Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

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:
key 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
Reason / purpose:
read-across: supporting information

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2005

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals 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.