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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A negative in vitro gene mutation assay and a positive in vitro chromosome aberration test are available.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP. Mistakes, e.g. concerning the concentrations used, caused repetitions of individual experiments.

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

Mistakes, e.g. concerning the concentrations used, caused repetitions of individual experiments.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

Mistakes, e.g. concerning the concentrations used, caused repetitions of individual experiments.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: Human lymphocytes.

Details on mammalian cell type (if applicable):

Whole blood was obtained from five healthy females and from three healthy males into Na-heparinized Vacutainers (Becton-Dickinson). The volunteers were non-smokers and were not suspected of any virus infection nor had been exposed to high levels of radiation or toxic chemicals.
Primary cultures were established within one hour after venipuncture by adding 0. 7 ml of whole blood and 0.2 ml of phytohemagglutinin HA 15 (Murex Diagnostics Ltd., UK, reconstituted by addition of 5 ml sterile aqua dest.) to 9.3 mL of complete culture medium.

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from Aroclor 1254 induced rat livers.

Test concentrations with justification for top dose:

Concentration range in the main tests (with metabolic activation), where metaphases were analysed: 0.0023 to 0.11 mg/mL.
Concentration range in the main tests (without metabolic activation), where metaphases were analysed: 0.0017 to 0.09 mg/mL.
9 Experiments were performed. For concentrations and treatment length of each experiment see the attachment.

For each experiment a stock solution was prepared by dissolving appropriate amounts of T15-AE with DMSO. The test substance solutions were then obtained by diluting this stock solution with DMSO and further with the appropriate culture medium (RPMI or- for 20 hours of incubation - complete culture medium) to achieve a final concentration of 1 % DMSO in the medium. All preparations were made freshly before adding them to the cell cultures.

Vehicle / solvent:

DMSO.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

The negative control substance was the culture medium without and with the metabolic activation system and with 1 % (v/v) DMSO, as the test substance was sufficiently soluble in DMSO and as, according to experience, DMSO at a concentration of 1 % in the medium does not adversely affect cell survival or the activity of the metabolic activation system used.
Medium: RPMI 1640 medium with L-glutamine (Gibco BRL Life Technologies, UK), used for incubation periods of 3 hours.
Complete culture medium: used for the incubation period of 20 hours.

Nine experiments were performed: 5 of them without and 4 with the use of a metabolic activation system. For details of each experiment see the attachment.
Primary lymphocyte cultures were incubated for 48 hours, afterwards the test substance was added. In the experiments with the use of a metabolic activation system the test substance was washed out three hours later, and the cultures were cultivated for another 15 hours. Colcemid was added for 2 hours, and then cells were fixed and slides prepared.
For cultures without addition of a metabolic activation system, the treatment time was 3 hours in the first 3 experiments and 20 hours in the last 2 experiments. For each concentration of the test substance two cultures were established.

Evaluation criteria:

Mitotic indices:
The mitotic indices were determined by counting a total of 2000 lymphocytes per cell culture and by recording the number of lymphocytes in any stage of mitosis.

Selection of cultures for analysis:
In all experiments the test cultures with the highest scorable test substance concentration and the two next lower concentrations (or the next lower concentration, if there were only two concentrations set up or scorable) were analysed. In experiment D with the use of a metabolic activation system no marked cytotoxic effect was noted at any concentration tested. Therefore experiment F with higher test substance concentrations was performed and the metaphases of experiment D were not analysed.

Coding of slides
Slides from the selected treatments were coded by assigning random numbers before analysing for aberrations. Self adhesive labels covered the identification marks on the slides. The code list was not available to the analysing persons until the last slide was analysed.

Chromosome analysis
ZEISS or Nikon microscopes were used for analysis (magnification factor of about 600 to 1000). In general, apart from cultures with obviously high numbers of meta phases with aberrations, 100 metaphases per culture (i.e. 200 per concentration) were analysed for structural and numerical chromosomal aberrations. Only well spread cells with 44 to 47 chromosomes, polyploid and endoreduplicated cells were acceptable for analysis.

