Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report date:
1987

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Version / remarks:
1983 version
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EEC Directive 84/449
Version / remarks:
1984 version
Deviations:
not specified
GLP compliance:
yes
Type of assay:
mammalian bone marrow chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
1,4-naphthoquinone
EC Number:
204-977-6
EC Name:
1,4-naphthoquinone
Cas Number:
130-15-4
Molecular formula:
C10H6O2
IUPAC Name:
1,4-naphthoquinone
Test material form:
solid

Test animals

Species:
hamster, Chinese
Strain:
not specified
Details on species / strain selection:
Detailed as EMD (brother-sister inbreeding)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: E. Merck, Darmstadt
- Age at study initiation: 7 to 9 weeks
- Weight at study initiation: 44.2 g (males) and 37.6 g (females)
- Assigned to test groups randomly: yes
- Fasting period before study: No
- Housing: Housed individually in Makrolon cages type 1 on softwood chippings under conventional conditions.
- Diet (e.g. ad libitum): Ad libitum (free access to ssniff hamster mixed food.
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 29°C
- Humidity (%): 33 to 71%
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.25% aqueous Methocel K4M Premium
- Justification for choice of solvent/vehicle: Standard vehicle
- Concentration of test material in vehicle: Not specified
- Amount of vehicle (if gavage or dermal): Not specified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

Dosed via oral gavage
Duration of treatment / exposure:
Single dose for all relevant animals (1 day)
Frequency of treatment:
Once on day of treatment
Post exposure period:
6 to 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day
Dose / conc.:
40 mg/kg bw/day
Dose / conc.:
120 mg/kg bw/day
No. of animals per sex per dose:
5 males and 5 females per dose group (70 in total). Any animals that died during the study were replaced with those from the same stock. At 3 hours before sacrifice and bone marrow sampling, the animals were treated with colcemide to arrest dividing bone marrow cells in the metaphase.
Control animals:
yes, concurrent vehicle
Positive control(s):
- Name: Endoxan (Cyclophosphamide).
- Justification for choice of positive control(s): Standard positive control
- Route of administration: Oral gavage
- Doses / concentrations: 20 mg dissolved in 10 mL Aqua pro injectione directly before use. A dose of 20 mg/kg was used on study.

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on results of a preliminary study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Bone marrow sampled at 24 hours post dose in animals receiving 15 or 40 mg/kg and 6, 24 and 48 hours post dose in animals receiving 120 mg/kg. Positive control animals were sampled at 24 hours post dose.

DETAILS OF SLIDE PREPARATION: Immediately after the hamsters were euthanized by CO2, both femora of each animal were dissected, cleaned from the rest of teh muscles and the epiphyses removed. bone marrow cells from both the femora per animal were flushed out withs aline and collected in a centrifuge tube and suspended. After centrifugation the supernatant was drawn off and the sediment suspended in KCl solution at 37°C. After hypotonic treatment for 10 minutes, the cells were futher centrifuged and the supernatant discarded. The cells were fixed twice in a mixture of acetic acid and methanol, dropped onto the slides in fresh fixative and flame dried. After over-night air drying at room temperature the slides were stained with aceto-orcein solution, differentiated in ethanol dipped twice in xylene and mounted in Eukitt.

METHOD OF ANALYSIS: All slides were coded before microscopic examination to ensure a lack of bias. 100 metaphases per animal were socred for structural chromosome aberrations using zeiss light microscopes with phase optics.

Morphological observations were scored as follows:
Gap - achromatic region in chromatid(s) not greater than the width of teh chromatid.
Break - achromatic region in chromatid(s) greater than the width of a chromatid or a discontinuity with displacement.
Exchange - aberrations arising from an exchange between one or two chromatids. These may be chromosome or chromatid interchanges. Only asymmetrical or chromatid exchanges will normally be recognised.
Multiple aberration - cells with more than 5 aberrations, gaps excluded.
Specific aberration - atypic chromosomes and pulverised metaphases were scored as specific aberrations.

