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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
Remarks:
This study was performed for the purposes of a registration outside the European Economic Area.
Adequacy of study:
key study
Study period:
2011-05-04 to 2011-06-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
unscheduled DNA synthesis

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Specific details on test material used for the study:
EC Number: 939-946-2

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Strain: Rat (Wistar)
Number of animals: 24 males
Acclimation: Minimum 5 days
Age of the animals: 5 - 6 weeks
Initial body weight at start of treatment: 180.8 g (SD* ± 10.8 g) (body weight range 161.1 g – 204.4 g)
According to the suppliers assurance the animals were in healthy condition. The animals underwent acclimation in the animal house for at least five days after their arrival. During this period the animals did not show signs of illness or altered behaviour. The animals were distributed into the test groups at random and identified by cage number and tail tags.
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions. The diet and water are routinely analysed to ensure the absence of any contaminant that could reasonably be expected to affect the purpose or integrity of the study.
Certificates of analysis are retained.
Housing: Group
Cage type: Makrolon Type III/IV, with wire mesh top
Bedding: Granulated soft wood bedding
Feed: Pelleted standard diet, ad libitum
Water: Tap water, ad libitum
Environment: Temperature 22 ± 2°C
Relative humidity 30 - 65 %
Artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
For genotoxicity investigations it is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly. The administered dose volume was 10 mL/kg b.w.. The maximum tolerated dose is determined to be the dose that causes signs of toxicity (e.g. reduced spontaneous activity, eyelid closure, apathy, etc.) without having major effects on survival within 24 hours. If no signs of toxicity are observed the highest dose recommended by the OECD guideline to be used is 2000 mg/kg b.w. The low dose was 50 % of the high
dose. Four male rats (except the negative and positive control with 2 males only) were assigned to each test group
Duration of treatment / exposure:
The animals received the test item once orally and were examined for acute toxic symptoms at intervals of approx. 1 h, 2 h and 4 h for the 4 hours treatment and 1 h, 2-4 h, and 16 h for the 16 hours treatment after administration of the test item.
Frequency of treatment:
once
Post exposure period:
4 and 16 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
250 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
500 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
4
Control animals:
yes, concurrent vehicle
Positive control(s):
for 4 hour interval : N,N´-dimethylhydrazinedihydrochloride (DMH) 80 mg/kg bw single oral dose
for 16 hour interval: 2-acetylaminofluorene (2-AAF) 100 mg/kg bw single oral doe

