2-{3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazol-3-ium-5-yl}ethyl hydrogen phosphate

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 (RA CAS 68684-55-9)

Genetic toxicity (cytogenicity) in mammalian cells in vitro (OECD 473, GLP) negative in cultured peripheral human lymphocytes with and without metabolic activation

Genetic toxicity (mutagenicity) in mammalian cells in vitro (OECD 476, GLP): negative in V79 cells with and without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 March - 12 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and β-naphtoflavone.

Test concentrations with justification for top dose:

4 hours treatment with and without S9 mix: 132.0, 263.0, 526.0, 1053.0, and 2105.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 hours with and without metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: > 1.5 x 10 exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concen¬trations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.
However, both, biological and statistical significance were considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not affected (pH 7.68 in the solvent control versus pH 7.31 at 2105.0 µg/mL).
- Effects of osmolality: No relevant increase (465 mOsm in the solvent control versus 501 mOsm at 2105.0 µg/mL).
- Precipitation: Precipitation did not occur.

RANGE-FINDING/SCREENING STUDIES:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 2 mg/mL, 2 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20% relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up to or beyond their limit of solubility. Precipitation or phase separation should be evaluated at the beginning and at the end of treatment by the unaided eye.
The pre-experiment was performed in the presence and absence of metabolic activation. Test item concentrations between 16.4 µg/mL and 2105 µg/mL were used. The highest concentration was chosen with respect to the current OECD Guideline 476 regarding the purity of the test item (95%).
In the pre-experiment no relevant toxic effects were observed after 4 hours treatment up to the maximum concentration with and without metabolic activation.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) before the test item was removed. No precipitation or phase separation occurred up to the maximum concentration with and without metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
The concentrations used in the main experiment were selected based on the data of the pre-experiment. Again, the maximum concentration was 2105 µg/mL. The individual concentrations were spaced by a factor of 2.

COMPARISON WITH HISTORICAL CONTROL DATA: Complies

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation.

Summary Table

	conc. µg/mL	S9 mix	relative CE I	relative cell density	rel. adj. CE I	mutant colonies per 10^6 cells	95% conf. interval
Culture I
Solvent control (DMSO)		-	100.0	100.0	100.0	26.0	1.7 - 30.2
Positive control (EMS)	300.0	-	112.4	89.6	100.7	293.3	1.7 - 30.2
Test Item	65.8	-	124.9	100.9	126.1	#	1.7 - 30.2
Test Item	132.0	-	109.3	102.5	112.1	33.5	1.7 - 30.2
Test Item	263.0	-	102.6	81.0	83.1	36.4	1.7 - 30.2
Test Item	526.0	-	97.6	101.8	99.4	20.6	1.7 - 30.2
Test Item	1053.0	-	111.8	104.3	116.6	28.3	1.7 - 30.2
Test Item	2105.0	-	117.6	111.1	130.7	21.4	1.7 - 30.2
Solvent control (DMSO)		+	100.0	100.0	100.0	24.1	2.0 - 29.4
Positive control (DMBA)	2.3	+	107.3	95.9	102.9	82.4	2.0 - 29.4
Test Item	65.8	+	107.6	86.9	93.6	#	2.0 - 29.4
Test Item	132.0	+	90.0	86.4	77.8	21.8	2.0 - 29.4
Test Item	263.0	+	98.6	127.1	125.3	21.3	2.0 - 29.4
Test Item	526.0	+	100.7	97.4	98.1	25.1	2.0 - 29.4
Test Item	1053.0	+	109.8	89.3	98.1	16.7	2.0 - 29.4
Test Item	2105.0	+	96.2	84.2	81.0	32.3	2.0 - 29.4

