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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

600-039-9

Cas Number:

10023-48-0

Molecular formula:

C12H17N4O4PS

IUPAC Name:

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

Test material form:

solid

Method

Target gene:

HPRT

Species / strain

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

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Controls 2

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative