Reaction mass of 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-(2-{[hydroxy(phosphonooxy)phosphoryl]oxy}ethyl)-4-methyl-1,3-thiazol-3-ium chloride and 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-5-[2-(phosphonooxy)ethyl]-1,3-thiazol-3-ium chloride dihydrate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Nov - 01 Dec 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

600-039-9

Cas Number:

10023-48-0

Molecular formula:

C12H17N4O4PS

IUPAC Name:

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

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: SkinEthic™ RHE-model

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model (Episkin/SkinEthic Laboratories, Lyon, France)
- Tissue batch number(s): 16-RHE-122
- Expiration date: 28 Nov 2016
- Date of initiation of testing: 23 Nov 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: At room temperature for 42 ± 1 min
- Temperature of post-treatment incubation: At 37 °C for 42 ± 1 h

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Test material and controls were removed immediately by gently rinsing with a minimum volume of 25 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min
- Spectrophotometer: Microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the SkinEthic™ RHE-model was assessed by undertaking an MTT cell viability test.
- Barrier function: According to the Quality Control Data, the barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) using 1% Triton X-100. The ET-50 value was determined to be 5.2 h.
- Histological observations (HES stained vertical paraffin sections): 6 cell layers, absence of significant histological abnormalities.

NUMBER OF REPLICATE TISSUES: Triplicate tissues; from each tissue, 3 absorbance measurements after MTT incubation were performed

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: The test item has the ability to directly reduce MTT. To evaluate the extent of non-specific interaction, three killed tissues were treated with the test item and the negative control, respectively. The treatment and MTT assay of the killed tissues was similar to the handling of the living tissues.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3 independent tissues

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to beirritant to skin if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 16 mg
- Other: Before application of the test substance, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test substance and the epidermis.

NEGATIVE CONTROL
- Amount(s) applied: 16 µL

POSITIVE CONTROL
- Amount(s) applied: 16 µL
- Concentration: 5% aqueous solution of sodium dodecyl sulfate

Duration of treatment / exposure:

42 ± 1 min

Duration of post-treatment incubation (if applicable):

42 ± 1 h

Number of replicates:

Triplicate tissues; from each tissue, 3 absorbance measurements after MTT incubation were performed

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three tissues

Value:

90.39

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: For correct interpretations of results it is necessary to assess the ability of the test substance to directly reduce MTT. This pre-test for direct MTT-reducing capacity of the test item did result in blue color, i.e. the test item is a direct MTT reducer.
- Colour interference with MTT: In the pre-test medium coloration by the test item was observed, but no tissues were stained during the study. Therefore, no additional tissues for color control were treated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (2.450, 2.313 and 2.318) was in the range of ≥ 0.8 and ≤ 3.0 for all three tissues. The mean OD was 2.318 which is higher than the historically established threshold of 1.436. As the negative control result fell within the range defined in the acceptance criteria, the result is considered to be valid.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control to 1.13% which is lower than the historically established threshold of 3.12%.
- Acceptance criteria met for variability between replicate measurements: The Standard deviations between the three tissue replicates of the negative control and the positive control were 5.57% and 2.95%, respectively, which is ≤ 18%.

Any other information on results incl. tables

Table 1: Summary or results

	Tissue 1		Tissue 2		Tissue 3		Mean		Standard Deviation
Group	OD	Viability (%)	OD	Viability (%)	OD	Viability (%)	OD	Viability (%)	Viability (%)
Negative control	2.450	105.68	2.192	94.55	2.313	99.77	2.318	100.00	5.57
Positive control	0.027	1.16	0.026	1.11	0.026	1.11	0.026	1.13	2.95
Test substance	2.110*	86.12	2.117*	96.58	2.046*	88.46	2.091	90.39	6.07

* corrected optical density after metabolic conversion

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Conclusions:

Under the conditions of the RHE test method the test substance did not show irritant properties.
CLP: not classified