Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-04-01 to 2016-08-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
additive
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT., Cserkesz u. 90.,H-1103 Budapest, Hungary
- Age at study initiation: 55-60 days (Young adult rats, less than 9 weeks old at the commencement of the treatment).
- Weight at study initiation: 258-273 g (randomization), 262-284 g (start of the test)
- Assigned to test groups randomly: yes: All animals were sorted according to body weight by computer and grouped according to weight ranges.
- Housing: 3 animals / cage; at the positive control group 2 animals / cages
- Diet: Animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum.
- Water: Animal received tap water from watering bottles (from municipal supply, as for human consumption, from 500 mL bottles), ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
Light: Artificial, 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: In the range of 22 ± 3 °C (the actual values: 21.2-23.2oC from 30 March to 04 April, 2016).
Relative humidity: In the range of 30 – 70 % (the actual values: 33-48 % from 30 March to 04 April, 2016).
Ventilation: Provided by central air-condition system. The numbers of air changes per hour is higher than 10.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Vehicle used: Sunflower oil
- Lot/batch no.: BH7683855
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations were prepared before each treatment. The test item was formulated in the vehicle in nominal concentrations of 400, 200 and 100 mg/mL. The nominal concentration values of 400, 200 and 100 mg/mL (and the corresponding dose levels: 2000, 1000 and 500 mg/kg body weight) were applied and referred throughout the study.

Duration of treatment / exposure:
Administration on day 0 and on 24 hours thereafter
Frequency of treatment:
twice
Post exposure period:
3-4 hours after the second treatment
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
corresponding to 400 mg/mL
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
corresponding to 200 mg/mL
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
corresponding to 100 mg/mL
No. of animals per sex per dose:
Numbers of Treated Animals: (males only)
6 animals in the dose groups and negative control group;
4 animals in the positive control group.

Numbers of Analysed Animals, Cells:
5 animals in the dose groups and negative control group;
3 animals in the positive control group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl methanesulfonate (EMS)
- Route of administration: oral (gavage)
- Concentration: This positive control was investigated at a concentration of 200 mg/kg body weight. It was dissolved and applied in physiological saline at a concentration of 20 mg/mL.

Examinations

Tissues and cell types examined:
liver and glandular stomach
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
For dose selection the guideline proposal and the information obtained from a previously performed acute oral toxicity study with the test item in rats (LD50 >2000 mg/kg bw) was taken into consideration. Based on this information the maximum dose was chosen to be 2000 mg/kg bw/day, and in addition to the maximum dose two additional doses, i.e. 1000 and 500 mg/kg bw/day were selected.
For the correct identification of the maximum tolerated dose (MTD) where no death, evidence of pain, suffering or distress occurs, a range finding test was performed in the testing laboratory, using the same species, strain, sex, and treatment regimen to be used in the main study.
In the range-finding test (based on the available information) two animals (male rats, CRL (WI) BR of Wistar origin) were treated with the test item by oral administration at 5 mL/kg body weight treatment volume at the concentration of 2000 mg/kg bw/day. The treatment was performed on two consecutive days with 24h interval. At the chosen concentration level mortality was not observed. Any clinical sign, any suffering of animals were not observed one hour after the treatment, and reduced activity, incoordination and laxity were observed after two and four hours after the first treatment. Any clinical signs or suffering of animals were not observed before the second treatment and reduced activity, incoordination laxity and, at one animal, salivation were observed after two and three hours after the second treatment. The symptoms decreased and eliminated about 7 hours after the second treatment and due to the noticed intensity thereafter the elimination of the clinical signs; 2000 mg/kg body weight/day was chosen as the highest dose level in the present study.

SAMPLING TIMES:
In this particular test the sampling was performed 3-4 hours after the second treatment (the animals were euthanized consistent with the effective animal welfare legislation and 3Rs principles using Isofluran CP®, humanely killed and the cells of the target tissues were isolated) and care was taken to necropsy all animal at the same time after the last dose.

