of treatment. Cytotoxicity was measured as Day 1 relative survival (RS). After exposure to Alpha-pinene multiconstituent at concentrations from 15.63 to 2000 mg/mL RS values ranged from 98 to 0% and from 90 to 0%, in the absence and presence of S9 mix, respectively.

In the second additional main mutation test in the absence of S9 mix, cells were exposed to alpha-pinene multiconstituent at concentrations from 1 to 50 µg/mL. No precipitate was observed by eye at the end of treatment. RS values ranged from 111 to 19% relative to the vehicle control. Alpha-pinene 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 second additional main mutation test in the presence of S9 mix, cells were exposed to alpha-pinene multiconstituent at concentrations from 20 to 100 µg/mL. No precipitate was observed by eye at the end of treatment. RS values ranged from 92 to 0% relative to the vehicle control. Alpha-pinene multiconstituent did not induce a statistically significant increase in mutant frequency. The positive control, 3-methylcholanthrene, induced a significant increase in mutant frequency.

It was concluded that alpha-pinene 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

Description of key information

alpha-Pinene multiconstituent did not increase the frequency of micronucleated normochromatic erythrocytes in an in vivo micronucleus assay performed on peripheral erythrocytes of mice exposed for 90 days by inhalation.

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

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 (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli (WP2uvrA) were exposed to alpha-pinene multiconstituent, with and without metabolic activation system. Vehicle and positive control groups were also included in these mutagenicity tests.

No signs of toxicity towards the tester strains were observed in the first experiment following exposure to the test item. Toxicity, observed as a reduction in revertant colony numbers, and slight to severe thinning of the background lawn, were obtained in all strains in the second experiment following exposure to the test item at 15 µg/plate in the absence of S9 mix. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to alpha-pinene multiconstituent, at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix. The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study.

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured human lymphocytes were exposed to the test item, alpha-pinene multiconstituent. Vehicle and positive control groups were also included in mutagenicity tests. In both the absence and presence of S9 mix, following 3-hour treatment, and in the absence of S9 mix, following 20-hour treatment, alpha-pinene multiconstituent did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared to 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. It was concluded that alpha-pinene multiconstituent 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 the test item for 3 hours, with and without metabolic activation (25% S9 [v/v] fraction of male Sprague Dawley rats liver induced with phenobarbital and 5,6-benzoflavone). Vehicle and positive control groups were also included in these mutagenicity tests.

In the absence of S9 mix, cells were exposed to alpha-pinene multiconstituent at concentrations from 1 to 50 µg/mL. No precipitate was observed by eye at the end of treatment. RS values ranged from 111 to 19% relative to the vehicle control. alpha-Pinene 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 presence of S9 mix, cells were exposed to alpha-pinene multiconstituent at concentrations from 20 to 100 µg/mL. No precipitate was observed by eye at the end of treatment. RS values ranged from 92 to 0% relative to the vehicle control. alpha-Pinene multiconstituent did not induce a statistically significant increase in mutant frequency. The positive control, 3-methylcholanthrene, induced a significant increase in mutant frequency.

It was concluded that alpha-pinene multiconstituent did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay.

Also, in a peripheral blood micronucleus test conducted similarly to OECD Guideline 474, alpha-pinene was administered through inhalation to groups of B6C3F1 mice (5/sex/dose) at dose levels of 0, 25, 50, 100, 200 or 400 ppm, 5 days/week for 13 weeks. No increase in the frequency of micronucleated normochromatic erythrocytes (NCE)/1000 NCEs and %NCEs were observed in peripheral blood samples in male or female B6C3F1 mice exposed to alpha-pinene.

Justification for classification or non-classification

In a reverse mutation assay in bacteria (Ames test) performed according to OECD Guideline 471 and in compliance with GLP, alpha-pinene multiconstituent was not mutagenic in S. typhimurium strains TA1535, TA 1537, TA 98 and TA100 and in Escherichia coli strain WP2 uvrA (pKM101) in presence and absence of metabolic activation, up to limit or cytotoxic concentrations.

The resultst were also negative in a chromosome aberation test performed in human lymphocytes according to OECD Guideline 487 and in compliance with GLP, in presence and absence of metabolic activation.

In addition, negative results were obtained in a gene mutation test (HPRT) in CHO cells, performed on alpha-pinene multiconstituent according to OECD Guideline 476 and in complinace with GLP, in presence and absence of metabolic activation.

Moreover, negative results were obtained in a chromosome aberation test performed on alpha-pinene multiconstituent in mice according to OECD Guideline 474.

As the results obtained with alpha-pinene multiconstituent were negative in a Ames test, in a gene mutation test (HPRT) in CHO cells and in two chromosome aberration tests (one in Human lymphocytes and one in vivo in mice), (-)-alpha-pinene is not classified according to CLP Regulation (EC) n° 1272/2008.