Registration Dossier

Administrative data

Description of key information

In vitro skin irritation (OECD 439): Non-irritant

In vitro skin corrosion (OECD 431): Non-corrosive

In vitro eye irritation (OECD 437): No prediction of eye irritation can be made.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to
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.
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. certificate)
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
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
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.

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%

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 EPISKINTM 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).

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 18 July 2018 and 20 July 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
yes
Remarks:
These deviations were considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. certificate)
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
Test system:
human skin model
Source species:
human
Cell type:
other: epithelial cells
Cell source:
other: not specified
Source strain:
not specified
Details on animal used as source of test system:
EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier : MatTek
Date received : 17 July 2018
EpiDermTM Tissues (0.63cm2) lot number : 28634
Assay Medium lot number : 071218MSA
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use.
Justification for test system used:
Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.
Vehicle:
unchanged (no vehicle)
Details on test system:
Negative Control
Information as provided by the Supplier.
Identification: Sterile distilled water
Batch: 17L04BB1A
Purity: Unknown
Expiry Date: 01 November 2020
Storage Conditions: Room temperature
Supplier: Baxter

Positive Control
Information as provided by the Supplier.
Identification: 8.0N Potassium Hydroxide
Batch: SLBM9898V
Purity: 7.92M
Expiry Date: 07 April 2020
Storage Conditions: Room temperature
Supplier: Sigma-Aldrich

Preparation of Negative and Positive Control Items and MTT
The negative control item was used as supplied.
The positive control item was used as supplied.
A 1.0 mg/mL MTT solution was prepared from a MatTek MTT-100 kit immediately prior to usage.

Study Design
Pre-Test Procedures
Assessment of Direct Test Item Reduction of MTT
MTT Dye 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 is still a sufficient amount 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.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below:
25 mg of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.
If the MTT solution containing the test item turned blue/purple relative to the control, the test item was presumed to have reduced the MTT.
An assessment of the test item’s capability to directly reduce MTT was inconclusive due to the intrinsic color of the test item and therefore, an additional procedure using freeze-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 freeze-killed tissues.
This step was a functional check which employs freeze-killed tissues that possess no metabolic activity but may absorb and bind the test item in the same way as viable tissues.
Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERMTM tissues in an empty 12-well plate and storing in a freezer (−14 to −30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature.
In addition to the normal test procedure, the test item was applied to two freeze-killed tissues per exposure period. In addition, two freeze-killed tissues per exposure period remained untreated. The untreated freeze-killed control normally show 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 or if it becomes colored when in wet or aqueous conditions. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
25 mg of test item was added to 300 μL of sterile water. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.
The test item was found to produce a colored solution which may interfere with the MTT assay. Therefore, viable color correction tissues were incorporated into the test to correct for this possibility. These tissues were treated the same as the main test with the exception of being placed into assay medium for 3 hours post-exposure instead of MTT. Two tissues were dosed with the test item and two remained untreated to act as negative controls for the 3 minute exposure group. The same was also performed for the 60 minute exposure group.

Double Correction Check
A third set of controls was also used to prevent a double correction from a colored test item that also reduces MTT using killed tissues.
Intrinsically colored test items may bind to both living and killed tissues and therefore the non-viable freeze-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 correct for color interference arising from the binding of the test item to living tissues.
Two freeze-killed tissues were dosed with the test item and two freeze-killed tissues remained untreated to act as the negative control for the 3 minute exposure, with the same number being used for the 60 minute exposure group. These tissues were incubated with assay medium instead of MTT post-exposure.