The position of each aberrant metaphase was recorded by the Vernier reading and photographs taken of each aberrant metaphase. The frequency of polyploid cells were based on scoring of 1000 mitoses per animal. The estimation of the mitotic index was based on scording 1000 cells per animal.

Evaluation criteria:
Statistical significance compared with control groups would indicate a positive response.
Statistics:
The percentage of structural aberrant metaphases per animal, number of mitotic cells per 1000 cells per animal and the number of polyploid cells per 1000 mitoses per animal were used as parameters for statistical analysis.

Regarding structural aberrations two evaluations were conducted; one in which cells carrying gaps and isogaps as the only aberration are included and one where such cells are excluded.

The evaluation was performed applying a non parametric test (i.e. a modification of the Mann-Whitney-Wilcoxon test for tied values. Testing was performed against the one-sided alternative.

As more than one group was compared with controls, the level of significance was modified by the Bonferroni correction according to the number of groups. The values of positive controls were treated in the same way.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Chinese Hamsters were dosed at 100, 120, 150, 200 or 250 mg/kg. Mortality was observed at the three highest doses. The dose of 120 mg/kg showed clear toxic effects (dyspnea and diarrhoea). Consequently this dose was chosen as the highest dose in the main study.

RESULTS OF DEFINITIVE STUDY

Structural aberrations: No chromosamal damaging affect was observed.
Polyploid cells: No statistically signifcant increase of polyploid cells were observed.
Mitotic index: No treatment-related variation was observed.
Bodyweight: No treatment-related variation was observed.
Clinica signs: 15 mg/kg (one animal observed to be prone), 120 mg/kg (three animals showed dyspnea, two showed ptosis, one had diarrhoea and one animal showed titubation. Most of the hamsters showed bluish urine.
Mortality: One animal died in each dose group.
Positive control: For the positive control, a statistically significant increase of aberrant metaphases for both evaluations (gaps included and excluded) was found.
Evidence of exposure: Clear evidence of systemic exposure and absorption of the test substance was apparent based on the observed clinical signs and mortality. Based on this obvious exposure it is very likely that the bone marrow was exposed to the test substance allowing for a full assessment of the clastogenic potential of the test substance.

The mean percentage of aberrant metaphases found 24 hours after treatment (gaps included) was 1.6% (negative control), 1.4% (15 mg/kg), 1.2% (40 mg/kg) and 1.3% (120 mg/kg). For the 120 mg/kg group the values were 2.0 and 1.3% at 6 and 48 hours respectively.
The corresponding values (gaps exlcuded) were 1.2, 1.0, 0.9 and 1.2% for the 24 hour groups and 1.7 and 1.1% for the 6 and 48 hour groups.

In the positive control the values were 6.1% (gaps included) and 5.9% (gaps excluded).

Any other information on results incl. tables

Aberrations per 100 cells (mean values)

Dose (mg/kg)

Time (hours)

Gaps

Iso-gaps

Breaks

Iso-breaks

Exchanges

Multiple aberr.

Specific aberr.

Total aberr. Incl.

Total aberr. Excl.

0

24

0.4

0

0.7

0.

0

0

0

1.8

1.4

15

24

0.4

0

0.6

0.4

0

0

0

1.4

1.0

40

24

0.3

0

0.6

0.3

0

0

0

1.2

0.9

120

6

0.3

0

0.8

1.0

0

0

0

2.1

1.8

120

24

0.1

0

0.6

0.6

0

0

0

1.3

1.2

120

48

0.2

0

0.6

0.7

0

0

0

1.5

1.3

20 (EMD)

24

0.2

0

3.5

2.1

1

0.5

0

7.3

7.1

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, no chromosome damaging effects were observed in the bone marrow metaphases of male and female Chinese hamsters after a single oral dose at 15, 40 or 120 mg/kg of the test substance.