	conc. µg/mL	S9 mix	relative CE I	relative cell density	rel. adj. CE I	mutant colonies per 10^6 cells	95% conf. interval
Culture II
Solvent control (DMSO)		-	100.0	100.0	100.0	23.5	1.7 - 30.2
Positive control (EMS)	300.0	-	84.1	114.9	96.6	292.4	1.7 - 30.2
Test Item	65.8	-	82.3	95.5	78.6	#	1.7 - 30.2
Test Item	132.0	-	78.5	131.4	103.2	11.3	1.7 - 30.2
Test Item	263.0	-	91.4	109.1	99.8	14.1	1.7 - 30.2
Test Item	526.0	-	57.9	75.4	43.7	8.7	1.7 - 30.2
Test Item	1053.0	-	66.7	89.5	59.7	11.8	1.7 - 30.2
Test Item	2105.0	-	93.0	119.6	111.2	8.2	1.7 - 30.2
Solvent control (DMSO)		+	100.0	100.0	100.0	20.7	2.0 - 29.4
Positive control (DMBA)	2.3	+	106.0	77.9	82.6	58.0	2.0 - 29.4
Test Item	65.8	+	122.3	78.4	95.9	#	2.0 - 29.4
Test Item	132.0	+	97.2	87.4	84.9	10.9	2.0 - 29.4
Test Item	263.0	+	111.7	87.5	97.7	30.9	2.0 - 29.4
Test Item	526.0	+	111.2	78.6	87.4	9.5	2.0 - 29.4
Test Item	1053.0	+	121.6	73.9	89.8	16.6	2.0 - 29.4
Test Item	2105.0	+	108.0	83.4	90.1	15.4	2.0 - 29.4

CE = Cloning Efficiency

#  culture was not continued as a minimum of only four analysable concentrations is required

DMBA: 7,12-dimethylbenzanthracene

EMS: ethylmethanesulphonate

Conclusions:

Interpretation of results: negative

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 May 2017 - 21 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted Jul 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test (migrated information)

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Sex, age and number of blood donors: healthy volunteers (approximately 18 - 35 years of age, non-smoker, female)
- Whether whole blood or separated lymphocytes were used: whole blood
- Methods for maintenance in cell culture: After blood sampling in heparin-coated tubes, the blood was kept under cooled conditions until start of experiment. The pre-treatment of blood with Phytohemagglutinin (PHA) E was done under cooled conditions (1.5 hours incubation). Mitotic stimulation of lymphocytes in chromosome medium B (ready-to-use, purchased from Biochrom) was done for 48 h at 37 °C and 5% CO2.

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Concentrations ranging from 1.95 to 2000 μg/mL were applied in the present study. For evaluation, slides of the following concentrations were chosen:
5 h treatment without metabolic activation: 250, 500, 1000 and 2000 μg/mL
5 h treatment with metabolic activation: 125, 250, 500, 1000 and 2000 μg/mL
29 h treatment wihout metabolic activation: 250, 500, 1000 and 2000 μg/mL

Vehicle / solvent:

- Solvent used: ultrapure water
- Justification for choice of solvent/vehicle: Preliminary experiments to evaluate the solubility of the test item in various solvents showed that ultrapure water was the only suitable solvent. The final concentration of ultrapure water present in cell culture medium was 1%.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 h with and without metabolic activation and 29 h without metabolic activation
- Expression time (cells in growth medium): 21 h for the short-term exposure time, the long-term exposure time were continuously exposed

SPINDLE INHIBITOR (cytogenetic assays): 0.1 µg/mL colchicine (3 h)

STAIN (for cytogenetic assays): aceto-orcein

NUMBER OF REPLICATIONS: duplicates in two independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: On the day of preparation, the cell cultures were centrifuged and the culture medium was replaced by KCl solution (0.2%) for hypotonic treatment. For fixation of cells, the tubes were placed into an ice bath and fixative (methanol:acetic acid, 3:1) was added. Cultures were incubated for 20 min at room temperature, then centrifuged. Two further fixation/centrifugation cycles followed. After the third fixation cycle, the supernatant was aspirated and the cell pellet carefully resuspended. From each parallel culture, two slides were prepared. The cell suspension was dropped onto wet, pre-cooled glass slides. The slides were dried for at least three days, then stained in aceto-orcein, immersed in xylene and made permanent with Entellan®.

