Oligomerisation products of beta-pinene

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

Mouse bone marrow micronucleus test

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Nu-Film-17

IUPAC Name:

Nu-Film-17

Constituent 2

Reference substance name:

Oligomerisation products of beta-pinene

EC Number:

701-246-8

Molecular formula:

Variable (dimer = C20-H34)

IUPAC Name:

Oligomerisation products of beta-pinene

Details on test material:

- Batch number: 154293
- pinene oilgomers content 96 wt%

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK), Margate, Kent, England.
- Age: 4 - 5 weeks old on arrival
- Weight: 14.9 - 21.6 g on arrival
- Assigned to test groups randomly: yes, using computer generated random numbers
- Fasting period before study:
- Housing: Animals were housed in polypropylene cages with stainless steel tops.
- Diet: Laboratory animal diet RM1 (E)SQC (Special Diet Services Ltd., Witham, Essex, England) ad libitum
- Water: Drinkning water from the public supply, ad libitum
- Acclimation period: At least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 45 - 60
- Air changes (per hr): 15
- Photoperiod: 12 hour light / 12 hour dark

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle: sterile water

Details on exposure:

The test material formed a doseable emulsion when mixed with sterile water (purified by reverse osmosis) at a maximum concentration of 200 mg/ml. Dosing formulations were prepared with sterile water on the day of dosing. The test material or dosing vehicle (sterile water) was administered once in a dose volume of 10 ml/kg via intraperitoneal injection to groups of male and female mice.

Duration of treatment / exposure:

Preliminary study: 48 hours
Main study: 24 and 48 hours

Frequency of treatment:

Single dose (both preliminary and main study)

Post exposure period:

24 hours after treatment, 5 test animals of each sex from each dose group were killed and smears were prepared from bone marrow cells of the femur. 48 hours after treatment the remaining 5 animals (per sex) from the 2000 mg/kg dose group and vehicle control group were killed and smears prepared from the bone marrow cells of the femur.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Main study: 500, 1000 and 2000 mg/kg
Basis:
nominal in water
intraperitoneal injection

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Preliminary study: 500, 1000, 1500 and 2000 mg/kg
Basis:
nominal in water
intraperitoneal injection

No. of animals per sex per dose:

Preliminary test: 2 animals/sex/dose
Main test: For the 500 and 1000 mg/kg test substance dose groups used 5 animals of each sex. For the 2000 mg/kg test substance dose group and vehicle control group, 10 animals of each sex were used.

Control animals:

yes

Positive control(s):

Chlorambucil (reference substance) was administered by intraperitoneal injection at a dosage of 30 mg/kg in aqueous 10% ethanol.

Examinations

Tissues and cell types examined:

Smears were prepared from the bone marrow cells of the femur.

Details of tissue and slide preparation:

Following carbon dioxide inhalation, animals were killed by cervical dislocation. The femurs of each animal were dissected out and cleaned of adherent tissue. The epiphyses were cut off to gain access to the marrow canal. Marrow cells were washed out with 2.5 ml foetal calf serum using a syringe and needle. These cells were centrifuged and the majority of the supernantant fluid was discarded, the remaining fluid was used to resuspend the cell pellet. Single drops of the cell suspension were placed onto clean dry slides, three smears (two smears for the preliminary study) were prepared and the slides left to dry. Cells were then fixed with methanol and stained using 5% Giemsa stain. After staining, slide were washed with buffer, air-dried and cleared in xylene, coverslips were applied using a DPX mountant.

Evaluation criteria:

The test substance can be considered to have caused clastogenic or other damage leading to micronucleus production if the following criteria are met:
- A statistically significant increase in micronucleated polychromatic erythrocyte are observed at least one dose levels in either or both sex.
- the increases are reproducible in animals of the same sex and same treatment group(s)
- the increases exceed the lab's historical range of the control group
- Evidence of a dose-response relationship increases in both sexes or increases at more than one sacrifice time will be considered to support the conclusion.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

weight loss seen in all mice at 1000 and 2000 mg/kg was of uncertain significance

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary study: All animals survived to the scheduled termination. Treatment related effect were observed at all test concentrations and included: underactivity (14 mice), piloerection (4 mice) and flat posture (1 mouse). One male of the 2000 mg/kg dose group was unclean at the base of the tail (observed from day 2 until scheduled termination).