Statistics:

The Chi2-Test (two-tailed, p=0.05) was used for the comparison between the negative control and the test substance cultures. If the results were positive, comparisons were made separately between the negative control and each concentration. If conditions for the Chi2-Test were not met, Fisher's Exact Test was used. Chi2-Test or Fisher's Exact Test were also used for the comparison between the negative and the positive controls.
The following parameters were evaluated:
• the number of metaphases with numerical aberrations ( < 46 chromosomes, > 46 chromosomes, tetraploidy, endoreduplication)
• the number of metaphases with structural aberrations, excluding gaps
• the number of chromatid-type aberrations, excluding gaps
• the number of chromosome-type aberrations, excluding gaps
• the number of gaps (chromatid- and isochromatid-type).
If there were more than one structural aberration of the same type within one metaphase, these aberrations were referred to as "multiple". For statistical comparisons of the numbers of a specific aberration, such aberrations were counted as two aberrations.
A statistically significant increase in the number of meta phases with aberrations or a concentration-related increase in this number is considered as a positive result. However, a result can also be regarded as positive when other than merely statistical considerations, for example the kind of aberrations observed, are taken into account.

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

No mitosis at 0.33 mg/mL; 43 % at 0.11 mg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Mitotic index reduced to 63 % at 0.09 mg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

negative but outside the historical control

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Mitotic index reduced to 63 % at 0.09 mg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Mitotic index reduced to 27 % at 0.11 mg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

For more details see the attachment.
Cytotoxicity
Experiments without a metabolic activation system:
After 3 hours of incubation no mitoses at all were found at test substance concentrations of 0.33 mg/ml and higher. The mitotic indices were reduced to 42.6% of the corresponding negative control at 0.11 mg/ml and to 59.9% at 0.09 mg/ml. After 20 hours of incubation and at 0.09 mg/ml the mitotic indices were reduced to 60.3 %of the corresponding negative control. At test substance concentrations equal to or lower than 0.06 mg/ml medium the mitotic indices were between 72.6 % and 120.3 % of the respective negative controls.
Experiments with a metabolic activation system:
As in the experiments without a metabolic activation system no mitoses were found at test substance concentrations of 0.33 mg/ml and higher. At 0.11 mg/ml the mitotic indices were reduced to 26.9 % of the corresponding negative controls. At test substance concentrations equal to or lower than 0.09 mg/ml medium the mitotic indices were between 67.8% and 134.2 % of the respective negative controls.

Numerical aberrations
In experiment A (no metabolic activation system, 3 hours of test substance treatment) the number of metaphases with endoreduplications was statistically significantly higher than in the respective negative controls at 0.11 mg test substance per ml medium and the number of metaphases with less than 46 chromosomes was statistically significantly lower. No other statistically significant differences in the number of meta phases with numerical aberrations were noted in any experiment performed at any other concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.

Structural aberrations
In experiment A (no metabolic activation system, 3 hours of test substance treatment) the number of meta phases with structural aberrations as well as the number of chromosome-type aberrations were at 0.11 mg test substance per ml medium statistically significantly higher than in the corresponding negative control. These figures as well as the number of chromatid-type aberrations were also outside the range of historical negative control data.
In experiment E (no metabolic activation system, 20 hours of test substance treatment) the number of metaphases with structural aberrations as well as the number of chromosome-type and of chromatid-type aberrations were at 0.09 mg test substance per ml medium outside the range of historical negative control data.
No statistically significant increases in the number of metaphases with structural aberrations were noted in any other experiment performed at any other concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not. All other figures were within the range of historical negative controls.

Gaps
The number of gaps was in three experiments statistically significantly higher than in the corresponding negative control and it was also outside the range of historical negative control data: In experiments A (no metabolic activation system, 3 hours of test substance treatment) and F (metabolic activation system, 3 hours of test substance treatment) at 0.11 mg test substance per ml medium and in experiment E (no metabolic activation system, 20 hours of test substance treatment) at 0.09 mg/ml.
No statistically significant increases in the number of meta phases with gaps were noted in any other experiment performed at any other concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not. All other figures were within the range of historical negative controls.