NUMBER OF CELLS EVALUATED: 1000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: A total of 150 well spread metaphases were examined per slide (300 metaphases per test item concentration, 600 in the solvent control)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is concentration-related when evaluated with an appropriate trend test, or occurs across at least two concentrations
c) any of the results is outside the distribution of the historical negative control data (e.g. 95% control limits).
When all of these criteria are met, the test item is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) all results are inside the distribution of the historical negative control data (e.g. 95% control limits).
The test item is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly positive or negative response.

Statistics:

The statistical evaluations were performed using the current version of the SPSS Base software at the testing facility.
Descriptive Statistics: For all groups, mean values were calculated for the following parameters:
• the percentage of observed aberrant metaphases/culture (gaps included),
• the percentage of observed aberrant metaphases/culture (gaps excluded),
• the number of mitoses per 1000 cells/culture
• the number of polyploid cells per 1000 mitotic cells/culture
Statistical Tests:
For further statistical analysis, the numbers of aberrant metaphases (gaps excluded) were used.
Pairwise comparisons within each experiment were performed. Each treatment group was compared to the concurrent negative control. For these comparisons, the Fisher’s Exact Test was performed against one-sided alternatives.
As no relevant increases in chromosomal aberrations occurred, no trend test was applied.

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

After a prolonged exposure period of 29 hours, a clear reduction in the mitotic index was seen at the highest concentration applied

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: A change in the pH value of the cell culture medium did not occur in the concentration range tested.
- Effects of osmolality: A change in the osmotic value of the cell culture medium did not occur in the concentration range tested.
- Precipitation: Precipitation of the test substance was not observed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: A clear increase of aberrant metaphases was found for the positive controls, i.e. MMC and CPA. Statistical analysis in all cases yielded significant results. An appropriate number of cells and concentrations were analyzable. The experiments were therefore accepted as valid.
- Negative (solvent/vehicle) historical control data: The mean values of aberrant metaphases (gaps excluded) in the negative controls of all experiments ranged from 1.50% to 3.00% and, thus, were within the usual range of the experiments performed in the testing laboratory.

Table 1: Summary of Results

Treatment					Concentr.									Rel. mitotic							Polyploid									Endomitot.						Aberrant metaph. [%]
Group					[µg/mL]									Index [%]							metaph. [%]^a									metaph.[%]						incl.gaps								excl.gaps								exchanges
Without S9 mix: Exposure: 5 hours
Solvent control														100							0.025									0.000						1.67								1.50								0.00
Test substance					250									92.3							0.000									0.050						1.33								1.00^ns								0.33
					500									68.2							0.050									0.000						1.33								1.33^ns								0.00
					1000									81.5							0.100									0.000						1.67								1.33^ns								0.00
					2000									69.2							0.050									0.000						1.33								1.33^ns								0.00
MMC					0.30									89.7							0.050									0.000						6.33								6.33**								0.67
Without S9 mix: Exposure: 29 hours
Solvent control														100							0.025									0.050						4.00								3.00								0.00
Test substance					250									82.0							0.000									0.000						5.33								3.33^ns								0.33
					500									92.5							0.050									0.000						4.00								2.67^ns								0.00
					1000^S									68.3							0.050									0.000						5.33								3.33^ns								0.00
					2000^S									37.9							0.050									0.000						5.33								4.33^ns								0.00
MMC					0.15									67.7							0.050									0.000						13.7								13.0**								4.00
With S9 mix: Exposure: 5 hours
Solvent control														100							0.100									0.025						3.00								3.00								0.00
Test substance					125									89.2							0.100									0.000						1.33								1.33^ns								0.00
					250									83.5							0.150									0.000						2.33								2.33^ns								0.00
					500									94.2							0.100									0.100						3.00								3.00^ns								0.33
					1000									79.1							0.000									0.100						0.67								0.33^ns								0.00
					2000									65.7							0.050									0.000						2.00								2.00^ns								0.00
CPA					1.50									56.1							0.100									0.050						12.0								12.0**								4.33

a: % of solvent control

MMC:  Mitomycin C

CPA: Cyclophosphamide

Statistical significance: **: p ≤ 0.01; *: 0.01 < p ≤ 0.05; ns: p > 0.05 (not significant)