Examinations

Tissues and cell types examined:
After terminally anaesthetising the rats the liver was perfused through
the vena portae with Hanks' balanced salt solution (HBSS) supplemented with collagenase (0.05 % (w/v), adjusted to pH 7.4 and maintained at 37° C
The isolated hepatocytes from the liver were washed twice with HBSS. The crude cell suspension was filtered through a stainless steel mesh to yield a single cell suspension. The quality of the perfusion was determined by the trypan blue dye exclusion method. In addition, the number of the isolated cells was determined. The washed hepatocytes were centrifuged and transferred into Williams medium E. This complete medium was adjusted to pH 7.6.
Three cultures were established from each animal. Aliquots of 2.5 mL with freshly isolated hepatocytes in complete culture medium (2.0 × 105 viable cells/mL) were added to 35 mm six-well dishes containing one 25 mm round plastic coverslip per well coated with gelatine. After an attachment period of approximately 1.5 h in a 95 % air/ 5 % CO2 humidified incubator at 37° C the culture medium was discarded. The cell layer was then rinsed once with PBS to remove non-adherent cells (9). Subsequently, 3HTdR (5 [Ci/mL, specific activity 20 Ci/mmol) in 2.0 mL culture medium
(WME, 1 % (v/v) FCS) was added to the cultures. After a labeling time of 4 h the cells were washed twice with WME supplemented with 1 % (v/v) FCS and 0.25 mM unlabeled thymidine. Cultures were incubated overnight using the same medium. To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % (w/v) sodium citrate for 10 minutes to swell the nuclei for better grain detection. The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3:1 v/v) for 20 minutes each, rinsed with 96 % (v/v) ethanol, and air-dried.
Details of tissue and slide preparation:
The cover slips were mounted on glass slides, cell side upwards and coated with KODAK NTB photographic emulsion in the dark The coated slides were stored in light-proof boxes in the presence of a drying agent for 14 days at 4° C. The photographic emulsion was then developed at room temperature, fixed in Fixer and stained with hematoxylin/eosin
Evaluation criteria:
Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. Slides were examined to ensure sufficient cells of normal morphology were present before analysis. The cells for scoring were randomly selected according to a fixed scheme. The number of silver grains in the nuclear area was counted automatically using the Sorcerer UDS device version 2.0 DT3152. In addition, the number of grains of the most heavily labeled nuclear-sized cytoplasm area adjacent to the nucleus was counted. Two slides per animal and 50 cells per slide were
evaluated. Heavily radio-labeled cells undergoing replicative DNA synthesis were excluded from counting.
The nuclear and cytoplasmic grain counts, the net grain counts (nuclear minus cytoplasmic grains) as well as the mean and percentage of cells in repair (cells with a net grain count larger than 5) were recorded. Individual slide and animal data are provided. The mean counts with standard deviation are used to describe the distribution of 3HTdR incorporation in the nucleus, the cytoplasm and for the net grains, respectively
Statistics:
Nuclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts. A test item is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points. A group average between 0 and 5 net grains is considered as a marginal response. A doserelated increase in nuclear and net grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive response with less than 5 net grains. Statistical significance may give further evidence for a positive evaluation. Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test. A test item producing net grains not greater than 0 or not significantly greater than the concurrent control, at anyone of the test points is considered non-effective in this system.
Biometry: A statistical evaluation of the results was not necessary to perform as the number of net grain counts of the groups treated with the test item were in the range of the corresponding controls.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clinical signs of toxicty at highest dose
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The highest dose level used was estimated in the pre-experiment to be the maximum tolerable dose. The treated animals expressed clinical signs of toxicity. The viability of the hepatocytes was not substantially affected by the in vivo treatment with the test item at any of the treatment periods or dose groups. The interindividual variations obtained for the numbers and the viabilities of the isolated hepatocytes are in the range of the laboratory historical control data reported previously. No UDS induction in the hepatocytes of the treated animals as compared to the concurrent vehicle controls was observed at either 250 or 500 mg/kg b.w. Neither the nuclear grains nor the resulting net grains were distinctly enhanced due to the in vivo
treatment of the animals with the test item for 4 hours or 16 hours, respectively. Therefore, the mean net grain counts obtained after treatment with the test item were consistently negative. As no UDS response was observed statistical analysis of the data was not performed.
In addition, no substantial shift to higher values was obtained in the percentage of cells in repair.
Appropriate reference mutagens [DMH, 80 mg/kg b.w. and 2-AAF, 100 mg/kg b.w.] were used as positive controls. In vivo treatment with DMH or 2-AAF induced distinct increases in the number of nuclear and net grain counts as well as in the percentage of cells in repair indicating the test system was working

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions reported, i.e. oral administration up to 500 mg/kg, the tolerable dose, the test item did not induce DNA-damage
leading to increased repair synthesis in the hepatocytes of the treated rats. Therefore, the test item is considered to be non-genotoxic in this in vivo UDS test system.
The study is considered to be relevant, reliable, adequate for risk assessment, and adequate for classification purposes.
Executive summary:

Study Design

The test item was assessed in the in vivo unscheduled DNA synthesis (UDS) assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats. The test item was formulated in corn oil, which was also used as the vehicle control. The volume administered orally was 10 mL/kg body weight. After a single oral treatment and a post-treatment period of 4 or 16 hours the animals were terminally anaesthetised and liver perfusion was performed. Primary hepatocyte cultures were established and exposed for 4 hours to 3HTdR (methyl-3H-thymidine), which is incorporated if UDS occurs.

Results

The test item was tested at the following dose levels: 4 and 16 hours preparation intervals: 250 and 500 mg/kg b.w.. The highest dose used was estimated in a pre-experiment to be the maximum tolerable dose. No substantial gender specific differences in toxicity were observed so the study was performed using male animals only. For each experimental group hepatocytes of four treated animals were assessed for the occurrence of UDS. For the negative and positive control groups two animals were assessed for the occurrence of UDS. The viability of the hepatocytes was not substantially affected by the in vivo treatment with the test item.

None of the dose levels tested revealed UDS induction in the hepatocytes of the treated animals as compared to the corresponding vehicle controls. Appropriate reference mutagens [DMH, 80 mg/kg b.w. and 2-AAF, 100 mg/kg b.w.] were used as positive controls. Treatment with the positive control substances revealed distinct increases in the number of nuclear and net grain counts.

Conclusion

In conclusion, it can be stated that under the experimental conditions reported, i.e. oral administration up to 500 mg/kg, the test item did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats. Therefore, the test item is

considered to be non-genotoxic in this in vivo UDS test system