1-(2,4-dimethylcyclohex-3-en-1-yl)propan-1-ol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 07 February 2017 and 17 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1. The study is considered reliable and according to the SkinEthic Reconstructed Human Corneal Epithelial Model and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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)

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

1-(2,4-dimethylcyclohex-3-en-1-yl)propan-1-ol

EC Number:

944-553-4

Cas Number:

1632042-40-0

Molecular formula:

C11H20O

IUPAC Name:

1-(2,4-dimethylcyclohex-3-en-1-yl)propan-1-ol

Test material form:

solid

Remarks:

White

Specific details on test material used for the study:

Identification: FRET 11-0571
Batch: PDJ378-75
Purity: 96.0%
Appearance: White, solid
Expiry Date: 01 June 2018
Storage Conditions: At room temperature
Stability in Solvent: Stable in water (not quantified)

Test animals / tissue source

Species:

other: Reconstructed Human Corneal Epithelial Model

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

Supplier: MatTek Corporation (82105 Bratislava, Slovakia)
Test Syetem Lot No.: 23764
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) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm diam).
EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test Item Preparation
Approximately 50 mg of the test item was tested topically on duplicate EpiOcular™ tissues. The tissue was placed back into the culture medium after dosing and incubated at standard culture conditions (37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air, 95% RH ) for 6 hours.

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2 replicates per treatment (test item, positive and negative controls)

Details on study design:

Pre-Study preparation
Cell Culture
EpiOcular™ kits and MTT-100 kits are 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) are 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 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. 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 were incubated at standard culture conditions overnight (19 hours).

Assessment of Direct MTT Reduction by the Test Item
Test items may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement. Therefore, it was necessary to assess this ability for the test item prior to conducting any assays with viable tissues. For this purpose approximately 50 mg 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 at standard culture conditions for three hours. A control (50 µL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was run concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
Since the MTT solution colour did not turn blue/purple, the test item was not presumed to be a MTT reducer, and an additional test with freeze-killed tissues to determine a correction factor for calculating the true viability was not necessary.

Assessment of Coloured or Staining Materials
Coloured test items or test items which become coloured after application to the tissues may interfere with the quantitative photometric MTT measurement, if the colourant binds to the tissue and is extracted together with MTT. Therefore, each test item has to be checked for its colourant properties.
Since the test item was non-coloured additional tests had to be performed to assess, if it became colorant after contact with water or isopropanol. For this purpose each approximately 50 mg of the test item was 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 for 2 to 3 hours at room temperature.
Since the test item did not become coloured either in water or isopropanol, it was not considered as possibly interacting with the MTT measurement and an additional test with viable tissues (with medium instead of MTT addition) to determine a correction factor for calculating the true viability did not have to be performed.

EXPERIMENTAL DESIGN AND STUDY CONDUCT
Experimental Performance
After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.
Test item exposure: After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control item were tested by applying approximately 50 mg (test item) or 50 µL (controls) topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 6 hours.
At the end of the 6 hours 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. The test item utilized a different set of three beakers. The inserts containing the tissue 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 (paper towel, gauze, etc.) 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 are 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. Decanting was most efficiently performed 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 about 25 minutes immersion incubation (post-soak) at room temperature. This incubation in assay medium was intended to remove any test item or control 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 insert was 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 approximately 18 hours at standard culture conditions (post-treatment incubation). 

MTT Assay
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Inserts were removed from the 24-well plate after 180 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. The plates were sealed with parafilm (between the plate cover and upper edge of the wells) or a standard plate sealer, and were extracted overnight (about 20 hours) at room temperature afterwards. The tissues were not pierced. The corresponding negative, positive, and additional viable tissues (without MTT addition) were treated identically without piercing. For this procedure it was necessary to seal the plates particularly thorough since a higher evaporation rate had to be expected due to the larger surface of wells in 6-well plates.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate(s).
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.

Results and discussion

In vitro

Results

Irritation parameter:

other: mean relative absorbance value, corresponding to the cell viability

Value:

4.2

Negative controls validity:

valid

Remarks:

100.0%

Positive controls validity:

valid

Remarks:

17.8%

Remarks on result:

other: eye irritating potential

Other effects / acceptance of results:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not lead to a change in colour. Therefore, an additional test with viable tissues without MTT addition was not necessary.
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour. Therefore, an additional test with freeze-killed tissues was not necessary.
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 4.2% (threshold for irritancy: ≤ 60%), consequently the test item was irritant to eye.
Concerning acceptance criteria:
• The negative control OD is > 0.8 and < 2.5 (1.697 and 1.709).
• The mean relative viability of the positive control is below 50% of the negative control viability (17.8%).
• The difference of viability between the two relating tissues of a single item is < 20% (values between 0.7% to 1.2%) in the same run (for positive and negative control tissues and tissues of single test items).

Discussion
This in vitro study was performed to assess the eye irritation potential of FRET 11-0571 by means of the Human Cornea Model Test.
Additional tests with viable or freeze-killed tissues were not performed, since the test item was not coloured intensively, did not dye water or isopropanol, and did not prove to be a MTT reducer.
About 50 mg of the test item and each 50 µL of the controls, respectively, were applied to each of duplicate EpiOcular™ tissue for 6 hours.
Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 17.8%, thus the validity of the test system is ensured.
The acceptance criteria were met.
Relevant irritating effects were observed following 6 hours incubation with FRET 11-0571. The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 4.2% compared with the value of the negative control (threshold for irritancy: ≤ 60%).

Any other information on results incl. tables

RESULTS

Results after treatment for 6 hours withFRET 11-0571and the controls

Dose Group	Absorbance Well 1 (Tissue 1/2)	Absorbance Well 2 (Tissue 1/2)	Mean Absorbance (Tissue 1/2)	Mean Absorbance* Tissue 1 and 2 minus Mean Blank	Mean Absorbance of 2 Tissues*	Rel. Absobance [%] Tissue 1 and 2**	Absolute Value of the Difference of the Rel. Absorbances [%] Tissue 1 and 2	Mean Rel. Absorance [% of Negatie Control]**
Blank	0.038	0.038	0.038	0.000
Negative Control	1.742	1.751	1.747	1.709	1.703	100.3	0.7	100.0
	1.767	1.703	1.735	1.697		99.7
Positive Control	0.340	0.363	0.351	0.313	0.303	18.4	1.2	17.8
	0.332	0.331	0.332	0.293		17.2
Test Item	0.102	0.101	0.101	0.063	0.071	3.7	0.9	4.2
	0.117	0.117	0.117	0.078		4.6

The presented values are rounded values.

*         Mean of two replicate wells after blank correction
**       Relative absorbance [rounded values]: (100 x (absorbance_{test item/positive control}) / absorbance_{negative control}

 

Assessment of Eye Irritation Potential – Viability of HCE Tissues

 

Item	OD562of Individual Tissue	Mean OD562	Mean OD562of tissues corrected for MTT direct reduction (-0.191)	Relative Mean Viability (%)
Negative Control	1.x	1x	na	100*
	1.x
	1.x
Positive Control	0.x	0.x	na	xx.x
	0.x
	0.x
Test Item	0.x	0.x	0.x	xx.x
	0.x
	0.x

 

Corrected viability of treated killed tissues

=

0.215 (tkt)-0.024 (ukt) = 0.191

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

na   =   Not applicable

OD₅₆₂    = Optical Density at 562 nm

tkt  =   treated killed tissue

ukt  =   untreated killed tissue 

In view of the viability of being > 60% the substance is not considered to be an irritant.

Applicant's summary and conclusion

Interpretation of results:

other: possesses an eye irritating potential

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, FRET 11-0571 possesses an eye irritating potential.

Executive summary:

This in vitrostudy was performed to assess the eye irritation potential of FRET 11-0571 by means of the Human Cornea Model Test.

The test item did not prove to be an MTT reducer in the MTT pre-test. And it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

Each 50 mg of the test item were applied to each of duplicate tissue for 6 hours. Each 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each.

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

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

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

Irritating effects were observed following incubation with FRET 11-0571. Compared with the value of the negative control, the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (4.2%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, FRET 11-0571 possesses an eye irritating potential.