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

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

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.

Test guideline 2

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

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

Remarks:

migrated information: powder

Details on test material:

Name: T15-AE
Chemical name: syn-2-(2-Aminothiazol-4-yl)-2-(methylcarbonyloximino) aceticacid-mercaptobenzthiazolylester.
Supplier: Sponsor.
Batch No.: 36429032.
Molecular formula: C14 H10 N4 O3 S3.
CAS No.: 104797-47-9
Appearance: Yellow powder.
Purity: 92.9 % (HPLC)
Conditions of storage: In the deep freezer, in the dark; may be used under light.
Stability at conditions of storage: 12 months.
Stability at room temperature: Ca. 2 weeks.
Expiry date: 8 April 2005 according to the Certificate of Analysis, but extended to October 2005 based on investigations by the sponsor.

Method

Species / strain

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.

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Test results 6

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'.

Applicant's summary and conclusion

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.