Positive controls
The positive control substances caused clearly higher numbers of metaphases with structural aberrations (statistically significant) than found in the negative controls, without as well as with the use of a metabolic activation system, thus demonstrating that the test systems were adequate and that the metabolic activation system functioned properly.

Remarks on result:

other: other: Experiment A, 3 h exposure, 0.11 mg/mL

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
positive without metabolic activation

The test substance induced structural chromosomal aberrations in cultured human lymphocytes when no metabolic activation was used.

Executive summary:

Possible mutagenic properties of T15 -AE were investigated by an in vitro mammalian chromosome aberration test in human lymphocytes, according to the EC-method B.10 and OECD 473.

Methods

Nine experiments were performed: 5 of them without and 4 with the use of a metabolic activation system. (liver microsomes from Aroclor 1254 induced rats, with a co-factor solution).

Primary lymphocyte cultures were established from whole blood freshly obtained from donors. After 48 hours of incubation, the test substance was added. In the experiments with the use of a metabolic activation system the test substance was washed out three hours later, and the cultures were cultivated for another 15 hours. Colcemid was added for 2 hours, and then cells were fixed and slides prepared. For cultures without addition of a metabolic activation system, the treatment time was 3 hours or 20 hours.

For each concentration of the test substance two cultures were established. One negative control (medium) and one positive control (methanesulfonic acid methyl ester (MMS) for cultures without metabolic activation system and cyclophosphamide (CP) for cultures with a metabolic activation system) were set up.

The concentrations of T15 -AE ranged from 0.0023 to 0.11 mg/mL (with metabolic activation) and from 0.0017 to 0.09 mg/mL (without metabolic activation). 100 metaphases were analysed for structural and numerical chromosomal aberrations, i.e. 200 per concentration.

Results

The substance caused marked cytotoxic effects at concentrations of 0.11 mg/ml and higher at least in one experiment (reduction of the mitotic indices to less than 50 % of the respective negative controls).

There was, under the conditions of this study, relevant evidence that T15-AE is able to induce structural chromosomal aberrations in cultured human lymphocytes when no metabolic activation system is used. The conclusion is based on a statistically significant increase in the number of meta phases with structural aberrations in one experiment at a very high and cytotoxic test substance concentration, and on the finding that the figures were outside the range of historical negative controls in the respective experiment and also in another experiment at a high test substance concentration.

Statistically significantly higher numbers of metaphases with gaps, compared to the concurrent negative controls, in a total of three experiments (two of them without and one with the use of a metabolic activation system), being also outside the range of historical negative controls, may additionally indicate a potential to induce structural chromosome aberrations in cultured human lymphocytes.

Conclusion

The test substance induced structural chromosomal aberrations in cultured human lymphocytes when no metabolic activation was used.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Principles of method if other than guideline:

Direct plate incorporation method.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102, TA1535

Metabolic activation:

with and without

Metabolic activation system:

Microsomal fraction of rat liver, induced with Aroclor 1254.

Test concentrations with justification for top dose:

2.5 to 600 µg/plate.
In a preliminary experiment the strain TA100 was mixed with phosphate buffer, the test substance solutions and top-agar and plated on standard agar. The growth of the bacterial lawn was recorded. The test substance prevented the growth of the bacterial background lawn at concentrations of 556 µg/plate and above.

Vehicle / solvent:

The test substance solution for the highest concentration group was prepared by dissolving 60 mg of T15-AE in 10 ml of DMSO. The solutions for the lower concentrations were prepared by subsequent dilution.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

sodium azide

other: 2-aminoanthracene; 1,8-dihydroxy-anthraquinone; 4-nitro-o-phenylenediamine; t-butyl-hydroperoxide; 2-nitrofluorene

Details on test system and experimental conditions:

The exposure was performed according to the 'Plate Incorporation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state. The number of viable cells in the overnight-cultures is in the range of 2 x 10^8 cells per mL.
The plates were incubated at 37 °C for 2 days and the growth of the bacterial background and the density of revertant colonies was determined.
The test substance was tested without as well as with an external metabolising system (S9-mix). The results were verified by a second, independent experiment. Three plate were used per concentration and per strain for the test substance; 6 for the negative control.

