Pin-2(3)-ene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

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

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

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

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

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

September- November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hemizygous hypoxanthine phosphoribosyl transferase (HPRT) gene

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Source: European Collection of Cell Cultures
- CHO-KI cells are functionally hemizygous at the HPRT locus.
- Type and identity of media: Ham’s Nutrient Mixture F12 medium
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No; karyotype was assumed to be stable.
- Other details: Prior to exposure to test item, spontaneous mutants were eliminated from the stock cultures by incubating the cells in H10 containing 15 μg/mL hypoxanthine, 0.3 μg/mL amethopterin and 4 μg/mL thymidine for three days. All cell cultures were maintained at 37 °C in an atmosphere of 5 % CO2 in air.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 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
Conc^n.of test item   (µg/mL)	Cell Count Day 1 (x10^6/mL)	No. of colonies on plate			Total no. of Colonies	Cloning Efficiency (%)	Adjusted Cloning Efficiency (%)	RS (%)	Mean RS (%)	No. of colonies on plate			Total no. of Colonies	Cloning Efficiency in non‑selective medium (%)	No. of colonies on plate					Total no. of Colonies	Cloning Efficiency in selective medium (%)	Mutant Frequency^a	Mean Mutant Frequency^a
		Plate 1	Plate 2	Plate 3						Plate 1	Plate 2	Plate 3			Plate 1	Plate 2	Plate 3	Plate 4	Plate 5
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
Conc^n.of test item   (µg/mL)	Cell Count Day 1 (x10^6/mL)	No. of colonies on plate			Total no. of Colonies	Cloning Efficiency (%)	Adjusted Cloning Efficiency (%)	RS (%)	Mean RS (%)	No. of colonies on plate			Total no. of Colonies	Cloning Efficiency in non‑selective medium (%)	No. of colonies on plate					Total no. of Colonies	Cloning Efficiency in selective medium (%)	Mutant Frequency^a	Mean Mutant Frequency^a
		Plate 1	Plate 2	Plate 3						Plate 1	Plate 2	Plate 3			Plate 1	Plate 2	Plate 3	Plate 4	Plate 5
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.

 

Reference 3

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 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.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2005

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted similarly to OECD Guideline 474 with deviations: no data on test material purity; age, body weight, housing and exposure conditions of animals; duration of exposure/day; positive/negative controls; polychromatic erythrocytes not scored

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

no data on test material purity; age, body weight, housing and exposure conditions of animals; duration of exposure/day; positive/negative controls; polychromatic erythrocytes not scored

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

None

Route of administration:

inhalation

Vehicle:

- Vehicle(s)/solvent(s) used: Air

Details on exposure:

None

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days/week

Post exposure period:

No data

Remarks:

Doses / Concentrations:
0, 25, 50, 100, 200 or 400 ppm
Basis:
no data

No. of animals per sex per dose:

Five

Control animals:

yes, concurrent vehicle

Positive control(s):

No data

Tissues and cell types examined:

Peripheral blood samples

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Doses extend up to the maximum tolerated dose

TREATMENT AND SAMPLING TIMES: Sample collection time: 24 hours

DETAILS OF SLIDE PREPARATION: Slides were air-dried, fixed and stained with a fluorescent DNA-specific stain (acridine orange).

METHOD OF ANALYSIS: Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) per animal. In addition, the percentage of normochromatic erythrocytes (%NCEs) and micronucleus cells in a population of 1000 erythrocytes was determined.

Evaluation criteria:

No data

Statistics:

Significance of micronucleated NCEs/1000 NCEs tested by trend test (significant at P ≤ 0.025), followed by Pairwise comparison with the vehicle controls (significant at P ≤ 0.008)

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Frequency of micronuclei in peripheral blood erythrocytes: See table 1 and 2

Table 1: Frequency of micronuclei in peripheral blood erythrocytes of male mice following administration of α-pinene

Start Date	Sample Collection Time	Sex	Dosing Regimen
07/25/2005	24 Hours	Male	INHAL x 5, 13 Weeks
		Dose (ppm)	Animal Number	Normochromatic Erythrocytes
				Trend P = 0.742
				No. Examined	Total MN Cells	Percent NCE	MN Cells
							per 1000
Vehicle Control	Air	0	XM0001	2000	3	98.7	1.5
		0	XM0002	2000	5	97.9	2.5
		0	XM0003	2000	1	97.6	0.5
		0	XM0004	2000	3	96.5	1.5
		0	XM0005	2000	4	96.8	2.0
Average ± SEM						97.50 ± 0.39	1.60 ± 0.33
Test Chemical		25	AM0201	2000	3	97.7	1.5
		25	AM0202	2000	5	97.0	2.5
		25	AM0203	2000	3	98.1	1.5
		25	AM0204	2000	2	97.6	1.0
		25	AM0205	2000	5	97.9	2.5
Average ± SEM						97.66 ± 0.19	1.80 ± 0.30
Pairwise P							0.366
Test Chemical		50	BM0401	2000	3	98.5	1.5
		50	BM0402	2000	3	97.1	1.5
		50	BM0403	2000	2	98.1	1.0
		50	BM0404	2000	8	97.3	4.0
		50	BM0405	2000	3	98.0	1.5
Average ± SEM						97.80 ± 0.26	1.90 ± 0.53
Pairwise P							0.306
Test Chemical		100	CM0601	2000	4	96.8	2.0
		100	CM0602	2000	5	96.9	2.5
		100	CM0603	2000	2	96.4	1.0
		100	CM0604	2000	3	98.2	1.5
		100	CM0605	2000	7	97.3	3.5
Average ± SEM						97.12 ± 0.31	2.10 ± 0.43
Pairwise P							0.205
Test Chemical		200	DM0801	2000	5	97.1	2.5
		200	DM0802	2000	4	96.7	2.0
		200	DM0803	2000	2	97.8	1.0
		200	DM0804	2000	5	97.5	2.5
		200	DM0805	2000	3	97.2	1.5
Average ± SEM						97.26 ± 0.19	1.90 ± 0.29
Pairwise P							0.306
Test Chemical		400	EM1001	2000	3	97.3	1.5
		400	EM1002	2000	1	96.7	0.5
		400	EM1003	2000	5	96.8	2.5
		400	EM1004	2000	1	97.4	0.5
		400	EM1005	2000	4	96.3	2.0
Average ± SEM						96.90 ± 0.20	1.40 ± 0.40
Pairwise P							0.643