DETAILS OF SLIDE PREPARATION:
- Liver Single Cell Preparation:
A portion of the left lateral lobe of the liver was removed and washed in the cold mincing buffer until as much blood as possible was removed; thereafter placed in mincing buffer (ice cold Hank’s Balanced Salt Solution (HBSS) containing 20 mM EDTA and 10 % DMSO), minced with a pair of fine scissors to release the cells. The cell suspension was kept on ice for about 30 seconds to allow the large clumps to settle. The supernatant was pipetted into an Eppendorf tube and used for comet slides.

- Glandular Stomach Single Cell Preparation:
The stomach was open and washed free shortly from food using cold phosphate buffered saline. The forestomach was removed and discarded. The glandular stomach was then placed into cold mincing buffer and incubated on ice for about 15 minutes. After the incubation the surface epithelia was gently scraped about two times using a scalpel blade. This layer was discarded and the gastric mucosa was rinsed with cold mincing buffer. Thereafter the stomach epithelia was carefully scraped for 4-5 times with scalpel blade. The obtained cell suspension was kept on ice for about 30 seconds to allow the large clumps to settle. The supernatant was pipetted into an Eppendorf tube and used for comet slides.

- Preparation of the Comet Assay Slides:
The slide preparation was done within one hour after single cell preparation. Four slides were prepared for each animal for each tissue sample, with six animals per dose group and vehicle control and four animals for the positive control groups. In summary 24 slides per treatment per tissue for the treatment groups and vehicle control and 16 slides per tissue for the positive controls. The slides were adequately coded (with number: animal number, parallel number and M letter for liver and G letter for stomach).

Pre-treatment of slides:
Conventional (superfrost) slides were dipped in hot 0.5 % normal melting point agarose in water. After gently remove the underside of the slides were wiped in order to remove the excess of agarose. The slides were then laid on a flat surface and were let allow drying.

Embedding the cells:
Before the use a volume of 130 μL of 0.5 % normal melting point agarose (NMA) was added on a microscope slide pre-layered with 0.5 % NMA (see above) and covered with a glass coverslip. The slides were placed on a tray until the agarose hardens (~ 5 minutes). After the cell isolations each cell suspension was mixed with 0.5 % or 1.0 % Low Melting Point Agarose (LMPA).

Thereafter 85-165 μL (~1-9 x 104 cells) of this mixture was added on the microscope slide after gentle slide off the coverslip*.
The microscope slides were covered with a new coverslip. After the LMPA-cell mixture hardens an additional 70 μL of NMA was dropped on the microscope slide after a gentle slide off the (second) coverslip and an additional new coverslip was laid on the slide. After the repeated NMA layer hardens the coverslip was removed.

* The aim of applying different volumes and concentrations of cell suspension and LMPA was the keeping of a relatively constant LMPA concentration and cell number (at ~104 order of magnitude). There were four cases where the cell number on the slides was ~1 x 105 order. At two liver samples: at one animal in 1000 mg/kg body weight dose and in the EMS control; furthermore at two stomach samples at one animal in the vehicle control and in the 1000 mg/kg body weight dose.

- Lysis:
After the top layer of agarose solidifies and the last glass coverslip was removed the slides were immersed in chilled lysing solution. The slides were kept overnight in lysing solution at 2-8 °C (in refrigerator) in the dark.
After the incubation period, the slides were rinsed to remove residual detergents and salts prior to the alkali unwinding step. This rinsing procedure was performed in electrophoresis buffer.

METHOD OF ANALYSIS:
- Unwinding and Electrophoresis:
The slides were removed from the lysing solution and randomly placed on a horizontal gel electrophoresis unit. The unit was filled up with freshly prepared electrophoresis solution until the surfaces of the slides are completely covered with the solution (to about 1-2 mm above the slides).
During the unwinding and electrophoresis a balanced design was used to place slides in the electrophoresis tank to mitigate the effects of any trends or edge effect within the tank and to minimize batch to batch variability.
The slides were left for 30 min. for the DNA to unwind. Thereafter the electrophoresis was conducted for 30 min. by applying a constant voltage of 25V and an electric current of about 300 mA (250-290 mA). The same volume of the electrophoresis solution was used at every run, therefore at constant voltage slight change in the electric current was noticed. All of these steps were sheltered from the daylight to prevent the occurrence of additional DNA damage.
The temperature of the electrophoresis solution (before the unwinding the electrophoresis solution was kept in refrigerator, its temperature was noticed: 3.5-4.2 °C)through unwinding and electrophoresis was maintained at a low temperature, at 5°C using a special cooler designed for Comet electrophoresis tank. The temperature of the electrophoresis solution before the unwinding, during the unwinding and electrophoresis was kept at 5°C, and recorded once during the procedure.

