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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:
Start of Experiment: 05 December 1997; End of Experiment: 16 December 1997; Study completion date: 13 January 1997.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium [2-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]-5-nitrobenzene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)
EC Number:
282-316-0
EC Name:
Disodium [2-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]-5-nitrobenzene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)
Cas Number:
84145-95-9
Molecular formula:
C32H17CrN6O11S.2Na
IUPAC Name:
disodium [2-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]-5-nitrobenzene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)
Test material form:
solid: particulate/powder
Details on test material:
Identity FAT 20013/D TE
Batch 1502011 (China)
Purity determined in this study
Appearance black powder at 20°C
Smell neutral
pH-Value pH-value of a solution of 2% (w/w) = 9.2
Expiration date August 03rd, 2020
Storage to be stored at room-temperature
Specific details on test material used for the study:
Identification: FAT 20'013/C
Description: Grey solid
Batch Number: 220
Purity / Formulation: 53.15 %
Stability of Test Article: Stable under specified storage conditions; expiration date: DEC-1999
Storage Conditions: In the original container at room temperature (approx. 20 °C), away from direct sunlight.
Safety Precautions: Gloves, goggles and face mask were obligatory to ensure the health and safety of the personnel.

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source: BRL, CH-4414 Füllinsdorf
Number of Animals: 72 (36 males/36 females)
Initial Age at Start of Acclimatization: 8-12 weeks
Acclimatization: minimum 5 days
Initial Body Weight at Start of Treatment: males mean value 31.5 g (SD ± 3.2 g); females mean value 25.1 g (SD ± 1.8 g)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Vehicle: Deionised water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test article was formulated in deionised water. The vehicle was chosen to its non-toxicity for the animals. All animals received a single standard volume of 20 mL/kg body weight orally.
Duration of treatment / exposure:
48 hours
Frequency of treatment:
single treatement
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
24 h interval
Dose / conc.:
670 mg/kg bw/day (nominal)
Remarks:
24 h interval
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
24h and 48h interval each
No. of animals per sex per dose:
6 animals per sex per dose
Control animals:
yes
Positive control(s):
Cyclophosphamide;
- Justification for choice of positive control(s): recommended by the guidelines
- Route of administration: oral
- Doses: 40 mg/kg b.w.
- Frequency: once

Examinations

Tissues and cell types examined:
Bone marrow - polychromatic erythrocytes (PCE)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours. Three adequate spaced dose levels extending over a single log range were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

TREATMENT AND SAMPLING TIMES: Single dose treatment; 24h for 200 mg/kg bw and 670 mg/kg bw 24 and 48 hours for 2000 mg/kg bw.

DETAILS OF SLIDE PREPARATION: The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and
normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides.
Five animals per sex and group were evaluated as described. The remaining 6th animal of each test group was evaluated in case an animal had died in its test group.
Evaluation criteria:
The study is considered valid if the following criteria are met:
- the vehicle controls are in the range of our historical control data (0.03 - 0.26 % PCEs with micronuclei).
- the positive controls show substantially increased values
- more than 80 % of animals are évaluable
Statistics:
Nonparametric Mann-Whitney test

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The mean number of normochromatic erythrocytes was increased after treatment with the highest dose of the test article as compared to the mean value of NCEs of the corresponding vehicle control, indicating that FAT 20013/C had cytotoxic properties in the bone marrow.

Applicant's summary and conclusion

Conclusions:
FAT 20013/C is considered to be non-mutagenic in this micronucleus assay.
Executive summary:

An in vivo study was performed to investigate the potential of FAT 20013/C to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. This study was conducted in accordance with OECD test guideline 474 and EEC Directive 92/69, L 383, Annex V, B 12. The test article was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally was 20 ml/kg b.w.. 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose levels of the test article were investigated: 24 h preparation interval: 200, 670 and 2000 mg/kg b.w.. 48 h preparation interval: 2000 mg/kg b.w.. The highest guideline-recommended dose (2000 mg/kg) was estimated by a pre-experiment to be suitable. None of the animals expressed toxic reactions. After treatment with the highest dose of test article the number of NCEs was increased as compared to the corresponding vehicle controls thus indicating that FAT 20013/C had cytotoxic effectiveness in the bone marrow. In comparison to the corresponding vehicle controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used. 40 mg/kg b.w. cyclophosphamide was used as positive control which showed a statistically significant increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 20013/C is considered to be non-mutagenic in this micronucleus assay.

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