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

Endpoint summary

Description of key information

Skin irriation (OECD 439, GLP): negative

Eye irritation (OECD 437, GLP): negative

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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 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

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: The 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%.

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

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Oct 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted Jul 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, Brensbach, Germany
- Characteristics of donor animals: 19 - 59 months old
- Storage, temperature and transport conditions of ocular tissue: Freshly isolated bovine eyes of cattle were collected from the slaughterhouse. Excess tissue was removed from the eyes and the eyes were kept and transported in transport medium cooled on ice.
- Time interval prior to initiating testing: The corneas were prepared immediately after delivery of the eyes to the laboratory.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: 20% (w/v) in 0.9% sodium chloride solution

VEHICLE
- Amount(s) applied: 750 µL

POSITIVE CONTROL
- Amount(s) applied: 750 µL
- Concentration: 20% (w/v) in 0.9% sodium chloride solution

Duration of treatment / exposure:

240 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

90 min at 32 ± 1 °C

Number of animals or in vitro replicates:

Triplicates for each treatment and control group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. Each cornea was mounted in a specially designed cornea holder.

QUALITY CHECK OF THE ISOLATED CORNEAS
The baseline opacity was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany). Any corneas that showed macroscopic tissue damage (scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

NUMBER OF REPLICATES
Triplicates for each treatment and control group

TREATMENT METHOD
Each cornea was mounted in a cornea holder (CiToxLAB, Veszprem, Hungary) with the endothelial side against the sealing ring (0-ring) of the posterior part of the holder. The cornea was gently flattened over the 0-ring without stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex form. For equilibration, the corneas in the holder were incubated (BSS 160, Grumbach Brutgeräte GmbH, Asslar, Germany) in a vertical position at 32 ± 1 °C for about one hour.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The corneal surface was washed three times with wash medium. Incubation medium was used as final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurements.
- POST-EXPOSURE INCUBATION: After the rinsing step, the corneas were incubated again in an incubator in a horizontal position at 32 ± 1 °C for 90 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The light transmission through the corneas, given as lux value, was recorded and thereafter converted into an opacity value (baseline opacity values).
- Corneal permeability: The amount of fluorescein that crossed the cornea was measured spectrophotometrically (OD490) in the medium from the posterior chamber.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as Category 1.
Test substance with an IVIS ≤ 3 was regarded as No Category.
Test substance with an IVIS > 3 ≤ 55: no prediction can be made.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean value of 3 corneae

Value:

1.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
No observations (tissue peeling, residual test chemical, non-uniform opacity patterns) were seen in a visually inspection of the corneas after treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.0 and, therefore within three standard deviations of the current historical mean of the negative control (IVIS: -1.5 to 3.5).
- Acceptance criteria met for positive control: After treatment with the positive control (20% imidazole) the calculated IVIS was 107.7 and therefore falls within two standard deviations of the current historical mean of the positive control (IVIS 78.2 to 135.3).

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 BCOP assay, the test substance did not show irritant properties towards eyes.
CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The skin irritancy potential of 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-4-methyl-5-[2-(phosphonooxy)ethyl]-thiazolium was determined in an in vitro skin irritation study using a human skin model according to OECD guideline 439 and under GLP conditions (reference 7.3.1-1). The negative control OD (2.450, 2.313 and 2.318) was in the acceptance criteria range of ≥ 0.8 and ≤ 3.0 for all three tissues. Exposure to the positive control dodecyl sulfate sodium salt induced a decrease in the relative absorbance as compared to the negative control to 1.13%. Thus, negative and positive controls met the acceptance criteria and can be considered as valid. The mean tissue viability after treatment with the test substance was determined to be 90.39% for all three tissues, thus indicating a clear negative result and the test substance did not show any irritating properties towards human-derived epidermal keratinocytes.

Under the specific circumstances it is possible to conclude that the test substance is not expected to cause skin irritating properties and does not require classification.

 

Eye irritation

To exclude corrosive properties towards the eyes, a bovine corneal opacity and permeability (BCOP) test according to OECD guideline 437 and in compliance with GLP was conducted with the registered substance (reference 7.3.2-1). After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.0 and, therefore within three standard deviations of the current historical mean of the negative control (IVIS: -1.5 to 3.5). After treatment with the positive control (20% imidazole) the calculated IVIS was 107.7 and therefore falls within two standard deviations of the current historical mean of the positive control (IVIS 78.2 to 135.3). Thus, negative and positive controls met the acceptance criteria and can be considered as valid. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 1.4.

According to the OECD guideline 437, it can be concluded that the test substance is not expected to cause eye irritating properties and does not require classification.

Justification for classification or non-classification

The available data on skin and eye irritation do not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.