Reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane

Endpoint summary

Description of key information

Skin Corrosion

In vitro, OECD 431, 2017 - The skin corrosivity potential of reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 431 and GLP.

Duplicate tissues were treated with the test item for exposure periods of 30 and 60 mins. At the end of the exposure period the test item was rinsed from each tissue before being loaded with MTT. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. After extraction, each tissue was pierced and the extraction solution aliquoted for absorbance measurements. Absorbency at 562 nm of each well was measured using a Anthos 2001 microplate reader.

Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 107.6 and 72.9 %, respectively. The quality criteria required for acceptance of the resuts was met.

Under the conditions of this study the test substance is not considered to be corrosive to the skin.

Skin Irritation

In vitro, OECD 439, 2017 - The skin irritation potential of reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane was assessed using an EpiSkin Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 439 and GLP.

Triplicate tissues were exposed to the test item for 15 minutes. At the end of the exposure period the test item was rinsed and the tissues incubated for a further 42 h in the presence of maintenance solution which would be used for possible inflammatory mediator determination. Each tissue was then loaded with MTT. After incubation and extraction, the solutions were aliquoted for absorbance measurements. Absorbency at 562 nm of each well was measured using a Anthos 2001 microplate reader.

Mean viability of tissues exposed to the test substance after 15 minutes were 86.2 %. It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal. The quality criteria required for acceptance of the resuts was met.

Under the conditions of this study the test substance is not considered to be irritant to the skin.

Eye Irritation (OECD 438)

In vitro/ ex vivo, OECD 438, 2017 - The Isolated Chicken Eye (ICE) test was conducted using reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane in accordance with OECD Guideline 438 "Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage" (2013).

Undiluted test item was applied evenly to the surface of three corneas before being washed off with isotonic saline solution after 10 second test item contact time. A negative and positive control group containing 2 and 3 corneas, respectively, were also prepared.

Observations for corneal opacity and corneal thickness were made at 0, 30, 75, 120, 180 and 240 minutes (after rinsing). Fluorescein retention was measured 30 minutes after rinsing.

Corneal opacity, fluorescein retention and corneal thickness were each classified as ICE Class II for the test item group, concluding that no prediction for eye irritation could be madefollowing assessment of the data for all endpoints.

Eye Irritation (OECD 492)

In vitro, OECD 492, 2017 - The eye irritation potential of Reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane was assessed by means of the Human Cornea Model Test in accordance with OECD Guideline 492 (2015) "Reconstructed human Cornea-Like Epithelium (RhCE) test method for identifying chemicals not requiring classificationand labelling for eye irritation or serious eye damage"

Additional tests with viable or freeze-killed tissues were not performed, since the colourless test item did not dye water or isopropanol, and did not prove to be a MTT reducer.

Tissues of the human cornea model EpiOcular^™were treated with the test item, the positive and the negative control for 30 minutes each in duplicate.

Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 17.9%, thus the validity of the test system is ensured.

Relevant irritating effects were observed following 30 minutes incubation with Reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane. The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 56.6% compared with the value of the negative control (threshold for irritancy: ≤ 60%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, Reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane possesses an 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:

21 - 23 September 2016

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:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark under nitrogen
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS: N/A

Test system:

human skin model

Source species:

other: MatTek EpiDerm Reconstructed Epidermis Model Kit (EpiDerm tissues Lot no.: 23356 and Assay medium Lot no.: 091516SLC)

Cell type:

other: epithelial

Cell source:

other: MatTek EpiDerm Reconstructed Epidermis Model Kit (EpiDerm tissues Lot no.: 23356 and Assay medium Lot no.: 091516SLC)

Source strain:

not specified

Details on animal used as source of test system:

MatTek EpiDerm Reconstructed Epidermis Model Kit
Date recieved: 20 Sep 2016
EpiDermTM Tissues (0.63cm2) lot number: 23356
Assay Medium lot number: 091516SLC
Stored refrigerated until use.

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: Tissues were incubated for 1 hour at 37 °C, 5% CO2 in 0.9 mL of pre-warmed assay medium.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 60 minute samples were incubated at 37 °C, 5% CO2 during the exposure phase. 3 min samples were not incubated (as the exposure period was too short).
- Temperature of post-treatment incubation (if applicable): Tissues were incubated at 37 °C, 5% CO2 for 3 hours post test item rinsing in the presence of MTT.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Trays were filled and emptied under a constant soft stream of DPBS until test item was removed.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: N/A
- Filter bandwidth: N/A

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
6 tissues were prepared for each of the 3 minute and 60 minutes exposure groups (2 x test item, 2 x negative control and 2 x positive control samples prepared per testing group).

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60‑Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean aborbency of test item / mean absorbency of negative control) x 100
Classification of corrosivity potential is based on relative viabilities for both exposure times according to Table 1.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): N/A

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): N/A

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): N/A

Duration of treatment / exposure:

Tissues exposed to the test item, positive control or negative control for 3 minutes or 60 minutes.

Duration of post-treatment incubation (if applicable):

Tissues incubated for 3 hours post-rinsing.

Number of replicates:

Duplicate replicates for each test group.

Species:

other: N/A

Strain:

other: N/A

Type of coverage:

other: N/A

Preparation of test site:

other: N/A

Vehicle:

other: N/A

Amount / concentration applied:

N/A

Duration of treatment / exposure:

N/A

Observation period:

N/A

Number of animals:

N/A

Details on study design:

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 DPBS 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.

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 562nm (OD562) of each well was measured using the Anthos 2001 microplate reader

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure period

Value:

107.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 % viability

Positive controls validity:

valid

Remarks:

4.0 % viability

Remarks on result:

other: no indication of corrosivity

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure period

Value:

72.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 % viability

Positive controls validity:

valid

Remarks:

3.4 % viability

Remarks on result:

other: no indication of corrosivity

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No.

- Direct-MTT reduction: 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.

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

- Colour interference with MTT: 50 µL of test item was added to 300 µL of sterile water. The solution was incubated in the dark at 37 oC, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.

The solution containing the test item did not become coloured. This was taken to indicate the test item did not have the potential to cause color interference.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 2.060 for the 3‑Minute exposure period and 2.017 for the 60‑Minute exposure period. The negative control acceptance criteria were therefore satisfied.

- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 3.4% relative to the negative control following the 60‑Minute exposure period. The positive control acceptance criterion was therefore satisfied.

- Acceptance criteria met for variability between replicate measurements: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.

- Range of historical values if different from the ones specified in the test guideline: N/A

Irritant / corrosive response data:

Details of cell viability after 3 and 60 minutes exposures are described in Table 2.

Table 2: Mean OD₅₆₂Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue	Exposure Period	MeanOD562of individual tissues	Mean OD562of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	2.017	2.060	0.061	3.0	100*
		2.103
	60 Minutes	2.082	2.017	0.093	4.6
		1.951
Positive Control	3 Minutes	0.091	0.082	0.013	na	4.0
		0.073
	60 Minutes	0.075	0.069	0.009	na	3.4
		0.062
Test Item	3 Minutes	2.235	2.217	0.025	1.1	107.6
		2.199
	60 Minutes	1.373	1.471	0.139	9.4	72.9
		1.569

OD = Optical density

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

na= Not applicable

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study the test substance is not considered to be corrosive in the in vitro skin corrosion test using the EpiDerm Skin Model.

Executive summary:

The skin corrosivity potential of reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 431 and GLP.

Duplicate tissues were treated with the test item for exposure periods of 30 and 60 mins. At the end of the exposure period the test item was rinsed from each tissue before being loaded with MTT. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. After extraction, each tissue was pierced and the extraction solution aliquoted for absorbance measurements. Absorbency at 562 nm of each well was measured using a Anthos 2001 microplate reader.

Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 107.6 and 72.9 %, respectively. The quality criteria required for acceptance of the resuts was met.

Under the conditions of this study the test substance is not considered to be 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:

05 October - 10 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark under nitrogen
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS: N/A

Test system:

human skin model

Source species:

other: EPISKIN™ Reconstructed Human Epidermis Model Kit Supplier: SkinEthic Laboratories, Lyon, France

Cell type:

other: Epitheleal

Details on animal used as source of test system:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 04 October 2016
EpiSkinTM Tissues (0.38cm2) lot number: 16-EKIN-040
Maintenance Medium lot number: 16-MAIN3-067
Assay Medium lot number: 16-ESSC-043

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert. 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.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37 °C, 5% CO2 in air for 42 hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 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.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT; A 3 mg/mL stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: N/A
- Filter bandwidth: N/A

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement): Classification of irritation potential is based upon relative mean tissue viability following the 15‑Minute exposure period followed by the 42‑Hour post‑exposure incubation period according to Table 1.

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): N/A

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): N/A

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): N/A

Duration of treatment / exposure:

Tissues were treated with the test item for an exposure period of 15 minutes.

Duration of post-treatment incubation (if applicable):

Rinsed tissues were incubated for 42 hours.

Number of replicates:

3 per test group.

Details on test animals or test system and environmental conditions:

N/A

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

N/A

Duration of treatment / exposure:

N/A

Observation period:

N/A

Number of animals:

N/A

Details on study design:

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/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.

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.

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 was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Replicate 1

Value:

84

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Replicate 2

Value:

87.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Replicate 3

Value:

86.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No. Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert.

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

- Colour interference with MTT: 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 colour was made. The solution containing the test item was colourless. It was therefore unnecessary to run colour correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 0.970 and the standard deviation value of the viability was 2.6%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 8.2% relative to the negative control treated tissues and the standard deviation value of the viability was 1.6%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 1.9%. The test item acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: N/A

Irritant / corrosive response data:

Details of cell viability after 15 minutes of test substance exposure are described in Table 2.

Table 2: Mean OD₅₆₂Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562of tissues	Mean OD562of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.956	0.970	0.025	98.6	100*	2.6
	0.999			103.0
	0.954			98.4
Positive Control Item	0.097	0.080	0.015	10.0	8.2	1.6
	0.071			7.3
	0.071			7.3
Test Item	0.815	0.836	0.019	84.0	86.2	1.9
	0.852			87.8
	0.840			86.6

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:

Under the conditions of this study the test item is considered to be non-irritating to skin.

Executive summary:

The skin irritation potential of reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane was assessed using an EpiSkin Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 439 and GLP.

Triplicate tissues were exposed to the test item for 15 minutes. At the end of the exposure period the test item was rinsed and the tissues incubated for a further 42 h in the presence of maintenance solution which would be used for possible inflammatory mediator determination. Each tissue was then loaded with MTT. After incubation and extraction, the solutions were aliquoted for absorbance measurements. Absorbency at 562 nm of each well was measured using a Anthos 2001 microplate reader.

Mean viability of tissues exposed to the test substance after 15 minutes were 86.2 %. It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal. The quality criteria required for acceptance of the resuts was met.

Under the conditions of this study the test substance is not considered to be irritant to the skin.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark under nitrogen
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS: N/A

Species:

chicken

Strain:

other: Gallus Gallus e.g. Ross 308 Broiler

Remarks:

Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Baileys Turkeys Ltd., Cheshire, UK
- Age at study initiation: approximately 56 days pre kill.
- Weight at study initiation: 3 kg pre kill.

Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 mL
- Concentration (if solution): N/A

Duration of treatment / exposure:

See "details on study design"

Observation period (in vivo):

See "details on study design"

Duration of post- treatment incubation (in vitro):

See "details on study design"

Number of animals or in vitro replicates:

The test item and positive control item groups consisted of three eyes and the negative control item consisted of two eyes.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES

Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.

Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are < 0.5.

Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt-tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner.

Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32 ±1.5 °C.

Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit-lamp microscope at the center of each cornea.

Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.

EQUILIBRATION AND BASELINE RECORDINGS

After the approval process the eyes were incubated for 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES

The test item and positive control item groups consisted of three eyes and the negative control item consisted of two eyes.

NEGATIVE CONTROL USED

Sodium chloride solution (0.9 % v/v)

SOLVENT CONTROL USED (if applicable)

Not applicable

POSITIVE CONTROL USED

Benzalkonium chloride solution (5 % v/v)

APPLICATION DOSE AND EXPOSURE TIME

0.03 mL of test item was applied to the cornea. The entire surface of the cornea was evenly covered. The test item remained in place for 10 seconds.

OBSERVATION PERIOD

Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL of isotonic saline
- Indicate any deviation from test procedure in the Guideline: N/A

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item.
- Damage to epithelium based on fluorescein retention: The mean fluorescein retention scores for all test eyes are calculated at the 30 minute time interval only. These measurements are used for the overall classification for each test item using a Haag-Streit BQ 900 (Switzerland) microscope.
- Swelling: Percentage corneal swelling was assessed from corneal thickness measurements. Mean percentage of corneal swelling for all test eyes was calculated for all the time points. The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit-lamp microscope at the center of each cornea.
- Macroscopic morphological damage to the surface: Pitting, sloughing, roughening of the corneal surface, and adhering of test item are all morphological effects that can be noted on the cornea. The classification of these findings was subject to interpretation.
- Others (e.g, histopathology): N/A

SCORING SYSTEM:
- Mean corneal swelling (%): 0 - 5, >5 - 12, >12 - 18 (>75 mins after test item application), >12 - 18 (<75 mins after test item application), >18 - 26, >26 - 32 (>75 mins after test item application), >26 - 32 (<75 mins after test item application), >32
- Mean maximum opacity score: 0 (no opacity), 0.5 (very feint opacity), 1 (scattered or diffuse areas; details of the iris are clearly visible), 2 (easily discernible translucent area; details of the iris are slightly obscured), 3 (severe corneal opacity; no specific details of the iris are visible; size of the pupil barely discernible), 4 (complete corneal opacity; iris invisible)
- Mean fluorescein retention score at 30 minutes post-treatment: 0 (no fluorescein retention), 0.5 (very minor single cell staining), 1 (single cell staining scattered throughout the treated area of the cornea), 2 (focal or confluent dense single cell staining), 3 (confluent large areas of the cornea retaining fluorescein)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

Irritation parameter:

cornea opacity score

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Remarks:

Scattered or diffuse areas; details of the iris clearly visible was noted in all test item treated eyes. Severe corneal opacity/Complete corneal opacity was noted in the positive control treated eyes. Very faint opacity was noted in the negative control treated eyes. No morphological effects were noted in the test item or control item treated eyes. Sloughing was noted in one of the positive control treated eyes.

Irritation parameter:

fluorescein retention score

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Remarks:

Very minor single cell staining/Single cell staining scattered throughout the treated area of the cornea. Focal or confluent dense single cell staining/Confluent large areas of the cornea retaining fluorescein were noted in the positive control treated eyes. Very minor single cell staining/No fluorescein retention was noted in the negative control treated eyes.

Irritation parameter:

percent corneal swelling

Value:

9.43

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Table 1: Individual Scores and Mean Scores for Corneal Efects - Test Item

End Point	Eye Number	Time (after decontamination) (mins)
		0	30	75	120	180	240
Corneal Opacity	3A	0.0	0.5	1.0	1.0	1.0	1.0
	6A	0.0	0.5	1.0	1.0	1.0	1.0
	8A	0.0	0.5	1.0	1.0	1.0	1.0
Mean		0.0	0.5	1.0	1.0	1.0	1.0
ICE Class		II
Fluoroscein Retention	3A		1.0
	6A		0.5
	8A		1.0
Mean			0.8
ICE Class		II
Corneal Thickness	3A	0.72	0.70	0.74	0.76	0.74	0.74
	6A	0.70	0.76	0.76	0.72	0.74	0.80
	8A	0.70	0.70	0.76	0.77	0.72	0.78
Mean		0.71	0.72	0.75	0.75	0.73	0.77
Mean % Corneal Swelling			1.89	6.60	6.13	3.77	9.43
ICE Class		II
ICE Classes Combined		3 x II
Classification		No prediction for eye irritation can be made.

Mean scores for corneal effects in the positive control group were; corneal opacity (Class IV), fluorescein retention (Class IV), corneal thickness (Class IV). 3 x IV Classes = Category 1 classification.

Mean scores for corneal effects in the negative control group were; corneal opacity (Class I), fluorescein retention (Class I), corneal thickness (Class I). 3 x I Classes = No category classification.

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction for eye irritation can be made following assessment of the data for all endpoints.

Executive summary:

The Isolated Chicken Eye (ICE) test was conducted using reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane in accordance with OECD Guideline 438 "Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage" (2013).

Undiluted test item was applied evenly to the surface of three corneas before being washed off with isotonic saline solution after 10 second test item contact time. A negative and positive control group containing 2 and 3 corneas, respectively, were also prepared.

Observations for corneal opacity and corneal thickness were made at 0, 30, 75, 120, 180 and 240 minutes (after rinsing). Fluorescein retention was measured 30 minutes after rinsing.

Corneal opacity, fluorescein retention and corneal thickness were each classified as ICE Class II for the test item group, concluding that no prediction for eye irritation could be made following assessment of the data for all endpoints.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 November - 01 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducte in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

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:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark under nitrogen
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS: N/A

Species:

human

Details on test animals or tissues and environmental conditions:

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 shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt (29 November 2016) 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 of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (about 18 hours).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL applied topically
- Concentration (if solution): N/A (test item applied unchanged)

Duration of treatment / exposure:

30 minutes

Observation period (in vivo):

N/A

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

2 tissue replicates per group

Details on study design:

- Details of the test procedure used

Pre Test Assessments

The test items ability to directly reduce MTT was assessed. 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. The result indicated that the test item did not have MTT reducing effects. An additional test with freeze-killed tissues did not have to be performed.

The photometric properties of the test item after contact with water and isopropanol were assesed. For this purpose each 50 µL of the test item were added to 1.0 ml of water and to 2 ml isopropanol in a glass tube. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for at least one hour, the isopropanol mixture or for 2 to 3 hours at room temperature. The result indicated that the test item did not dye the water or isopropanol and would therefore not interfere with the photometric assessment of MTT during the main test.

Main Test

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, 95% RH) 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 beakers 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 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 glass tube containing 1 ml of warm Assay Medium. The tissues are incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

After post-treatment incubation of 120 minutes the MTT assay was performed.

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 are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was 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 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark and then shaken for 2-3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
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, version 4.7.1). No reference wavelength measurement was used.

- RhCE tissue construct used, including batch number: EpiOcular kit (Lot: 23574). Supplied by MatTek Coorp., Slovakia.

- Doses of test chemical and control substances used: 50 µL applied topically.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Tissues exposed for 30 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH. Tissues were immersed for 2 seconds during the rinsing process. Post exposue incubation was 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2.

- Justification for the use of a different negative control than ultrapure H2O (if applicable): N/A

- Justification for the use of a different positive control than neat methyl acetate (if applicable): N/A

- Description of any modifications to the test procedure: N/A

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): N/A

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 tissues per test group (test item, positive and negative control). 1 blank tissue prepared with no treatment.

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm

- Description of the method used to quantify MTT formazan: See above.

- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): N/A

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The percent viability of each of the two relating tissues for each control and test item relative to the negative control (100% control) were calculated. The difference of the viability between duplicate tissues was calculated. If the difference is >20% the test is considered as non-qualified. The mean test item viability (TI viability) was calculated and the test item was classified according to the prediction model. If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labelled non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Yes

- Complete supporting information for the specific RhCE tissue construct used: Yes

- Reference to historical data of the RhCE tissue construct: Yes

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Yes

- Positive and negative control means and acceptance ranges based on historical data: Yes

- Acceptable variability between tissue replicates for positive and negative controls: Yes

- Acceptable variability between tissue replicates for the test chemical: Yes

Irritation parameter:

other: Spectrophotometric adsorbance assessment of MTT.

Value:

56.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 56.6% (threshold for irritancy: ≤ 60%), consequently the test item was irritant to eye.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: N/A

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD is > 0.8 and < 2.5 (values between 1.617 and 1.690).

- Acceptance criteria met for positive control: The mean relative viability of the positive control is below 50% of the negative control viability (17.9%).

The difference of viability between the two relating tissues of a single item is < 20% (values between 0.8% and 2.3%) in the same run (for positive and negative control tissues and tissues of single test items)

- Range of historical values if different from the ones specified in the test guideline: N/A

Table 1       Results after treatment for 30 minutes withReaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexaneand the controls

Dose Group	Ab-sorbance Well 1 (Tissue 1/2)	Ab-sorbance Well 2 (Tissue 1/2)	Mean Absor-bance (Tissue 1/2)	Mean Absorbance* Tissue 1 and 2	Mean Absorbance of 2 Tissues*	Rel. Absorbance [%] Tissue 1 and 2**	Absolute Value of the Difference of the Rel. Absorbances [%] Tissue 1 and 2	Mean Rel. Absorbance [% of Negative Control]**
Blank	0.039	0.038	0.038	0.000
Negative Control	1.617	1.626	1.622	1.583	1.602	98.9	2.3	100.0
	1.690	1.626	1.658	1.620		101.1
Positive Control	0.339	0.328	0.334	0.295	0.287	18.4	1.0	17.9
	0.318	0.316	0.317	0.279		17.4
Test Item	0.936	0.942	0.939	0.901	0.907	56.2	0.8	56.6
	0.953	0.951	0.952	0.913		57.0

*         Mean of two replicate wells after blank correction
**        Relative absorbance [rounded values]

Interpretation of results:

study cannot be used for classification

Remarks:

The EpiOcularTM EIT is not intended to differentiate between UN GHS Category 1 (serious eye damage) and UN GHS Category 2 (eye irritation). This differentiation will need to be addressed in a weight-of-evidence for this endpoint.

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, Reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane possesses an eye irritating potential.

Executive summary:

This in vitro study was performed to assess the eye irritation potential of Reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane by means of the Human Cornea Model Test.

Additional tests with viable or freeze-killed tissues were not performed, since the colourless test item did not dye water or isopropanol, and did not prove to be a MTT reducer.

Tissues of the human cornea model EpiOcular^™were treated with the test item, the positive and the negative control for 30 minutes each in duplicate.

Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 17.9%, thus the validity of the test system is ensured.

Relevant irritating effects were observed following 30 minutes incubation with Reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane. The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 56.6% compared with the value of the negative control (threshold for irritancy: ≤ 60%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, Reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane possesses an eye irritating potential.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin Corrosion/Irritation

OECD 431 EPIDERM: Non-corrosive

Following 3 minute and 30 minute exposures, percentage viabilities of tissues were 107.6 and 72.9 % respectively.  All relevant validity criteria were met for this study and it was concluded that the substance is not corrosive to the skin.

OECD 439 EPISKIN: Non-irritant

The relative mean viability of the test item treated tissues was 86.2% after the 15 Minute exposure period and 42 Hours post exposure incubation period.  All relevant validity criteria were met for this study and it was concluded that the substance is not irritating to the skin.

The substance does not meet the GHS or CLP criteria for skin corrosion/irritation.

Eye Damage

OECD 438 ICE: Inconclusive

The study was incconclusive "no prediction could be made".  Mean COP scores ranged from 0.2 - 1.3 at 0 - 240 minutes exposure time respectively (NC = 0.3 - 0.5, PC = 0.2 - 3.7).

The result from the ICE study demonstrate some corneal effect – very faint opacity with scattered or diffused areas although the iris is clearly visible, the faint opacity was also noted in the negative control.  Based on historical data both positive and negative control data are within the acceptable criteria affirming the suitability of the test system.  No prediction could be made based on the results i.e. ICE Class II – the maximum mean score for both corneal swelling (9.43%) and opacity (score of 1) are well within the >5 to 12 % and 0.6-1.5, respectively.

OECD 492 EpiOcular: Positive (GHS 2A, CLP 2)

The test item did dye water or isopropanol (colour interference pre-test) and did not prove to be a MTT reducer (MTT interference pre-test). Therefore, addidtional tests with freeze-killed tissues or viable tissues (without MTT reduction) did not have to be performed.

In the main test, the 30 minutes exposure of the tissues to the test item caused a decrease of the viability value to 56.6% compared with the negative control (=100%). Since this value is below the threshold for irritancy (≤ 60%), the test item has to be classified as eye irritant.

Data from the ICE and EpiOcular studies are not able to sub-classify and we can only state GHS Eye Irritation Category 2A and/or CLP Regulation (EC) 1272/2008 category 2 based on a positive EpiOcular (OECD 492) and an inconclusive (but not GHS ECI category 1 from ICE (OECD 438)).