Registration Dossier

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:
14 January 1991 - 17 January 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The analogue isobornyl acetate which shares the same functional groups with dextro alpha fenchyl acetate also has comparable values for the relevant molecular properties. Test method according to OECD 474. GLP study.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl acetate
EC Number:
204-727-6
EC Name:
Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl acetate
Cas Number:
125-12-2
Molecular formula:
C12H20O2
IUPAC Name:
(1S,2S,4S) 1,7,7-trimethylbicyclo[2.2.1]hept-2-yl acetate
Details on test material:
- Name of test material (as cited in study report): Isobornylacetat-Extra
- Molecular formula (if other than submission substance): C12H20O2
- Molecular weight (if other than submission substance): 196.286
- Smiles notation (if other than submission substance): CC(=O)O[C@H]1C[C@@H]2CC[C@@]1(C)C2(C)C
- InChl (if other than submission substance): 1/C12H20O2/c1-8(13)14-10-7-9-5-6-12(10,4)11(9,2)3/h9-10H,5-7H2,1-4H3
- Structural formula attached as image file (if other than submission substance): see Fig.
- Physical state: Liquid
- Analytical purity: 93.5 – 95.0 %
- Impurities (identity and concentrations): other terpene esters and camphene
- Lot/batch No.: 249/90
- Storage condition of test material: dark, at room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation: 28.7 g (males) and 22.8 g (females)
- Housing: in fully air-conditioned rooms in Macrolon cages (Type 3), on softwood granulate in groups of 5 animals
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: sesame oil
- Concentration of test material in vehicle: 20 % (w/v)
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test sample dilutions were prepared fresh each day. 500 mg test item were weight in a 25 mL flask, mixed with sesame oil and topped up to the calibration mark.
Frequency of treatment:
Once
Post exposure period:
24, 48 and 72 hours (killing times)
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
70 animals (35 male and 35 female):
Group 1: 0 mg/kg bw (5 males and 5 females) killing time: 24 h post administration
Group 2: 2000 mg/kg bw (5 males and 5 females) killing time: 24 h post administration
Group 4: 0 mg/kg bw (5 males and 5 females) killing time: 48 h post administration
Group 5: 2000 mg/kg bw (5 males and 5 females) killing time: 48 h post administration
Group 6: 0 mg/kg bw (5 males and 5 females) killing time: 72 h post administration
Group 7: 2000 mg/kg bw (5 males and 5 females) killing time: 72 h post administration
Control animals:
yes, concurrent vehicle
Positive control(s):
Endoxan(R). Group 3 (5 males and 5 females) killing time: 24 h post administration
- Route of administration: oral, gavage
- Doses / concentrations: 50 mg/kg bw (0.5% w/v in distilled water , 10 mL/kg bw)

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes cells (from both femora)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Preliminary studies were conducted to determine the highest administrable non lethal dose level. 3 mice per sex and per dose were exposed to 5000, 4000, 3000 and 2000 mg/kg bw test item.

TREATMENT AND SAMPLING:
After treatment, animals were killed by carbon dioxide asphyxiation 24, 48 and 72 hours after application.

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 (5 min, 1200 rpm) and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide and air-dried for 24 hours. The slides were then stained.

METHOD OF ANALYSIS:
1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) was determined.
Statistics:
The number of polychromatic erythrocytes with micronuclei and the number of normocytes with micronuclei were evaluated statistically. The comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase). The results of the treatment groups were compared with the corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided). All statistical results were based on a 95% level of significance.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
(see below)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 5000, 4000, 3000 and 2000 mg/kg
- Mortality:
At 5000 mg/kg bw: 1/3 males and 2/3 females died;
At 4000 mg/kg bw: 0/3 males and 1/3 females died;
At 3000 mg/kg bw: 0/3 males and 3/3 females died;
At 2000 mg/kg bw: No death were observed.
- Clinical signs of toxicity in test animals: Several clinical signs were observed at 3000-5000 mg/kg bw doses. At 2000 mg/kg bw, uncoordinated gait, increased spontaneous activity and stilted gait was observed.

RESULTS OF DEFINITIVE STUDY
- Mortality: All animals survived.
- Signs of toxicity: uncoordinated gait, increased spontaneous activity. These signs were fully reversible by 5-6 hours after application.
- Induction of micronuclei (for Micronucleus assay): The number of polychromatic (PCE) and normochromatic (NCE) erythrocytes containing micronuclei was not increased.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of PCE/NCE in both male and female animals remained unaffected by the treatment, and was statistically not different from the control values.
- Positive control: Endoxan induces 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 PCE/NCE was not changed to a significant extent.

Any other information on results incl. tables

Summary of findings in bone marrow erythrocytes:

Sex

Dose (mg/kg)

Sample time

No. animals

Erythrocytes

Erythrocytes with micronuclei

Poly mean

Normo

mean

 mean

No

%

 

Mut I

No

%

 

Mut I

Male

0

24 h

5

1000

1000

0.92

2

0.16

I

1.0

1

0.08

I

1.0

2000

5

1000

1000

1.07

2

0.18

-I

1.1

1

0.08

-I

1.0

Endoxan

5

1000

1000

0.82

23

2.32

*A

14.5

1

0.14

-I

1.7

Female

0

5

1000

1000

1.03

1

0.12

I

1.0

1

0.08

I

1.0

2000

5

1000

1000

0.86

1

0.08

-I

0.7

0

0.04

-I

0.5

Endoxan

5

1000

1000

0.79

21

2.10

*A

17.5

2

0.22

*A

2.8

Male

0

48 h

5

1000

1000

0.90

2

0.20

I

1.0

2

0.16

I

1.0

2000

5

1000

1000

0.78

1

0.08

-I

0.4

0

0.04

-I

0.2

Female

0

5

1000

1000

0.96

1

0.12

I

1.0

1

0.08

I

1.0

2000

5

1000

1000

0.86

1

0.14

-I

1.2

0

0.04

-I

0.5

Male

0

72 h

5

1000

1000

1.06

2

0.18

I

1.0

0

0.04

I

1.0

2000

5

1000

1000

1.08

1

0.10

-I

0.6

1

0.10

-I

2.5

Female

0

5

1000

1000

1.05

2

0.20

I

1.0

1

0.08

I

1.0

2000

5

1000

1000

1.20

1

0.14

-I

0.7

0

0.04

-I

0.5

Mut. I = Mutagenic index

- = No difference from control (P>0.05

I = within the normal range

* = Significantly different from control (P<0.05)

A = Outside the normal range

The data matrix is included in the reporting format attached.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Based on the read-across approach from experimental data with the analogue isobornyl acetate, dextro alpha fenchyl acetate was determined to be not mutagenic in the micronucleus test.
Executive summary:

An in-vivo micronucleus test was performed with the analogue isobornyl acetate according to OECD 474. Five mice per sex and per group (70 in total) were exposed to a single dose of test item at 2000 mg/kg bw, based on preliminary results. Cyclophosphamide was used as a positive control (5 mice per sex). Animals were killed 24, 48 and 72 hours after administration of the test compound. Cyclophosphamide was used as a positive control. For each animal, bone marrow smears were flushed from both femora and the slides were prepared for erythrocyte micronuclei observation. 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 and was statistically not different from the control values, indicating no mutagenicity. Based on these results, the read-across approach was applied and dextro alpha fenchyl acetate was determined to be not mutagenic in the micronucleus test.