Evaluation criteria:

The plates with less than about 50 revertant colonies, i.e. the plates of TA98 and TAI535 with the exception of the positive controls, were counted visually by marking the colonies with a felt tipped pen. The other plates were photographed with a video camera and the picture files were scanned for colonies by a computer program.
The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
• For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2.5 fold of the amount of the spontaneous revertants.
• For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 1.67 fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of the historic data of the Ames test.

Statistics:

Means and standard deviation were calculated for the number of mutants in every concentration group.

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100, TA102, TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The used strains of Salmonella typhimurium showed the expected genetic properties and were sensitive against several mutagenic chemicals.

All positive control substances increased the mutation frequency to more than the threshold values. As 2-aminoanthracene, 1,8-dihydroxy-anthraquinone and 7,12-dimethyl-benz[a]anthracene require metabolic activation for mutagenicity, the results of these substances demonstrate also the efficiency of the metabolising system.

Solubility
A precipitate was visible when the test substance was mixed with the agar at the 600 µg/plate samples. In the preliminary experiment, at higher concentrations, the precipitate was also visible when the colonies were counted and impeded the counting.

Toxicity
In the preliminary test the test substance prevented the growth of the bacterial background lawn at concentrations of 556 µg/plate and above.
In the main test without metabolisation a reduced bacterial background lawn and reduced numbers of revertants were observed in all plates of the 600 µg/plate group. With metabolisation toxicity was reduced, resulting in only a decreased number of revertants at the 600 µg/plate samples.

Mutagenicity:
No increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations was observed. The addition of an external metabolising system did not change these results.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The substance is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 up to the concentration of 600 µg per plate.

Executive summary:

T15 -AE was tested for mutagenic activity with the Salmonella typhimurium reverse mutation test (Ames Test). The study was conducted in accordance with the OECD guideline 471 and EU-method B.13/14. The test substance, dissolved in DMSO was tested at concentrations ranging from 2.5 µg to 600 µg per plate according to the "direct plate incorporation method" without external metabolisation as well as with an external metabolising system (S9-mix). As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA102 and TAI535 were used. Negative and positive controls were included. An independent repetition of the experiment was performed.

Results

Positive controls: All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system.

Test substance:

Toxicity: In the preliminary test the test substance prevented the growth of the bacterial background lawn at concentrations of 556 µg/plate and above. In the main test without metabolisation a reduced bacterial background lawn and reduced numbers of revertants were observed in all plates of the 600 µg/plate group. With metabolisation toxicity was reduced, resulting in only a decreased number of revertants at the 600 µg/plate samples.

Mutagenicity:

No increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations was observed. The addition of an external metabolising system did not change these results.

 

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The test item was investigated in the in vivo comet assay on isolated liver and stomach cells under alkaline conditions in the rat, administered orally twice with 2000, 1000 and 500 mg/kg body weight/day. The test item did not induce 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.

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to other study

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

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.

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.

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

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)

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.

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.

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).

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Cytogenetic study: The decision of the authors of the report, to designate the outcome of the in vitro cytogenetic test as positive is based on a weak evidence. Only one out of 8 analysed experiments yielded a positive result and only at a high dose that already caused clear cytotoxicity (mitotic index reduced to 43 %). The other 7 analysed experiments were negative. This included a repetition of the first experiment at concentrations up to 0.09 mg/mL, still causing cytotoxicity (mitotic index reduced to 63 %), that did not show a significant increase of the aberration rate.

The only other evidence of the authors for deciding on a positive result was that the historical rate of aberrations was exceeded in 2 experiments.

Justification for classification or non-classification

No classification is derived from the results.