Sulisobenzone

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study report

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)

Principles of method if other than guideline:

The purpose of this study is to provide classification of dermal corrosion potential of a chemical by using a three-dimensional human epidermis model, according to OECD Test Guideline No. 431 “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method”. The EpiDermTM SCT allows identification of non-corrosive and corrosive substances according to the United Nations (UN) Globally Harmonized System of Classification and Labeling of Chemicals (GHS).

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sulisobenzone

EC Number:

223-772-2

EC Name:

Sulisobenzone

Cas Number:

4065-45-6

Molecular formula:

C14H12O6S

IUPAC Name:

5-benzoyl-4-hydroxy-2-methoxybenzenesulfonic acid

Details on test material:

Name of test substance : 2-Hydroxy-4-methoxybenzophenone- 5-sulfonic acid (Sulisobenzone)- Substance type: Organic- Physical state: Solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek In Vitro Life Science Laboratories

Source strain:

not specified

Details on animal used as source of test system:

Test System
The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (with a surface of 0.5 cm2). The keratinocytes were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native epidermis. Each lot of tissues was Quality Assured by MatTek In Vitro Life Science Laboratories according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.

Vehicle:

water

Remarks:

the tissues were moistened with sterile water prior to application of the solid test chemical

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
MatTek EpiDermTM tissue cultures

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
For the 3-minute exposure, the dosed tissues were placed in the sterile hood at room temperature. For the 60-minute exposure, the dosed tissues were placed in an incubator at 37deg C, 5% CO2 for the remainder of the 60-minute exposure period
- Temperature of post-treatment incubation (if applicable):
37deg C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:

For the 3-minute exposure, four tissues at a time were dosed with an interval of 45 seconds. When a treatment of 3 minutes was reached for the set of four tissues, the dosed tissues were gently rinsed with a constant stream of sterile DPBS by filling and emptying the tissue inserts 20 times to remove any residual test article. The bottom of the inserts was gently botted on a sterile cotton compress and the tissues were then placed in 0.3 mL of fresh maintenance medium (in 24-well plates) until all tissues were treated and washed. After the 1-hour exposure, the tissues were gently rinsed with a constant stream of sterile DPBS by filling and emptying the tissue inserts 20 times to remove any residual test article. The bottom of the inserts was gently botted on a sterile cotton compress and the tissues were then placed in 0.3 mL of fresh maintenance medium (in 24-well plates) until all tissues were washed.
- Observable damage in the tissue due to washing:
none
- Modifications to validated SOP:
none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
24-well plates containing 300 microliters MTT medium (1.0 mg/mL).
- Incubation time:
3 hours of incubation at 37 deg C, 5% CO2 in a humified incubator
- Spectrophotometer:
Thermo Scientific™ Multiskan™ FC Microplate Photometer
- Wavelength:
570 nm
- Viability:
All data were background subtracted before analysis. MTT data are represented as % viable compared to negative control. Data was generated as follows:
Blanks:
The optical density (OC) mean from all replicates for each plate (ODblank)

Negative Controls (NC):
The blank corrected value was calculated: ODNC = ODNCraw – ODblank
The OD mean per NC tissue was calculated.
The mean OD for all tissues corresponds to 100% viability
The mean, standard deviation (SD) and the percent coefficient of variation (% CV)

Positive Controls (PC):
Calculation of the blank corrected value: ODPC = ODPCraw – ODblank
The OD mean per PC tissue was calculated.
The viability per tissue was calculated: %PC = [ODPC / mean ODNC] x 100
The mean viability for all tissues was calculated: Mean PC =%PC / number of tissues
The standard deviation (SD) and the percent coefficient of variation (% CV)

Tested Compound (TC):
Calculation of the blank corrected value: ODTC = ODTCraw – ODblank
The OD mean per treated tissue was calculated.
The viability per tissue was calculated: %TC = [ODTC / mean ODNC] x 100
The mean viability for all tissues was calculated: Mean TC = %TC / number of tissues
The standard deviation (SD) and the percent coefficient of variation (% CV)

Data Correction Procedure for MTT Interfering Compounds (if applicable)
True viability = Viability of treated tissue – Interference from test article = ODtvt – ODkt where ODkt = (mean ODtkt – mean ODukt)

ODtvt = optical density of treated viable tissue
ODkt = optical density of killed tissue
ODtkt = optical density of treated killed tissue
ODukt = optical density of untreated killed tissue (NC treated tissue)

Data Correction Procedure for Colored Compounds (if applicable)
True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in media without MTT = ODtvt – ODvt

Skin Corrosion Prediction
A chemical is regarded to be corrosive if the relative tissue viability after 3 min treatment with a test chemical is decreased below 50%. In addition, materials are regarded to be non-corrosive after 3 min (viability< 50%) are regarded to be corrosive if the relative tissue viability after 1-hour treatment with a test chemical is decreased below 15%.

Mean Tissue Viability Prediction

3 min < 50% Corrosive
3 min > 50% and 1 hour: < 15% Corrosive
3 min > 50% and 1 hour: > 15% Non-corrosive

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Before dosing, the tissues were moistened with 25 microliters of sterile water to improve the contact of the tissue surface to the test article. Approximately 25 mg of each test article was evenly applied to the apical surface of each tissue.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
50microliters

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
50 microliters

Duration of treatment / exposure:

3 minute and 60 minute exposure

Duration of post-treatment incubation (if applicable):

37deg C, 5% CO2

Number of replicates:

duplicates

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 1

Value:

85.7

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 3 min. endpoint; mean of OD: 2.098

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 2

Value:

13.4

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 1 hrs. endpoint; mean of OD: 0.315

Other effects / acceptance of results:

The assay passed all acceptance criteria as the ODs of the negative control exposed tissues were > 0.8, the positive control exposed tissues were less than 15% viable and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was <30 (except for where indicated as incompatible or with an asterisk).

Any other information on results incl. tables

Name	CAS	Tissue		3 min endpoint								1 hour endpoint
			Aliquote 1	Aliquote 2	mean	OD Mean	viability	Mean	Diff	Diff /2	Aliquote 1	Aliquote 2	mean	OD Mean	viability	Mean	Diff
							[%]	[%]	[%]	[%]					[%]	[%]	[%]
NC	N/A	tissue 1	2.646	2.615	2.630	2.449	107.4	100.0	14.8	7.42	2.293	2.173	2.233	2.358	94.7	100.0	10.6
		tissue 2	2.266	2.268	2.267		92.6				2.499	2.468	2.483		105.3
PC	1310-58-3	tissue 1	0.401	0.433	0.417	0.432	17.0	17.6	1.2	0.62	0.064	0.063	0.063	0.064	2.7	2.7	0.1
		tissue 2	0.447	0.448	0.447		18.3				0.064	0.065	0.065		2.7
K1#4	4065-45-6	tissue 1	2.146	2.184	2.165	2.098	88.4	85.7	5.5	2.73	0.419	0.409	0.414	0.315	17.6	13.4	8.4
		tissue 2	1.999	2.064	2.031		82.9				0.221	0.213	0.217		9.2

Chemical name		CAS	3min	Diff	1hour	Diff
K1#4		4065-45-6	85.7	5.5	13.4	8.37

	Chemical name	CAS	Supplier	purity	In vivo	In vitro	MTT	pH	Remark
4	K1#4	4065-45-6						1.63	Corrosive

Applicant's summary and conclusion

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

The dermal corrosion potential of the test chemical was determined by using a three-dimensional human epidermis model, according to OECD Test Guideline No. 431 “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method”. The mean of OD for test chemical was determined to be 2.098 and 0.315 for 3 min. enpoint and 1 hour endpoint, respectively. The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 85.7 % and 13.4 % for 3 min. endpoint and 1 hour endpoint,respectively. Based on these values, the test chemical can be considered to corrosive to skin. It can be further classified under the category "Category 1" as per CLP Regulation.

Executive summary:

The dermal corrosion potential of the test chemical was determined by using a three-dimensional human epidermis model, according to OECD Test Guideline No. 431 “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method”.

The EpiDerm^TMSCT allows discrimination between non-corrosive and corrosive chemicals according to the UN Globally Harmonized System of Classification and Labeling of Chemicals (GHS). The MatTek EpiDerm^TMtissue cultures were obtained from MatTek In Vitro Life Science Laboratories. The MatTek EpiDerm^TMtissue cultures were placed in the refrigerator at 5°C. Prior to use, the tissues were incubated (37±1°C, 5±1% CO₂) in 0.9 mL of fresh maintenance medium in 6 -well plates for~one hour.

Before dosing, the tissues were moistened with 25microliter of sterile water to improve the contact of the tissue surface to the test article. Approximately 25 mg of solid test article was evenly applied to the apical surface of each tissue. Each treatment with test article or control was conducted in duplicate. The exposure period for the test articles and controls was 3 and 60 minutes. For the 60-minute exposure, the dosed tissues were placed in an incubator at 37±1°C, 5±1% CO₂for the remainder of the 60-minute exposure period.

Following the rinsing period, the MTT assay was performed by transferring the tissues to 24-well plates containing 300microliter MTT medium (1.0 mg/mL). After 3 hours of incubation at 37±1°C, 5±1% CO₂in a humified incubator, the blue formazan salt was extracted by submerging the tissues in 2 mL isopropanol (Lot 28677, kit A; provided by MatTek, Slovakia) in a 24-well plate. The extraction time was approximately 2 hours and 05 minutes with gentle shaking. The optical density of the extracted formazan (200mL/well if a 96-well plate) was determined using a Thermo Scientific™ Multiskan™ FC Microplate Photometer at 570 nm. Relative cell viability was calculated for each tissue as % of the mean negative control tissue. A chemical was regarded to be corrosive if the relative tissue viability after 3 min treatment with a test chemical was decreased below 50%. In addition, materials were regarded to be non-corrosive after 3 min (viability>50%) were regarded to be corrosive if the relative tissue viability after 1-hour treatment with a test chemical was decreased below 15%.

The mean of OD  for test chemical was determined to be 2.098 and 0.315 for 3 min. endpoint and 1 hour endpoint, respectively. The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 85.7 % and 13.4 % for 3 min. enpoint and 1 hour endpoint,respectively. Based on these values, the test chemical can be considered to corrosive to skin. It can be further classified under the category "Category 1" as per CLP Regulation.