Registration Dossier

Toxicological information

Eye irritation

Currently viewing:

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-05-14 - 2018-06-13
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:
OECD Guideline 491 (Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Molecular formula: C8H14O4
Molecular Mass:
174.20 g/mol
Characteristics (Physical Appearance):
White Crystalline powder
CAS No.:
505-48-6
Batch Number:
308
Purity:
100%

Test animals / tissue source

Species:
rabbit
Details on test animals or tissues and environmental conditions:
Cell line:
SIRC (Statens Seruminstitut Rabbit Cornea) cell line.
Medium:
Minimum essential medium (MEM) supplemented with
L-glutamine containing 10% fetal bovine serum (FBS) and Penicillin Streptomycin Solution (1X).
Culture Conditions:
37 ± 1 °C, ~5% CO2, 90 to 100% Humidity.
Cell refreshing Agent:
Dulbecco’ Phosphate Buffer Saline.
Cell Dispersion Agent:
0.25% Trypsin EDTA.

Test system

Vehicle:
other: mineral oil
Controls:
yes
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
200 μL
Test item was suspended uniformly in the mineral oil at 5% (w/w) concentration and further diluted by serial 10-fold dilution to 0.5% then to 0.05% concentration. Test item was tested at both 5% and 0.05% concentrations.0.01% Sodium lauryl sulfate (SLS) in normal saline was used as a positive control.
Duration of treatment / exposure:
five minutes at room temperature
Duration of post- treatment incubation (in vitro):
After exposure, cells were washed twice with 200 μL of PBS and 200 μL of MTT solution (0.5 mg MTT per ml of culture medium) was added. After two-hour reaction time in an incubator (37 ± 1˚C, ~5% CO2 and 90 to 100% humidity), the MTT solution was decanted.
Number of animals or in vitro replicates:
Three independent experiments, each containing three replicate wells (i.e., n=9) were performed along with concurrent medium, positive and vehicle controls.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: cell viability
Remarks:
5%
Value:
59.69
Vehicle controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: cell viability
Remarks:
0.05%
Value:
81.03
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
other: No prediction can be made
Conclusions:
'Short Time Exposure In-Vitro Study Using SIRC Cell Line for Identification of Eye Irritation or Serious Eye Damage of Suberic Acid' was carried out in compliance with the Organization for Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Section 4, No. 491 - Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage, adopted by the council on 09 October 2017 and as per mutually agreed Study Plan.
Under the given experimental conditions of 'Short Time Exposure In-Vitro Study Using SIRC Cell Line for Identification of Eye Irritation or Serious Eye Damage of Suberic Acid' (OECD Guideline No. 491)', it is concluded that this test method has successfully classified the test item into ‘No prediction can be made’ category as per the United Nations (UN) Globally Harmonised System (GHS) for classification of chemicals.
Executive summary:

'Short Time Exposure In-Vitro Study Using SIRC Cell Line for Identification of Serious Eye Damage by Suberic Acid' was carried out in compliance with the Organization for Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Section 4, No. 491 - Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage, adopted by the council on 09 October 2017 and as per mutually agreed Study Plan.

Suberic Acid was evaluated for its irritancy potential based on its ability to induce cytotoxicity in Short Time Exposure (STE) Test method. The cytotoxic effect of test item on corneal epithelial cells is an important mode of action (MOA) leading to

corneal epithelium damage and eye irritation.

After extraction from cells, cell viability in the STE test method is assessed by the quantitative measurement of blue formazan salt produced by the living cells. It is due to enzymatic conversion of the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide), also known as Thiazolyl Blue Tetrazolium Bromide. The obtained cell viability is compared to

the solvent control (relative viability) and used to estimate the potential eye hazard of test item.

The relative cell viability of each solvent control, medium control and test item expressed as a percentage.

Optical density of the medium control was found to be 0.47 which was higher than 0.3.

The relative cell viability obtained for solvent control (mineral oil) was 90.64%, which was greater than 80% relative to

medium control.

The relative cell viability obtained for 0.01% sodium lauryl sulfate (Positive control) was 23.54%, i.e. between 21.1% and 62.3% according to laboratory historical data established by the method developer.

Standard deviations of the final cell viability derived from three experiments were 2.21 and 8.59 for both 5% and 0.05% concentrations of test item respectively, which is less than 15%.

Cell viability in both the concentrations i.e. 5% and 0.05% was 59.69% and 81.03% respectively, , which was less than 70%

for 5% test concentration and greater than 70% for 0.05% test concentration.

Under the given experimental conditions of this study, it is concluded that the test item is considered to fall under

‘No prediction can be made’ category of UN GHS classification.