Pin-2(3)-ene

Genetic toxicity: in vitro

Administrative data

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

(+)-pin-2(3)-ene

EC Number:

232-087-8

EC Name:

(+)-pin-2(3)-ene

Cas Number:

7785-70-8

Molecular formula:

C10H16

IUPAC Name:

(1R,5R)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

Constituent 2

Reference substance name:

(-)-pin-2(3)-ene

EC Number:

232-077-3

EC Name:

(-)-pin-2(3)-ene

Cas Number:

7785-26-4

Molecular formula:

C10H16

IUPAC Name:

(1S,5S)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

impurity 1

Reference substance name:

(1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

EC Number:

227-336-2

EC Name:

(1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

Cas Number:

5794-03-6

Molecular formula:

C10H16

IUPAC Name:

(1R,4S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

impurity 2

Reference substance name:

(1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

EC Number:

227-337-8

EC Name:

(1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

Cas Number:

5794-04-7

Molecular formula:

C10H16

IUPAC Name:

(1S,4R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

impurity 3

Reference substance name:

(-)-pin-2(10)-ene

EC Number:

242-060-2

EC Name:

(-)-pin-2(10)-ene

Cas Number:

18172-67-3

Molecular formula:

C10H16

IUPAC Name:

(1S,5S)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane

impurity 4

Reference substance name:

1,7,7-trimethyltricyclo[2.2.1.02,6]heptane

EC Number:

208-083-7

EC Name:

1,7,7-trimethyltricyclo[2.2.1.02,6]heptane

Cas Number:

508-32-7

Molecular formula:

C10H16

IUPAC Name:

1,7,7-trimethyltricyclo[2.2.1.0~2,6~]heptane

impurity 5

Reference substance name:

7,7-dimethyl-2-methylene-(1R,4S)-bicyclo[2.2.1]heptane

Cas Number:

116724-26-6

Molecular formula:

C10H16

IUPAC Name:

7,7-dimethyl-2-methylene-(1R,4S)-bicyclo[2.2.1]heptane

impurity 6

Reference substance name:

7,7-dimethyl-2-methylene-, (1S,4R)-bicyclo[2.2.1]heptane

Cas Number:

7378-37-2

Molecular formula:

C10H16

IUPAC Name:

7,7-dimethyl-2-methylene-, (1S,4R)-bicyclo[2.2.1]heptane

impurity 7

Reference substance name:

(1R,5R)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane

Cas Number:

19902-08-0

Molecular formula:

C10H16

IUPAC Name:

(1R,5R)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane

Test material form:

liquid

Details on test material:

Batch No. : FAB-29062016
Purity : 96.2% (sum of the two main constituents)
Name of test material (as cited in study report): alpha-pinene multiconstituent
Physical state: colourless liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 28 June 2017

Method

Target gene:

His+ for S. typhimurium; trp+ for E. coli

Species / strain

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

Controls

Controls 1

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-Aminoanthracene

Controls 2

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Test results 6

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

Test results 7

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

Test results 8

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

Test results 9

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

Test results 10

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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