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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Apr 2001 - 04 Jul 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 8-10 weeks
- Weight at study initiation: Males: 27.4 – 31.0 g; Females: 24.6 – 29.0 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: five per sex per polycarbonate cage
- Diet: standard pelleted laboratory diet (Altromin, code VRF-1, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: none
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was weighed on the day of use and the weighed test substance was kept at ambient temperature under nitrogen until use. The test substance was used within 4 h after weighing.
Duration of treatment / exposure:
Not applicable
Frequency of treatment:
Single treatment
Post exposure period:
24 and 48 h
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 per sex per dose at any sampling time point
Control animals:
other: yes (0.9% NaCl)
Positive control(s):
- cyclophosphamide (CP)
- Justification for choice of positive control(s): none given in report - standard control
- Route of administration: by oral gavage
- Doses / concentrations: 50 mg salt/kg bw

Examinations

Tissues and cell types examined:
Bone marrow smear of the femurs (polychromatic erythrocytes with and without micronucleus, normochromatic erythrocyes)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on a range finding assay in 4 animals with a dose of 2000 mg/kg bw

TREATMENT AND SAMPLING TIMES:
500, 1000, 2000 mg/kg bw: 24 h post-administration
2000 mg/kg bw: 48 h post-administration

DETAILS OF SLIDE PREPARATION: cells were washed, re-suspended, fixed on glass slides with methanol, stained using the "Wright-stain procedure"

METHOD OF ANALYSIS: light microscope - area selected at x100, analysis performed at x 1000
2000 PCEs scored for presence of micronuclei. The ratio of PCE to NCEs, (normochromatic erythrocytes) was determined by counting and differentiating the first 1000 erythrocytes.
Evaluation criteria:
The test article would have been considered to induce a positive response if a biologically and statistically significant increase in the frequency of micronucleated polychromatic erythrocytes is observed at one or more doses (Wilcoxon Rank Sum Test, two-sided test at p= 0.05) at any sampling time in the combined data for both sexes or in the male or female animals separately.
The test article was judged negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control values was observed at any sampling time.
Statistics:
Wilcoxon Rank Sum Test

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: No abnormalities were observed within 3 days.
- Evidence of cytotoxicity in tissue analyzed: No abnormalities were observed.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: No induction was observed.
- Ratio of PCE/NCE: No change of the ratio was observed after treatment with test substance. A decrease of the PCE/NCE ratio was observed after treatment with cyclophosphamide.

Any other information on results incl. tables

Summary of results of bone marrow micronuleus analysis following single ip administration

Treatment (10 ml/kg bw)

Sex

Time (hr)

No. of animals

PCE/total erythrocytes

Micronuclei/1000 PCE

0.9% NaCl

M

24

5

1.00 ± 0.06

1.2 ± 1.6

 

F

24

5

1.02 ± 0.04

3.8 ± 0.8

500

M

24

5

0.96 ± 0.05

1.6 ± 1.8

 

F

24

5

0.93 ± 0.01

2.4 ± 1.8

1000

M

24

5

0.98 ±0.08

0.8 ± 1.1

 

F

24

5

0.99 ± 0.07

1.6 ± 1.1

2000

M

24

5

0.98 ± 0.07

2.6 ± 1.5

 

F

24

5

0.96 ± 0.04

1.6 ± 1.8

Positive control

M

48

5

0.53 ± 0.11

*23.8 ± 9.3

 

F

48

5

0.408 ± 0.01

*18.0 ± 4.2

2000

M

48

5

0.99 ± 0.04

3.8 ± 3.6

 

F

48

5

0.98 ± 0.06

2.2 ± 1.9

*Statistically significant increase, p < 0.05

Applicant's summary and conclusion

Conclusions:
The test material has been tested according to OECD 474 and under GLP conditions. No significant increase in the incidence of micronucleated polychromatic erythrocytes was observed in the bone marrow of male or female rats 24 or 48 hours after a single oral dose of up to and including 2000 mg/kg bw. A reduction in the PCE/total erythrocyte ratio was only observed in the 48 hour positive control group. It is concluded that the test substance is negative for the induction of micronuclei under the conditions of the study.