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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001

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:
1997
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Proxel Press Paste
Batch BX103

The Test Substance employed was pre-dried technical grade active substance.
Purity 93.1%

Test animals

Species:
rat
Strain:
other: Han Wistar rats
Sex:
male

Administration / exposure

Route of administration:
oral: gavage
Duration of treatment / exposure:
one dose administered by oral gavage
Doses / concentrations
Remarks:
Doses / Concentrations:
560 mg/kg and 1400 mg/kg


Control animals:
yes, concurrent vehicle
Positive control(s):
• Acetamidofluorene (2-AAF) suspended in corn oil at a concentration of 7.5 mg/L
• Dimethylnitrosamine (DMN) dissolved in purified water at a concentration of 1.0 mg/L

Examinations

Tissues and cell types examined:
100 hepatocytes were analysed per animal, where possible using two out of three slides in each case.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

When treated orally once with PROXEL Press Paste at doses up to 1400 mg/kg, male rats showed no induction of UDS in hepatocytes isolated ex vivo approximately 12-14 or 2-4 hours after dosing.  It is concluded that PROXEL Press Paste had no genotoxic activity detectable in this test system under the experimental conditions employed.

Applicant's summary and conclusion

Conclusions:
Materials and methods
In an initial toxicity range-finder study, groups of three male rats were dosed once with 700, 1000. 1400 and 2000 mg/kg PROXEL Press Paste (BIT). Mortalities were seen at 2000 mg/kg and therefore a dose of 1400 mg/kg was considered representative of a maximum tolerated dose.
Groups of at least three male rats were treated once with the solvent 0.5% methyl cellulose; PROXEL Press Paste (BIT) at 560 mg/kg or 1400 mg/kg; or the required positive control, by oral gavage, at a dose volume of 10 mL/kg. The positive controls used were 75 mg/kg 2 acetamidofluorene (2-AAF) suspended in corn oil (12-14 hour experiment) and 10 mg/kg dimethylnitrosamine (DMN) dissolved in purified water (2-4 hour experiment).
Approximately 12-14 hours (Experiment 1) or 2-4 hours (Experiment 2) after dosing, animals were killed and their livers perfused with collagenase to provide a primary culture of hepatocytes. Cultures were made from three animals in each dose group (or in the case of the 1400 mg/kg dose group in the 12-14 hour experiment, from the two surviving animals) and were treated with [3H] thymidine. Six slides from each animal were prepared with fixed hepatocytes and of these three were dipped in photographic emulsion to prepare autoradiograms. Slides were examined microscopically after development of the emulsion and staining, and the net grain count (NNG), the number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm, was determined for one hundred cells with normal morphology (from two of the three slides prepared). For each slide, animal and dose point, the population average NNG and percentage of cells responding or in repair (NNG of ≥ 5) was calculated.
The data were evaluated for indication of PROXEL Press Paste induced UDS. The criteria for a positive result were if the PROXEL Press Paste (at any dose at either post dose time point) yielded group mean NNG values of zero or above and ≥ 20% of cells had mean NNG values of ≥ 5. The data would also be indicative of a positive result if an increase above the solvent control levels was seen in both NNG and the percentage in repair.

Results and discussion
In the preliminary study during the 2 day post-dose observation period piloerection, weight loss, lethargy and abnormal breathing (1000 mg/kg dose level only) were observed at 700, 1000 and 1400 mg/kg dose levels. At 2000 mg/kg, piloerection, eye closure, abnormal breathing and lethargy were observed. One animal was killed in extremis shortly after dosing at 2000 mg/kg and one animal from this dose group was found dead one day after dosing. In the remaining animal some weight loss was observed.
As mortalities were seen at 2000 mg/kg, a dose of 1400 mg/kg was considered representative of a maximum tolerated dose. The maximum dose level of 1400 mg/kg was therefore selected for the main study which was tested together with a dose rate of 560 mg/kg.
In the main study, clinical signs of piloerection and lethargy were observed at the top dose tested, 1400 mg/kg, in both experiments. On the day after dosing with 1400 mg/kg PROXEL Press Paste in the 12-14 hour experiment, two animals (out of four animals) were found dead. No clinical signs were observed in the 560 mg/kg dose group.
Negative (vehicle) control animals gave group mean NNG values of 2.3 and 0 in experiments 1 and 2, respectively. These values did not exceed the upper limit of the historical negative control range and only 0 to 0.3% cells in experiments 1 and 2 were in repair. Group mean NNG values were increased by 2-AAP and DMN treatment to ≥ 5 and more than 50% cells found to be in repair. The vehicle control NNG value was consistent with both published and historical control data, and the system was shown to be sensitive to two known DNA damaging agents requiring metabolism for their action. The assay was therefore accepted as valid.
Treatment with PROXEL Press Paste at doses up to 1400 mg/kg yielded NNG values less than zero, producing group mean NNG values over the two experiments in the range of -2.5 to -0.1, values below the threshold of zero NNG required for a positive response. No cells were seen in repair at any dose of PROXEL Press Paste (BIT).
It may be noted that due to mortalities seen at the top dose tested in the 12-14 hour experiment, it was only possible to analyse UDS from two animals. In view of the clear negative results obtained in this assay, this was not considered to have prejudiced the validity of the study.
The data obtained in this study indicate that oral treatment of male rats dosed once with 540 or 1400 mg/kg PROXEL Press Paste did not result in increased UDS in hepatocytes isolated approximately 12-14 or 2-4 hours after dosing.

Conclusion
When treated orally once with PROXEL Press Paste at doses up to 1400 mg/kg, male rats showed no induction of UDS in hepatocytes isolated ex vivo approximately 12-14 or 2-4 hours after dosing. It is concluded that PROXEL Press Paste had no genotoxic activity detectable in this test system under the experimental conditions employed.
The study can be considered to be compatible with OECD 486. Although UDS analysis was not performed on three animals in the top dose group (due to a mortality) in the 12-14 hour test, it is not considered to affect the validity of the study since the results from the UDS analysis performed were clearly negative.
Executive summary:

A study was conducted to determine the in vivo genotoxic potential of the substance according to OECD Guideline 486. Groups of three male rats were treated with the test substance at 0, 560 and 1400 mg/kg bw by oral gavage. The positive controls used were 75 mg/kg bw 2 acetamidofluorene (2 -AAF) suspended in corn oil (12 -14 hour experiment) and 10 mg/kg bw dimethylnitrosamine (DMN) dissolved in purified water (2 -4 hour experiment). Approximately 12 -14 hours (experiment 1) or 2 -4 hours (experiment 2) after dosing, animals were killed and their livers were perfused with collagenase to provide a primary culture of hepatocytes and the cultures were treated with [3H] thymidine. Slides from the cultures were evaluated to determine the net grain count as a measure of the unscheduled DNA synthesis. Negative (vehicle) control animals gave group mean net grain count of  2.3 and 0 in experiments 1 and 2, respectively. These values did not exceed the upper limit of the historical negative control range. Group mean net grain count values were increased by 2 -AAP and DMN treatment. Overall, the system was shown to be sensitive to two known DNA damaging agents requiring metabolism for their action. The assay was therefore accepted as valid. Treatment with the test substance up to 1400 mg/kg bw yielded net grain values less than zero, producing group mean values in the range of -2.5 to -0.1, values below the threshold required for a positive response. Under the study conditions, the substance was not considered to be genotoxic in the primary hepatocyte cultures in rat (Howe, 2001).