S-2-Benzothiazolyl (Z)-2-(2-aminothiazol-4-yl)-2-acetyloxyiminothioacetate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Comet Assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016

Deviations:

no

Principles of method if other than guideline:

The comet assay (single cell gel electrophoresis assay) can indicate the mutagenic potential of the test item by measuring its ability to induce DNA damage in the target organs or tissues.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

457-660-1

EC Name:

-

Cas Number:

104797-47-9

Molecular formula:

C14H10N4O3S3 (Hill formula) C14H10N4O3S3 (CAS Formula)

IUPAC Name:

(Z)-[1-(2-amino-1,3-thiazol-4-yl)-2-(1,3-benzothiazol-2-ylsulfanyl)-2-oxoethylidene]amino acetate

Test material form:

solid: particulate/powder

Details on test material:

Test item: t15-AE
Chemical name: S-2-Benzothiazolyl (Z)-2-(2-aminothiazol-4-yl) -2-acetyloxyiminothioacetate
Batch No.: 31139692
CAS No.: 104797-47-9
Appearance: Yellowish light brown, weakly odorous powder
Manufacturing date: 15 June 2015
Retest date: May 2017
Storage: Refrigerator (2-8ºC), protected from light

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species / Strain: Rat, CRL (WI) BR of Wistar origin
Justification of the species: Rats are routinely tested in this test and the chosen Wistar rat was selected due to a wide range of experience with this strain of rat in corresponding toxicity studies.
Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90.

Sex:

male

Details on test animals or test system and environmental conditions:

Hygienic level at arrival: SPF
Body weights at arrival: 240-250 g
Hygienic level during the study: Good conventional environment
Acclimatization time: 7 days (from 04 to 11 January, 2017)
Age at the treatment: 57-60 days (Young adult rats, less than 9 weeks old at the commencement of the treatment).
Body weight range at the randomization: 282.3-323.6 g
Body weight range at the start of the test (on day 0, before the first treatment): 289.9-337.3 g, the weight variation in animals involved at the randomization and starting point of the study did not exceed ± 20 %.

Housing: 3 animals / cage; at the positive control group 2 animals / cages
Cage type: Type III polypropylene/polycarbonate (Size: 22 x 32 x 19 cm (width x length x height).
Bedding: Certified laboratory wood bedding (Lignocel Hygienic Animal Bedding)

Illumination: Artificial light, 12 hours daily, from 6 a.m. to 6 p.m.
Temperature: In the range of 22 ± 3 °C (the actual values: 20.1-23.3 °C).
Relative humidity: In the range of 30 – 70 % (the actual values: 31-57 %).
Ventilation: Provided by central air-condition system. The numbers of air changes per hour is higher than 10.

Animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, ad libitum.
Animals received tap water from watering bottles (from municipal supply, as for human consumption, from 500 mL bottles), ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Methylcellulose (1 % in ultrapure water).

Details on exposure:

A treatment volume of 10 mL dose preparation/kg body weight was administered in all test item treatment groups and in the vehicle control group and in the positive control group.

Duration of treatment / exposure:

2 treatments: once on Day 0 and 24 hours thereafter.

Frequency of treatment:

2 treatments: once on Day 0 and 24 hours thereafter.

Post exposure period:

Expression Time (Sampling Time):
The sampling time is considered as a critical variable because it is determined by the period needed for the test chemicals to reach maximum concentration in the target tissue and for DNA strand breaks to be induced but before those breaks are removed, repaired or lead to cell death. A suitable compromise for the measurement of genotoxicity is to sample at 2-6 hour after the last treatment. 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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Number of animals treated: 28. Six animals in the dose groups and negative control group; 4 animals in the positive control group.
Number of animals investigated (cell preparations Comet Assay): 23 animals; 5 animals per test item dose or vehicle control groups and 3 animals in the positive control group. The required minimum is: 5 scorable animals/group in the vehicle (negative) control and 3 scorable animals/group in the positive control group; therefore correspondently 6 and 4 animals were treated to avoid difficulties from possible mortality during the treatments and from problem during the cell isolations.

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulfonate (EMS)

Examinations

Tissues and cell types examined:

Liver cells and glandular stomach cells.

Details of tissue and slide preparation:

Liver: 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 Cells: 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 epithelium 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.

