Disodium 4,5-dihydroxybenzene-1,3-disulphonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

RhCE test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 - 08 Dec 2016

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:

Adopted in 2015

Deviations:

no

Test guideline 2

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:

Adopted in 2017

GLP compliance:

yes (incl. QA statement)

Remarks:

Slovak National Accreditation Service, Bratislava, Slovak Republic

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4,5-dihydroxybenzene-1,3-disulphonate

EC Number:

205-741-5

EC Name:

Disodium 4,5-dihydroxybenzene-1,3-disulphonate

Cas Number:

149-45-1

Molecular formula:

C6H6O8S2.2Na

IUPAC Name:

disodium 4,5-dihydroxybenzene-1,3-disulphonate

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
- in vitro test method: Eye Irritation Test (EIT) with reconstructed human cornea-like epithelium (RhCE) tissue, EpiOcular™ (MatTek). The EpiOcular™ human cell construct for eye irritation testing is a nonkeratinized epithelium prepared from normal human keratinocytes (MatTek, In Vitro Life Science Laboratories, SR). It models the corneal epithelium with progressively stratified, but not cornified cells. The ability to expose the tissue topically is essential to model the same kind of progressive injury expected in vivo. In this assay, the test item is applied to the surface of the corneal epithelial construct for a fixed period, removed, and the tissue allowed to express the resulting damage. Two construct tissues (replicates) are used for each test treatment and each control group; a 6-h exposure with 18 hours incubation post-exposure. Relative tissue viability post-exposure is determined against the negative control-treated constructs by evaluating the reduction of MTT to a formazan product, determined spectrophotometrically (optical density). A concurrent positive control is used with each assay to determine validity of the test. Based on the "depth of injury model," the EpiOcular eye irritation test (EIT) is intended to differentiate those materials that are nonirritants (would not require a warning label in the European chemical classification systems) from those that would require labelling as GHS eye irritant category 1 or 2.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The EpiOcular™ human cell construct for eye irritation testing (OCL-200-EIT) (Lot No. 23755) was obtained from MatTek, In Vitro Life Science Laboratories, Slovak Republic.

- Quality control (QC): The defined production release criteria (Barrier function, Morphology) of the RhCE tissue were not given in the study report.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg applied topically per tissue

Duration of treatment / exposure:

6 h at 37 ± 1°C

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

Two tissue replicates were exposed per treatment with the test material, positive control and negative control, respectively.

Details on study design:

- Details of the test procedure used: EpiOcular™ was delivered one day before the pre-incubation of tissues, stored overnight in original packaging at 2-8°C. At Day 1, each cultured tissue was removed, inspected and transferred to plates containing assay medium and pre-incubated overnight at 37 ± 1°C, in a humidified atmosphere of 5 ± 1% CO2. At Day 2, the tissues were pre-treated by wetting with DPBS and incubated at standard culture conditions for 30 min. The negative and positive controls were tested by applying 50 µL topically on the tissues. The test material was tested by applying it topically onto the tissue surface at 50 mg per tissue; two tissues (replicates) were used per treatment with test material, negative and positive controls. The cultures were returned to the incubator for 6 hours. After the 6-hour exposure time, tissues were rinsed with DPBS (three times) to remove any residual test material. After rinsing, the tissues were immediately transferred to and immersed in previously-warmed assay medium for a 25-minute immersion incubation (post-soak) at room temperature. The tissues were then blotted, returned to pre-warmed medium, and post-incubated for an additional 18 hours. Then the cultures were transferred to 0.3 mL of MTT reagent (1 mg/mL) and incubated at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air for 3 h. After incubation, the cultures were blotted and transferred to new tissue well-plates, and extracted in 1 mL of isopropanol for 2 h, with shaking, at room temperature. At the end of this non-submerged extraction, the tissues were discarded, and 1 mL of isopropanol was added into each respective well and mixed. Duplicate volumes of 200 μL of each extraction solution were transferred to a 96-well plate and their absorbances (ODs) were recorded.
- RhCE tissue construct used, including batch number: The EpiOcular™ human cell construct for eye irritation testing (OCL-200-EIT) (Lot No. 23755), MatTek, In Vitro Life Science Laboratories, Slovak Republic.
- Doses of test chemical and control substances used: test material: 50 mg of AF-378; negative control: 50 µL of water for injection (Aqua pro injectione, Lot 153248091, B Braun, expiry date 07/2018); positive control: 50 µL of Methyl Acetate (Lot 091916MHB, MatTek, expiry date 09/2017)
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Exposure: 6 h at 37 ± 1°C; Post-exposure immersion: 25 min at room temperature; Post-exposure incubation: 18 h not further specified.
- Justification for the use of a different negative control than ultrapure H2O (if applicable): Water for injection (Aqua pro injectione) was used for the negative control article
- Justification for the use of a different positive control than neat methyl acetate (if applicable): Methyl Acetate was used
- Description of any modifications to the test procedure: None
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): The test material was tested for direct MTT-reduction and colouring test chemicals, with results being negative in both procedures
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): Two replicates per treatment: test material, positive and negative controls
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): Wavelength of 540 nm without a reference filter, measured on a spectrophotometer
- Description of the method used to quantify MTT formazan: Incubation in 0.3 mL of MTT reagent (1 mg/mL) at 37 ± 1°C, in a humidified atmosphere of 5 ± 1% CO2.
- Acceptable variability between tissue replicates for positive and negative controls: The difference must be <20% in the same run
- Acceptable variability between tissue replicates for the test material: The difference must be <20% in the same run

Results and discussion

In vitro

Results

Irritation parameter:

other: percent viability

Run / experiment:

two tissue replicates per treatment

Value:

ca. 54.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Negative control OD >1.0 and <2.6
- Acceptance criteria met for positive control: Mean relative viability of the positive control at 6 h exposure is <60% of the Negative control viability
- Acceptance criteria for all materials tested: The difference of viability between the two replicate tissues of the test material, positive control, and negative control is <20% in the same run

Any other information on results incl. tables

Eye irritation potential of test item after 6-hour exposure in the human eye irritation test model: EpiOcular^TM

Test Item	OD Mean	SD of OD	Viability Mean (%)	SD of Viability	in vivo Prediction
Negative control^a	1.074	0.187	100.0	17.41	NI
Positive control^b	0.295	0.037	27.5	3.40	I
Test substance	0.584	0.150	54.4	13.96	I

^aH2O

^bmethyl acetate

NI = non-irritant

I = irritant

Applicant's summary and conclusion

Interpretation of results:

other: irritating potential

Conclusions:

Under the conditions of the conducted test, the test substance is considered to possess an irritating potential towards human cornea in the EpiOcular™ model but the result is not conclusive with respect to classification of the test substance as eye irritant (Eye Irritant Cat. 2) or serious eye damage (Eye Damage Cat. 1) and therefore requires further evaluation and/or data generation.

Executive summary:

Considering both, the irritant potential observed in the EpiOcular™ model and the IVIS of -0.40 in the BCOP not requiring classification for eye irritation or serious eye damage (Cat. 1), the test substance is classified as Eye Irrit. 2 (H319) in a worst case approach.