2[2'-Hydroxy-5'-(phenyl)ureylenphenyl]benzothiazole

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Jan 2018 - 12 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

(adopted 29 July 2016

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:EpiDerm Skin Model (EPI-200, MatTek , kits H and G)
- Tissue batch number: 27667
- CoA date: 10/01/2018
- Date of initiation of testing: 10/01/2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature , (3 min exposure) , 37.0 ± 1.0°C (60 min exposure)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.
- Incubation time: 3h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.708 ± 0.053 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 8.47 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for potential biocontaminants. (viruses, bacteria, yeast and other fungi.): no contamination was observed

NUMBER OF REPLICATE TISSUES: 2 replicates for each treatment condition (3 min and 60 min experiment)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test substance did not directly reduce MTT and showed no colouring as compared to the solvent.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
- Concentration (if solution): used as supplied

VEHICLE
No vehicle was used but 25 µL of sterile water was added for wetting of the test item to increase tissue surface contact

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL Milli-Q water
- Concentration (if solution):
used as supplied

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL 8N KOH
- Concentration (if solution): used as supplied

Duration of treatment / exposure:

3 and 60 minutes

Number of replicates:

duplicates were used

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test - negative control - 3 and 60 minutes

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test - positive control - 3 minutes

Value:

15

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test - Positive control - 60 minutes

Value:

8.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test - Test item - 3 minutes

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 5

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test - Test item - 60 minutes

Value:

104

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:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: The test item was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The positive control condition showed that the test system has the proficiency to detect decrease of cellular viability.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The mean OD570 for the negative control treated tissues was 1.8 for the 3 Minute exposure period and 1.82 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control:
The relative mean tissue viability for the positive control treated tissues was 8.9 % relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: IIn the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was =< 8.0%, indicating that the test system functioned properly. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table1 :Mean OD570Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item
Tissue	Exposure Period	OD570of individual tissues	Mean OD570of duplicate tissues	Standard Deviation	Coefficient of Variation	Relative Mean Viability (%)
					(%)
Negative Control	3 Minutes	1,554	1,547	0,004	0,4	100
		1,55
	60 Minutes	1,565	1,616	0,073	6,2
		1,668
Positive Control	3 Minutes	0,267	0,231	0,051	27	15
		0,195
	60 Minutes	0,152	0,144	0,011	10	8,9
		0,136
Test Item	3 Minutes	1,583	1,546	0,052	4,6	100
		1,51
	60 Minutes	1,756	1,685	0,1	8	104
		1,615
OD = Optical density

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, tes item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate test item for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)).

The possible corrosive potential of test item was tested through topical application for 3 minutes and 1 hour. The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 17J31PR252A of test item was a slight yellow powder with a purity of 98%. Skin tissue was moistened with 25 μL of Milli-Q water and at least 25 mg of Test item was applied directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 8.9% after the 1-hour exposure.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit =<2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was =< 8.0%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with test item compared to the negative control tissues was 100% and 104%, respectively. Because the mean relative tissue viability for test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment test item is considered to be not corrosive.

In conclusion, test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.