1,8-Naphthalenediol, 1,8-dibenzoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well-documented GLP study, OECD draft guideline validated for distinguishing non-irritants from irritants

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

There are no official national or international guidelines for the EpiOcularTM test yet; however, the study was performed according to the methods described in the following publications:
- MatTek Corporation, Ashland, MA 01721, USA: EpiOcularTM human cell construct: Procedure details, Version 3.1a of February 10, 2010.
- Harbell J.W. et al. (2009): COLIPA Program on Optimization of Existing In Vitro Eye Irritation Assays for Entry into Formal Validation: Technology Transfer and Intra/Inter Laboratory Evaluation of EpiOcular Assay for Chemicals. Poster # 378, Society of Toxicology, March 2009.

In addition the study follows the testing strategy for determination of eye irritation/corrosion as given in the following OECD guideline:
- OECD Guideline for Testing of Chemicals No. 405, October 2, 2012 (“Acute Eye Irritation/Corrosion”)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Test material

Test material information

Constituent 1

Reference substance name:

8-(benzoyloxy)naphthalen-1-yl benzoate

EC Number:

810-797-4

Cas Number:

331711-99-0

Molecular formula:

C24 H16 O4

IUPAC Name:

8-(benzoyloxy)naphthalen-1-yl benzoate

Details on test material:

- Name of test material (as cited in study report): Thane 003-6
- Physical state: solid
- pH: ca. 5 (undiluted test substance, moistened with water)
- Analytical purity: approx. 98.153%
- Test-substance No.: 13/0254-1
- Batch identification: LJ-008-09-136
- Homogeneity: The test substance was homogeneous by visual inspection.
- Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Test animals / tissue source

Species:

other: not applicable (in vitro study)

Strain:

other: not applicable (in vitro study)

Details on test animals or tissues and environmental conditions:

not applicable (in vitro study)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable (in vitro study)

Amount / concentration applied:

bulk volume of 50 μL (using a sharp spoon)

Duration of treatment / exposure:

90 minutes

Observation period (in vivo):

18 hours

Number of animals or in vitro replicates:

not applicable (in vitro study)

Details on study design:

Test system
The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinozytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200).

Direct MTT reduction
To assess the ability of the test material to directly reduce MTT a pretest was performed as described below. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (de-ionized water) was tested concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.

Basic procedure
Two tissues were treated with the test substance, the PC and NC, respectively.
There are two separate protocols for liquids and solids, differing in exposure time and postincubation period. Due to the physical condition of the test substance the protocol for solids was applied.
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at standard culture conditions for 16 – 24 hours (pre-incubation).
After the pre-incubation the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.
Using a sharp spoon, a bulk volume of 50 μL of the test material was applied covering the whole tissue surface. Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC). After application, the tissues were placed into the incubator until the total exposure time of 90 minutes was completed.
To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium (post-soak immersion) in order to remove residual test substance. After 12 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 18 hours (postincubation period).
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

Negative control (NC): De-ionized water, sterile
Positive control (PC): Methyl acetate (98+%, CAS No.: 79-20-9, Merck KGaA, Germany)

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: mean tissue vialitiy

Basis:

other: Thane 003-6

Score:

97

Remarks on result:

other: no eye irritation potential

Results 2

Irritation parameter:

other: mean tissue viability (%)

Basis:

other: negative control

Score:

100

Remarks on result:

other: no eye irritation potential

Results 3

Irritation parameter:

other: mean tissue viability

Basis:

other: positive control: methyl acetate

Score:

17

Remarks on result:

other: eye irritation potential

Other effects:

The substance was not able to reduce MTT directly.

Any other information on results incl. tables

Results of EpiOcular(TM) Eye irriation test:

Test substance		Tissue 1	Tissue 2	Mean	SD
NC	mean OD570	1.576	1.788	1.682
	viability (% of NC)	93.7	106.3	100	12.6
Thane 003 -6	mean OD570	1.580	1.689	1.634
	viability (% of NC)	93.9	100.4	97	6.5
PC	mean OD570	0.248	0.340	0.294
	viability (% of NC)	14.7	20.2	17	5.5

NC, negative control; PC, positive control