1,8-Naphthalenediol, 1,8-dibenzoate

Endpoint summary

Description of key information

Epiderm, in vitro skin irritation test (BASF, 2014): not irritating (mean tissue viability >100%)
BCOP, in vitro eye corrosion test (BASF, 2014): not corrosive, not severely irritating (in vitro irritation score 6.7)
EpiOcular, in vitro eye irritation test (BASF, 2014): not irritating (mean tissue viability 97%)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin 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: Guideline and GLP study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Species:

other: not applicable (in vitro test)

Strain:

other: not applicable (in vitro test)

Type of coverage:

other: not applicable (in vitro test)

Preparation of test site:

other: not applicable (in vitro test)

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable (in vitro test)

Amount / concentration applied:

see "Any other information on materials and methods incl. tables"

Duration of treatment / exposure:

see "Any other information on materials and methods incl. tables"

Observation period:

see "Any other information on materials and methods incl. tables"

Number of animals:

not applicable (in vitro test)

Details on study design:

TEST SYSTEM:
Three dimensional human epidermis model
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.

Irritation / corrosion parameter:

other: other: mean tissue viability (%)

Value:

111

Remarks on result:

other:

Remarks:

Basis: other: Thane 003-6. Remarks: no potential for skin irritation. (migrated information)

Irritation / corrosion parameter:

other: other: mean tissue viability (%)

Value:

100

Remarks on result:

other:

Remarks:

Basis: other: negative control. Remarks: no potential for skin irritation. (migrated information)

Irritation / corrosion parameter:

other: other: mean tissue viability

Value:

3

Remarks on result:

other:

Remarks:

Basis: other: positive control: 5% SDS. Remarks: skin irritation potential. (migrated information)

Irritant / corrosive response data:

The substance is not able to reduce MTT directly. The mean viability of the test substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 111%.

Results from EpiDerm(TM) Skin irritation test:

Test substance		Tissue 1	Tissue 2	Tissue 3	Mean	SD
NC	mean OD570	2.340	2.311	2.223	2.291
	viability (% of NC)	102.1	100.9	97.0	100	2.66
Thane 003 -6	mean OD570	2.500	2.476	2.622	2.533
	viability (% of NC)	109.1	108.1	114.5	111	3.42
PC	mean OD570	0.075	0.066	0.0836	0.075
	viability (% of NC)	3.3	2.9	3.6	3	0.37

NC, negative control; PC, positive control

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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.

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)

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)

Irritation parameter:

other: mean tissue vialitiy

Basis:

other: Thane 003-6

Score:

97

Remarks on result:

other: no eye irritation potential

Irritation parameter:

other: mean tissue viability (%)

Basis:

other: negative control

Score:

100

Remarks on result:

other: no eye irritation potential

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.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation was assessed with an in vitro skin irritation test (EpiDerm), according to OECD TG 439. Testing of MTT reduction by the substance itself showed that it was not able to reduce MTT directly. The substance was applied for 1 hour onto the tissue, then rinsed off and tissue viability was determined after 42 h incubation. Mean tissue viability of 1,8 -Naphthalenediol, 1,8 -dibenzoate (Thane 003-6) treated tissues was 111%, for a chemical to be considered "irritant", tissue viability needs to be equal to or less than 50%. Therefore, Thane 003-6 is considered not irritant to the skin.

Eye irritation and corrosion was addressed by two in vitro test methods. A "Bovine Corneal Opacity and Permeability" (BCOP) Test (according to OECD TG 437) was used to assess corrosion and severe irritation potential of Thane 003-6. A 20% aqueous solution of the test substance was applied to bovine corneas for 4 hours, after which corneal opacity and permeability were assessed, creating an "in vitro irritation score" (IVIS). The IVIS for Thane 003-6 was 6.7, the negative control was at 5.0, and the positive control at 103.9. According to the evaluation criteria, Thane 003-6 was determined to be not corrosive or severely irritant. However, since the IVIS score was >3 and <55, no prediction on mild or moderate eye irritation potential could be made, further tests were required.

Therefore, an EpiOcular^TM in vitro eye irriation test was performed. The EpiOcular^TM model is a 3D non-keratinized tissue construct composed of normal human-derived keratinocytes used to model human corneal epithelium. The testing was conducted. The tissues were incubated with the test substance for 90 minutes, the substance was then washed off. After an 18-hour post-incubation period, the mean tissue viability was determined using MTT. The mean tissue viablity for Thane 003-6 was found to be 97%, while the negative control was 100% and the positive control (methyl acetate) was 17%. A chemical is considered as "irritant", if the mean tissue viability after incubation with test material is less than or equal to 50%. Therefore, Thane 003-6 did not show an eye irritation potential under the test conditions chosen.

Justification for selection of skin irritation / corrosion endpoint:
guideline and GLP study

Justification for selection of eye irritation endpoint:
GLP study

Justification for classification or non-classification

Based on the available data, 1,8-Naphthalenediol, 1,8-dibenzoate (Thane 003-6) does not need to be classified for skin or eye irritation according to Regulation (EC) No 1272/2008 (CLP/GHS).