Main Test
Pre-Incubation
The assay medium was brought to room temperature before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3-Minute and 60-Minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

Application of Test Item and Rinsing
Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-Minute and 60-Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT-loading. Another 24-well plate was prepared for the MTT-loading. 300 μL of either pre-warmed assay medium (holding plate) or MTT medium (MTT-loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-Minute exposure period was returned to the incubator, while the other was being dosed for the 60-Minute exposure. For the 60-Minute exposure period, 50 μL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 25 mg of the test item and 50 μL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 μL of sterile water was added for wetting of the test item to increase tissue surface contact. The plate was returned to the incubator (37 °C, 5% CO2) for the 60-Minute exposure period.
When dosing for the 60-Minute exposure period was complete, the same procedure was repeated for the 3-Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT-loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60-Minute exposure period was complete, the same rinsing and MTT-loading procedure was repeated.
After the 3-Hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

Absorbance/Optical Density Measurements
After extraction, each tissue was pierced with a pipette fitted with a 1000 μL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 μL aliquots of the extract were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader and LT-com analysis software.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 mg of test item
50 μL of sterile distilled water (negative control)
50 μL of 8.0 N Potassium Hydroxide (positive control)
Duration of treatment / exposure:
3 minutes and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
Duplicate
Irritation / corrosion parameter:
% tissue viability
Remarks:
3-minute exposure period
Run / experiment:
Mean
Value:
91.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Remarks:
60-minute exposure period
Run / experiment:
Mean
Value:
88.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Other effects / acceptance of results:
The mean OD570 for the negative control treated tissues was 2.017 for the 3-Minute exposure period and 1.875 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 3.3% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Direct MTT Reduction

An assessment of the test item’s capability to directly reduce MTT was inconclusive due to the intrinsic color of the test item. Therefore, an additional procedure using freeze‑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 freeze‑killed tissues for quantitative correction of results or for reporting purposes.

Assessment of Color Interference with the MTT endpoint

The solution containing the test item was a black color, therefore additional color correction tissues were incorporated into the testing procedure. However, the results obtained showed that no 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 viabilities for each treatment group were as follows:

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Item

3 minute

100*

3.9

91.2

60 minute

100*

3.3

88.7

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

Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue

Exposure Period

MeanOD570 of individual tissues

Mean OD570 of duplicate tissues

Standard Deviation

Coefficient of Variation
(%)

Relative Mean Viability (%)

Negative Control

3 Minutes

2.041

2.017

0.034

1.7

100*

1.993

60 Minutes

1.907

1.875

0.045

2.4

1.843

Positive Control

3 Minutes

0.087

0.078

0.013

na

3.9

0.069

60 Minutes

0.070

0.061

0.013

na

3.3

0.051

Test Item

3 Minutes

1.754

1.839

0.120

6.5

91.2

1.923

60 Minutes

1.598

1.664

0.093

5.6

88.7

1.730

OD = Optical density

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

na = Not applicable

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used: EU CLP
Conclusions:
The test item was considered to be non-corrosive to the skin.
Executive summary:

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to have the potential to cause color interference and therefore additional tissues were incorporated for color correction purposes. An assessment of the test item’s capability to directly reduce MTT was inconclusive due to the intrinsic color of the test item and therefore, an additional procedure using freeze‑killed tissues was performed. A third set of controls was included, comprising freeze‑killed tissues, in order to prevent a double correction from a colored test item that also reduces MTT. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT-loading each tissue was placed in 2 mL of Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD570).

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 viabilities for each treatment group were as follows:

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Item

3 minute

100*

3.9

91.2

60 minute

100*

3.3

88.7

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

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

Conclusion

The test item was considered to be non-corrosive to the skin.

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:
The study was conducted between 04 September 2018 and 04 September 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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
Purity: See Certificate of Analysis
Sponsor Description: Dark brown powder
Envigo Description: Black powder
Expiry Date: 31 May 2019
Storage Conditions: Room temperature in the dark
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
Vehicle:
other: 20% w/v solution in sodium chloride 0.9% w/v
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Test item: 0.75 mL of 20% w/v solution in sodium chloride 0.9% w/v
Negative control: 0.75 mL of Sodium chloride 0.9% w/v
Positive control: 0.75 mL of Imidazole, 20% w/v solution in sodium chloride 0.9% w/v
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
Triplicate
Details on study design:
Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 65 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer.
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed.

Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of media representing each cornea was dispensed into the appropriate wells of a pre labeled 96 well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.
No histopathology was required for this study.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
24.2
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The positive control In Vitro Irritancy Score was within the range of 71.2 to 132.9. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤2.3 and permeability ≤0.44. The negative control acceptance criteria were therefore satisfied.

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD492)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

1

5

5

0

 

0.002

 

 

2

5

5

0

 

0.002

 

 

3

3

4

1

 

0.001

 

 

 

 

 

0.3*

 

0.002¨

 

0.4

Positive
Control

4

7

90

83

82.7

1.830

1.828

 

5

4

85

81

80.7

1.700

1.698

 

6

3

54

51

50.7

0.908

0.906

 

 

 

 

 

71.3·

 

1.478·

93.5

Test Item

7

5

27

22

21.7

0.039

0.037

 

9

5

41

36

35.7

0.005

0.003

 

10

5

20

15

14.7

0.002

0.000

 

 

 

 

 

24.0·

 

0.014·

24.2

OD= Optical density           * = Mean of the post-treatment -pre‑treatment values             ¨= Mean permeability                     ·= Mean corrected value

Corneal Epithelium Condition Post Treatment

Treatment

Cornea Number

Observation
Post Treatment

Negative Control

1

Clear

2

Clear

6

Clear

Positive Control

4

Cloudy

5

Cloudy

6

Cloudy

Test Item

7

Clear with some black staining

9

Clear with some black staining

10

Clear with some black staining

 

Corneal Epithelium Condition

The corneas treated with the test item were clear with some black staining post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment

In Vitro Irritancy Score

Test Item

24.2

Negative Control

0.4

Positive Control

93.5

Interpretation of results:
study cannot be used for classification
Conclusions:
No prediction of eye irritation can be made.
Executive summary:

Introduction

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short‑term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

Method

The test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). 

Data Interpretation

The test item is classified according to the prediction model as follows:

IVIS

UN GHS

≤ 3

No Category

>3; ≤ 55

No prediction can be made

> 55

Category 1

 

Results

The In Vitro irritancy scores are summarized as follows:

Treatment

In Vitro Irritancy Score

Test Item

24.2

Negative Control

0.4

Positive Control

93.5

Conclusion

No prediction of eye irritation can be made.

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro skin irritation (OECD 439):

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTMreconstructed 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 was103.6% 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).

In vitro Skin corrosion (OECD 437):

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to have the potential to cause color interference and therefore additional tissues were incorporated for color correction purposes. An assessment of the test item’s capability to directly reduce MTT was inconclusive due to the intrinsic color of the test item and therefore, an additional procedure using freeze‑killed tissues was performed. A third set of controls was included, comprising freeze‑killed tissues, in order to prevent a double correction from a colored test item that also reduces MTT. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT-loading each tissue was placed in 2 mL of Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD570).

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 viabilities for each treatment group were as follows:

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Item

3 minute

100*

3.9

91.2

60 minute

100*

3.3

88.7

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

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

Conclusion

The test item was considered to be non-corrosive to the skin.

In vitro Eye irrritation (OECD 437):

Introduction

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short‑term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

Method

The test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). 

Data Interpretation

The test item is classified according to the prediction model as follows:

IVIS

UN GHS

≤ 3

No Category

>3; ≤ 55

No prediction can be made

> 55

Category 1

 

Results

The In Vitro irritancy scores are summarized as follows:

Treatment

In VitroIrritancy Score

Test Item

24.2

Negative Control

0.4

Positive Control

93.5

 

Conclusion

No prediction of eye irritation can be made.

Justification for classification or non-classification

Based on the negative results in the skin irritation tests the substance does not need to be classified for this endpoint according to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Based on the negative results in the skin corrosion tests the substance does not need to be classified for this endpoint according to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Preventative Cat. 1 classification for eye irritation/damage due to unsuitability of the substance toward in silico methods and further in vitro methods do not permit assessment of reversibility of corneal staining . Staining was observed in the BCOP study; which needs to be considered according to EU CLP.