Registration Dossier

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:
From 14.01.1991 to 25.03.1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD guideline 474 and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Camphen techn. rein
- Physical state: clear wax-like solid
- Analytical purity: 78 %
- Purity test date: September 28th, 1990.
- Lot/batch No.: 54/90 dated May 31st, 1990
- Stability in solvent: Proved for 4 hours, dated November 16th, 1990
- Storage condition of test material: dark at room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation: males: 28.7 g (25.33 g); females: 23.5 g (21.27 g)
- Housing: In fully air-conditioned rooms in Macrolon cages (Type 3), on softwood granulate in groups of 5 animals
- Diet (e.g. ad libitum): rat/mice diet Altromin 1324 (Altromin-GmbH, Lage/Lippe), ad libitum
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 ºC
- Humidity (%): 55 ± 10 %
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 14.01.1991 To: 17.01.1991

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: sesame oil (Oleum Sesami Ph.Eur.III, Fa. Pharm. Fabrik GmbH, Ffm.)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test compound dilutions were prepared fresh each day. 10000 mg Camphen techn. rein were weight in a beaker, mixed with sesame oil (Oleum Sesami Ph.Eur.III, Fa. Pharm. Fabrik GmbH, Ffm.), washed out in a 25 ml flask and topped up to the calibration mark. A solution was formed.
Duration of treatment / exposure:
A single dose was administered.
Frequency of treatment:
A single dose was administered.
Post exposure period:
Test compound and negative control: Animals were killed 24, 48 or 72 hours after administration.
Positive control: Animals were killed 24 h after administration.
Doses / concentrations
Remarks:
Doses / Concentrations:
4000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5 males and 5 females in each dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
Endoxan (Cyclophosphamid) was used as positive control substance and was administered orally at a dose of 50 mg per kg bodyweight.

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
For each animal, about 3 mL foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 24 hours.
Evaluation criteria:
1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation are coded to ensure that the group which they belonged to remains unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically; comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase).
Statistics:
The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided). The statistical evaluations were performed using the "Diamant" computer program Version 2.0, supplied by the Department of Information and Communication Hoechst AG. All statistical results are based on a 95 % level of significance. Actual data were also compared with historical controls.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
The 4000 mg per kg bodyweight dose level was chosen since a preliminary study had shown it to be the maximum applicable dose.

RESULTS OF DEFINITIVE STUDY
All animals survived after application of 4000 mg Camphen techn. rein per kg bodyweight. The following signs of toxicity were observed: narrowed palpebral fissures, diarrhea, uncoordinated gait and stilted gait.
Within 3 hours after application all animals were free of clinical signs of toxicity.
The bone marrow smears were exami ned for the occurance of micronuclei in red blood cells. The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with Camphen techn. rein and was statistically not different from the control values.
Endoxan induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extent.

Any other information on results incl. tables

Under the conditions of the present study, Camphene is not mutagenic in the micronucleus test.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the present study, Camphene is not mutagenic in the micronucleus test.
Executive summary:

Camphene was tested in the micronucleus test. The study described was performed according to the OECD guideline 474. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0 and 4000 mg/kg bw. The 4000 mg/kg bw dose level was chosen since a preliminary study had shown it to be the maximum applicable dose.

The animals were treated once with the test compound and according to the test procedure the animals were killed 24, 48 or 72 hours after administration of the test compound.

Endoxan was used as positive control substance and was administered orally at a dose of 50 mg/kg bw.

The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with Camphene and was statistically not different from the control values.

Endoxan induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extent.

The results indicate that, under the conditions of the present study, Camphene is not mutagenic in the micronucleus test.