Monalazone disodium

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 18-Jan 25, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes (MatTek). It models the cornea 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. It also allows both solid and liquid test materials to be applied directly to the tissue.

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Monalazone disodium

EC Number:

262-810-2

EC Name:

Monalazone disodium

Cas Number:

61477-95-0

Molecular formula:

C7H4ClNO4S.2Na

IUPAC Name:

disodium 4-[(chloroazanidyl)sulfonyl]benzoate

Test material form:

solid

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcular™ human cell construct for eye irritation testing (OCL-200-EIT) (Lot No.27020) was obtained from MatTek In Vitro Life Science Laboratories, SR.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

Two

Details on study design:

Treatment
After pre-incubation tissues were pre-wetted with 20 L of Dulbecco's Phosphate Buffered Saline (DPBS), then approximately 50 mg of the test item and 50 μL control items were applied topically onto the tissue surface for 6 hours. Two tissues were used per treatment, negative and positive controls. After exposure period, each tissue was rinsed gently with DPBS to remove any residual test item. After post-soak immersion at room temperature for
25 minutes, tissues than were transferred to fresh medium and incubated for 18 hours. Afterwards, the MTT assay was performed by transferring the tissues to 24-wells plate containing MTT medium (1 mg/mL) and incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 3 hours.
After incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2 mL/tissue of isopropanol for 2 hours at room temperature.
Each extraction solutions in a volume of 200 μL were transferred to a 96-well plate and the optical density of extracted formazan was determined using a spectrophotometer at 540 nm. Before testing, to identify the possible interference with MTT endpoint, test item was checked for its ability to reduce MTT directly.

Assessment of Direct Test Item Reduction by MTT
Test items may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement. Therefore, it is necessary to assess this ability for each test item prior to conducting any assays with viable tissues. For this purpose, a 1.0 mg/mL MTT solution (in DMEM) was prepared. Approximately 50 mg of the test item was added to 1 mL of the MTT solution in a 6-well plate and the
mixture was incubated in the dark at 37  1°C in a humidified atmosphere of 5  1% CO2 in air (standard culture conditions) for three hours. A negative control (50 μL of aqua pro injectione) was run concurrently. At the end of the exposure time the colour of treated MTT remained unchanged and it was concluded that the test item did not reduce MTT directly.

Assessment of Colored or Staining Materials
Colored test items or test items which become colored after application to the tissues may interfere with the quantitative photometric MTT measurement if the colorant binds to the tissue and is extracted together with MTT. Therefore, each test item has to be checked for its colorant properties.
For this purpose, test item in the amount approximately 50 mg was added to 2 mL of isopropanol, the same amount as used for MTT extraction and incubated in 6-well plates for 3 hours at room temperature. Two 200 μL aliquots of isopropanol solutions and of pure isopropanol were transferred to a 96-well plate and the absorbance was measured with a plate reader at the MTT measurement wavelength. After subtraction of the OD for isopropanol, the OD of the test item solution was < 0.08 and the test item has not to be considered as possibly interacting with the MTT measurement. The test item in the amount approximately 50 mg was added into 1 mL of purified water. The mixture was incubated in 6-well plate in the incubator at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 60 min. At the end of the exposure time the presence of the staining was evaluated. The colour of mixture was unchanged and the test item had not the potential to stain tissue.

Receipt and Preparation of Cultures
EpiOcular™ was delivered one day before the pre-incubation of tissues and was stored in original package at 2-8°C. At day 1, each culture was removed from the agarose gel using a sterile forceps, inspected and transferred to a pre-labeled 6-well plates containing 1 mL of assay medium per well. The EpiOcularTM cultures were pre-incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 for 60 min. At the end of the first pre-incubation
period, the medium was replaced by 1 mL of fresh assay medium. Further, the tissues were pre-incubated in incubator overnight prior to dosing for release of transport stress related compounds and debris.

Assay Procedure
After the overnight incubation, the tissues were pre-wetted with 20 uL of DPBS. The tissues were incubated at standard culture conditions for 30 minutes. After pre-wetting, the negative and positive controls were tested by applying 50 uL topically on the tissues. The test item was applied topically onto the tissue surface at amount approximately 50 mg. Two tissues were used per treatment, negative and positive controls. The cultures were returned to the incubator for 6 hours. After treatment time, tissues were rinsed with DPBS (in three glass beakers) to remove any residual test material. After rinsing, the tissue was immediately transferred to and immersed in 5 mL of previously-warmed assay medium in 12-well plate for a 25-minute immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion, each insert was removed from the medium and the insert was blotted on absorbent material and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm medium. The tissues were post-incubated for an additional 18 hours. Then, the cultures were transferred to 24-well plate containing 0.3 mL/well of MTT reagent (1 mg/mL) and incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 3 hours. After incubation, the cultures were blotted on absorbent paper and transferred to a pre-labeled 6-well plate and extracted in 2 mL of isopropanol at room temperature for 2 hours. Volume of 2x 200 μL of each extraction solution were transferred to a 96-well plate and the absorbances (ODs) were recorded.

Handling of Results
Data include cytotoxicity and viability determination. The optical densities (ODs) were read in a 96-well plate spectrophotometer using a wavelength 540 nm without a reference filter.Data files of optical densities (ODs) generated by spectrophotometer (without blank substraction) were manually transported into the first spreadsheet of the EXCEL workbook (Import). The control of data transmission was performed by the responsible person. The
blank corrections, calculation of results and statistical parameters were done automatically in the second part of the workbook (Results). For each individual tissue treated with test item, PC and NC, the individual relative tissue viability was calculated. For each test item, PC and NC, the mean relative viability of the two individual tissues were calculated and used for classification according to the Prediction Model. The spreadsheet was shown a graph of the results (% of relative viability ± SD).

Results and discussion

In vitro

Results

Irritation parameter:

other: % viability

Value:

2.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The assay is considered valid if the following criteria were met:
Negative Control (NC):
The negative control OD > 0.8 and < 2.5.

Positive Control (PC):
The mean relative viability of the positive control is at 6 hr exposure below 50% of control viability.

Standard Deviation (SD):
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.

Any other information on results incl. tables

Table 1. Eye irritation potential of monalazone disodium after 6hr exposure in human model.

Substance	OD mean	SD of OD	Viability mean (%)	SD of viabilities	in vivo prediction
Negative control (H2O)	1,560	0,102	100,0	6,54	NI
Postive control	0,552	0,019	35,4	1,19	I
Monalazone disodium	0,045	0,007	2,9	0,42	I

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, Monalazone disodium was irritating to the eye in the EpiOcular™ test under the experimental conditions described in this report. The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1).
This Test Guideline does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B), as defined by UN GHS. For these purposes, further testing with other in vitro test methods is required.

Executive summary:

Monalazone disodium was examined for eye irritation potential in EpiOcularTM Eye Irritation Test in compliance with OECD Guideline 492. MatTek EpiOcular™ tissue samples were treated in duplicate with the test article, negative control and postive control (exposure time of 6 hours). The magnitude of viability was quantified by MTT test. The absolute mean OD of negative control (OD570 = 1.560) was within the laboratory historical control data range. The positive control had a mean cell viability of 35.4% after 6 hours exposure. The positive control met the acceptance criterion: mean tissue viability less than 50%. The viability of culture treated by Monalazone disodium was 2.9%. Based on the results of the study (mean tissue viability ≤60%)

, the test item Monalazone disodium is considered to be irritant (I).