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in vivo mammalian cell study: DNA damage and/or repair
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
GLP compliance:
yes (incl. QA statement)
Type of assay:
unscheduled DNA synthesis

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
solid - liquid: suspension
Details on test material:
- Name of test material (as cited in study report): FAT 70' 884/C
- Physical state: Solid, light yellow powder
- Analytical purity: 97.6%
- Impurities (identity and concentrations): Not reported.
- Lot/batch No.: 02464CL3
- Expiration date of the lot/batch: Reported as "unknown"
- Stability under test conditions: Reported as "unknown"
- Storage condition of test material: At room temperature, protected from light.

Test animals

other: Fischer IFFA CREDO (Saint-Germain-sur-l'Arbresle-France)
Details on test animals or test system and environmental conditions:
- Source: Saint-Germain-sur-l'Arbresle-France
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: ca. 200g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: 42.5 x 26.5 x 15 cm polypropylene cages with dust-free sterilized wood shavings as bedding
- Diet (e.g. ad libitum): AO4C10 irradiated rat/mouse feed from U.A.R.
- Water (e.g. ad libitum): Drinking water, softened, treated by osmosis, and filtered on 0.2 micron membrane ad libitum
- Acclimation period: 5 days

- Temperature (°C): 22, +-2
- Humidity (%): 55, +-15
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE PHASE (Initiated - Completed): 13 Jan 2004 to 9 March 2004

Administration / exposure

Route of administration:
oral: gavage
5% carboxymethylcellulose (CMC) in distilled water
Details on exposure:
- Test compound was suspened in the vehicle at 200 mg/L
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
Assay I: 12 - 16 hour expression time
Assay II: 2 - 4 hour expression time
Doses / concentrations
Doses / Concentrations:
2000 and 1000 mg/kg-bw (Dose Volume: 10 mL/kg-bw)
other: Oral gavage dosage and volume
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Positive control(s):
Assay I: 2-acetylaminofluorene
- Justification for choice of positive control(s): Not reported
- Route of administration: Oral gavage
- Dose: 25 mg/kg-bw

Assay II: Dimethylhydrazine
- Justification for choice of positive control(s): Not reported
- Route of administration: Oral gavage
- Dose: 10 mg/kg-bw


Tissues and cell types examined:
Liver cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: High dose is maximum recommended by guideline, plus a lower mid-dose.

TREATMENT AND SAMPLING TIMES: Single treatment. Assay I: 12-16 hours Assay II: 2 - 4 hours

DETAILS OF SLIDE PREPARATION: The culture was diluted to provide approximately 1.5E005 viable cells/mL. 3 mL is transferred to each well of 6-well multiplates containing round plastic coverslips. Twelve replicate wells per animal are prepared. The cultures were incubated at 37°C in an atmosphere of 5% CO2 for approximately 90 minutes to allow attachment to the coverslips. Each multiplate was identified by animal number, treatment, administred dose and sampling time.

METHOD OF ANALYSIS: Six of the 12 slides from each animal are coated in Kodak NTB -2 liquid emulsion (the remainder is kept in reserve and autoradiographied if required). Each slide is dipped individually into the emulsion, ensuring that no air-bubbles are generated. After gelling, the slides are incubated in a light-tight box and left refrigerated for 10-14 days. At the end of this time, the emulsion is developed and fixed. The cell nuclei and cytoplasm are stained with Meyers hemalum. Slides are dehydrated in ethanol, cleared in xylene and mounted with coverslips for microscopic examination. Where possible, nuclear and cytoplasmic grain counts are obtained from 50 cells per slide, 3 slides per animal (test compound treatments, solvent and positive controls). Each slide is examined to ensure that the culture was viable. Each slide is scanned and cells scored such that there is no risk that the same cell is counted more than once. Nuclear (NC) and cytoplasmic (CC) grain counts are recorded, and the net nuclear grains (NNG) per cell is determined (NNG = NC - CC).

OTHER: Liver cells radiolabeled with 10 µCi/mL of 3H Thymidine
Evaluation criteria:
The following criteria will be used for analysis of slides:
1. Only cells with normal morphology will be scored (50/slide, where possible).
2. Isolated nuclei with no surrounding cytoplasm will not be scored.
3. Cells with unusual staining artefacts will not be scored.
4. Heavily labelled cells in S-phase will not be scored.
5. All other normal cells, 50/slide, will be scored where possible.

Acceptance criteria for the results: The study will be considered valid if the negative control mean had zero NNG counts or less (i.e. a negative value, within the historical range). The positive control treatments should have values of 5 or more NNG with 50% or more cells having NNG counts of 5 or greater. Acceptance under any other criteria will be discussed in the report.

Criteria for a genotoxic activity: The test compound is considered as positive in this assay, if at any dose and at either time point: (1) The test compound yields group mean NNG values greater than 0 NNG and 20% or more of cells are responding (NNG values = 5). (2) An increase is observed in both NNG and the percentage of cells in repair. If the test compound fails to induce UDS at any dose in either time point, it will be considered clearly negative in this system. If neither situation occurs, the results are discussed case by case and another independent study may be envisaged after modifying the dose range taking account the findings obtained using other test systems.

Results and discussion

Test results
no effects
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
The test substance was not genotoxic.
Executive summary:

The test substance was investigated at two expression times (2-4 hours and 12-16 hours) in the in vivo Unscheduled DNA Synthesis (UDS) test in hepatocytes from male Fischer rats, treated orally once with 2000 and 1000 mg/kg. As a conclusion, the test substance did not reveal any genotoxic activity under these test conditions.