Acetylcysteine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 17, 2018 - April 19, 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

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:

October 9, 2017

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EpiOcular Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; For use with MatTek Corporation´s Reconstructed Human EpiOcular Model; MatTek Corporation

Version / remarks:

June 29, 2015

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol No. 164: Ocular Irritation Assay for Chemicals using EpiOcular EIT

Version / remarks:

September 14, 2015

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Acetylcysteine

EC Number:

210-498-3

EC Name:

Acetylcysteine

Cas Number:

616-91-1

Molecular formula:

C5H9NO3S

IUPAC Name:

N-acetylcysteine

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 mg per tissue

Details on study design:

- Details of the test procedure used
The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential.

- RhCE tissue construct used, including batch number
EpiOcular Tissue (OCL-200, OCL-212), Lot No. 27033, MatTek In Vitro Life Science Laboratories

- Doses of test chemical and control substances used
Solid test item: 50 mg per tissue
Negative control: 50 µL per tissue
Positive control: 50 µL per tissue

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
6 hours treatment at 37°C and 5% CO2, 25 min post-exposure immersion followed by 18 hours post-exposure incubation at 37°C and 5% CO2

- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2

- Wavelength used for quantifying MTT formazan: 570 nm

- Description of the method used to quantify MTT formazan
After the post-treatment incubation period, the treated tissues were transferred in a 24-well plate filled with 300 µL MTT solution (1.0 mg/mL MTT). Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes (± 10 minutes) at 37°C and 5% CO2. The inserts were removed from the 24-well plate after 180 minutes (± 10 minutes). The bottom of the inserts was blotted on absorbent material, and then transferred to a 6-well plate containing 2 mL isopropanol so that no isopropanol was flowing into the inserts. The plate was sealed with a standard plate sealer. To extract the MTT, the plates was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The corresponding negative and positive controls were treated identically. The extract solution was mixed and 2 x 200 µL were transferred into a 96-well plate. The OD was read using a spectrophotometer at 570 nm wavelength.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The test item is identified as not requiring classification and labeling according to UN GHS (No Category) if the mean percent tissue viability is more than 60%. In this case no further testing in other test methods is required. If the mean percent tissue viability is less than or equal 60%, no prediction can be made. In this case, further testing with other test methods will be required because RhCE test methods show a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.

- Complete supporting information for the specific RhCE tissue construct used
Tissue viability: OD [540-570] 1.996
Barrier function: ET-50 = 27.45 min
Sterility: no contamination

- Acceptance Criteria: The results are acceptable if:
1. The negative control OD >0.8 and <2.5
2. The mean relative viability of the positive control is:
a) 30 minute exposure: below 50% of control viability
b) 6 hour exposure: below 50% of control viability
3. 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

Irritation parameter:

other: % viability

Run / experiment:

1st Experiment

Value:

2.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

17.8%

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: non

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

The test item is not a direct MTT reducer and the test item has no colorant properties.

Applicant's summary and conclusion

Interpretation of results:

other: no prediction can be made

Conclusions:

Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.

Executive summary:

A study according OECD TG 492 was conducted to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model.

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.

After treatment with the negative control (sterile deionized water) the mean OD was 1.686 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 17.8% (study acceptance criterion: <50%). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 2.4% and thus, lower than 60%. i.e. according to OECD TG 492, no prediction can be made regarding the eye hazard potential of the test.