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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:
From 28-Nov-2002 to 23-Jan-2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
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

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstrasse 27, 33178 Borchen
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: Male animals-mean = 181.4 g (= 100 %) (min = 173 g, max = 191 g); female animals- mean = 146.5 g (=100 %) (min = 140 g, max = 154 g)
- Assigned to test groups randomly: Yes
- Housing: Five animals per cage in transparent macrolon cages (type IV) on soft wood granulate in an air conditioned room.
- Diet (e.g. ad libitum): Rat/mice diet ssniff R/M-H (V 1534), ad libitum, ssniff® GmbH, Postbox 2039, 59480 Soest
- Water (e.g. ad libitum): Tap water in plastic bottles, ad libitum
- Acclimation period: 5 d under study conditions
- Animal identification: Fur marking with KMnO4 and cage numbering

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C (except short lasting deviations due to disturbances of air condition)
- Humidity (%): 50% (except short lasting deviations due to disturbances of air condition)
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

IN-LIFE DATES: From December 03, 2002 to December 05, 2002

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: deionised water
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SUSPENSION: On the day of first administration the test substance was dissolved in deionized water at an appropriate concentration.
A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

Stability and homogeneity in the vehicle : guaranteed for 96 hours in deionized water by the sponsor (archived with the raw data)


Duration of treatment / exposure:
2 d
Frequency of treatment:
twice at an interval of 24 h
Doses / concentrations
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
5/sex/group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control: Cyclophosphamide
Dissolved in: distilled water
Dose: 40 mg/kg bw
Route and frequency of administration: Oral (gavage), once
Volume Administered: 10 mL/kg bw

Examinations

Tissues and cell types examined:
- 2,000 polychromatic erythrocytes were counted for each animal.
- The number of cells with micronuclei was recorded, not the number of individual micronuclei.
- The ratio of polychromatic erythrocytes to 200 normochromatic erythrocytes was determined.
- Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: No preliminary experiments were performed, as corresponding toxicological information was available. Since 5000 mg per kg body weight resulted in no lethality in acute oral toxicity testing a limit test with 2000 mg per kg body weight was performed.
.

-Extraction of the bone marrow: Animals were killed by carbon dioxide asphyxiation 24 h after dosing. One femora was removed and the bone freed of muscle tissue. The proximal end of the femora was opened, the bone marrow flushed into a centrifuge tube containing about 3 mL of fetal bovine serum and a suspension was prepared. The mixture was then centrifuged for 5 minutes at approximately 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 h.

-Staining procedure: The slides were stained as follows:-
-5 minutes in methanol
-5 minutes in May-Grunwald's solution
-brief rinsing twice in distilled water
-10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
-rinsing in distilled water
-drying
-coating with Entellan
Evaluation criteria:
Both biological and statistical significances were considered together for evaluation purposes. A test substance is considered as positive if there is a significant dose- related increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant dose-related increase in the number of micronucleated polychromatic erythrocytes is considered non-clastogenic in this system.
Statistics:
Assuming the study is valid based on a monotone-dose-relationship, one-sided Wilcoxon tests were performed initially comparing control values with those of the highest dose group. A significance level of 5% is adopted for all tests.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
Exposure was assured by the presence of light grey coloured tissues at necropsy
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
All animals survived after treatment. No signs of toxicity were detected. All dosed animals showed black discoloured faeces and Body surface showed grey discolorations which were noted on hairless areas 24 hours after the first administration up to the end of study.
The dissection of the animals revealed a nearly black coloured content of the gastro-intestinal tract and light grey coloured tissues.
The bone marrow smears were examined for the occurrence of micronuclei in red blood cells. The incidence of micronucleated polychromatic erythrocytes in the test tem-treated groups was within the normal range of the negative control groups (mean of micronucleated polychromatic erythrocytes per 2000 cells: 1.7 — 4.9). No statistically significant increase in micronucleated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and differed less than 20% from the control values.
Cyclophosphamide (Endoxan®) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system.

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the rat. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay.
Executive summary:

A study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD Guideline 474, EPA OPPTS 870.5395 and EU Method B.12, in compliance with GLP.

The test compound was dissolved in deionized water and was given twice at an interval of 24 hours as oral doses of 2000 mg per kg body weight to male and female rats (Hsd:Sprague Dawley), based on available acute oral toxicity study data (see 5.6). At study start the animals were 6 weeks of age and had mean body weights of 181.4 g (M) and 146.5 g (F). According to the test procedure the animals were killed 24 hours after the last administration.

Endoxan®was used as positive control substance and was administered once orally at a dose of 40 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei was not significantly increased compared with the control. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with the test substance and differed less than 20% from the control value.

Endoxan®induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that the test substance is not clastogenic in the micronucleus testin vivo.