Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 01, 1996 to January 13, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None

Data source

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

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:
other: mammalian cell clastogenicity assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
None

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
Recommended by the guideline
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 31.2 g (SD ± 3.0 g)
females mean value 22.9 g (SD ± 1.5 g)
According to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of C C R for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour. The animals were distributed into the test groups at random and identified by cage number.

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 ±3°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: PEG 400 was used as vehicle control.
- Justification for choice of solvent/vehicle: Widely accepted.
- Catalogue No.: 9726 (gas chromatography grade)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test article was formulated in PEG 400. The vehicle was chosen to its relative non-toxicity for the animals. All animals received a single standard volume of 10 ml/kg body weight orally.
Duration of treatment / exposure:
once
Post exposure period:
24 h preparation interval: 200, 670 and 2000 mg/kg b.w..
48 h preparation interval: 2000 mg/kg b.w..
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day
Remarks:
24 h preparation interval
Dose / conc.:
670 mg/kg bw/day
Remarks:
24 h preparation interval
Dose / conc.:
2 000 mg/kg bw/day
Remarks:
24 as well as 48h preparation interval
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide;
- Justification for choice of positive control(s): recommended by the guideline
- Route of administration: orally, once
- Doses / concentrations: 40 mg/kg b.w.

Examinations

Tissues and cell types examined:
polychromatic erythrocytes (PCE)
Details of tissue and slide preparation:
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 centrifiiged 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.

Analysis of cells:
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:
A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.
However, both biological and statistical significance should be considered together.
Statistics:
Statistical significance was evaluated by means of the nonparametric 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:
not examined
Positive controls validity:
valid
Additional information on results:
After treatment with the test article the number of NCEs was not substantially increased as compared to the corresponding vehicle controls thus indicating that FAT 92'504/A had no cytotoxic effectiveness in the bone marrow.
In comparison to the corresponding vehicle controls there was no 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 administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Applicant's summary and conclusion

Conclusions:
FAT 92504/A can be considered to be not-clastogenic in vivo.
Executive summary:

The test article FAT 92504/A (mixture of DISPERSE BLUE 054 and DISPERSE BLUE 077) was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was formulated in PEG 400. PEG 400 was used as vehicle control. The volume administered orally was 10 ml/kg b.w.. Twenty four and 48 h after the 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 (exception animal no. 49 and 52 = 2000 PCEs) were scored for micronuclei.

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 mean number of normochromatic erythrocytes (NCEs) was not substantially increased after treatment with the test article as compared to the mean values of NCEs of the corresponding vehicle controls, indicating that FAT 92504/A had no cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test article. The mean values of micronuclei observed after treatment with FAT 92'504/A were in the range of the vehicle control group. 40 mg/kg b.w. cyclophosphamide administered per os 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 in the bone marrow cells of the mouse.