- Neutralization and Preservation of Slides:
After electrophoresis, the slides were removed from the electrophoresis unit, covered with neutralization solution left stand for about 5 minutes, thereafter blotted and covered again with neutralization solution. This procedure was repeated for three times. Subsequently the slides were exposed for additional 5 minutes to absolute ethanol in order to preserve all of the slides. The slides were stored at room temperature until scored.

- Staining:
The slides were air dried and then stored at room temperature until they were scored for comets. Just prior the scoring the DNA, the slides were stained using 50 μL of 20 μg/mL Ethidium bromide.

- Evaluation of slides:
Every animal was euthanized and its cells were isolated. For each animal and each tissue 4 slides were prepared (8 slides per animal, 48 slides per test item doses and negative control and 32 slides per positive controls).*

* One animal died at the highest dose group of 2000 mg/kg body weight, before the sampling. This animal was excluded from the sampling and subsequent analysis. At the highest dose of 2000 mg/kg body weight 40 slides were prepared.

Three slides of five animals per vehicle control and test item treatments were stained and analysed (6 slides per animal, 30 slides per dose) and three slides of three animals per positive controls were stained and analysed (6 slides per animal, 18 slides per dose). Coded slides were stained and blind scored. The slides were examined with an appropriate magnification (200x) using fluorescent microscope (Olympus BH-2) equipped with an appropriate excitation filter (TRITC) and with an Alpha DCM 510B CMOS camera. For image analysis the Andor Kinetic Imaging Komet 6.0 (Andor Technology) was used. For each tissue sample fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analyzed cells per test item treatment, per vehicle control and 450 per positive controls). DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, tail moment** and tail length.
** Olive Tail Moment (OTM): is expressed in arbitrary units, is calculated by multiplying the percentage of DNA (fluorescence) in the tail by the length of the tail in μm. The tail length is measured between the center of the comet head and the end of the comet tail.

The tail % DNA (also known as tail intensity) was applied for the evaluation and interpretation of the results and determined by the DNA fragment intensity in the tail expressed as a percentage of the cell’s total intensity. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells were excluded from the image analysis data collection, however determining of their frequency is useful for the data interpretation. The ghost cells were recorded for each slide per animal, per type of the treatment and per tissue. The ghost cells are also known as clouds or hedgehogs, are morphological indicative of highly damaged cells and their presence often associated with severe genotoxicity, necrosis and apoptosis.
Ghost cells results from a total migration of the DNA from the nucleus into the comet tail, reducing the size of the head to a minimum.
Evaluation criteria:
The test chemical is clearly negative if:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- there is no concentration-related increase when evaluated with an appropriate trend test;
- all results are inside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administration;
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) is demonstrated.

The test chemical is clearly positive if:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- the increase is dose-related when evaluated with an appropriate trend test;
- any of the results are outside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administration;
Statistics:
The heterogeneity of the obtained data was tested. The statistical significance of % tail DNA values, OTM and tail length values; furthermore the number of ghost cells was carried out using the appropriate statistical method, using SPSS PC+ software.
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. In case of a positive analysis, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data were not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. In case of a positive analysis result, the inter-group comparisons were performed using Mann-Whitney U-test.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw/day
- Clinical signs of toxicity in test animals: At the chosen concentration level mortality was not observed. Any clinical sign, any suffering of animals were not observed one hour after the treatment, and reduced activity, incoordination and laxity were observed after two and four hours after the first treatment. Any clinical signs or suffering of animals were not observed before the second treatment and reduced activity, incoordination laxity and, at one animal, salivation were observed after two and three hours after the second treatment. The symptoms decreased and eliminated about 7 hours after the second treatment and due to the noticed intensity and elimination of the observations; 2000 mg/kg body weight/day was chosen as the highest dose level in the present study.

