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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:
Test start date: March 27, 2001; Test end date: 24 April 2001; Study completion date: July 17, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
May 19, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-dihydro-6-hydroxy-1,4-dimethyl-2-oxo-5-[[3-[(phenylsulphonyl)oxy]phenyl]azo]nicotinonitrile
EC Number:
261-693-5
EC Name:
1,2-dihydro-6-hydroxy-1,4-dimethyl-2-oxo-5-[[3-[(phenylsulphonyl)oxy]phenyl]azo]nicotinonitrile
Cas Number:
59312-61-7
Molecular formula:
C20H16N4O5S
IUPAC Name:
3-[(5-cyano-2-hydroxy-1,4-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)diazenyl]phenyl benzenesulfonate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test material identified as: FAT 93529/A
- Source and batch No.of test material: 1-5 / 2000 laborgetrocknet
- Expiration date of the batch: March 2006

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability of the test substance in the solvent/vehicle: 24 hours in water

OTHER SPECIFICS:
- Aggregate state at room temperature: solid
- Colour: yellow
- Purity: technical purity

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: NMRI
- Source: RCC Ltd., Biotechnology and Animal Breeding Division; CH-4414 Füllinsdorf
- Number of animals: 72 (36 males/36 females)
- Initial age at start of acclimatization: 8-10 weeks
- Acclimatization: minimum 5 days
- Initial body weight at start of treatment: males mean value 33.6 g (SD ± 2.3 g); females mean value 25.5 g (SD ± 2.3 g)

Husbandry
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
- Housing: single
- Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
- Bedding: Granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe)
- Feed: Pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water: Tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Environment: temperature 21 ± 4°C
- Relative humidity 30-70%
- Artificial light: 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test article was formulated in corn oil.

- All animals received a single standard volume of 20 mL/kg bw orally.
Duration of treatment / exposure:
The animals received the test article, the vehicle or the positive control substance once.
Frequency of treatment:
Single treatment
Post exposure period:
Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
24 hour interval only
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
24 hour interval only
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
24 and 48 hour interval
No. of animals per sex per dose:
6 male and 6 female rats
Control animals:
yes, concurrent vehicle
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:
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-Griinwald (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.
Details of tissue and slide preparation:
Analysis of Cells:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 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 2000 the PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated.
Evaluation criteria:
- A test article is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.
- A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system..
- Statistical methods (nonparametric Mann-Whitney test (8)) will be used as an aid in evaluating the results.
- However, the primary point of consideratrion is the biological relevance of the results.
Statistics:
Non parametric Mann-Whitney test

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: A single dose of 2000 mg/kg bw was used
- Clinical signs of toxicity in test animals: The treated animals expressed no toxic reactions. The urine color was orange at 2-4 hours after treatment, while 6 hours after treatment, it was yellow orange.
- Rationale for exposure: Highest (limit dose) was used as recommended by the guideline.


RESULTS OF DEFINITIVE STUDY
- The mean number of normochromatic erythrocytes was not increased after treatment with the test item as compared to the mean value of NCEs of the vehicle control indicating that FAT 93'529/A had no cytotoxic properties in the bone marrow. However, the urine colour of the treated animals had taken on the colour of the test item indicating the systemic distribution of the test item and its bioavailability in the target organ.

- In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with FAT 93'529/A were below or near to the value of the vehicle control group.

- 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Applicant's summary and conclusion

Conclusions:
The test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.
Executive summary:

The test substance FAT 93529/A was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse as per the methodology described in OECD Guideline 474 and EU Method B.12. The test substance was formulated in corn oil. Corn oil was used as vehicle control. The volume administered orally was 20 mL/kg bw. 24 h and 48 h after a single administration of the test substance 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. At least 2000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs. The following dose levels of the test substance were investigated: 24 h preparation interval: 500, 1000 and 2000 mg/kg bw, 48 h preparation interval: 2000 mg/kg bw. After treatment with the test item the number of NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle control thus indicating that FAT 93529/A had no cytotoxic effectiveness in the bone marrow. However, the urine of the treated animals had taken over the colour of the test item indicating the systemic distribution and the bioavailability of the test item in the target organ. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated, that the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.