Disodium 2-(11-oxido-3-oxo-3H-dibenzo[c,h]xanthen-7-yl)benzoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

30 January - 03 February, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 2-(11-oxido-3-oxo-3H-dibenzo[c,h]xanthen-7-yl)benzoate

EC Number:

228-794-6

EC Name:

Disodium 2-(11-oxido-3-oxo-3H-dibenzo[c,h]xanthen-7-yl)benzoate

Cas Number:

6359-10-0

Molecular formula:

C28H14Na2O5

IUPAC Name:

disodium 2-(11-oxido-3-oxo-3H-dibenzo[c,h]xanthen-7-yl)benzoate

Details on test material:

- Physical appearance: Black powder
- Aggregate State at RT: Solid
- Bacht No./ Lot. No.: 5811022884
- Date of expiry: June 2019

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: As recommended in OECD Guideline for Eye Irritation, EpiOcular™ reconstructed human ocular tissue has been selected as test system for in vitro eye irritation. Supplied by MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, Bratislava - Slovakia
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: See "Any other information on materials and methods"

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

30 min post soak period, 18 h post treatment incubation

Number of animals or in vitro replicates:

2

Details on study design:

- RhCE tissue construct used, including batch number: EpiOcular™ cornea epithelial model (OCL-200-EIT). Lot No. 27021
- Doses of test chemical and control substances used:
Test item: 50 mg
Neg. Control: 50 µL
Pos. Control: 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
Exposure: 6 h at 37 °C
Post-exposure immersion: 30 min at room temperature
Post-exposure incubation: 18 h at 37 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals
Test item developed colour when dissolved in distilled water and isopropanol. Post incubation optical density of samples were measured at 570 nm and the OD readings were >0.08. Test item was considered as possibly interacting with the MTT measurement and an additional test on colorant controls was performed to determine the amount of color bound to and extracted from the tissues. For this purpose the colored test item is applied to two additional tissues, the colorant controls (CC).
The test item was pre checked for direct MTT reduction in quantitative MTT estimation. Post incubation, the test item with MTT formed black color. As there was color change, test item was considered as reducer of MTT.
As test item reduced MTT, a functional check using freeze-killed tissue controls (killed controls = KC) was performed in one definitive assay to evaluate whether the test item is not binding to the tissue and leading to a false MTT reduction signal.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled):
All controls were perfomed in dublicates
- Wavelength of measuring device: 570 nm, 96-well plate reader
- Description of the method used to quantify MTT formazan:
Optical density (OD) measurement of the MTT extracts.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
A test item is labeled as “non-irritant”, if the test item treated percent tissue viability determined by MTT assay is >60 % relative to the negative control treated tissue viability.
A test item is labeled as “irritant”, if the percent tissue viability determined by MTT assay is <60 % relative to the negative control treated tissue viability.

Results and discussion

In vitro

Results

Irritation parameter:

other: cell viability [%]

Value:

-2.75

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

Any other information on results incl. tables

Table 2: Summary of Optical Density (OD) and Viability (%)

Treatment		OD	Viability (%)	Viability difference between tissues	Classification
Negative Control (DBPS)	Mean	1.252	100.00	0.26	NI
	±SD	0.006	0.52
	n	2	2	2
Positive Control (Methyl acetate)	Mean	0.076	6.1	0.14	I
	±SD	0.004	0.28
	n	2	2	2
Test Item	Mean	-	-2.75	-	I
	±SD		-
	n		2
NI = Non Irritant; I = Irritant; n = No. of tissues

Applicant's summary and conclusion

Interpretation of results:

other: UN GHS Category 1 or Category 2

Conclusions:

Based on the results obtained in an in vitro eye irritation assay according to OECD guideline 492, the test item is categorised as irritant (UN GHS Category 1 or Category 2) as the mean percentage tissue viability is less than 60 % of the negative control.

Executive summary:

The eye irritating potential of the test item was determined in an in vitro eye irritation assay (RhCE) according to OECD guideline 492. Two replicates of EpiOcular™ tissues were treated with test item (50 mg) , positive control (methyl acetate, 50 µL) or negative control (sterile deionized water, 50 µL), respectively and incubated for 6 hours (± 15 min) at standard culture conditions (37 °C in an incubator with 5 % CO2, 90±10 % humidified atmosphere).

Exposure of test material was terminated by rinsing with Ca++Mg++Free-DPBS solution. After rinsing, the tissues were incubated for a 30 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). The viability of each tissue was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically (570 nm).

The test item has an intrinsic colour (dark black), therefore, two additional test item treated tissues were used for the non-specific OD evaluation. The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. Since the test item is a possible MTT-reducer and has an intrinsic colour (dark black) a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference.

The test item induced significantly reduced cell viability in comparison to the negative control (mean relative viability: -2.75 %). Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, indicated that the test item has an eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, the test item is considered as irritating to the eye (UN GHS Category 2 or 1). For a reliable assessment, further in vitro testing is necessary for a final classification.