RESULTS OF DEFINITIVE STUDY:
- Appropriateness of dose levels and route: The highest dose (2000 mg/kg bw/day) is selected according to the criteria required by the OECD 489 guideline. Exposure via oral route was chosen as this route was considered to be the most relevant exposure route.

Any other information on results incl. tables

Results of Analytical Measurements

Based on the OECD 489 guideline three doses: 2000, 1000 and 500 mg/kg body weight/day were selected in this study. For these doses the following corresponding treatment solutions were prepared: 400, 200 and 100 mg/mL (the treatment volume was 5 mL/kg body weight). The treatment solutions were prepared freshly before each treatment and corresponding analytical samples were taken and immediately measured or stored in refrigerator until analysis.Based on the results of the available validation study and partial validation study the test item proved to be adequately stable in Sunflower oil formulations at 1, 10 and 500 mg/mL concentration levels at least for 4 hours at room temperature and for 4 days in refrigerator (5 ± 3°C), (Study Nos.: 552.102.2994 and 552.102.3150). The homogeneity of the samples was good, the test item in Sunflower oil formulations was considered to be homogeneous. The measured concentration values remained within the ±5% of nominal range at all concentrations. The nominal concentration values of 400, 200 and 100 mg/mL (and the corresponding dose levels: 2000, 1000 and 500 mg/kg body weight) were applied and referred throughout the study.

Cytotoxicity, Ghost Cells

In this study a first indication of possible cytotoxicity was estimated by Trypan blue dye exclusion technique. This screening technique as an indicator provided preliminary information from the effectiveness and success of the single cell preparation. The cell concentrations of the isolated cell suspensions were in the 105 106 order of magnitude. The viability values of both the liver and the stomach cell suspensions remained in the same, vehicle control range at all test item treatment doses and positive controls. The screened average viability values varied between 81-85 % at the liver cell preparations and 79-83 % at the stomach cell preparations. The decrease of viability was not more than 30 % compared to the concurrent control in any case. In addition, each slide was examined for presence of ghost cells (possible indicator of cytotoxicity and/or apoptosis). Ghost cells results from a total migration of the DNA from the nucleus into the comet tail, reducing the size of the head to a minimum. However, the increased frequency of ghost cells may indicate that cells with severe DNA damage continue to be alive, possibly due to protective functions such as DNA repair. The interpretation of ghost cell data should always be done in the context of additional cytotoxicity assessments. According to the testing laboratory’s experience the number of ghost cells is in average about 3-8/slide in the liver preparations and 3-10/slide at the stomach preparations, higher numbers were observed at the 1,2-Dimethylhydrazine dihydrochloride positive control treatments in earlier studies. In the present study, in the stomach samples the number of ghost cells remained nearly in the same range (a clear dose-dependent change was not noticed) at the test item doses and positive control; however its statistical evaluation established significant differences between the vehicle control and the 1000 and 2000 mg/kg body weight/day doses. In these cases, the higher frequency of ghost cells was not accompanied with increased DNA migration. The relatively higher number of ghost cells in stomach samples was predominantly associated with toxic effects attributable to the test item, which was observed during macroscopic inspection of the tissue prior to isolation of cells and a generally decreased cell number in the cell isolations. According to the laboratory expertise at the cell isolations the average cell numbers are in 105-106/mL order of magnitude. In the present study most of the preparations were in 105/mL order of magnitude. At the evaluation of ghost cells a relatively poorer cell suspension quality was also found as it was described as possible explanation for higher frequency of ghost cells by the expert group in the JaCVAM validation trial.At the liver samples the numbers of ghost cells did not differ statistically significantly from that of the vehicle control at the examined doses but a statistically significant increase of ghost cells was noticed at the EMS treatments. At the EMS treatments all parameters that are relevant for the mutagenicity assessment (% tail DNA, tail length OTM values) were within the acceptable ranges of the testing laboratory and were in accordance with the referred literature. The relatively higher number of ghost cells at the EMS treatments is usually not observed in the testing laboratory. The referred JaCVAM validation trial report does not mention the known positive control EMS as ghost cell increasing chemical (such as e.g.: 1,2-Dimethylhydrazine dihydrochloride in the liver) either. In the present study the higher frequency of ghost cells at the EMS treatments was considered to be attributable to the severe DNA damage (genotoxicity) of the reference compound in the absence of any macroscopic tissue changes or a generally lower cell number in the cell isolates.

