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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From January 14 to 17, 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study following OECD guideline 474 with minor deviations: no data on evaluation criteria
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

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:
yes
Remarks:
no data on evaluation criteria
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Camphen techn. rein
- Ordered by: Gb. A, Werk Gersthofen, PA Terpene, Dr. Gscheidmeier
- Physical state: Clear wax-like solid
- Analytical purity: 78%
- Purity test date: September 28, 1990
- Lot/batch No.: 54/90
- Storage condition of test material: Dark at room temperature
- Stability: Until May 1991 at approx. 25 °C
- Stability in solvent: Stable for 4 hours
- Specific gravity: ca. 0.87 g/mL (20 °C); ca. 0.84 g/mL (50 °C)
- pH value in water: ca. 7

Test animals

Species:
mouse
Strain:
other: NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF breeding colony
- Strain: Hoe: NMRKf (SPF71)
- Age at study initiation: 7 weeks
- Weight at study initiation: Males: 25-33 g; females: 21-27 g
- Housing: Housed in groups of five in Macrolon cages
- Diet (e.g. ad libitum): Rat/mice diet Altromin 1324 (Altromin-GmbH, Lage/Lippe, Germany), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 10%
- Air changes (per hour): 10/hour
- Photoperiod (hours dark / hours light): 12 hours dark/12 hours light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Sesame oil
- Concentration of test material in vehicle: 40% w/v
- Amount of vehicle (if gavage): 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test material solutions were prepared fresh each day by mixing the test material with sesame oil.
Duration of treatment / exposure:
24, 48 or 72 hours (test compound and negative control); 24 hours (positive control)
Frequency of treatment:
Single
Post exposure period:
No
Doses / concentrations
Remarks:
Doses / Concentrations:
4000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Five
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamid - Endoxan (Charge 098492)
- Route of administration: Oral (gavage)
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:
Femur bone
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose selection was based on a preliminary range-finding test. Oral administration of the test substance at 5000 mg/kg bw caused heavy clinical symptoms in male and female mice. 4000 mg/kg bw was considered the maximum tolerated dose and was selected as dose level for the main study.

TREATMENT AND SAMPLING TIMES: Femora were removed for marrow extraction from the animals killed by CO2 asphyxiation at 24, 48 or 72 hours after exposure.

DETAILS OF SLIDE PREPARATION: Bone marrow cells extracted, preparations spread on slides and air-dried for 24 hours. The slides were fixed in methanol, stained with May-Grunwald solution and Giemsa and coded.

METHOD OF ANALYSIS: Slides were scanned to determine the number of polychromatic erythrocytes (PCEs) with micro nuclei per 1000 PCEs and number of normochromatic erythrocytes (NCEs) with micronuclei per 1000 NCEs per animal. In addition, PCE:NCE ratio was also determined.
Evaluation criteria:
No data
Statistics:
- Statistical analysis was performed using the ‘Diamant’ computer program Version 2.0.
- Proportion of PCE or NCE with micronuclei was evaluated using Wilcoxon method (paired, one-sided, increase).
- Ratio of PCE/NCE was evaluated using Wilcoxon method (paired, two-sided).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
observed signs of toxicity resolved within 3 hours of application
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose: 5000 mg/kg bw
- Clinical signs of toxicity in test animals: Stilted gait, increased tonus of the abdominal position, diarrhea, narrowed palpebral fissures back-arched position, reduced spontaneous activity, piloerection

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity including narrowed palpebral fissures, diarrhea, uncoordinated gait and stilted gait were observed at 4000 mg/kg bw and resolved completed with 3 hours of application.
- Frequency of micronuclei in PCEs and NCEs and PCE:NCE ratio: See table 1

Any other information on results incl. tables

Table 1: Micronucleus assay - Summary table

Sex

Dose

(mg/kg bw)

Sampling

time

Number

of

animals

Erythrocytes

Erythrocytes with micronuclei

PCE/NCE#

PCE (mean)

NCE (mean)

Mean

SD

No.

%

SD

 

Mutagenic

index

No.

%

SD

 

Mutagenic

index

Male

0 (vehicle

 control)

24 h

 5 

 0.92 

 0.16 

2

0.16

0.11

I

1

1

0.08

0.08

I

 1.0 

 

 4000 

 5 

 1.02 

 0.10 

2

0.18

0.08

 -I

1.1

1

0.08

0.08

-I

 1.0 

 

Positive

 control

 5 

0.82

 0.15 

23

2.32

0.36

*A

14.5

1

0.14

0.11

-I

 1.7 

Female

0 (vehicle

 control)

 5 

 1.03 

 0.15 

1

0.12

0.11

I

1

1

0.08

0.08

I

 1.0 

 

 4000 

 5 

 0.94 

 0.11 

1

0.06

0.05

-I

0.5

1

0.06

0.05

-I

 0.7 

 

Positive

 control

 5 

 0.79 

 0.07 

21

2.1

0.49

*A

17.5

2

0.22

0.04

*A

 2.8 

Male

0 (vehicle

 control)

48 h

 5 

 0.90 

 0.13 

2

0.2

0.1

I

1

2

0.16

0.09

I

 1.0 

 

 4000 

 5 

 0.69 

 0.17 

1

0.14

0.11

-I

0.7

1

0.1

0.07

-I

 0.6 

Female

0 (vehicle

 control)

 5 

 0.96 

0.18

1

0.12

0.04

I

1

1

0.08

0.08

I

 1.0 

 

 4000 

 5 

 0.92 

 0.19 

1

0.14

0.05

-I

1.2

1

0.1

0.1

-A

 1.3 

Male

0 (vehicle

 control)

72 h

 5 

 1.06 

 0.19 

2

0.18

0.08

I

1

0

0.04

0.05

I

 1.0 

 

 4000 

 5 

 1.02 

 0.15 

2

0.16

0.05

-I

0.9

1

0.1

0.1

-I

 2.5 

Female

0 (vehicle

 control)

 5 

 1.05 

 0.10 

2

0.2

0.1

I

1

1

0.08

0.08

I

 1.0 

 

 4000 

 5 

 1.09 

 0.12 

1

0.1

0

-I

0.5

1

0.08

0.08

-I

 1.0 

#Mean number of polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) = 1000; I = within the normal range; - = no difference from control (P > 0.5); * = significantly different from control (P < 0.05); A = outside the normal range

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the test conditions, camphene is not considered as mutagenic in the mouse bone marrow micronucleus test according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° 1272/2008.
Executive summary:

In a bone marrow micronucleus test, performed according to GLP and OECD guideline 474, NMRI mice (5/sex/dose) were given a single oral (gavage) dose of camphene in sesame oil at concentrations of 0 and 4000 mg/kg bw. Bone marrow was extracted after 24, 48 or 72 hours of exposure and the prepared slides were scanned to determine the number of polychromatic erythrocytes (PCEs) with micro nuclei per 1000 PCEs and number of normochromatic erythrocytes (NCEs) with micronuclei per 1000 NCEs per animal. In addition, PCE:NCE ratio was determined. A preliminary range-finding test was also conducted in which heavy clinical symptoms were observed in male and female mice at 5000 mg/kg bw.

 

Positive control (cyclophosphamide, 50 mg/kg bw) induced a marked and statistically significant increase of the number of polychromatic erythrocytes with micronuclei in both males and females indicating the sensitivity of the test system. No statistically significant increases in the frequency of micronucleated PCEs or NCEs and PCE:NCE ratios were observed at any dose levels with camphene.

 

Under the test conditions, camphene is not considered as mutagenic in the mouse bone marrow micronucleus test according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° 1272 /2008.