Weight loss was apparent in 16 mice 24 hours after treatment and in 14 mice after 48 hours.

The polychromatic:mature erythrocyte ratios are given in table 1 (below). Bone marrow toxicity was suggested by a reduction in the ratio of polychromatic to mature erythrocytes to 0.7 and 0.6 in mice of the 1500 and 2000 mg/kg dose groups respectively. In view of the small sample of animals used in this test and absence of concurrent control, this data should be treated with caution. From this data the highest dose selected for the main test was 2000 mg/kg.

Main test: although changes in bodyweight were observed in the majority of test animals in this study, these were not considered to be significant changes due to the short timespan between weighings. In the positive control group (chlorambucil) weight loss was observed in all mice during the 24 hour period between dosing and sacrifice. Clinical signs were observed at all concentrations tested, these included: underactivity (29 mice), piloerection (8 mice) and hunched postures (5 mice). Mice of the 2000 mg/kg dose group showed signs that included flat posture (2 mice) and unclean fur at the base of the tail (6 mice).

No statistically significant differences in the frequencies of micronucleated polychromatic cells were observed between sexes in any dose group (see table 2). The positive control group (chlorambucil) produced a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes and this increase confirms the sensitivity of the test system.

The polychromatic to mature erythrocyte ratio for each dose group was similar to corresponding vehicle control group values at each sacrifice time.

Any other information on results incl. tables

Table 1. Preliminary study: polychromatic: mature erythrocyte ratios

Group	Treatment (mg/kg)	Animal number	Polychromatic cells (P)		P:M ratio
				Mature cells (M)	By animal	By sex	By group
1	500	1M	1184	100	1.2	1	0.9
		2M	1001	1110	0.9
		9F	1013	1184	0.9	0.8
		10F	1042	1249	0.8
2	1000	3M	1023	1092	0.9	1	0.9
		4M	1017	1050	1
		11F	1006	1066	0.9	0.8
		12F	1030	1365	0.8
3	1500	5M	1000	1838	0.5	0.5	0.7
		6M	954	2054	0.5
		13F	1013	1090	0.9	0.8
		14F	1026	1419	0.7
4	2000	7M	1005	1702	0.6	0.6	0.6
		8M	1004	1827	0.5
		15F	1001	2022	0.5	0.6
		16F	1019	1593	0.6

P:M ratio = Ratio of polychromatic to mature erythrocytes

Table 2. Main study. Polychromatic: mature erythrocyte ratio

Group	Treatment (mg/kg)	Frequency of micronucleated polychromatic erythrocytes		P:M ratio
		Mean +/- SD	Range
24 hour sacrifice time
1	Sterile water	0.6 +/-0.8	0.0 - 1.8	1
2	Nu-Film-17 (500 mg/kg)	0.9 +/- 0.7	0.0 - 2.0	0.9
3	Nu-Film-17 (1000 mg/kg)	0.6 +/- 0.6	0.0 - 1.5	0.9
4	Nu-Film-17 (2000 mg/kg)	0.9 +/- 1.0	0.0 - 2.8	0.8
5	Chlorambucil (30 mg/kg)	45.6 +/- 18.9*	20.7 - 87.1	1
48 hour sacrifice time
1	Sterile water	1.0 +/- 0.8	0.0 - 2.0	1
4	Nu-Film-17 (2000 mg/kg)	0.4 +/- 0.6	0.0 - 1.8	0.9

* Highly significant (P<0.01)

P:M ratio: ratio of polychromatic to mature erythrocytes

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of this study, no evidence of chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes was obtained following intraperitoneal administration at high dosage.

Executive summary:

In this mouse bone marrow micronucleus assay, 5 animals/sex/dose (the highest dose group and negative control group used 10 animal/sex) were treated intraperitoneally at doses of 0, 500, 1000 and 2000  mg/kg bw.  Bone marrow cells were harvested at 24 h (for all dose groups) and 48 hours (high dose group only) post-treatment.  The vehicle was sterile water.

 

There were signs of toxicity during the study which included underactivity (29 mice), piloerection (8 mice) and hunched postures (5 mice). Mice of the 2000 mg/kg dose group showed signs that included flat posture (2 mice) and unclean at the base of the tail (6 mice).  The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow in test group animals.