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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-05-22 - 2008-06-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 486) performed under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Version / remarks:
adopted on 21st July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis

Test material

Constituent 1
Test material form:
other: Solid (bulk) and solid-liquid suspension (dispersion containing micronized ETH50 (nano form))
Details on test material:
Please refer to "Confidential details on test material".

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River France origin, Saint-Germain-sur-l'Arbresle, France
- Age at study initiation: 5 to 6 weeks old
- Weight at study initiation: 180 g to 215 g (12-16-hour expression time), 164 g to 207 g (2-4-hour expression time)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: polypropylene cages measuring 42.5 x 26.6 x 15 cm, covered by a stainless steel netted lid, in which male rats were placed in groups of 3 or 2; bedding consisted of dust-free, sterilized wood shavings
- Diet: A04C10 irradiated rat/mouse feed from SAFE
- Water: Drinking water softened, treated by osmosis and filtered on 0.2 µm membrane, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 55 ± 15 %
- Air changes: 20 changes/hour
- Photoperiod: 12 hours dark/ 12 hours light

IN-LIFE DATES: From: 2007-01-15 To: 2007-08-29 (in-life and slide reading phase)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicles/solvents used:
Distilled water (FAT 65’080/E and FAT 65’080/E Placebo)
Carboxymethylcellulose at 0.5% in distilled water (FAT 65’080/B)
- Justification for choice of solvent/vehicle: solubility of the test item
- Amount of vehicle: 10 mL/kg bw

Duration of treatment / exposure:
Single administration
Frequency of treatment:
Single administration
Post exposure period:
- Test item group animals dosed were sacrificed 2 or 12 hours after treatment for cell collection (2-4 hour or 12-16 hour expression time)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.
FAT 65’080/B
Remarks:
Doses / Concentrations:
1000, 2000 mg/kg bw
Basis:
nominal conc.
FAT 65’080/E
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
nominal conc.
FAT 65’080/E Placebo
No. of animals per sex per dose:
3 male rats
Control animals:
other: distilled water
Positive control(s):
- Dimethylhydrazine (10 mg/kg bw) for the 2-4-hour expression time
- 2-acetamidofluorene (25 mg/kg bw) for the 12-16-hour expression time
- Route of administration: oral route

Examinations

Tissues and cell types examined:
Liver - prepared hepatocyte cultures
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- From the results of a preliminary toxicity test.

TREATMENT AND SAMPLING TIMES:
- Normal expression time: 12-16 hours after dosing (first assay)
- Earlier expression time: 2-4 hours after dosing (second assay)

Preparation of hepatocyte cultures:
Three animals were assigned randomly for hepatocytes sampling in each group. Individual animals were anaesthetized and prepared for perfusion of the liver with Hepes buffer kept at 37°C at 40 mL/min for 5 minutes. The liver was then perfused using a collagenase buffer at 37°C at 20 mL/min for 5 minutes. At the end, the liver became 'spongy'. It was cut free into nylon 150-µm filter-funnel positionned on a sterile tube. The Glisson's capsule was carefully cut open and the hepatocytes were released by adding complete Williams's culture medium (WE-C). The hepatocyte suspension was centrifuged and the resulting pellet was resuspended in WE-C. The centrifugation and resuspension procedure was repeated twice and the hepatocyte pellets were resuspended in WE-C. The proportion of viable cells was determined with the help of a Malassez haemocytometer using trypan blue technique. A volume of 3 mL was transferred to each well of 6-well multiplates containing round plastic coverslips coated with collagen. Twelve replicate wells per animal were prepared. The cultures were incubated at 37°C in an atmosphere of 5 % CO2 for approximately 90 minutes to allow attachment to the coverslips.

Radiolabelling of hepatocyte cultures:
After this attachment period, the supernatant medium was removed and the cells gently washed with Williams E medium-Incomplete (WE-I) and then replaced with WE-I containing 10 µCi/mL of 3H Thymidine. Cultures were incubated for 3 - 8 hours at 37 °C in an atmosphere of 5 % CO2. At the end of the labelling period, the supernatant medium was removed and the cells were washed three times with WE-I medium containing 0.25 mM unlabelled thymidine to diminish unincorporated radioactivity ("cold-chase"). Then, the cultures were incubated overnight with 3 mL of the same solution.

DETAILS OF SLIDE PREPARATION:
The cells were rinsed with WE-I medium, fixed 3 times using a Carnoy mix (ethanol-acetic acid), and washed with distilled water at least 4 times. After that, the coverslips were dried at room temperature and finally, mounted on slides with EUKITT.
Autoradiography: Six of the 12 slides from each animal were coated in Kodak NTB-2 liquid emulsion. After gelling, the slides were incubated in a light-tight box and left refrigerated for 10-14 days. At the end of of this time, the emulsion was developed and fixed. The cell nuclei and cytoplasm were stained with Meyers hemalum. Slides were dehydrated in ethanol, cleared in xylene and mounted with coverslips for microscopic examination.

METHOD OF ANALYSIS:
Grain counting was performed using an image analysis system (Visilog, Noesis, France). Where possible, nuclear and cytoplasmic grain counts were obtained from 50 cells per slide, 3 slides per animal (test article treatments, solvent and positive controls). Each slide was examinated to ensure that the culture was viable. Each slide was scanned and cells scored such that there was no risk that the same cell was counted more than once. Nuclear (NC) and cytoplasmic (CC) grain counts were recorded, and the net nuclear grains (NNG) per cell was determined (NNG = NC-CC).
Evaluation criteria:
The following criteria were used for analysis of slides:
1. Only cells with normal morphology were scored.
2. Isolated nuclei with no surrounding cytoplasm were not scored.
3. Cells with unusual staining artefacts were not scored.
4. Heavily labelled cells in S-phase were not scored.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Remarks:
no unscheduled DNA synthesis in rat hepatocytes
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF THE RANGE-FINDING STUDY
- In the preliminary toxicity assay, 2 groups of 4 male Fischer rats were dosed once at 2000 mg/kg bw of the test item.
- Following dosing, the animals were observed regularly for a period of at least 48 hours and any clinical signs and any mortalities were recorded.
- No clinical signs and no incidences of mortality were noted in rats treated with the test item
- Under these conditions, the UDS test was performed with 2000 mg/kg bw of the test item

RESULTS OF THE DEFINITIVE STUDY
- In the UDS assay performed with 12 - 16 or 2 - 4-hour expression times, no significant increase in the mean net nuclear grain count values of cells in repair (NNG > 5) were noted when compared to control and the mean percentages of cells in repair were comparable with the solvent controls. Furthermore, frequencies of cells in S-phase were low.
- Thus, all items tested did not reveal any genotoxic activity under the test conditions.
- Regarding the animals treated with FAT 65`080/E and /B (at 2000 mg/kg bw), systemic exposure with the respective test substance was demonstrated.

Applicant's summary and conclusion