% Tail DNA, Olive Tail Moment and Tail Length Comparisons

The test item TBPIB-75-AL was investigated in three dose levels 2000, 1000 and 500 mg/kg body weight/day. The treatments were performed in two consecutive days (x2). Two target organs (tissues) of each animal were investigated: liver and stomach. For each tissue sample fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analyzed cells per test item treatment, per vehicle control and 450 per positive controls). DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, olive tail moment (OTM) and tail length. The tail % DNA (also known as tail intensity) was applied for the evaluation and interpretation of the results and determined by the DNA fragment intensity in the tail expressed as a percentage of the cell’s total intensity. The mean % tail DNA values of each dose remained in the vehicle control range at both, at the liver and stomach samples. Samples allowing the conclusion that the substance did not cause any effect. The slightly different (higher or lower) values did not differ statistically significantly from that of the vehicle control up to the limit dose of 2000 mg/kg body weight/day. Additionally the % tail DNA mean median values were calculated and investigated. In the liver and stomach samples the same tendencies were obtained as it was noticed at the mean values. Statistical significances were not obtained at the test item doses. The mean median value calculations were performed in the case of the % tail DNA parameter, only. The analysis of these values confirmed the results obtained at the mean value calculations. Additionally the olive tail moment (OTM) and tail length values of the vehicle control and each treatment were compared. Statistical significances at the OTM values of the liver and stomach samples were noticed at the 500 and 2000 mg/kg body weight/day doses. The statistical significances were considered as not relevant for mutagenicity assessment since the significance is linked to lower (but acceptable) values than the corresponding vehicle control value. In the case of the tail length comparisons statistical significance was noticed in the liver samples at the dose of 500 mg/kg body weight/day. The statistical significance was considered as not relevant for mutagenicity assessment since the significance is linked to a lower value which is well within the historical control data range.

Applicant's summary and conclusion

Conclusions:
The test item was investigated by the means of the in vivo comet assay on isolated liver and stomach cells under alkaline conditions in the male WISTAR rats administered orally twice with 2000, 1000 and 500 mg/kg body weight/day, with one sampling time of about 3 to 4 hours after the second treatment. Under the experimental conditions, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in liver or in stomach cells. The investigated test item is negative and did not show genotoxic activity in the examined tissues.
Executive summary:

The purpose of the comet assay (single cell gel electrophoresis assay) was to evaluate the mutagenic potential of the test item by measuring its ability to induce DNA damage in the target organs, tissues as specified and requested by ECHA. Formulations were prepared before each treatment. The test item was formulated in the vehicle in nominal concentrations of 400, 200 and 100 mg/mL. The measured concentration values remained within the ±5% of nominal range at all concentration levels examined. The nominal concentration values 400, 200 and 100 mg/mL (and the corresponding dose levels: 2000, 1000 and 500 mg/kg body weight) were applied and referred throughout the study. Analysis of formulations (for checking of each concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility according to the validated analytical method (Study codes: 552.102.2994 and 552.102.3150). The test substance was administered orally by gavage; twice: once on the day 0 and 24 hours thereafter at the test item doses and negative controls. The positive control animals were treated by oral gavage once during the experiment on the day 1. The target Tissues were the stomach and the liver. The Sampling time was 3-4 hours after the second treatment (doses and vehicle control) and 3-4 hours after the treatment (positive control) the animals were euthanized and the cells of the target tissues were isolated. Cytotoxicity was determined on a small sample of each isolated cell suspension following the Trypan blue dye exclusion technique, directly after sampling. Prior the scoring the DNA was stained with 50 μL of 20 μg/mL Ethidium bromide; The comets were measured via a digital camera linked to an image analyzer system using a fluorescence microscope equipped with an appropriate excitation filter at a magnification of 200X. For image analysis the Komet 6.0 F (Andor Technology) was used. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells were excluded from the image analysis data collection. The Numbers of treated Animals were 6 animals in the dose groups and negative control group; 4 animals in the positive control group. The Numbers of Analysed Animals, Cells: 5 animals in the dose groups and negative control group; 3 animals in the positive control group. For each tissue sample fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analyzed cells per test item treatment, per vehicle control and 450 per positive control).

