Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
None
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 018631.A8
- Expiration date of the lot/batch: August 2003

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: at least 24 hours
- Solubility and stability of the test substance in the solvent/vehicle: 100g/l (at 20 °C) in water

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, CH-4414 Füllinsdorf
- Age at study initiation: 8-12 weeks
- Weight at study initiation:
*males mean value 33.8 g (SD ± 3.0 g)
*females mean value 27.3 g (SD ± 1.4 g)


- Housing: single
- Cage type: Makrolon Type I, with wire mesh top
- Feed: pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Bedding: granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe)
- Water : tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 °C
- Humidity (%): relative humidity 30-70 %
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

The animals were distributed into the test groups at random and identified by cage number.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Deionised water
Details on exposure:
Approximately 18 hours before treatment the animals received no food but water ad libitum. At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.
Duration of treatment / exposure:
Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.
Frequency of treatment:
One treatment
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw (total dose)
Remarks:
Sampling time: 24 h
Dose / conc.:
670 mg/kg bw (total dose)
Remarks:
Sampling time: 24 h
Dose / conc.:
2 000 mg/kg bw (total dose)
Remarks:
Sampling time: 24 h
Dose / conc.:
2 000 mg/kg bw (total dose)
Remarks:
Sampling time: 48 h
No. of animals per sex per dose:
Twelve animals, six males and six females, were treated per dose group and sampling time.
Control animals:
yes, concurrent vehicle
Positive control(s):
- Name: CPA; Cyclophosphamide
- Route of administration: Once / Orally
- Doses / concentrations: 10 ml/kg b.w.
Solution prepared on day of administration.
The stability of CPA at room temperature is good. At 25 °C only 3.5 % of its potency is lost after 24 hours.

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
The epiphyses of femur 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 air dried smear then stained with May-Grünwald /Giemsa. At least one slide was made from each bone marrow sample.
Evaluation criteria:
Analysis of Cells:
Evaluation of the slides was performed with 100x oil immersion objectives. 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. Ten animals (5 males, 5 females) per test group were evaluated as described.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results
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:
Pre-Experiment for Toxicity
In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. FAT 40'069/C formulated in deionised water.
The volume administered was 10 ml/kg b.w..
Reduction of spontaneous activity:
-1/1 (after 1 hour post treatment male/female).
-1/1 (after 6 hours post treatment male/female).
-1/1 (after 24 hours post treatment male/female).
-0/0 (after 48 hours post treatment male/female).

Additionally, the urine of the treated animals was coloured: after 1 hour = blue; after 6 hours = green; after 24 hours = light green.
On the basis of these data 2000 mg/kg b.w. were estimated to be suitable.

RESULTS OF DEFINITIVE STUDY:
Summary of micronucleus test results:
- Test group / dose mg/kg b.w. / Sampling time (h)/ PCEs with micronuclei (%) / range / PCE/ NCE.
- vehicle / 0 mg/kg b.w. / 24 hours /0.150 % /0-7 /2000/ 1784.
- test article / 200 mg/kg b.w. / 24 hours /0.075 % /0-3 /2000/ 1885.
- test article / 670 mg/kg b.w. / 24 hours /0.045 % /0-2 /2000/ 1710.
- test article / 2000 mg/kg b..w / 24 hours /0.070 % /1-3 /2000/ 1983.
- Cyclo-phosphamide / 40 mg/kg b.w. / 24 hours /0.070 % /12-5 /2000/ 2064.
- test article / 2000 mg/kg b.w. / 48 hours /0.055 % /0-4 /2000/ 1828.


Any other information on results incl. tables

Biometry

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

 Vehicle contro vs test group  Significance
 200 mg FAT 40'069/C/kg b.w.; 24 h  n.t.  -
 670 mg FAT 40'069/C/kg b.w.; 24 h  n.t.  -
 2000 mg FAT 40'069/C/kg b.w.; 24 h  n.t.  -
 40mgCPA/kgb.\v.;24h  +  <0.0001
 2000 mg FAT 40'069/C/kg b.w.; 48 h  n.t.  -

+ = significant;

n.t = not tested, as the mean micronucleus frequency was not above the vehicle control value

Applicant's summary and conclusion

Conclusions:
FAT 40069/C is considered to be non-mutagenic in the micronucleus assay.
Executive summary:

An in vivo study was performed to investigate the potential of FAT 40069/C to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. This test was performed according to the OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) and follows GLP methodology. The test item was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally was 10 ml/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Ten animals (5 /sex) per test group were evaluated for the occurrence of micronuclei. 2000 polychromatic erythrocytes (PCEs) 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. 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 item and with any dose level used (Up to 2000 mg/kg/d). However the positive control at 40 mg/kg b.w. cyclophosphamide administered showed a substantial increase of induced micronucleus frequency. In conclusion under the experimental conditions reported, the test item did not induce micronuclei by the in-vivo mouse micronucleus test. So, FAT 40069/C is considered to be non- clastogenic in this micronucleus assay.