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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OECD guideline 474).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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): EP202
- Physical state: Liquid, colorless, clear
- Analytical purity: The test substance is a mixture containing different components.
- Composition of test material, percentage of components:
- Test substance No.: 06/0723-1
- Batch identification: F701501mH
- Stability under test conditions: The stability of the test substance under storage conditions throughout the study period is guaranteed.
- Storage condition of test material: Room temperature

Test animals

Species:
mouse
Strain:
other: Crl:NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: mean: 30.1 g
- Assigned to test groups randomly: yes, under following basis: appropriate computer program
- Housing: single
- Diet: Standardized pelleted feed
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle: corn oil
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in corn oil, corn oil was used as vehicle.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: All test substance formulations were prepared immediately before administration.


Duration of treatment / exposure:
48 h (test substance), 24 h (positive controls)
Frequency of treatment:
twice with a 24-hour interval between both administrations (the positive controls were administered only once)
Post exposure period:
24 h
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw (10 mL solution/kg bw)
Basis:
nominal conc.
No. of animals per sex per dose:
5 male animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide; vincristine
- Justification for choice of positive control(s): The stability of cyclophophamide and vincristin is well-defined under the selected conditions, since both substances are well-established reference clastogens and aneugens, respectively.
- Route of administration: cyclophosphamide: oral administration, vincristine: intraperitoneal administration
- Doses / concentrations: cyclophosphamide: 20 mg/kg bw, vincristine: 0.15 mg/kg bw

Examinations

Tissues and cell types examined:
2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The test doses were selected on the basis of the non-GLP range finding part of the study (see Additional informations on results).


DETAILS OF SLIDE PREPARATION: The bone marrow was prepared according to the method described by Schmid (1976, The micronucleus test for cytogenetic analysis, Hollaender, A. (ed), Chemical Mutagens, Principles and Methods for their Detection, Volume 4, Plenum Press, New York; 1977, The micronucleus test, Kilbey et al. (eds), Handbook of Mutagenicity Test Procedures, Elsevier Scientific Publishing Company, Amsterdam - New York - Oxford) and Salamone et al (1980, Towards an improved micronucleus test. Studies on 3 model agents, mitomycin C, cyclophosphamide and dimethylbenzanthracene. Mut. Res., 74, 347 - 356). The slides were stained with eosin and methylene blue (modified May-Gruenwald solution or Wrights solution). After briefly rinsing in purified water, the preparations were soaked in purified water. Subsequently, the slides were stained with Giemsa solution. After rinsing twice in purified water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.


Evaluation criteria:
The mouse micronucleus test is considered valid if the following criteria are met: - The quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 2000 PCEs per animal and a clear differentiation between PCEs and NCEs; - The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected; - The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical negative control data both for PCEs and for NCEs; - The two positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above;
A finding is considered positive if the following criteria are met: - Statistically significant and dose-related increase in the number of PCEs containing
micronuclei; - The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical negative control data;
A test substance is considered negative if the following criteria are met: - The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical negative control data.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF AG). The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
(The administration of 2000 mg/kg bw of the test substance led to distinct clinical signs of toxicity (squatting posture, staggering: 1-2 h after the administrations).)
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw (two treatments) was recommended as the highest dose according to the OECD guideline.
- Clinical signs of toxicity in test animals: All animals (male and female) survived. The clinical signs observed were hunched posture and staggering within one hour after administration only. However, there were no distinct differences in the symptoms between males and females (Thus, only male animals and a dose of 2000 mg/kg bw were selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg bw were administered as further doses)


RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): An inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected at the top dose of 2000 mg/kg bw.

Any other information on results incl. tables

Substance Dose (mg/kg) sacrifice interval [h] Micronuclei in PCE Number of NCEc
total [‰]a large MN [‰]b
vehicle solvent 24* 0.9 0.0 3678
test substance 500 24* 0.9 0.0 4287
test substance 1000 24* 1.1 0.0 4365
test substance 2000 24* 1.2 0.0 5952
positive ctrl 20 24** 9.7# 0.0 3127
positive ctrl 0.15 24** 48.6# 12.5# 6513
* twice administration: sacrifice 24 hrs after 2nd administration, respectively 48 hrs after 1st administration
** single administration: sacrifice 24 hrs after administration
# p</= 0.01
PCE = polychromatic erythrocyts (2000 were scored for micronuclei)
NCE = normochromatic erythrocyts
a: sum of small and large micronuclei
b: large micronuclei (indication for spindle poison effect)
c: number of NCEs observed when scoring 10000 PCEs

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions chosen here, the test substance EP202 does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any
impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.
Executive summary:

EP202 was administered to male Crl:NMRI mice (5 mice/dose). The study is reliable. Under the experimental conditions chosen here, the test substance EP202 does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

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