Table 2: Frequency of micronuclei in peripheral blood erythrocytes of female mice following administration of α-pinene

Start Date	Sample Collection Time	Sex	Dosing Regimen
07/26/2005	24 Hours	Female	INHAL x 5, 13 Weeks
		Dose (ppm)	Animal Number	Normochromatic Erythrocytes
				Trend P = 0.899
				No. Examined	Total MN Cells	Percent NCE	MN Cells
							per 1000
Vehicle Control	Air	0	XF0101	2000	3	97.4	1.5
		0	XF0102	2000	3	98.0	1.5
		0	XF0103	2000	2	97.0	1.0
		0	XF0104	2000	2	98.0	1.0
		0	XF0105	2000	4	97.6	2.0
Average ± SEM						97.60 ± 0.19	1.40 ± 0.19
Test Chemical		25	AF0301	2000	3	98.0	1.5
		25	AF0302	2000	7	97.0	3.5
		25	AF0303	2000	5	97.9	2.5
		25	AF0304	2000	2	97.7	1.0
		25	AF0305	2000	4	98.6	2.0
Average ± SEM						97.84 ± 0.26	2.10 ± 0.43
Pairwise P							0.118
Test Chemical		50	BF0501	2000	5	98.3	2.5
		50	BF0502	2000	2	98.2	1.0
		50	BF0503	2000	3	97.9	1.5
		50	BF0504	2000	4	97.6	2.0
		50	BF0505	2000	4	97.2	2.0
Average ± SEM						97.84 ± 0.20	1.80 ± 0.25
Pairwise P							0.240
Test Chemical		100	CF0701	2000	4	97.9	2.0
		100	CF0702	2000	4	97.7	2.0
		100	CF0703	2000	0	97.2	0.0
		100	CF0704	2000	4	97.6	2.0
		100	CF0705	2000	5	95.9	2.5
Average ± SEM						97.26 ± 0.36	1.70 ± 0.44
Pairwise P							0.295
Test Chemical		200	DF0901	2000	2	98.8	1.0
		200	DF0902	2000	5	97.7	2.5
		200	DF0903	2000	4	97.6	2.0
		200	DF0904	2000	2	98.4	1.0
		200	DF0905	2000	4	97.2	2.0
Average ± SEM						97.94 ± 0.29	1.70 ± 0.30
Pairwise P							0.295
Test Chemical		400	EF1101	2000	1	98.0	0.5
		400	EF1102	2000	2	97.7	1.0
		400	EF1103	2000	2	97.9	1.0
		400	EF1104	2000	3	97.9	1.5
		400	EF1105	2000	3	97.7	1.5
Average ± SEM						97.84 ± 0.06	1.10 ± 0.19
Pairwise P							0.726

Conclusions:

alpha-Pinene was not mutagenic in the mouse peripheral blood micronucleus test.

Executive summary:

In a peripheral blood micronucleus test conducted similarly to OECD Guideline 474, α-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. Peripheral blood samples were obtained 24 hours after the last dosing. Smear slides were air-dried, fixed, stained with fluorescent DNA-specific stain (acridine orange) and scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) per animal. In addition, the percentage of normochromatic erythrocytes (%NCEs) and micronucleus cells in a population of 1000 erythrocytes was determined.

 

No increase in the frequency of micronucleated NCEs/1000 NCEs and %NCEs were observed in peripheral blood samples in male or female B6C3F1 mice administered alpha-pinene.

 

Therefore, alpha-pinene was not mutagenic in the mouse peripheral blood micronucleus test.

 

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 according to OECD Guideline 476 and in complinace with GLP, in presence and absence of metabolic activation.

Moreover, alpha-pinene was negative in a chromosome aberation test performed in mice according to OECD Guideline 474.

As the results obtained with alpah-pinene multiconstituent were negative in an 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), the test item is not classified according to CLP Regulation (EC) n° 1272/2008.