Pin-2(3)-ene

Genetic toxicity: in vivo

Administrative data

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

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:

publication

Title:

Unnamed

Year:

2005

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Pin-2(3)-ene

EC Number:

201-291-9

EC Name:

Pin-2(3)-ene

Cas Number:

80-56-8

Molecular formula:

C10H16

IUPAC Name:

2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

Test material form:

liquid

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

None

Administration / exposure

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

Doses / concentrations

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

Examinations

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)

Results and discussion

Test results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.