Cytotoxicity Measurements: In this assay the proportion of viable cells was scored using a Trypan blue technique. Cytotoxicity was determined on a small sample (using 100 μL cell suspension) of each isolated cell suspension following the Trypan blue dye exclusion technique. The available volume of the cell suspension was diluted to 1:1 ratio with trypan blue dye and the number of living and compromised cells counted. This screening technique as an indicator provided preliminary information from the effectiveness and success of the single cell preparation. The results of this dye exclusion method were predominantly informative as ratios between the control and test item treated doses, because in this experiment a mincing and homogenization technique was used for the single cell preparations. The decrease of viability should not be more than 30 % when compared to the concurrent control.

In this study no histopathological examination is required due to the negative results observed.

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. 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.
Pre-treatment of slides: 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).
Embedding the cells: Thereafter 85 or 165 μL (~1-9 x 10E4 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.
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.
Unwinding, 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. 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: 6 °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, 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 twice. 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 2 μg/mL Ethidium bromide.

Evaluation criteria:

For each animal and each tissue 4 slides were prepared.
Three slides of five animals per vehicle control and test item treatments were stained and analysed and three slides of three animals per positive controls. 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. DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, tail length and tail moment.
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.

Statistics:

The heterogeneity of the obtained data was tested. The statistical significance of % tail DNA values, tail length and OTM values; furthermore the number of ghost cells was carried out using the appropriate statistical method, using SPSS PC+ software.
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.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Tables of the results are presented in the Attachment.
No mortality was observed during the treatments and expression period. Neither toxic symptoms nor any clinical signs were observed during the treatments. At the tissue isolation normal appearance, anatomy of target organs (liver, stomach) was observed in all dose levels and controls. Hyperaemia in the stomach and intestine; furthermore reddish discoloration of intestinal content and excrement was observed at 2000 mg/kg body weight/day. The average body weights increased in negative and positive controls when compared the weight values just before the first treatment and just before the sacrifice. The body weight increases were minimal 0.33 and 1.69 %. At 2000 mg/kg body weights/day 0.79 %, at 1000 mg/kg body weight/day 2.91 % and at 500 mg/kg body weight/day 3.83 % doses body weight decrease was noticed.

Cytotoxicity, Ghost Cells: A first indication of possible cytotoxicity was estimated by Trypan blue dye exclusion technique. 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 83-90 % at the liver cell preparations and 83-89 % at the stomach cell preparations. The decrease of viability was not more than 30% compared to the concurrent control in any case.
At the liver samples the numbers of ghost cells did not differ statistically significantly from that of the vehicle control at the examined test item treated doses but a statistically significant increase of ghost cells was noticed at the EMS treatments. In the stomach samples the percentage of ghost cells remained nearly in the same range (a clear dose-dependent change was not noticed) and did not differ statistically significantly from that of the vehicle control at the test item doses and positive control.

% Tail DNA, Olive Tail Moment and Tail Length Comparisons:
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 median % tail DNA values of each dose remained in the vehicle control range at both, the liver and the stomach samples.
The olive tail moment values in the liver and stomach samples did not differ statistically significantly from that of the vehicle control at the whole examined dose range.
The tail length values in the liver samples at the test item doses (500-2000 mg/kg bw/day) were slightly higher than the tail length value of the vehicle control. The higher values remained in the same range at the three test item doses, but the statistical evaluation established significant differences between the vehicle control and the test item treatments. The statistical significance at the tail length values of the liver samples was not linked with dose-relationship, and the slightly higher values remained well within the corresponding historical control data range of the vehicle control.
The tail length values did not differ statistically significantly from that of the vehicle control at the examined doses in the stomach samples.

Under the experimental conditions, the test item t15-AE 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 t15-AE is negative and did not show genotoxic activity in the examined tissues.

The concurrent negative control was considered as acceptable for addition to the laboratory historical control database.
The concurrent positive control induced responses that are compatible with those generated in the historical control database and produce a statistically significant increase compared with the concurrent negative control.

Any other information on results incl. tables

The test item proved to be adequately stable in 1 % Methylcellulose formulations at 1 and 200 mg/mL concentration levels at least for 1 day at room temperature and for 3 days in refrigerator (5 ± 3°C).

Applicant's summary and conclusion

Conclusions:

The test item t15-AE did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in liver or in stomach cells. The test item did not show genotoxic activity in the examined tissues.

Executive summary:

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.