[No public or meaningful name is available]

Endpoint summary

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.

In vivo eye irritation (OECD 405): Not classified according to EU CLP/GHS

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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

Guideline:

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

GLP compliance:

yes (incl. QA statement)

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 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).

Reference 2

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

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. QA statement)

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: non-corrosive

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

Reference 1

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

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

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. QA statement)

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.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 05 JUne 2019 and 14 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Adopted 02 October 2012.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item: Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide
Chemical name: Reaction mass of Magnesium dioxide(oxo)silane and Silicon dioxide (MgSiO3, Si and SiO2)
Appearance: Dark brown powder.
Storage conditions: Room temperature (15° to 25°C) protected from light.
Supplier: Sponsor
Batch number: E80-181130-11.
Expiry date: 30 November 2019.
Purity: 94%.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Animal Information:
Animals for this study were selected from a stock supply of healthy adult rabbits of the New Zealand White strain. They were in the weight range of 3.45 to 3.77 kg and at approximately 20 to 23 weeks of age, prior to treatment (Day 1). All rabbits were acclimatized to the experimental environment for a period of at least 10 weeks prior to the start of the study.

Animal Care and Husbandry:
Each animal was housed individually in a plastic cage with perforated floors and was offered 150 g of a standard laboratory rabbit diet per day; drinking water was provided ad libitum. The batch of diet used for the study is analyzed for nutrients, possible contaminants and micro-organisms likely to be present in the diet and which, if in excess of specified amounts, might have an undesirable effect on the test system. During acclimatization and the study period the animals were given a dietary supplement of hay daily and a small amount of chopped fresh vegetables were given twice weekly.

During the acclimatization and study period the animals were given small soft white untreated wood blocks for environmental enrichment. A stainless steel key ring was also attached to the front of each cage as environmental enrichment.

Results of routine physical and chemical examination of drinking water, as conducted by the supplier are made available.

Animal room environmental controls were set to maintain temperature within the range 15 to 21°C, and relative humidity within 45 to 70%. These environmental parameters were recorded and the permanent record archived with other departmental raw data. Lighting was controlled by means of a time switch to give 12 hours of artificial light (06:00 to 18:00 GMT) in each 24 hour period.

Each animal was identified by a numbered tag placed through the edge of one ear. Each cage was identified by a label displaying the study number and animal number.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

dose of a volume of 0.1 mL of the test item

Duration of treatment / exposure:

Single ocular dose

Observation period (in vivo):

The treated eyes were observed 1, 24, 48 and 72 after instillation, with an additional observation on Day 7 for the sentinel animal.

Number of animals or in vitro replicates:

3 rabbits

Details on study design:

Dose Administration:
The eyes of each animal were examined prior to instillation of the test item to ensure that there was no pre-existing corneal damage, iridial inflammation or conjunctival irritation. Each animal was gently restrained. The dose was instilled into the right eye by pulling the lower eyelid away from the eye ball to form a cup into which the test item was dropped. The eyelids were then gently held together for one second before releasing; the left eye remained untreated.

A single animal was treated in advance; in the absence of a severe effect in this animal two further animals were committed to the study.

Anaesthetic and analgesia regime:
The following topical anaesthetic and systemic analgesia regime was employed:
• one hour before administration: 0.01 mg/kg buprenorphine was administered subcutaneously.
• approximately 15 minutes before administration topical anaesthesia was administered: one/two drops of topical ocular anaesthesia were applied to each eye; the procedure was repeated approximately 5 minutes later. The test material was administered approximately 10 minutes after the last administration of anaesthesia.
• approximately eight hours after test material administration; 0.01 mg/kg buprenorphine and 0.5 mg/kg meloxicam were administered subcutaneously.

Serial Observations:
Clinical Observations:
The behavior of each rabbit was observed immediately following instillation of the test item to allow assessment of the initial pain response.

The animals were returned to their cages and checked at least twice during the first hour after dosing and at regular intervals throughout the day to ensure no severe injury passed unnoticed. Ocular reactions to treatment were assessed 1, 24, 48 and 72 hours and seven days after treatment, according to the criteria below.

Ocular Responses:
The untreated eye was used as a comparison with the treated eye during assessment of ocular lesions. Ocular irritation was assessed using the prescribed numerical system:

Cornea Opacity: degree of density (area most dense taken for reading)
No opacity: 0
Scattered or diffuse areas of opacity (other than slight dulling of normal luster), details of iris clearly visible: 1
Easily discernible translucent areas, details of iris slightly obscured: 2
Nacreous areas, no details of iris visible, size of pupil barely discernible: 3
Opaque cornea, iris not discernible through the opacity: 4

Area of cornea involved:
None: 0
One quarter (or less) but not zero: 1
Greater than one quarter, but less than half: 2
Greater than half, but less than three quarters: 3
Greater than three quarters, up to whole area: 4

The presence or absence of ulceration or stippling of the cornea will be designated by “+”; positive or “-”: negative. Redness of the cornea, such as pannus formation, will be described on an individual basis.

Iris
Normal: 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperemia or injection, any of these or combination of any thereof, iris still reacting to light (sluggish reaction is positive): 1
No reaction to light, hemorrhage, gross destruction (any or all of these): 2

Conjunctivae Redness (refers to the most severe reading of palpebral and bulbar conjunctivae, as compared to the control eye)
Blood vessels normal: 0
Some blood vessels definitely hyperemic (injected): 1
Diffuse, crimson color, individual vessels not easily discernible: 2
Diffuse beefy red: 3

Chemosis (lids and/or nictating membranes)
No swelling: 0
Any swelling above normal (includes nictating membranes): 1
Obvious swelling with partial eversion of lids: 2
Swelling with lids about half-closed: 3
Swelling with lids more than half-closed: 4

The presence or absence of necrosis or ulceration was designated by “+”; positive or “-”: negative.

Discharge
No discharge: 0
Any amount different from normal: 1
Discharge with moistening of lids and hairs adjacent to lids: 2
Discharge with moistening of lids and hairs and considerable area around eye: 3*
Extensive mucoid discharge: 4
*Maximum score permissible under system of Kay and Calandra

Any other lesion not covered by this scoring system was described.
An ophthalmoscope or pencil beam torch was available for use to facilitate inspection of the eyes.

Terminal Investigations:
Following completion of the observation period the animals were humanely killed by an intravenous injection of sodium pentobarbital.

Irritation parameter:

cornea opacity score

Basis:

animal: 195

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: 196

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: 197

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal: 195

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal: 196

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal: 197

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal: 195

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

fully reversible within: 24 hrs

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal: 196

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

fully reversible within: 24 hrs

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal: 197

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

fully reversible within: 24 hrs

Irritation parameter:

chemosis score

Basis:

animal: 195

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal: 196

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal: 197

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hrs

Irritant / corrosive response data:

Injection of the conjunctival blood vessels (Grade 1 redness) was evident in all animals, with slight discharge (Grade 1) in one, one hour after instillation. Slight chemosis (Grade 1) was apparent in one animal at the 1 hour and 24 hour examination. In all animals black staining (dose residue) was present around the right eye throughout the observation period.

The treated eyes of all animals were overtly normal 48 hours after installation.

Instillation of the test item gave no initial pain response.

Other effects:

Clinical signs:
There was no sign of toxicity or ill health in any rabbit during the observation period.

Mean values for ocular lesions for Kay and Calandra classification

Mean irritation scores after instillation of test item

Area of eye	1 hour	24 hours	48 hours	72 hours
Cornea	0	0	0	0
Iritis	0	0	0	0
Conjunctiva	2.67	0.67	0	0
Total mean score	2.67	0.67	0	0

Mean values for ocular lesions for EC (regulation 1272/2008) and GHS classification

24, 48 and 72 hours after instillation of test item

Animal number and sex	Corneal Opacity	Iridial Lesions	Redness of Conjunctiva	Chemosis
195 M	0.00	0.00	0.00	0.00
196 M	0.00	0.00	0.00	0.00
197 M	0.00	0.00	0.00	0.33

Interpretation of results:

GHS criteria not met

Conclusions:

The highest total mean score was 0.67 occurring at the 24 hour observation; accordingly under the criteria Kay and Calandra (1962) the test item was classified as “mildly irritating” to the eye.
The test item did not require labeling in accordance with European Commission regulation 1272/2008.

Executive summary:

The purpose of this study was to assess the eye irritation potential of reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide to the rabbit. The method followed was that described in:

- Method B5 Acute Toxicity (Eye Irritation) of Commission Regulation (EC) No. 440/2008

- OECD Guideline for Testing of Chemicals No.405 “Acute Eye Irritation/Corrosion”. Adopted 02 October 2017.

Three rabbits were each administered a single ocular dose of a volume of 0.1 mL of the test item, as supplied; the treated eyes were observed 1, 24, 48 and 72 after instillation, with an additional observation on Day 7 for the sentinel animal.

Injection of the conjunctival blood vessels (Grade 1 redness) was evident in all animals, with slight discharge (Grade 1) in one, one hour after instillation. Slight chemosis (Grade 1) was apparent in one animal at the 1 hour and 24 hour examination. In all animals black staining (dose residue) was present around the right eye throughout the observation period.  

The treated eyes of all animals were overtly normal 48 hours after installation.

Instillation of the test item gave rise to practically no initial pain response.

The total mean scores according to the system of Kay and Calandra (1962) are summarized below:

Area of eye	Time after instillation
	1 hour	24 hours	48 hours	72 hours
Cornea	0.0	0.0	0.0	0.0
Iritis	0.0	0.0	0.0	0.0
Conjunctiva	2.67	0.67	0.0	0.0
Total mean score	2.67	0.67	0.0	0.0

The highest total mean score was 0.67 occurring at the 24 hour observation; accordingly under the criteria of Kay and Calandra (1962) Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide was classified as “mildy irritating” to the eye.

The means of scores for the ocular reactions at approximately 24, 48 and 72 hours after administration, calculated separately for each animal, are summarized below:

Animal number and sex	Corneal opacity	Iridial lesions	Conjunctival
			Redness	Chemosis
195M	0.0	0.0	0.0	0.0
196M	0.0	0.0	0.0	0.0
197M	0.0	0.0	0.0	0.33
EC Category 1 trigger values*	≥3	>1.5
EC Category 2 trigger values*	≥1	≥1	≥2	≥2

*             Classification triggered if any value is attained by two or more animals.

M           Male

The test item did not require labeling in accordance with European Commission regulation 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

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 EPISKIN^TMreconstructed 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.

In vivo eye irritation (OECD 405):

Three rabbits were each administered a single ocular dose of a volume of 0.1 mL of the test item, as supplied; the treated eyes were observed 1, 24, 48 and 72 after instillation, with an additional observation on Day 7 for the sentinel animal.

Injection of the conjunctival blood vessels (Grade 1 redness) was evident in all animals, with slight discharge (Grade 1) in one, one hour after instillation. Slight chemosis (Grade 1) was apparent in one animal at the 1 hour and 24 hour examination. In all animals black staining (dose residue) was present around the right eye throughout the observation period.  

The treated eyes of all animals were overtly normal 48 hours after installation.

Instillation of the test item gave rise to practically no initial pain response.

The means of scores for the ocular reactions at approximately 24, 48 and 72 hours after administration, calculated separately for each animal, are summarized below:

Animal number and sex	Corneal opacity	Iridial lesions	Conjunctival
			Redness	Chemosis
195M	0.0	0.0	0.0	0.0
196M	0.0	0.0	0.0	0.0
197M	0.0	0.0	0.0	0.33
EC Category 1 trigger values*	≥3	>1.5
EC Category 2 trigger values*	≥1	≥1	≥2	≥2

*             Classification triggered if any value is attained by two or more animals.

M           Male

The test item did not require labeling in accordance with European Commission regulation 1272/2008.

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.

Based on the result of an in-vivo eye irritaiton study (OECD 405), the test item does not require labelling in accordance with European Commission Regulation 1272/2008.