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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:
From September 6, 1989 to October 18, 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
yes
Remarks:
No exposure time of 12-16 h was taken into account.
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 89-009-1
- Substance type: Light yellow liquid
- Physical state: Liquid
- Purity: responsibility of the sponsor and recorded in the sponsor's file
- Stability: responsibility of the sponsor and recorded in the sponsor's file
- Storage condition of test material: At ambient temperature
- Other: specific gravity 1.1359 g/ml

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Kingston, NY
- Age at study initiation: twelve week old young adult Fischer 344 rats
- Weight at study initiation: 194-244 grams
- Fasting before study
- Housing: Animals were housed in accordance with the "Guide for the Care and Use of Laboratory Animals"
- Acclimation period: 20 days prior to initiation of the assay

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 3°C
- Humidity (%): 30 to 70%
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water
- Amount of vehicle: 10 ml/kg
Details on exposure:
Dilutions were made just prior to use.
Duration of treatment / exposure:
3 hours
Frequency of treatment:
Single dose
Post exposure period:
Not applicable
Doses / concentrationsopen allclose all
Dose / conc.:
920 mg/kg bw/day (nominal)
Dose / conc.:
2 300 mg/kg bw/day (nominal)
Dose / conc.:
4 600 mg/kg bw/day (nominal)
Dose / conc.:
9 200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Positive control(s):
Dimethylnitrosamine, via oral gavage at 10 mg/kg.

Examinations

Tissues and cell types examined:
The liver was excised and examined.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Sponsor recommended that the high dose be 1.0 x LD50 in rats (9200 mg/kg) as an estimate of the maximum tolerated dose. Three lower doses were also evaluated at 920, 2300 and 4600 mg/kg as well as the solvent control, dH2O and the positive control, DMN, at 10 mg/kg.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Eighteen male Fischer 344 rats were anesthetized with 50 mg/kg of sodium pentobarbital by intraperitoneal injection. The liver was excised and placed in 50 mL of warm serumless WME in a sterile culture dish. The liver was trimmed of fat, excess connective tissue and any sections of liver that still showed signs of blood. The liver was then transferred to a sterile (100 x 25 mm) culture dish containing 50 mL of warm Medium B. The liver was held by the connective tissue in the porta hepatis and the capsular membrane of the liver was opened and removed at several points on the dorsal surface with the aid of a scalpel and forceps. Cells were detached by gently combing the liver with a 3/4" camel's hair brush. Detachment was complete when only fibrous tissue remained. The cells were then aliquotted into a 50 mL centrifuge tube by pipetting gently with a sterile 10 mL polystyrene pipet. The volume of the tube was adjusted to 35-40 mL with WME supplemented with 10% v/v calf serum. The tube was allowed to stand in a vertical position for 10 minutes to flocculate the hepatocytes. Viability of the resuspended hepatocytes was measured by trypan blue dye exclusion.

DETAILS OF SLIDE PREPARATION: 1 x 1E05 viable hepatocytes were inoculated into 12 well cluster dishes containing 15 mm diameter Thermanox plastic coverslips in WME containing 10% calf serum. The hepatocytes were allowed to attach for approximately 2hours in a 37°C incubator. The cultures were rinsed with serum-free medium and refed with WME containing 10 uC/mL of 3H-thymidine and incubated. Four hours after exposure, the cultures were washed once with 2 mL WME and incubated overnight in 3 mL of WME supplemented with 0.25 mM unlabeled thymidine. Fourteen to sixteen hours after incubation, the cultures were washed three times with 3 mL volumes of phosphate buffered saline by aspiration.

Cell fixation: The cells on coverslips were swelled in 1% sodium citrate for 10-15 minutes and fixed in three 10-minute changes of 100% ethanol:glacial acetic acid (3:1). The fixed cultures were then washed twice with approximately 2 mL volumes of dH2O. The coverslips were air dried and mounted cell surface up on glass slides with permount. Slides were dipped in NTB-2 photographic emulsion in the dark, allowed to dry overnight and stored at 4°C in light-proof slide boxes containing desiccant for one week.

Staining: After seven days of exposure time, the autoradiographs were developed in D19 at approximately 15°C for 4 minutes, washed in deionized water with 5 mL glacial acetic acid for 30 seconds, immersed in Fixer for 10 minutes, washed in running tap water for 5 minutes and dried. They were then stained in Harris Alum hematoxylin followed by a dip rinse in acid alcohol, rinsed in running tap water for 2-5 minutes and a dip rinse in ammonium water. The slides were then rinsed in running tap water for 2-5 minutes, dipped in 70% ethyl alcohol followed by a 10-60 second dip in eosin solution. The slides were then rinsed in 3 separate baths of 95% ethyl acohol for 2 minute intervals, followed by rinsing in 3 separate baths of 100% ethyl alcohol for 2 minute intervals. The slides were air dried, and coverslipped in permount. Excess emulsion was scraped off.

METHOD OF ANALYSIS: Unscheduled DNA repair synthesis, evidenced by a net increase in black silver grains over the nucleus, is quantified by determining nuclear and cytoplasmic grain counts using an Artek 880 automated colony counter with microscopic/video camera attachment interfaced to an Apple II computer for data acquisition. A total of 450 cells/dose point (50 cells/coverslip, 3 coverslips/rat and 3 rats/dose point) were counted for autoradiographic UDS determination. Slides were coded before being counted. A correction coefficient was calculated by th following method. An area/grain ratio was obtained by visually scoring an area of each slide containing 3-5 nuclear grains and then obtaining an object area count with Artek Model 880 Colony Counter. Five such areas were scored, the differences summed and the mean obtained. This value serves as a correction coefficient. The cytoplasimc grain count was quantitated by randomly selecting the highest of three nuclear-sized areas adjacent to each nucleus. This value was subtracted from the uncorrected nuclear grain count to determine the net nuclear grain count. Both nuclei of binucleated cells were recorded separately. Replicative DNA synthesis was evidenced by nuclei blackened with grains too numerous to count. The data of the HPC/DNA Repair Assay are reported as mean grains/nucleus from the triplicate wells. The solvent control should have a net nuclear grain count of < or = 0 or the study will be repeated. Also the positive control, DMN, should yield a mean net nuclear grain count > or = 5.
Evaluation criteria:
The test article is reported positive when the minimum net grain count of 5 per nuclei is consistently observed in triplicate wells. Where possible a dose response profile should be observed. When an adequate dose response is not attained, the chemical may be classified as a "suspect" genotoxicant. To resolve the genotoxic potential of the chemical, the sponsor may choose to initiate a second experiment with dose levels closely bracketing the positive response which resulted in classifying the chemical as a "suspect" genotoxicant.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
None of the treated cultures produced mean net nuclear grain counts that were substantially greater than the solvent control. The positive control DMN gave a mean net nuclear grain count greater than the solvent control.

Applicant's summary and conclusion

Conclusions:
The substance was negative in inducing unscheduled DNA synthesis at doses of 920, 2300, 4600 and 9200 mg/kg.