2-[3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazoniol-5-yl]ethyl dihydrogen diphosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity (mutagenicity) in bacterial cells (OECD 471, GLP): negative in TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA with and without metabolic activation

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Apr - 10 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted Jul 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate with and without metabolic activation
Experiment II: 33, 100, 333, 1000, 2500 and 5000 μg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine: -S9: 10 and 50 µg/plate for TA98 and TA1537, respectively; 2-aminoanthracene: +S9: 2.5 or 10 µg/plate for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation; Experiment I); preincubation (Experiment II)

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants

- OTHER: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

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:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to and including the highest dose.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment served as Experiment I, as all strains were tested in the pre-experiment and the results were valid.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control data fell within the range of the historical control data (please refer to Table 3 under "any other information on results incl. tables").
- Negative (solvent/vehicle) historical control data: The negative control data (solvent and untreated) fell within the range of the historical control data (please refer to Table 3 under "any other information on results incl. tables").

Table 1: Summary of Experiment I (plate incorporation)

Metabolic Activation	Test Group	Dose Level per group and per plate	Revertant Colony Counts (Mean of 3 plates ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2uvrA
Without Activation	Deionised water		12 ± 3	8 ± 3	30 ± 10	195 ± 8	40 ± 3
	Untreated		9 ± 2	9 ± 4	24 ± 7	195 ± 7	32 ± 8
	Test substance	3 µg	10 ± 3	10 ± 4	23 ± 3	191 ± 9	33 ± 6
		10 µg	13 ± 6	9 ± 2	25 ± 8	190 ± 22	39 ± 4
		33 µg	10 ± 3	11 ± 5	27 ± 3	188 ± 15	44 ± 5
		100 µg	13 ± 4	7 ± 3	29 ± 10	191 ± 17	38 ± 4
		333 µg	12 ± 4	7 ± 2	24 ± 5	198 ± 23	40 ± 3
		1000 µg	8 ± 3	9 ± 3	31 ± 6	209 ± 16	43 ± 5
		2500 µg	10 ± 3	10 ± 5	30 ± 5	196 ± 14	44 ± 4
		5000 µg	11 ± 3	9 ± 2	30 ± 6	182 ± 16	43 ± 7
	NaN3	10 µg	1327 ± 26			2265 ± 83
	4-NOPD	10 µg			354 ± 18
	4-NOPD	50 µg		69 ± 11
	MMS	2.0 µL					922 ± 16
With Activation	Deionised water		12 ± 3	17 ± 4	33 ± 3	165 ± 12	43 ± 5
	Untreated		12 ± 4	13 ± 2	46 ± 4	163 ± 4	52 ± 5
	Test substance	3 µg	15 ± 2	16 ± 2	37 ± 6	166 ± 26	47 ± 3
		10 µg	14 ± 3	18 ± 3	43 ± 5	171 ± 29	50 ± 4
		33 µg	11 ± 5	13 ± 3	32 ± 6	189 ± 10	44 ± 3
		100 µg	14 ± 5	12 ± 3	46 ± 4	175 ± 4	47 ± 10
		333 µg	12 ± 3	13 ± 4	42 ± 4	154 ± 16	39 ± 9
		1000 µg	11 ± 4	11 ± 1	37 ± 6	163 ± 8	45 ± 3
		2500 µg	8 ± 1	9 ± 3	29 ± 8	168 ± 11	50 ± 4
		5000 µg	11 ± 1	10 ± 5	33 ± 3	175 ± 11	50 ± 3
	2-AA	2.5 µg	475 ± 27	169 ± 28	3890 ± 419	4361 ± 172
	2-AA	10.0 µg					358 ± 35

NaN3 = sodium azide

4-NOPD = 4-nitro-o-phenylene-diamine

MMS = methyl methanesulfonate

2-AA = 2-aminoanthracene

Table 2: Summary of Experiment II (pre-incubation)

Metabolic Activation	Test Group	Dose Level per group and per plate	Revertant Colony Counts (Mean of 3 plates ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2uvrA
Without Activation	Deionised water		11 ± 3	10 ± 1	25 ± 5	173 ± 12	34 ± 3
	Untreated		11 ± 2	9 ± 3	25 ± 4	179 ± 8	39 ± 7
	Test substance	33 µg	10 ± 1	10 ± 5	27 ± 6	190 ± 6	37 ± 5
		100 µg	15 ± 4	8 ± 2	23 ± 8	182 ± 28	33 ± 3
		333 µg	14 ± 6	7 ± 3	26 ± 6	196 ± 23	46 ± 1
		1000 µg	14 ± 2	12 ± 3	25 ± 3	172 ± 14	35 ± 7
		2500 µg	13 ± 2	7 ± 0	33 ± 6	162 ± 19	35 ± 1
		5000 µg	8 ± 2	9 ± 1	28 ± 8	184 ± 7	47 ± 3
	NaN3	10 µg	1095 ± 33			1643 ± 143
	4-NOPD	10 µg			349 ± 5
	4-NOPD	50 µg		89 ± 17
	MMS	2.0 µL					797 ± 128
With Activation	Deionised water		10 ± 1	13 ± 3	39 ± 12	179 ± 13	55 ± 12
	Untreated		13 ± 6	17 ± 5	43 ± 10	137 ± 18	47 ± 11
	Test substance	33 µg	12 ± 2	14 ± 5	42 ± 13	150 ± 13	51 ± 5
		100 µg	11 ± 5	11 ± 5	35 ± 6	163 ± 13	54 ± 5
		333 µg	9 ± 1	15 ± 3	29 ± 4	172 ± 6	55 ± 12
		1000 µg	16 ± 8	12 ± 4	32 ± 6	144 ± 13	55 ± 7
		2500 µg	15 ± 4	15 ± 1	31 ± 6	157 ± 18	55 ± 6
		5000 µg	15 ± 6	14 ± 5	34 ± 4	141 ± 11	40 ± 10
	2-AA	2.5 µg	301 ± 5	126 ± 10	2920 ± 93	1842 ± 65
	2-AA	10.0 µg					329 ± 58