All of the validity criteria regarding the negative and positive control treatments as well as the number of analysed cells, and the investigated dose levels were met (See: Validity of the Study). No mortality was observed during the treatments and expression period in the 1000 mg/kg body weight/day, 500 mg/kg body weight/day doses and in the controls (negative, positive). After the second treatment one animal died at the highest dose group of 2000 mg/kg body weight/day. Toxic symptoms or any clinical signs were not observed during the treatments in the controls (negative and positive) and in the 1000 and 500 mg/kg body weight/day doses. At the highest dose group of 2000 mg/kg body weight/day changed motility, reduced activity and incoordination was principally noticed after the first treatment. The signs discontinued, the animals were asymptomatic before the second treatment. The second treatment was performed as planned. Beside reduced activity and incoordination, salivation and piloerection were observed after the second treatment. At the tissue isolation after the opening of the stomach a characteristic strong chemical smell was noticed at the test item treatments and the smell intensity increased dose-dependently. Macroscopic change of gastric mucosa layer furthermore bedding material in the stomach was noticed at 1000 and 2000 mg/kg body weight/day. Hyperaemia in of the stomach and intestine was additionally observed at 2000 mg/kg body weight/day. The average body weights increased in negative and positive control and test item treatments at 1000 and 500 mg/kg body weights/day. The body weight increases remained in the same range. At 2000 mg/kg body weight/day slight body weight decrease was noticed. At the screening cytotoxicity measurements (using Trypan blue dye exclusion method) significant cytotoxicity was not noticed in any test item and control item treatments. In the stomach samples the number of ghost cells remained nearly in the same range at the test item doses and positive control; however the statistical evaluation established significant differences between the vehicle control and the 1000 and 2000 mg/kg body weight/day doses. At the liver samples the numbers of ghost cells did not differ statistically significantly from that of the vehicle control at the examined doses and a statistically significant increase of ghost cells was noticed at the EMS treatments. The ghost cells are a possible indicator of cytotoxicity and/or apoptosis. According to the referred literature increased frequency of ghost cells may indicate cells with severe DNA damage (genotoxicity). To be conscious of the mutagenicity results and laboratory’s earlier experience, the relatively higher number of ghost cells in the stomach samples at the test item treated doses were considered to be a possible indicator of cytotoxicity. While ghost cells in the liver samples treated with EMS are rather considered to be a possible indicator of genotoxicity. DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, Olive Tail Moment (OTM) and tail length. The mean % tail DNA values of each dose remained in the vehicle control range at both, at the liver and stomach samples. The slightly different (higher or lower) values did not differ statistically significantly from that of the vehicle control up to the limit dose of 2000 mg/kg bw/day . Additionally the % tail DNA mean median values were calculated and investigated.In the liver and stomach samples the same tendencies were obtained as it was noticed at the mean values.Statistical significances were not obtained at all test item doses. The mean median value calculations were performed in the case of the % tail DNA parameter, only. The analysis of these values confirmed the results obtained at the mean value calculations. Additionally the olive tail moment (OTM) and tail length values of the vehicle control and each treatment were compared. All of the obtained statistical significances at the OTM values and tail length values of the liver and stomach samples were considered as not relevant for mutagenicity assessment since the significance is linked with lower (but acceptable) values than the corresponding vehicle control value. In the case of the tail length comparisons statistical significance was noticed in the liver samples at the dose of 500 mg/kg body weight/day. The statistical significance was considered as not relevant for mutagenicity assessment since the significance is linked to a lower value which is well within the historical control data range. The test item was investigated by the means of the in vivo comet assay on isolated liver and stomach cells under alkaline conditions in the male WISTAR rats administered orally twice with 2000, 1000 and 500 mg/kg body weight/day, with one sampling time of about 3 to 4 hours after the second treatment. Under the experimental conditions, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in liver or in stomach cells. The investigated test item is negative and did not show genotoxic activity in the examined tissues.