5-Thiazolecarboxamide, N-(2-chloro-6-methylphenyl)-2-[(6-chloro-2-methyl-4-pyrimidinyl)amino]-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 18, 2003 to September 30, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to OECD method and in accordance with GLP. Study material is well characterized.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[(6-chloro-2-methylpyrimidin-4-yl)amino]-N-(2-chloro-6-methylphenyl)-1,3-thiazole-5-carboxamide

EC Number:

695-686-7

Cas Number:

302964-08-5

Molecular formula:

C16 H13 C12 N5 O S

IUPAC Name:

2-[(6-chloro-2-methylpyrimidin-4-yl)amino]-N-(2-chloro-6-methylphenyl)-1,3-thiazole-5-carboxamide

Details on test material:

Beige solid

Method

Target gene:

The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-mi­ nus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.

Strain WP2 (5) and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent (Trp+) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain may be more readily mutated by agents.

When summarised the mutations of the TA strains and the E. coli strain, used in this study can be described as follows:

Salmonella typhimurium
Strains Genotvoe Tvoe of mutations indicated
TA 1537 his C 3076; rfa•; uvrs•: frame shift mutations
TA 98 his D 3052; rta•; uvrff;R-factor " "
TA 1535 his G 46; rta•; uvrff: base-pair substitutions
TA 100 his G 46; rta•; uvrff;R-factor " "
Escherichia coli
WP2 uvrA tro•; uvrA•: base-oair substitutions and others

Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in RCC Cytotest Cell Research according to B. Ames et al. (1)

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium: TA1535, TA1537, TA98, TA100 and Escherichia coli WPU2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix(2.5%) from Aclor 1254-induced rat liver

Test concentrations with justification for top dose:

Without metabolic activation:
TA 1535, TA 100; 10 µg/plate
TA 1537, TA 98; 10 µg/plate in TA 98, 50 µg/plate in TA 1537
WP2 uvrA; 4 µg/plate

With metabolic activation:
TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA; 2.5 µg/plate (TA 1535, TA 1537, TA 98, TA 100),
10 µg/plate (WP2 uvrA)

On the day of the experiment, the test item BMS 540268 was dissolved in DMSO (MERCK, D-64293 Darmstadt; purity > 99 %). The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria

Vehicle / solvent:

Without metabolic activation:
TA 1535, TA 100 Dissolved in: water deionised
TA 1537, TA 98 Dissolved in: DMSO (MERCK, D-64293 Darmstadt; purity > 99 %)
WP2 uvrA Dissolved in: water deionised

With metabolic activation:
TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA dissolved in: DMSO (MERCK, D-64293 Darmstadt; purity > 99 %)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent untreated and solvent controls were performed

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

10 ug/plate -TA 1535, TA 100; without metabolic activation

Controls 2

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD 9TA 1537, TA 98)

Remarks:

50 ug/plate - TA 1537, 10 ug/plate TA 98; without metabolic activation

Controls 3

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

4 uL/plateWP2uvrA; without metabolic activation

Controls 4

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2.5 ug/plate - TA 1535, TA 1537, TA 98, TA 100, and 10 ug/plate- WP2uvrA; ; with metabolic activation

Details on test system and experimental conditions:

The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-mi­ nus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.

Strain WP2 (5) and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent (Trp+) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain may be more readily mutated by agents.

When summarised the mutations of the TA strains and the E. coli strain, used in this study can be described as follows:

Salmonella typhimurium
Strains Genotvoe Tvoe of mutations indicated
TA 1537 his C 3076; rfa•; uvrs•: frame shift mutations
TA 98 his D 3052; rta•; uvrff;R-factor " "
TA 1535 his G 46; rta•; uvrff: base-pair substitutions
TA 100 his G 46; rta•; uvrff;R-factor " "
Escherichia coli
WP2 uvrA tro•; uvrA•: base-oair substitutions and others

Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in RCC Cytotest Cell Research according to B. Ames et al. (1) and D. Maron and B. Ames (4). In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

The bacterial strains TA 1535, TA 1537, and TA 100 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 98 was obtained from E. Merck (D-64293 Darmstadt). The Escherichia coli strain WP2 uvrA was obtained from RCC Ltd. (CH-4332 Stein).

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation of the data is required.

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with BMS 540268 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix) with the exception of strain TA 1535, where a slight increase in revertant colony numbers was observed. However, the required threshold of three times the number of the corresponding solvent control was not reached. Furthermore, no dose dependency was observed and the results could not be reproduced in the second experiment where the more sensitive pre-incubation method was applied. Therefore, this effect is judged to be based on biologically irrelevant fluctuations in the number of colonies. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In strain TA 100 with S9 mix, the data in the solvent control (exp. I) were slightly above our historical control range in the presence of metabolic activation. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

The historical range of positive controls was exceeded in strain TA 1535 (exp. I and II) and in TA 100 (exp. II) without metabolic activation and in strains TA 1537 (exp. I and II) and TA 98 (exp. I) with metabolic activation. This effect indicates the sensitivity of the strains rather than compromisi ng the assay.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­ crease of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

12.2   Summary of Results

 

Test item:           BMS 540268

S9 mix from:  Rat liver (Batch R 250703)

 

 

without S9 mix

 

Concentration µa/plate	TA 1535 I              II		Revertants/plate mean from three plates TA 1537               TA 98    TA 100 I              II            I              II            I              II						WP2 I	uvrA II
Negative control Solvent control Positive control" 33 100 333 1000 2500 5000	17 17 862 36 46 46 40 29 30	13 22 1058 23 29 26 29 25 21	6 10 73 8 5 7 9 7 6	9 10 76 10 9 14 12 6 10	26 22 157 24 28 32 25 31 25	29 25 166 27 26 28 27 27 23	166 182 715 216 180 164 170 146 128	179 179 1168 186 207 194 163 155 154	41 43 752 44 43 31 48 43 46	41 45 258 36 48 37 49 44 50

 

with S9 Mix

 

Concentration µg/plate	TA 1535 I              II		Revertants/plate mean from three plates TA 1537               TA 98    TA 100 I              II            I              II            I              II						WP2 uvrA I              II
Negative control Solvent control Positive control111 33 100 333 1000 2500 5000	19 19 423 14 17	14 16 357 16 13	11 10 367 11 13	14 12 197 7 10	36 25 1061 26 27	29 22 417 23 27	179 212 1183 200 267	183 190 749 214 193	36 34 172 55 48	36 43 281 43 37
	16	12	13	11	36	22	165	156	43	34
	17	15	10	14	26	29	179	163	44	51
	19	14	8	13	32	28	167	165	49	47
	16     14            7             12      15               21          140        163    49               57

 

 

•    Sodium azide (10.0 µg/plate) strains TA 1535 and TA 100

4-nitro-o-phenylene-diamine strains TA 1537 (50 µg/plate) and TA 98 (10.0 µg/plate) Methyl methane sultanate (4 µUplate) strain WP2 uvrA

•• 2-aminoanthracene (2.5 µg/plate) strains TA 1535, TA 1537, TA 98, and TA 100 2-aminoanthracene (10.0 µg/plate) strain WP2uvrA

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with BMS 540268 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.