NaN3 = sodium azide

4-NOPD = 4-nitro-o-phenylene-diamine

MMS = methyl methanesulfonate

2-AA = 2-aminoanthracene

Table 3: Historical Data

Strain		without S9 mix				with S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA 1535	Solvent control	12	2.5	6	25	12	2.5	7	26
	Untreated control	12	3.1	6	28	12	2.9	7	26
	Positive control	1130	143.1	334	1816	388	58.2	176	668
TA1537	Solvent control	10	2.2	6	19	13	3.5	7	30
	Untreated control	11	2.7	5	21	14	4.0	7	31
	Positive control	82	12.7	43	157	191	60.8	83	434
TA 98	Solvent control	25	4.4	13	43	34	6.2	15	58
	Untreated control	27	4.9	12	43	37	6.5	11	57
	Positive control	378	73.7	211	627	3949	771.8	360	6586
TA 100	Solvent control	156	26.0	78	209	148	32.3	73	208
	Untreated control	176	23.6	79	217	172	25.4	85	218
	Positive control	1966	293.2	498	2767	3798	830.4	536	6076
WP2uvrA	Solvent control	41	5.6	27	63	50	6.8	28	72
	Untreated control	42	5.8	30	63	52	6.8	36	88
	Positive control	798	362.7	319	4732	378	112.6	167	1265

Conclusions:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in bacteria

Mutagenicity of 2-[3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazoniol-5-yl]ethyl dihydrogen diphosphate tetrahydrate (CAS 68684-55-9) was tested in a bacterial reverse mutation assay performed according to OECD guideline 471 and under GLP conditions (reference 7.6.1-1). The assay was performed with a standard battery of Salmonella typhimurium tester strains including TA 1535, TA 1537, TA 98 and TA 100, and Escherichia coli WP2uvrA, with and without metabolic activation. The highest concentration of 5000 µg/plate was tested in all bacterial strains. 2-[3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazoniol-5-yl]ethyl dihydrogen diphosphate (CAS 136-09-4) did not exhibit mutagenic properties in the absence or presence of metabolic activation. Toxicity indicated by reduced number of revertants was not observed in any tester strains in the applied concentration range (from 3 to 5000 µg/plate using the plate incorporation method and from 33 to 5000 µg/plate using the pre-incubation method). The Positive control substances induced a distinct increase in the number of revertants in all strains with and without metabolic activation thereby showing the validity of the assay. The solvent control was also shown to be valid.

Based on the results of the conducted study, 2-[3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazoniol-5-yl]ethyl dihydrogen diphosphate tetrahydrate (CAS 68684-55-9) is not considered to exhibit mutagenic properties in bacterial cells.

Furthermore, data on cytogenicity and mutagenicity in mammalian cells was available for the source substance, 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-4-methyl-5-[2-(phosphonooxy)ethyl]-thiazolium (CAS 10023-48-0), which were not included in the dossier, since according to the data requirements specified in Annex VII of Regulation (EC) No 1907/2006, data on cytogenicity and mutagenicity in mammalian cells is not required for the tonnage band 1 - 10 t/a. The available data with 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-4-methyl-5-[2-(phosphonooxy)ethyl]-thiazolium (CAS 10023-48-0) revealed no mutagenic and clastogenic properties in V79 cells and in cultured peripheral human lymphocytes, respectively (reference 7.6.1-2 and 7.6.1-3, registration dossier 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-4-methyl-5-[2-(phosphonooxy)ethyl]-thiazolium).

Justification for classification or non-classification

Based on the available data on genetic toxicity, there is no indication that 2-[3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazoniol-5-yl]ethyl dihydrogen diphosphate induces genetic toxicity in bacteria.

Moreover, the available data with Thiazolium, 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-4-methyl-5-[2-(phosphonooxy)ethyl]- (CAS 10023-48-0) revealed no mutagenic and clastogenic properties in V79 cells and in cultured peripheral human lymphocytes, respectively. Based on the analogue approach, the same results were expected for the target substance. The available data do not meet the classification criteria according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.