[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 29 August 2018 and 03 September 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:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

These deviations were considered to have not affected the integrity or validity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Silicon

EC Number:

231-130-8

EC Name:

Silicon

Cas Number:

7440-21-3

Molecular formula:

Si

IUPAC Name:

Silicon

Constituent 2

Reference substance name:

Magnesium silicate

EC Number:

237-413-2

EC Name:

Magnesium silicate

Cas Number:

13776-74-4

Molecular formula:

H2O3Si.Mg

IUPAC Name:

magnesium(2+) ion oxosilanebis(olate), crystalline

Constituent 3

Reference substance name:

Silicon dioxide

EC Number:

231-545-4

EC Name:

Silicon dioxide

Cas Number:

7631-86-9

Molecular formula:

O2Si

IUPAC Name:

Silicon dioxide, amorphous

Constituent 4

Reference substance name:

Dimagnesium silicate

EC Number:

233-112-5

EC Name:

Dimagnesium silicate

Cas Number:

10034-94-3

Molecular formula:

Mg2O4Si

IUPAC Name:

dimagnesium silicate

impurity 1

Reference substance name:

Carbon

EC Number:

231-153-3

EC Name:

Carbon

Cas Number:

7440-44-0

Molecular formula:

C

IUPAC Name:

Carbon

impurity 2

Reference substance name:

Metallic impurities (Na and S)

IUPAC Name:

Metallic impurities (Na and S)

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide
Batch: Y180510A
Sponsor’s Description: Dark brown powder
Envigo’s Description: Black powder
Expiry Date: 31 May 2019
Storage Conditions: Room temperature in the dark

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN™ Reconstructed Human Epidermis Model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Cell source:

other: not specified

Source strain:

not specified

Details on animal used as source of test system:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : EpiSkin Laboratories, Lyon, France
Date received : 28 August 2018
EpiSkinTM Tissues (0.38cm2) lot number : 18-EKIN-035
Maintenance Medium lot number : 18-MAIN3-043
Assay Medium lot number : 18-ESSC-038

Justification for test system used:

The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13 Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.
Test items are applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man.

Vehicle:

unchanged (no vehicle)

Details on test system:

Negative Control
Information as provided by the Supplier.
Identification: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
Batch: 1754643
Purity: >98%
Expiry Date: 01 February 2019
Storage Conditions: Approximately 4 °C in the dark
Supplier: Gibco

Positive Control
Information as provided by the Supplier.
Identification: Sodium dodecyl sulphate
Batch: SLBT3991
Purity: 99.5%
Expiry Date: 01 March 2020
Storage Conditions: Room temperature
Supplier: Sigma-Aldrich

Preparation of Negative and Positive Control Items and MTT
The negative control item, Dulbecco’s Phosphate Buffered Saline (DPBS), was used as supplied.
The positive control item, Sodium dodecyl sulphate (SDS), was prepared as a 5% w/v aqueous solution. The positive control was formulated within 2 hours of being applied to the test system.
A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
A 0.04 N solution of hydrochloric acid in isopropanol was prepared when required.

Study Design
Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 mg of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and non-viable water killed tissues for quantitative correction of the results.
An assessment to determine if the test item was potentially able to directly reduce MTT proved inconclusive due to the black color of the test item. Therefore, as a precaution, an additional procedure using water-killed tissues was performed. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on viable and water killed tissues.
This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues.
Water-killed tissues were prepared prior to the study by placing untreated EPISKINTM tissues in a 12 well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37 °C, 5% CO2 in air for a minimum of 48 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (-14 to -30 °C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature.
In addition to the normal test procedure, the MTT reducing test item was applied to three water killed tissues. In addition, three water killed tissues remained untreated. The untreated water killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

Assessment of Color Interference with the MTT endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
10 mg of test item was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.
The test item was found to have the potential to cause color interference and therefore additional tissues were incorporated for color correction purposes. These tissues were treated identically to the tissues of the main test with the exception of being placed into assay medium for 3 hours post exposure instead of MTT. Three tissues were dosed with the test item and three remained untreated to act as negative controls.

Double Correction Check
A third set of controls, comprised of water-killed tissues, was also used to prevent a double correction from a colored test item that also reduces MTT.
Intrinsically colored test items may bind to both living and killed tissues and therefore the non viable water killed tissues may not only correct for potential direct MTT reduction by the test item, but also for color interference arising from the binding of the test item to the killed tissues. This could lead to a double correction for color interference since the viable color interference tissues already corrects for color interference arising from the binding of the test item to living tissues.
Three water killed tissues were dosed with the test item and three water killed tissues remained untreated to act as the negative control. These tissues were incubated with assay medium instead of MTT post exposure.

Pre-incubation (Day 0: Tissue Arrival)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory : Yes
Temperature Indicator Color Satisfactory : Yes
Agar Medium Color Satisfactory : Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre labeled 12 well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Main Test
Application of Test Item and Rinsing (Day 1)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12 well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis. Approximately 10 mg (26.3 mg/cm^2) of the test item was then applied to the epidermal surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7 Minute contact time the SDS solution was re spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
Following the 42 Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer at 14 to 30 ºC for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3 Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density (OD570) was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT 4500 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Approximately 10 mg(26.3 mg/cm^2) of test item
10 µL of DPBS (negative control)
10 µL of SDS 5% w/v (positive control)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Triplicate

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

103.6

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:

The relative mean tissue viability for the positive control treated tissues was 8.9% relative to the negative control treated tissues and the standard deviation value of the viability was 4.5%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.744 and the standard deviation value of the viability was 5.2%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 3.8%. The test item acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Direct MTT Reduction

An assessment to determine if the test item was potentially able to directly reduce MTT proved inconclusive due to the black color of the test item. Therefore, as a precaution, an additional procedure using water-killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

Assessment of Color Interference with the MTT endpoint

The test item was found to have the potential to cause color interference and therefore additional tissues were incorporated for color correction purposes. However, the optical density results obtained showed that negligible color interference occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.

Double Correction Check

The results of the color correction tissues were not used; therefore it was unnecessary to use the results of the killed color correction tissues as no double correction for color interference would have occurred.

Test Item, Positive Control Item and Negative Control Item

The relative mean viability of the test item treated tissues was103.6% after a 15‑Minute exposure period and 42‑Hour post‑exposure incubation period.

It was considered unnecessary to perform IL-1aanalysis as the results of the MTT test were unequivocal.

Mean OD₅₇₀Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570of tissues	Mean OD570of triplicate tissues	±SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.750	0.744	0.039	100.8	100*	5.2
	0.780			104.8
	0.703			94.5
Positive Control Item	0.061	0.066	0.033	8.2	8.9	4.5
	0.035			4.7
	0.101			13.6
Test Item	0.777	0.771	0.028	104.4	103.6	3.8
	0.740			99.5
	0.795			106.9

OD = Optical Density

SD = Standard deviation

* =  The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used: EU CLP

Conclusions:

The test item was classified as non-irritant. The following classification criteria apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN^TM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post‑exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. 

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. An assessment to determine if the test item was potentially able to directly reduce MTT proved inconclusive due to the black color of the test item. Therefore, as a precaution, an additional procedure using non-viable tissues was performed for potential correction purposes. The test item was found to have the potential to cause color interference and therefore additional tissues were incorporated for color correction purposes.   A third set of controls was included, comprising non-viable tissues, in order to prevent a double correction from a colored test item that also reduces MTT. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 570 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viability of the test item treated tissues was 103.6% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was classified as non-irritant. The following classification criteria apply:

EU CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 can not be determined).