1-naphthalenesulfonic acid, 5-[(4-hydroxyphenyl)amino]-8-(phenylamino)-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 February- 02 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

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:

2015 July 28

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) SOP; For the prediction of acute ocular irritation of chemicals. For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model (10 February 2017).

Version / remarks:

2017 February 10

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-naphthalenesulfonic acid, 5-[(4-hydroxyphenyl)amino]-8-(phenylamino)-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives

EC Number:

235-521-4

EC Name:

1-naphthalenesulfonic acid, 5-[(4-hydroxyphenyl)amino]-8-(phenylamino)-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives

Cas Number:

12262-32-7

Molecular formula:

not applicable

IUPAC Name:

Reaction product of 1-naphthalenesulfonic acid, 5-[(4-hydroxyphenyl)amino]-8-(phenylamino)- with sodium polysulfide, leuco derivatives

Test material form:

solid

Specific details on test material used for the study:

Expiration date: 26 March 2020
Storage: Room temperature (15-25°C)

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose. The EpiOcular™ EIT is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS No Category) without further testing, however, a drawback of this test is the inability to distinguish between Category 1 (corrosive to eye) and Category 2 (eye irritant). Thus, in case of a positive result further testing is required.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcular™ (OCL-200-EIT) kits are manufactured according to defined quality assurance procedures. All biological components of the EpiOcular™ tissue and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with Triton X-100 (100 μL of 0.3% (v/v) Triton X-100).

Details on environment
The EpiOcular™ human cell construct (MatTek Corporation) is used in this assay. This is a three-dimensional human cornea model.
Supplier: MatTek In Vitro Life Science Laboratories
Mlynské Nivy 73, 821 05 Bratislava, Slovakia
Lot No.: 23779
Expiry date: 12 May 2017

The EpiOcular™ (OCL-200-EIT) units were stored in refrigerator (2-8°C) until the preparation of tissues for treatment is started. The assay media supplied with the kits were stored at refrigerator (2-8°C).

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 hours (± 15 min) at standard culture conditions (37 °C in an incubator with 5 % CO2, 90±10% humidified atmosphere).

Duration of post- treatment incubation (in vitro):

18 hours ± 15 minutes at standard culture conditions.

Number of animals or in vitro replicates:

In this assay 2 replicates per test item and 2 replicates negative controls, 2 replicates positive controls, 2 replicates colour controls and 2 replicates non-specific colour control were used. Furthermore, 2 killed treated tissues and 2 killed negative control tissues are used for the MTT evaluation.

Details on study design:

- Details of the test procedure used :
The tissues were equilibrated to room temperature for about 15 minutes, while the Assay Medium was pre-warmed to 37±1°C. The appropriate number of an assay plate wells (6-well plates) was filled with the pre-warmed medium (1 mL per well). The insert was transferred aseptically into the 6-well plates and pre-incubated at 37°C in an incubator with 5 ± 1% CO2, 90 ± 10% humidified atmosphere for one hour in the Assay Medium, than the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37°C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (16 - 24 hours). During the pre-treatement the tissues were pre-wetted with approximately 20 μL of Ca++Mg++Free-DPBS. If the Ca++Mg++Free-DPBS did not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at standard culture conditions for 30 ± 2 minutes. Approximately 50 mg test item was applied (each test item treated insert) topically onto the EpiOcular™ tissues. The insert was gently shaken from side to side to ensure that tissue was completely covered by the test item. After this procedure the tissue was then returned to its medium.
After rinsing, the tissues were transferred to and immersed in 5 mL RT Assay Medium in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation (Post-Soak) at RT; after it, the medium was decanted off the tissue, and the insert was blotted on absorbent material, and afterwards transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of warm (37°C) Assay Medium. The tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation).
MTT Test
After the post-incubation the EpiOcular™ units were transferred into the MTT ready to use solution filled 24-well plate (300 µL of 1 mg/mL MTT per well) and then incubated for 3 hours (± 10 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, 90 ± 10% humidified atmosphere. Inserts were removed from the 24-well plate after 3 hours ± 10 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol was flowing into the insert. The plate was sealed with parafilm. To extract the MTT, the plates were placed on an orbital plate shaker and shaken (150 rpm) for approximately 2 hours at room temperature. At the end of the extraction period, the tissue was not pierced. The corresponding negative, positive, and colorant controls were treated identically.

- RhCE tissue construct used, including batch number :
EpiOcular™ (OCL-200-EIT), MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 821 05 Bratislava, Slovakia, Lot No 23779
- Doses of test chemical and control substances used
Test item: 50 mg
Control substances: 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
Exposure: 6 h
Post-exposure immersion: 25 min
Post-exposure incubation: 18 h
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
The test item has an intrinsic colour (bluish black).
The test item is a possible MTT-reducer.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled):
In this assay 2 replicates per test item and 2 replicates negative controls, 2 replicates positive controls, 2 replicates colour controls and 2 replicates non-specific colour control were used. Furthermore, 2 killed treated tissues and 2 killed negative control tissues are used for the MTT evaluation.
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan :
200 µL sample from each tube was placed into the wells of a 96-well plate and read Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at the wavelength of 570 nm using isopropanol solutions as the blank (8×200 µL).
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
The irritancy potential of test items is predicted by the mean tissue viability of tissues exposed to the test item. The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60% of the negative control. However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2, further testing with other test methods will be required to decide on its final classification
- Complete supporting information for the specific RhCE tissue construct used :
see "Any other information on materials and methods"
- Assay Acceptance Criteria:
The mean OD value of the two negative control tissues should be between 0.8 and 2.5.
The acceptable percentage viability for positive control (mean of two tissues) is: 6 hours exposure: below 50 % of control viability
The difference of viability between the two relating tissues of a single chemical is < 20% in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: cell viability mean %

Run / experiment:

1 and 2

Value:

83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

1 and 2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

Table 3: OD values and viability percentages of the controls

Controls	Optical Density (OD)		Viability (%)	Δ%
Negative Control: Sterile deionized water	1	1.740	104	7.5
	2	1.614	96
	mean	1.677	100
Positive Control: Methyl acetate	1	0.509	30	8.6
	2	0.364	22
	mean	0.437	26

  

Table 4: OD values and viability percentages of the test item (including corrected values)

Test Item	Optical Density (OD)		TODTT	Viability (%)	Relative Viability (%)	Δ%
Test Item	1	1.522	1.430	91	85	15.6
	2	1.260	1.169	75	70
	mean	1.391	1.299	83	77
	standard deviation (SD)			11..03	11.03

  

Table 5: OD values of additional controls for MTT-interacting test item

Additional controls	Optical Density (OD)
Negative control killed tissues: Sterile deionized water	1	0.042
	2	0.193
	mean	0.118
Test item treated killed tissues:	1	0.248
	2	0.171
	mean	0.209

  

Table 6: OD values and NSC % of additional control

Additional colour control	Optical Density (OD)		Non Specific Colour %(NSCliving %)	Δ%
Test item	1	0.014	0.82	0.1
	2	0.013
	mean	0.014

 

Remark: Δ% is the difference of viability between the two relating tissues

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro eye irritation assay according to OECD guideline 492, a relative mean cell viability of 77 % was determined. The test item is not considered as eye irritating (UN GHS: No Category).

Executive summary:

In an in vitro eye irritation assay (RhCE) according to OECD guideline 492, the eye irritating potential of the test item on three-dimensional RhCE tissue in the EpiOcular™ model was determined.

Before treatment the tissues were pre-wetted with ca. 20 μL of Ca++Mg++Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with 50 mg test item 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 25 ± 2 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 disk was assessed via MTT assay and quantified spectrophotometrically. Sterile deionized water or methyl acetate treated tissues were used as negative and positive controls, respectively.

The test item has an intrinsic colour (bluish 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.

The test item is a possible MTT-reducer and has an intrinsic colour (bluish black). Therefore, a third control for non-specific colour in killed tissues (NSCkilled) was performed with two killed treated tissues to avoid a possible double correction [TODTT (MTT and NSC)] for colour interference. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 77 %). Therefore the test item was considered to be non-irritant to eye (UN GHS No Category).