S: pipetted as suspension

Conclusions:

Interpretation of results: negative

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

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:

not examined

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:

not examined

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:

not examined

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:

not examined

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:

not examined

Positive controls validity:

valid

Remarks on result:

other: Source: CAS 136-09-4

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. Based on the analogue approach, same results are expected for the target substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for read-across

There are no data available on the genetic toxicity in bacterial cells for 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-4-methyl-5-[2-(phosphonooxy)ethyl]-thiazolium (CAS 10023-48-0). Read-across from a source substance is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. in order to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VII, 8.4. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

Genetic toxicity in bacteria

CAS 68684-55-9

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. The test substance 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.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

The clastogenic activity of Thiazolium, 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-4-methyl-5-[2-(phosphonooxy)ethyl]- (CAS 10023-48-0) was investigated in an in vitro mammalian chromosome aberration test in cultured peripheral human lymphocytes performed according to OECD guideline 473 and under GLP conditions (reference 7.6.1-2). The test substance was dissolved in ultrapure water and two independent experiments were performed. In the first experiment, the test substance was tested up to 2000 μg/mL for a 5-h exposure time in the absence and presence of metabolic activation (S9 mix) followed by a 21-h post-incubation period and thereafter by a 3-h exposure with colchicine to arrest the lymphocytes in mitosis. In the second experiment, the test substance was tested up to 2000 μg/mL for a 29-h continuous exposure time in the absence of S9 mix. The test substance did not precipitate in the culture medium at this dose level. No clear cytotoxic effects, i.e. reduction in mitotic index were induced by the test substance in the absence or presence of metabolic activation (S9-mix) after an exposure period of 5 hours. After a prolonged exposure period of 29 hours, a clear reduction in the mitotic index was seen at the highest concentration applied. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9 mix) functioned properly. The test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9 mix, in either of the two independently repeated experiments. No relevant effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed in the absence and presence of S9 mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that the test substance is not clastogenic in human lymphocytes under the experimental conditions described.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

An in vitro mammalian cell gene mutation assay was performed according to OECD guideline 476 and under GLP conditions with the test substance 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-4-methyl-5-[2-(phosphonooxy)ethyl]-thiazolium (CAS 10023-48-0) in Chinese hamster lung fibroblasts (V79) (reference 7.6.1-3). The cells were treated with the test substance for 4 h with and without metabolic activation (cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with phenobarbital andβ-naphthoflavone). The test substance was tested at concentrations of 65.8, 132, 263, 526, 1053 and 2105 µg/mL in two independent mutation experiments, both with and without metabolic activation. Precipitation and cytotoxicity were not observed up to the highest tested concentration of 2105 µg/mL. 7,12-dimethylbenzanthracene and ethylmethanesulphonate were used as positive controls with and without S9 mix, respectively and caused a distinct increase in the number of mutation colonies. The mean of mutants observed after treatment with the test substance and the vehicle control in both experiments were within the range of the 95% confidence interval of the vehicle control, with the exception of the tested concentrations of 132 and 263 µg/mL in experiment I without metabolic activation and the tested concentrations of 263 and 2105 µg/mL in experiment II and I with metabolic activation, respectively. As these increases were not concentration-related and not reproducible, the effects were considered to be incidental and not test item related. Therefore, n hydrochloride

Conclusion for genetic toxicity

The available data show that the appropriate source substance 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 mutagenic to bacteria. In addition, the available in vitro data with the target substance Thiazolium, 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-4-methyl-5-[2-(phosphonooxy)ethyl]- (CAS 10023-48-0) revealed no mutagenic properties in V79 cells and no clastogenic properties in cultured peripheral human lymphocytes.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met.Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-4-methyl-5-[2-(phosphonooxy)ethyl]-thiazolium (CAS 10023-48-0), data will be generated from information on reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

 

The available data on genetic toxicity with the source and the target substance do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.