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 based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
10 Feb-28 Mar 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo 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): only trade name given
- Physical state: pale yellow liquid
- Analytical purity: no data
- Lot/batch No.: N567701
- Storage condition of test material: at room temperature in the dark
- Other: density approximately 860 kg/m³ (25 ºC)

Test animals

Species:
mouse
Strain:
other: Swiss, CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 31.2-32.4 (males, range of mean group values), 24.2-25.8 (females, range of mean group values)
- Assigned to test groups randomly: yes, assigned to treatment groups as they came to hand from delivery boxes
- Fasting period before study: no
- Housing: Five animals per sex per group were housed in labelled polycarbonate cages containing purified sawdust as bedding material (Woody SPF, supplied by B.M.I., Helmond, the Netherlands)
- Diet: standard pelleted laboratory animal diet (Carfil Quality BVBV, Oud-Turnhout, Belgium)
- Water: tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
dosing solutions were prepared directly prior to administration by dissolving the test substance in corn oil. The dosing volume was 10 mL/kg bw.
Duration of treatment / exposure:
Not applicable
Frequency of treatment:
Single treatment
Post exposure period:
24 or 48 h
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control animals were dosed with cyclophosphamide.
- Route of administration: intraperitoneal injection
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:
Tissue: bone marrow
Cell type: bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A suitable dose range for the micronucleus test was selected based on the results of a dose range-finding study. Two dose groups, each comprising 3 males and 3 females, received a single dose of the test substance. The animals were observed for 3 days, during which mortality and physical condition were recorded daily.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
The animals were dosed once and sacrificed by cervical dislocation at 24 or 48 h (2000 mg/kg bw test substance and vehicle control) or 24 h only (500 and 1000 mg/kg bw test substance, and positive control group)

DETAILS OF SLIDE PREPARATION:
The femur bone was removed, and the bone marrow canal exposed and flushed with approximately 2 mL of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue) and marked (with the NOTOX study identification number and the animal number). The drop was spread by moving a clean slide at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were then air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.The slides were automatically stained using the 'Wright-stain-procedure' in an 'Ames' HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

METHOD OF ANALYSIS:
Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.
Evaluation criteria:
A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range.

A test substance is considered positive in the micronucleus test if:
It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.
A test substance is considered negative in the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups alone.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.
Statistics:
The Wilcoxon Rank Sum Test; two-sided test (P < 0.05) was for all statistical calculations.

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
- Dose range: 1000 and 2000 mg/kg bw
- Clinical signs of toxicity in test animals: no effects were observed

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes from the bone marrow of test substance treated animals (see Table 1 and 2). The positive control substance (cyclophosphamide) induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes, showing the positive control was valid.
- Ratio of PCE/NCE (for Micronucleus assay): The animals of the groups that were treated with the test substance did not show a decrease in the ratio of polychromatic to normochromatic erythrocytes compared with the vehicle controls. As the highest dose of the test substance was the recommended limit dose according to the guideline, the study result is acceptable, despite the lack of toxicity at the highest dose level. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared with the vehicle controls.

Any other information on results incl. tables

Table 1: Results of the in vivo micronucleus assay in male animals

Exposure group

Sampling time (h)

Number of animals

Number of micronucleated PCEs/ 2000 PCEs (mean±SD)

Ratio PCEs/ NCEs (mean±SD)

Vehicle (corn oil)

24

5

0.4±0.5

0.99±0.02

Vehicle (corn oil)

48

5

0.2±0.4

1.01±0.04

500 mg/kg bw

24

5

0.8±0.8

1.00±0.03

1000 mg/kg bw

24

5

0.2±0.4

0.99±0.01

2000 mg/kg bw

24

5

0.6±0.9

1.01±0.04

2000 mg/kg bw

48

5

0.4±0.5

1.00±0.01

Cyclophosphamide

48

5

25.0±8.0*

0.51±0.20

PCE = polychromatic erythrocyte

NCE = normochromatic erythrocytes

*Significantly different from corresponding control group (Wilcoxon Rank Sum Test, P < 0.05)

 

 

Table 2: Results of the in vivo micronucleus assay in female animals

Exposure group

Sampling time (h)

Number of animals

Number of micronucleated PCEs/ 2000 PCEs (mean±SD)

Ratio PCEs/ NCEs (mean±SD)

Vehicle (corn oil)

24

5

0.6±0.9

1.01±0.02

Vehicle (corn oil)

48

5

0.0±0.0

0.99±0.02

500 mg/kg bw

24

5

1.2±0.8

1.02±0.02

1000 mg/kg bw

24

5

0.6±0.5

0.98±0.02

2000 mg/kg bw

24

5

0.4±0.5

0.99±0.07

2000 mg/kg bw

48

5

0.4±0.5

1.00±0.02

Cyclophosphamide

48

5

13.2±2.8*

0.56±0.05

PCE = polychromatic erythrocyte

NCE = normochromatic erythrocytes

*Significantly different from corresponding control group (Wilcoxon Rank Sum Test, P < 0.05)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative