5H-Cyclopenta[h]quinazoline, 6,6a,7,8,9,9a-hexahydro-7,7,8,9,9-pentamethyl-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

The study was conducted between 27 May 2014 and 29 May 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2015

Materials and methods

Test guideline

Qualifier:

equivalent or similar 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:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

7,7,8,9,9-pentamethyl-5H,6H,6aH,7H,8H,9H,9aH-cyclopenta[h]quinazoline

EC Number:

801-694-5

Cas Number:

1392325-86-8

Molecular formula:

C16H24N2

IUPAC Name:

7,7,8,9,9-pentamethyl-5H,6H,6aH,7H,8H,9H,9aH-cyclopenta[h]quinazoline

Test material form:

other: Solid

Details on test material:

Identification: IFF TM 12-206 (FRET 10-0199)
Description: White solid block
Storage conditions: Approximately 4 °C, in the dark

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

Human Corneal Epithelium (HCE)
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 27 May 2014
HCE Tissues (0.5cm2) batch number: 14-HCE-019
Maintenance Medium lot number: 14-MIMA-019

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test material: 30 mg
Positive control: 30 µL
Negative control: 30 µL

Duration of treatment / exposure:

10 minutes

Number of animals or in vitro replicates:

3 for the test material, positive control and negative control, each.

Details on study design:

Pre-Test – Assessment of Direct Test Item reduction of MTT
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 formazan dye 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 to formazan, thus mimicking dehydrogenase activity of the cellular mitochondria of viable cells. This property of the test item is only a problem, if at the time of the MTT test (after the chemical has been rinsed off) there are still sufficient amounts of the test item on or in the tissues. To identify this possible interference, the test item was checked for its ability to reduce MTT directly.
30 mg of test item was added to 1 mL of a 0.5 mg/mL MTT solution and incubated at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution turned blue, the test item was presumed to have reduced the MTT.
The test item was found to have the ability to directly reduce MTT and therefore the following procedure, employing freeze-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues, was required.
In addition to the normal test procedure, the MTT reducing test item was applied to one freezekilled tissue. One freeze-killed tissue remained untreated. The untreated freeze-killed tissue demonstrates a small amount of MTT reduction due to residual reducing enzymes. Freeze-killed tissues were prepared by placing untreated SkinEthic HCE tissues in a freezer (-14 to -30 ºC) overnight. Once killed, the tissues were stored in the freezer.

Receipt and Preparation of Tissues
On arrival, the SkinEthic HCE tissues (Day 6 cultures), were stored at room temperature prior to transferring into 24-well plates designated ‘arrival plates’ containing 300 μL of maintenance medium. It was important to ensure that there were no air bubbles present under the tissue inserts. The tissues were incubated overnight at 37 °C, 5% CO2 in air.

Main Test
Using sterile techniques, 1 mL of maintenance medium at room temperature, was dispensed into the appropriate number of wells of 6-well plates designated ‘treatment plates’. Each well was labeled with details of the treatment and the appropriate exposure time. Separate treatment plates were used for the test item, negative and positive controls to avoid the possibility of cross contamination occurring. Before treatment, the 7 day old tissues were transferred from the ‘arrival plates’ into the wells of the ‘treatment plates’ containing the maintenance medium.
Triplicate tissues were treated with 30 mg of the test item for 10 minutes. The tissues were dosed at regular timed intervals to allow for the period taken to rinse each insert following exposure and to ensure each tissue received an equal exposure time. Triplicate tissues were treated with 30 μL of solution A to serve as negative controls and triplicate tissues were treated with 30 μL of 2% w/v SDS to serve as positive controls. The plates were incubated at 37 °C, 5% CO2 in air during the exposure time.
At the end of the relevant exposure period, each tissue insert was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco’s Phosphate Buffered Saline (DPBS) without Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labeled 24-well plate designated ‘holding plate’ containing 300 μL of maintenance medium (at room temperature) until all the tissues were rinsed. Following rinsing, the tissues were transferred to a pre-labeled 24-well plate designated ‘MTT Loading plate’ containing 300 μL of a 0.5 mg/mL MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for approximately three hours at 37 °C, 5% CO2 in air.

At the end of the incubation period the inserts were rinsed twice with phosphate buffered saline and blotted on absorbent paper to remove residual MTT solution and transferred to a pre-labeled 24-well plate designated ‘MTT extraction plate’ containing 0.75 mL of isopropanol in each of a sufficient number of wells. An extra 0.75 mL of isopropanol was added onto each tissue and the plate sealed to prevent isopropanol evaporation. The plate was wrapped in aluminum foil (to protect from light) and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue.
At the end of the extraction period, each tissue insert was pierced with a pipette fitted with a 1000 μL tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded. For each tissue triplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate.
200 μL of isopropanol alone was added to three wells designated as ‘blanks’. The optical density was measured (quantitative measurement of tissue viability) at 562nm (OD562) using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Results

Irritation parameter:

other: Relative Mean Viability (%)

Run / experiment:

10 minute exposure

Value:

100.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Assessment of Direct Test Item Reduction of MTT
The MTT solution containing the test item turned purple which indicated that the test item directly reduced MTT and therefore the MTT viability assay was performed in parallel on viable and freeze-killed tissues. However, the results obtained showed a negligible degree of interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes.

Assessment of Eye Irritation Potential
The relative mean viability of the test item treated tissues after a 10-Minute exposure period was 100.7%.

Assay Acceptance Criterion
The relative mean tissue viability for the positive control treated tissues was 16.1% relative to the negative control treated tissues. The quality criterion required for the acceptance of results in the test was satisfied.

Any other information on results incl. tables

Assessment of eye irritation potential - viability of HCE tissues

Item	OD562of individual tissue	Mean OD562	Relative mean viability (%)
Negative control	0.964	1.021	100*
	1.057
	1.041
Positive control	0.163	0.164	16.1
	0.132
	0.198
Test item	1.080	1.028	100.7
	0.976
	1.027

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

Applicant's summary and conclusion

Interpretation of results:

other: Not eye irritating

Remarks:

in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

The substance is not an eye irritant in the eye irritation test using the SkinEthic reconstructed Human Corneal Epithelium model (OECD TG 492).

Executive summary:

The substance was tested for eye irritation using the SkinEthic reconstructed Human Corneal Epithelium model (OECD TG 492). The tissues were exposed to 30 mg of the substance for 10 minutes in triplicate. Phosphate buffered saline and sodium dodecyl saline were used as negative and positive control, respectively, and tested in triplicate to confirm the validity of the test. The substance directly reduced MTT and therefore the viability assay was performed in parallel on viable and freeze-killed tissues. However, the results obtained showed a negligible degree of MTT interference and therefore it was considered not necessary to correct the results. The relative mean viability of the test substance treated tissues was 100.7%. Based on this result, the substance is not eye irritating.