Registration Dossier

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Administrative data

Description of key information

Skin corrosion (OECD 431): The test item does not possess any skin corrosion potential.

Skin corrosion (OECD 439): The test item does not possess any skin irritation potential.

Eye irritation (OECD 437): The test item does not possess any eye irritation potential.

Eye irritation (OECD 492): The test item does not possess any eye irritating potential.


Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Experimental start date 07 November 2018. Experimental completion date 09 November 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: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: PG-RAW-0004
CAS Number: 2109705-93-1; 2111193-86-1
Batch: RDRW004-3
Purity: 95.5%
Physical state/Appearance: Clear colorless liquid
Expiry Date: 01 September 2019
Storage Conditions: Room temperature in the dark
Test system:
human skin model
Remarks:
EpiDerm Reconstructed Human Wpidermis Model Kit
Source species:
other: EpiDerm Reconstructed Human Epidermis Model Kit
Cell type:
other: Epidermal
Cell source:
other: EpiDerm Reconstructed Human Epidermis Model Kit
Type of coverage:
other: Substance added by pipette
Preparation of test site:
other: The test item was applied topically to the corresponding tissues ensuring uniform covering.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
50 µL 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.
Duration of treatment / exposure:
3 minutes
60 minutes
Observation period:
Immediate observation following exposure
Number of animals:
Two x 24-well plates
Details on 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:

50 µL 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.

The test item was shown to directly reduce MTT in the direct MTT reduction test. 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 absorb and bind the test item like 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 MTT reducing 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 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 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.

50 µL 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.

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. 50 µL of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. 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.

Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
103.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure
Value:
90.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Direct MTT Reduction

An assessment found the test item was able to directly reduce MTT.  Therefore, an additional procedure using freeze killed tissues was performed.  However, the results obtained showed that negligible 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 did not become colored.  This was taken to indicate the test item did not have the potential to cause color interference.

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

Tissue

Exposure Period

MeanOD570of individual tissues

Mean OD570of duplicate tissues

Standard Deviation

Coefficient of Variation
(%)

Relative Mean Viability (%)

Negative Control

3 Minutes

1.968

1.981

0.018

0.9

100*

1.994

60 Minutes

1.899

1.864

0.050

2.7

1.828

Positive Control

3 Minutes

0.086

0.078

0.012

na

3.9

0.069

60 Minutes

0.068

0.066

0.003

na

3.5

0.064

Test Item

3 Minutes

1.982

2.042

0.085

4.2

103.1

2.102

60 Minutes

1.684

1.684

0.001

0.0

90.3

1.683

 

 


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 for interpretation of results: EU
Conclusions:
The test item, PG-RAW-004, was classified as non-corrosive to the skin. The following classification criteria apply:
EU CLP (1272/2008/EC)/UN GHS: Not classified for corrosivity.
EU DSD (67/548/EEC): Not classified for corrosivity.
UN Packing Group: Non-Corrosive.
Executive summary:

The skin corrosivity of the test substance was determined according to OECD Guideline 431 using the EPISKIN™ Reconstructed Human Epidermis Model.

Quality Criteria

The mean OD570for the negative control treated tissues was 1.981 for the 3‑Minute exposure period and 1.864 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 0.066% 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.

Conclusion

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

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 14 November 2018 and 03 December 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.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: PG-RAW-0004
Physical state/Appearance: Clear colorless liquid
Storage Conditions: Approximately 4 °C in the dark
Test system:
human skin model
Source species:
human
Cell type:
other: Reconstructed Human Epidermis
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: 27 November 2018
EpiSkinTM Tissues (0.38cm^2) lot number: 18-EKIN-048
Maintenance Medium lot number: 18-MAIN3-059
Assay Medium lot number : 18-ESSC-053
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.
Vehicle:
unchanged (no vehicle)
Details on test system:
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 µL 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 water killed tissues for quantitative correction of the results.
The MTT solution containing the test item did not turn blue/purple but was an inconclusive dark yellow color. 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 thus give rise to a false negative result. Therefore, an additional procedure was conducted in parallel on viable and non-viable, water-killed tissues, in order to perform quantitative correction procedures to determine the quantity of true viability versus false viability.
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 µL 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.

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. 10 µL (26.3 µL/cm^2) of the test item was applied to the epidermis 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:
Reference Items
Negative Control
Information as provided by the Supplier.
Identification: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
Purity: >98%
Storage Conditions: Approximately 4 °C in the dark

Positive Control
Information as provided by the Supplier.
Identification: Sodium dodecyl sulphate
Purity: 99.5%
Storage Conditions: Room temperature

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.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Triplicate
Irritation / corrosion parameter:
other: relative mean viability
Run / experiment:
Mean
Value:
75
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 9.3% relative to the negative control treated tissues and the standard deviation value of the viability was 9.3%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.808 and the standard deviation value of the viability was 2.6%. 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 18.3%. The test item acceptance criterion was therefore not satisfied. This is reported as a deviation.

Repeat Experiment
The main test of this assay was run on two occasions. Due to a technician error the incorrect program template was selected on the microplate reader during the first run of this assay. The consequence was that the optical densities of the non-viable, water-killed tissue groups, included in the assay for the purposes of quantitative correction of results, were not measured. The main test was therefore repeated.

Direct MTT Reduction

The MTT solution containing the test item did not turn blue/purple but was an inconclusive dark yellow color. 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 thus give rise to a false negative result. Therefore, an additional procedure was conducted in parallel on viable and non-viable, water-killed tissues, in order to perform quantitative correction procedures to determine the quantity of true viability versus false viability. However, the results obtained showed that no interference due to direct reduction of MTT in the main test occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results. 

Assessment of Color Interference with the MTT endpoint

The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

Test Item, Positive Control Item and Negative Control Item

The relative mean viability of the test item treated tissues was 75.0% 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

OD570 of tissues

Mean OD570 of triplicate tissues

±SD of OD570

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.815

0.808

0.021

100.9

100*

2.6

0.824

102.0

0.784

97.0

Positive Control Item

0.162

0.075

0.075

20.0

9.3

9.3

0.035

4.3

0.028

3.5

Test Item

0.663

0.606

0.147

82.1

75.0

18.3

0.439

54.3

0.717

88.7

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
Conclusions:
The relative mean tissue viability after 15 minutes treatment with the substance compared to the negative control tissue was 75%. Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment, the substance is considered to be non-irritant.
Executive summary:

The possible skin irritation potential of PG-RAW-0004 was tested through topical application for 15 minutes. The study procedures described in this report were based on the OECD TG 439. Skin tissue was treated by topical application of 10 µL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 75%.

Since the mean relative tissue viability for PG-RAW-0004 was higher than 50% after 15 minutes treatment the substance is considered to be non-irritant. The positive control had a mean cell viability of 9.3% after 15 minutes exposure. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was18.3%. The test item acceptance criterion was therefore not satisfied. This is reported as a deviation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between the 03 January 2019 and 03 January 2019.
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 Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: PG-RAW-0004
Physical state/Appearance: Clear colorless liquid
Storage Conditions: Room temperature in the dark
Species:
other: Eyes from adult cattle
Strain:
other: Not applicable
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:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the test item or control items were applied to the cornea
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 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 60 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 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 10 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.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
Following the final 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 (4 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.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
1.9
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 positive control In Vitro Irritancy Score was within the acceptance range. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity and permeability values below the established upper limits. The negative control acceptance criteria were therefore satisfied.

Corneal Epithelium Condition

The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment

In Vitro Irritancy Score

Test Item

1.9

Negative Control

1.1

Positive Control

53.5

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD492)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post Incubation

Post-Incubation - Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

2

3

3

4

1

 

0.000

 

 

3

6

6

8

2

 

0.012

 

 

4

6

6

6

0

 

0.000

 

 

 

 

 

 

1.0*

 

0.004¨

 

1.1

Positive Control

5

6

52

50

44

43.0

0.572

0.568

 

6

3

48

45

42

41.0

0.988

0.984

 

9

3

32

34

31

30.0

1.545

1.541

 

 

 

 

 

 

38.0·

 

1.031·

53.5

Test Item

10

4

6

7

3

2.0

0.045

0.041

 

11

3

5

6

3

2.0

0.006

0.002

 

12

3

4

5

2

1.0

0.001

0.000

 

 

 

 

 

 

1.7·

 

0.014·

1.9

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

Corneal Epithelium Condition Post Treatment and Post Incubation

Treatment

Cornea Number

Observation

Post Treatment

Post Incubation

Negative Control

2

Clear

Clear

3

Clear

Clear

4

Clear

Clear

Positive Control

5

Cloudy

Cloudy

6

Cloudy

Cloudy

9

Cloudy

Cloudy

Test Item

10

Clear

Clear

11

Clear

Clear

12

Clear

Clear

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test item was considered not to be an ocular corrosive or severe irritant.
Executive summary:

The eye irritancy potential of the PG-RAW-0004 was assessed according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and is not classified, according to EU CLP criteria.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 31 October 2018 and 29 November 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: PG-RAW-0004
Appearance: Colourless liquid
Storage Conditions: At +2 to + 8 °C, light protected
Stability in Solvent: Not indicated by the Sponsor
Species:
other: Reconstructed Human Corneal Epithelial Model
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
EpiOcular Kit Components Needed for the Assay
Lot No.: 27082
Sealed 24-well plate Contains 12/24 inserts with EpiOcular™ tissues on agarose
Serum-free test medium DMEM-Medium
Positive control Methyl Acetate (CAS#79-20-9)
12-well plate Holding plate
24-well plates For MTT viability assay
6-well plates For storing inserts, or for topically applying test agents
Ca++Mg++-Free DPBS Dulbecco´s Phosphate Buffered Saline

MTT-100 Assay Kit Components
1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM) For diluting MTT concentrate prior to use in the MTT assay
1 bottle, 60 mL Extractant solution (Isopropanol) For extraction of formazan crystals

Cell Culture
EpiOcular™ kits and MTT-100 kits were purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELL®, 10 mm Ø).
EpiOcular™ tissues were received at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt (28 November 2018) of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL fresh Assay Medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) overnight (about 17 hours).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied (volume with unit): 50 µL

CONTROLS:
- Amount applied (volume with unit): 50 µL
Duration of treatment / exposure:
30 minutes
Duration of post- treatment incubation (in vitro):
12 minute immersion incubation followed by 120 minute post-treatment incubation
Number of animals or in vitro replicates:
Duplicates
Details on study design:
Test Item Preparation
50 µL (83.3 µL/cm² according to guideline) of the test item were dispensed directly atop duplicate EpiOcular™ tissue for 30 minutes.

Study Controls
Concurrent controls were used for several Envigo CRS GmbH studies performed simultaneously. Each 50 µL were applied to duplicate tissues for 30 minutes.

MTT-Solution
On the day of the experiment a MTT solution 1.0 mg/mL in DMEM was prepared.

Assessment of Direct MTT Reduction by the Test Item
Test items may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement. Therefore, it was necessary to assess this ability for the test item prior to conducting any assays with viable tissues. For this purpose approximately 50 µl of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 µL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was performed concurrently. If the MTT solution colour turned blue/purple, the test item will be presumed to have reduced the MTT.
Since the test item proved to reduce MTT, a functional check using freeze-killed tissue controls (killed controls = KC) had to be performed in at least one definitive assay to evaluate whether the test material was not binding to the tissue and leading to a false MTT reduction signal.

Assessment of Coloured or Staining Materials
Coloured test items or test items which become coloured after application to the tissues could interfere with the quantitative photometric MTT measurement if the colorant bound to the tissue and would be extracted together with MTT. Therefore, each test item had to be checked for its colouring properties.
Since the test item was non-coloured, additional tests had to be performed to assess, if it becomes coloured after contact with water or isopropanol. For this purpose, 50 µL of the test item were added to 2.0 mL of isopropanol (glass tube) and shaken for 3 hours at room temperature. 2.0 mL of isopropanol were used as control. Additionally 50 µL of the test item were added to 1.0 mL of deionised water (glass tube) and incubated at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for at least 1 hour. 1 mL of deionised water was used as control.
Since the test item did not dye water or isopropanol, additional tests with viable tissues did not have to be performed.

EXPERIMENTAL DESIGN AND STUDY CONDUCT
Experimental Performance
After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) for 30 minutes.
After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control items were tested by applying 50 µL topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 30 minutes.
At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test item. Each test item utilized a different set of three beakers. The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beaker of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for a 12 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm Assay Medium. The tissues were incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

MTT Assay
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Inserts were removed from the 24-well plate after 180 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2 mL isopropanol in each well so that isopropanol was flowing into the insert. The plates were sealed with parafilm and a standard plate sealer, and were immediately extracted. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for 2 hours at room temperature. The tissues were pierced. The corresponding negative, positive, and additional viable tissues (without MTT addition) were treated identically with piercing.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.
Irritation parameter:
other: Tissue viability (%)
Run / experiment:
Mean
Value:
72.82
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Concerning acceptance criteria:
• The negative control OD is > 0.8 and < 2.5 (2.167 - 2.243).
• The tissue viability of the positive control is below 50% of the negative control viability (42.88%).
The difference of viability between the two relating tissues of a single item is < 20% (values between 0.57 p.p and 7.56 p.p) in the same run (for positive and negative control tissues and tissues of single test items). This applied also to the killed controls (items and negative control) which were calculated as percent values related to the viability of the relating negative control.

Direct MTT reduction

Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent showed purple colour. Therefore, an additional test with freeze-killed tissues was necessary.

Assessment of Color Interference with the MTT endpoint

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not lead to a change in colour. Therefore, an additional test with viable tissues without MTT addition was not necessary.

Treatment Group

OD 570 nm
Well 1

OD 570 nm
Well 2

Mean OD of
2 Wells

Mean OD

of 2 Wells blank

corrected

Evaluation Mean OD570(blank corrected)                  > 0.08

Blank
Aqua Deion.

0.036

0.044

0.040

 

Test Item + Aqua Deion.

0.045

0.046

0.045

0.005

no

Blank Isopropanol

0.037

0.038

0.037

 

Test Item+

Isopropanol

0.038

0.037

0.038

0.000

no

The mean OD was < 0.08 and therefore viable tissues were not necessary in the main experiment.

Main experiment

Results after treatment for 30 minutes with PG-RAW-0004 and the controls

Test Group

Tissue No.

Well 1 [OD570]

Well 2 [OD570]

Mean [OD570] (Well 1 and

well 2)

Mean [OD570] blank corr. (Well 1 and

well 2)

Mean [OD570] of T1 and T2

Tissue viabil.* [%]

rel. viabil. of T1 and T2**

Diff. of viabil. between T1 and T2 [p.p.]

Mean viability of test item after data correct. procedure [%]

Blank

 

0.035

0.035

0.035

 

73.40***

Negative

Control

1

2.243

2.192

2.218

2.182

2.158

100.0

101.2

2.30

2

2.169

2.167

2.168

2.133

98.8

Positive

Control

1

1.062

1.021

1.042

1.007

0.925

42.88

46.7

7.56

2

0.892

0.865

0.879

0.844

39.1

Test Item

1

1.614

1.556

1.585

1.550

1.571

72.82

71.8

1.96

2

1.640

1.615

1.627

1.592

73.8

Blank

 

0.035

0.035

0.035

 

Negative Control
Freeze killed

Tissues

1

0.118

0.118

0.118

0.083

0.076

3.53

3.8

0.61

2

0.107

0.103

0.105

0.070

3.2

Test Item Freeze

killed Tissues

1

0.106

0.104

0.105

0.070

0.063

2.94

3.2

0.57

2

0.092

0.093

0.092

0.057

2.7

*Tissue viability = (100 x meanOD of T1 & T2test item/positive control/negative control)/meanOD of T1 & T2negative control

**Relative Tissue Viability = (100 x meanOD blank correctedtest item/positive control/negative control)/meanOD of T1 & T2negative control

***Corrected mean viability = Tissue viabilitytest item– (Tissue viabilityKC test item– Tissue viabilityKC negative control)

Interpretation of results:
other: The test item does not possess any eye irritating potential
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, PG-RAW-0004 does not possess any eye irritating potential.
Executive summary:

This in vitro study was performed to assess the eye irritation potential of PG-RAW-0004 by means of the Human Cornea Model Test.

The test item proved to be an MTT reducer in the MTT pre-test. Its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed tissues had to be performed. The viability values resulted in these additional tests were used to correct the values gained in the normal tests.

Each 50 µL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissues for 30 minutes.

After treatment with the negative control, the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system.

The difference of relative viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Irritating effects were not observed following incubation with PG-RAW-0004. Compared with the value of the negative control, the mean absorption value corresponding to the tissue viability did not decrease below 60% (determined value for the test item: 73.40%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, PG-RAW-0004 does not possess any eye irritating potential.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Skin corrosion OECD TG 431:

This in vitro study was performed to assess the skin corrosion potential of PG-RAW-0004 by means of the EPIDERM Skin Corrosion Test.

The test item was able to directly reduce MTT in the MTT pre-test. Therefore, an additional procedure using freeze-dried tissues was performed. However, the results obtained showed that negligible 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.

The test solution containing the test item did not become coloured. This was taken to indicate that the test item did not have the potential to cause colour interferference.

The mean OD570for the negative control treated tissues was 1.981 for the 3‑Minute exposure period and 1.864 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 0.066% 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.

In conclusion, the test item was considered to be non-corrosive to the skin.

Skin irritation OECD TG 439:

The possible skin irritation potential of PG-RAW-0004 was tested through topical application for 15 minutes. The study procedures described in this report were based on the OECD TG 439. Skin tissue was treated by topical application of 10 µL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 75%.

Since the mean relative tissue viability for PG-RAW-0004 was higher than 50% after 15 minutes treatment the substance is considered to be non-irritant. The positive control had a mean cell viability of 9.3% after 15 minutes exposure. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 18.3%. The test item acceptance criterion was therefore not satisfied. This is reported as a deviation.

Eye irritation OECD TG 437:

The eye irritancy potential of the PG-RAW-0004 was assessed according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and is not classified, according to EU CLP criteria.

Eye irritation OECD TG 492:

Thisin vitrostudy was performed to assess the eye irritation potential of PG-RAW-0004 by means of the Human Cornea Model Test.

The test item proved to be an MTT reducer in the MTT pre-test. Its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed tissues had to be performed. The viability values resulted in these additional tests were used to correct the values gained in the normal tests.

Each 50 µL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissues for 30 minutes.

After treatment with the negative control, the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system.

The difference of relative viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Irritating effects were not observed following incubation with PG-RAW-0004. Compared with the value of the negative control, the mean absorption value corresponding to the tissue viability did not decrease below 60% (determined value for the test item: 73.40%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, PG-RAW-0004 does not possess any eye irritating potential.

Justification for classification or non-classification

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

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

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