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Toxicological information

Genetic toxicity: in vivo

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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:
2003-01-07 to 2003-03-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study (OECD 486), GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Dose Range Finding: Charles River Laboratories, Raleigh, North Carolina, USA (males and females)
Main Study: Harlan, Dublin, Virginia, USA (males)
- Age at study initiation: approximately 8 weeks (dose range finding assay) or 10 weeks (UDS assay)
- Assigned to test groups randomly: yes
- Housing: up to 2 per cage (acclimation), singly after randomization in suspended stainless-steel cages
- Diet: PMI certified Rodent Diet® 5002, ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days prior to the initiation of dosing

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 4°C, recorded at least once daily
- Humidity: 55 ± 15%, recorded at least once daily
- Photoperiod: 12-hour Iight - 12-hour

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: PEG 400
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing suspensions were prepared for each treatment group by adding a weighed amount of test item to a measured volume PEG 400 and mixing well to obtain homogeneous suspensions. Suspensions were obtained over the entire target concentration range used of 25 to 200 mg/mL. The dosing suspensions were prepared no more than 3 hours prior to dosing and were held at room temperature.
Duration of treatment / exposure:
not applicable (single oral gavage administration)
Frequency of treatment:
single administration
Post exposure period:
Two timepoints for UDS were employed, one at 2 to 4 hours after administration and another at 14 to 16 hours after administration.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 500, 1000, 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
4 males per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
N-dimethylnitrosamine (DMN)
- Justification for choice of positive control(s): The positive control article, N-dimethylnitrosamine, is known to induce UDS in rat hepatocytes in vivo.
- Route of administration: gavage
- Doses / concentrations: administered at approximately 10 mg/kg and 15 mg/kg for the 2- to 4-hour and 14- to 16-hour timepoints, respectively (no control articles were used in the dose range finding assay).

Examinations

Tissues and cell types examined:
cultured rat liver hepatocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The highest dose selected for the UDS assay was 2000 mg/kg, based on the results of the dose range finding assay. Two additional dose levels were selected, using dilution steps of 50% and 25%.

TREATMENT AND SAMPLING TIMES:
Two timepoints for UDS were employed, one at 2 to 4 hours after administration of a single dose of the test article and another at 14 to 16 hours after administration. For the 2- to 4-hour timepoint, the animals ranged in weight from 201-219 grams. For the 14- to 16-hour timepoint, the weight range of the animals used was 203-226 grams.

DETAILS OF SLIDE PREPARATION:
All animals from each group were perfused for the collection of hepatocytes and establishment of cultures. However, cultures from only three animals per group were evaluated for UDS. After attachment of the cells, the cultures were labeled with 10 µCi/mL 3H-TdR for 4 hours. The cultures were prepared for analysis of nuclear labeling by autoradiography after washing out the unincorporated label.

METHOD OF ANALYSIS:
Three animals from the vehicle, positive control and test article dose groups were analyzed for nuclear labeling.
The cells were examined microscopically. Only normally-appearing nuclei were scored, and any occasional nuclei blackened by grains too numerous to count were excluded as cells in which replicative DNA synthesis occurred rather than repair synthesis. UDS was measured by counting nuclear grains and subtracting the average number of grains in three nuclear-sized areas adjacent to each nucleus (cytoplasmic count). This value is referred to as the net nuclear grain count.
The net nuclear grain count was routinely determined for 50 randomly selected cells on triplicate coverslips (150 total nuclei) for each animal. The average mean net nuclear grain count (± standard deviation) was determined from the triplicate coverslips (150 total nuclei) for each animal and averaged for each treatment condition.
Evaluation criteria:
The test article is considered active in the UDS assay at applied concentrations that cause:
- An increase in the group average of the mean net nuclear grain count to at least three grains per nucleus above the concurrent vehicle control group average leading to a positive number, or;
- An increase in the group average of the percent of nuclei with five or more net grains such that the percentage of these nuclei in test cultures is 10% above the percentage observed in the group average of the concurrent vehicle controls.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 250-2000 mg/kg bw
- Clinical signs of toxicity in test animals:
All animals were normal (no signs of toxicity) within 1 hour of dosing. During the next two days of observations, toxic signs included soft feces, discolored (purple) urine, discolored (purple) feces, brown or purple stains of the anal/genital area, and purple stains on the tails and paws. No lethality occurred. Both sexes behaved very similarly, which justified the use of males only for the UDS assay.

RESULTS OF DEFINITIVE STUDY
- Clinical sings: Toxic signs were the same as those obtained in the dose range finding study. No deaths occurred.
- Mutagenicity results:
None of test article treatment groups yielded a positive mean net nuclear grain count (-1.22 and -0.28 for the early (2 to 4 hours after treatment) and late timpoint (14 to 16 hours after treatment), respectively, and the highest percent cells with >= 5 grains was only 4.00 % and 4.22 % for the early and late timpoint, respectively, well below the criterion for a positive response.
- Controls:
The vehicle and positive control results were well within the historical data ranges. The DMN positive control treatments induced large increases in nuclear labeling that clearly exceeded both criteria used to indicate UDS.
Since the positive control animals were responsive, the test results were considered to provide conclusive evidence for the lack of UDS induction by the test article.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative