Reaction mass of (E)-1-(3,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one and (E)-1-(4,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one

Endpoint summary

Description of key information

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

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

Eye irritation (OECD 437): Not requiring classification for eye irritation and serious eye damage.

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

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 27 March 2019 Experimental completion date 28 March 2019

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:

no

Qualifier:

according to guideline

Guideline:

other: • Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: PG-RAW-90-032
Chemical Name: Reaction mass of (E)-1-(3,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one and (E)-1-(4,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one
CAS Number: 2248118-61-6; 2248118-60-5
Batch: RDRW 534-93
Purity: 99.0%
Molecular Weight; 206.33
Physical state/Appearance: Clear colorless liquid
Expiry Date: 01 November 2020
Storage Conditions: Approximately 4°C in the dark

Test system:

human skin model

Remarks:

EpiDerm™ Human Skin Model

Source species:

other: EpiDerm™ Human Skin Model

Cell type:

other: Epithelial

Cell source:

other: EpiDerm™ Human Skin Model

Source strain:

other: EpiDerm™ Human Skin Model

Details on animal used as source of test system:

EpiDerm™ Human Skin Model Kit


Supplier : MatTek
Date received : 26 March 2019
EpiDermTM Tissues (0.63cm2) lot number : 28689
Assay Medium lot number : 032119MSC
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. Viable cells are able to reduce MTT to formazan whereas non-viable cells cannot. The results are used to make a prediction of the corrosivity potential of the test item (increased cytotoxicity is indicative of corrosion potential).

This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

Vehicle:

unchanged (no vehicle)

Details on test system:

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.

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.

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 tissue inserts were blotted and transferred to 24 well plates for formazan (reduced MTT) extraction. The formazan was extracted from the top and bottom of the tissue by completely immersing the tissue insert in 2 mL of isopropanol. The plate was covered with plate sealer, to prevent isopropanol evaporation, and stood overnight at room temperature, to allow extraction to proceed.

Control samples:

yes, concurrent no treatment

yes, concurrent positive control

Amount/concentration applied:

50 µL of test item, Purity: 7.92M

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2 x 24-well plates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

118.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

123.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Direct MTT Reduction
The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.

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.

Quality Criteria

The mean OD₅₇₀for the negative control treated tissues was 1.577 for the 3‑Minute exposure period and 1.622 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 4.4% 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.

Interpretation of results:

GHS criteria not met

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. Viable cells are able toreduce MTT to formazanwhereas non-viable cells cannot. The results are used to make a prediction of the corrosivity potential of the test item (increased cytotoxicity is indicative of corrosion potential).

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. 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*	4.6	118.5
60 minute	100*	4.4	123.9

*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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 03 April 2019. Experimental completion date: 08 April 2019

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:

when applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: PG-RAW-90-032
Chemical Name: Reaction mass of (E)-1-(3,6-dimethylcyclohex-3en-1-yl)-2-methylpent-1-en-3-one and (E)-1-(4,6dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3one
CAS Number: 2248118-61-6; 2248118-60-5
Batch: RDRW 534-93
Purity: 99.0%
Molecular Weight: 206.33
Physical state/Appearance: Clear colorless liquid
Expiry Date: 01 November 2020
Storage Conditions: Approximately 4°C in the dark

Species:

other: EPIKSIN in vitro Reconstructed Human Epidermis (RHE) Model

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : EpiSkin Laboratories, Lyon, France
Date received : 02 April 2019
EpiSkinTM Tissues (0.38cm2) lot number : 19-EKIN-014
Maintenance Medium lot number : 19-MAIN3-014
Assay Medium lot number : 19-ESSC-013

Type of coverage:

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:

Test material
- Applied volume: 10 ul
- Concentration (if solution): undiluted

Positive control
- Applied volume: 10 ul
- Concentration (if solution): 5% w/v

Negative control
- Applied volume: 10 ul
- Concentration (if solution): undiluted

Duration of treatment / exposure:

15-Minute exposure period and 42 hours post-exposure incubation period.

Number of animals:

Triplicate tissues were treated with: test substance, positive control and negative control.

Details on study design:

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..

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 2):
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/cm2) 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.

Irritation / corrosion parameter:

other: relative mean viability

Run / experiment:

Mean

Value:

58.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Direct MTT Reduction

The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.  

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 58.5% after a 15-Minute exposure period and 42-Hour post-exposure incubation period.

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

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 4.4% relative to the negative control treated tissues and the standard deviation value of the viability was 0.7%.  The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control treated tissues was 0.857 and the standard deviation value of the viability was 1.0%.  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 0.8%.  The test item acceptance criterion was therefore satisfied.

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

Item	OD570 of tissues	Mean OD570of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.866	0.857	0.008	101.1	100*	1.0
	0.852			99.4
	0.853			99.5
Positive Control Item	0.045	0.038	0.006	5.3	4.4	0.7
	0.034			4.0
	0.035			4.1
Test Item	0.509	0.501	0.007	59.4	58.5	0.8
	0.495			57.8
	0.499			58.2

OD = Optical Densit

SD = Standard deviation

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

Interpretation of results:

other: Not irritating

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The relative mean tissue viability after 15 minutes treatment with the substance compared to the negative control tissue was 58.5%. 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 the substance 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 58.5%.

Since the mean relative tissue viability for PG-RAW-90 -032 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 4.4% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was 0.8%, indicating that the test system functioned properly.

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:

11 April 2019 - 11 April 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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

GLP compliance:

yes (incl. QA statement)

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.

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.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent negative control

Amount / concentration applied:

0.75 mL of the test item or control items were applied to the appropriate corneas.

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

Not specified

Number of animals or in vitro replicates:

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.

Details on study design:

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.

SCORING SYSTEM: In vitro irritancy score (Table1)
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

TOOL USED TO ASSESS SCORE: 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.

Irritation parameter:

in vitro irritation score

Run / experiment:

PG-RAW-90-032

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

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.

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.
(Table 2)

Table 2. Overview of irritancy scores

The In Vitro irritancy scores are summarized as follows:

Treatment	In VitroIrritancy Score
Test Item	0.0
Negative Control	0.5
Positive Control	53.8

Interpretation of results:

GHS criteria not met

Conclusions:

No category. Not requiring classification to UN GHS.

Executive summary:

The In Vitro irritancy score of PG-RAW-90-032, was 0.0 according to OECD Test Guideline No. 437 (updated 09 October 2017) “Bovine Corneal Opacity and Permeability Assay.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 01 April 2019 Experimental completion date: 04 April 2019

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

Qualifier:

according to guideline

Guideline:

other: • MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model; 29 June 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: PG-RAW-90-032
Batch: RDRW 534-93
Purity: 99.0%
Appearance: Colourless liquid
Expiry Date: 01 November 2020
Storage Conditions: At +2 to +8 °C, light protected*
Stability in Solvent: Stable in water (not quantified)
Purpose of Use: Industrial chemical
* only valid for storage condition, not for test performance

Species:

other: Human cornea model

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

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 ).

EpiOcular™ tissues were received at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt (02 April 2019) 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:

50 µL (83.3 µL/cm² according to guideline)

Duration of treatment / exposure:

30 minutes pre-treatment

Duration of post- treatment incubation (in vitro):

30 minutes

Number of animals or in vitro replicates:

Three beakers

Details on study design:

Main Experiment
Pre-Incubation
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.

Treatment
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 PBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of PBS 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 PBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with PBS, 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 PBS 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).

Post-Incubation
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.

The inserts were removed from the 24-well plate after 180 minutes. Since the test item was colourless, 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 stored about 16.5 h at 2-8 °C in the dark. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for about 3 hours at room temperature. Then, the tissues were pierced.

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: Viability value (%)

Run / experiment:

Main experiment

Value:

106.35

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The mean absorbance value of the test item, corresponding to the relative cell viability, increased to 106.35% (threshold for irritancy: ≤ 60%), consequently the test item was not irritant to eye.

Concerning acceptance criteria:
• The OD of the tissues treated with the negative control is > 0.8 and < 2.5 (1.911 and 1.968).
• The tissue viability of the positive control is below 50% of the negative control viability (21.74%).

The difference of viability between the two relating tissues of a single item is < 20% (values between 0.41 p.p and 2.96 p.p) in the same run (for positive and negative control tissues and tissues of single test items).

ResultsAND DISCUSSION

Pre-Experiment

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.

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.	0.037	0.037	0.037
Test Item + Aqua Deion.	0.045	0.045	0.045	0.009	no
Blank Isopropanol	0.037	0.037	0.037
Test Item+ Isopropanol	0.038	0.038	0.038	0.001	no

The mean OD was < 0.08 and therefore, an additional test with viable tissues without MTTaddition was not necessaryin the main experiment.

Direct MTT reduction by the test item

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

Main experiment

Results after treatment for 30 minutes withPG-RAW-90-032and 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		0.037	0.037	0.037						106.35***
Negative Control	1	1.958	1.938	1.948	1.911	1.940	100.0	98.5	2.96
	2	2.047	1.964	2.006	1.968			101.5
Positive Control	1	0.473	0.490	0.482	0.445	0.422	21.74	22.9	2.36
	2	0.429	0.443	0.436	0.399			20.6
Test Item	1	2.160	2.056	2.108	2.071	2.064	106.39	106.8	0.74
	2	2.132	2.055	2.094	2.056			106.0
Blank		0.037	0.037	0.037
Negative Control Freeze killed Tissues	1	0.101	0.099	0.100	0.063	0.069	3.54	3.2	0.62
	2	0.113	0.111	0.112	0.075			3.9
Test Item Freeze killed Tissues	1	0.109	0.112	0.111	0.074	0.070	3.59	3.8	0.41
	2	0.105	0.101	0.103	0.066			3.4

* Tissue viability = ((100 x meanOD of T1 & T2 test item) / positive control/negative cpntrol) / mean OD of T1 and T2 neagtive control

** Relative Tissue viability = ((100 x meanOD blank corrected test item) (positive control/ negative control)) / mean OD of T1 and T2 negattive contol

*** Corrected mean viability = Tissue viability of test item - (Tissue viability KC test item - Tissue viability KC negative control)

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, PG-RAW-90-032 does not need to be classified according UN GHS.

Executive summary:

This in vitro study was performed to assess the eye irritation potential of PG-RAW-90-032 by means of the Human Cornea Model Test.

The test item proved to be an MTT reducer in the MTT 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.

The OD of the tissue replicates treated with the negative control was > 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-90-032. 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: 106.35%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, PG-RAW-90-032does notneed to be classified according UN GHS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

In vitro skin corrosion (OECD TG 431)

Mean tissue viability was 118.5% after 3 -min exposure to PG-RAW-90 -032, and 123.9% after 60 -min exposure. PG-RAW-90 -032 is considered to be non-corrosive to the skin.

In vitro skin irritation (OECD TG 439)

Mean tissue viabiity was 58.5% after 15 -min exposure to PG-RAW-90 -032 and 42-Hour post-exposure incubation period. PG-RAW-90 -032 is considered to be non-irritating to the skin.

In vitro eye irritation (OECD TG 492)

When compared with the value of the negative control, after 30 -min exposure to PG-RAW-90 -032, the mean absorption value corresponding to the tissue viability was 106.35%. PG-RAW-90 -032 is considered to be non-irritating to the eye.

Bovine corneal opacity and permeability test (OECD TG 437)

The in Vitro irritancy score of PG-RAW-90-032 was 0.0. It is considered to be of no category, not requiring classification according to UN GHS.

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.