Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 03, 2017 to September 08, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Assay was conducted by a GLP accredited laboratory using OECD Guideline 437

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
yes
Remarks:
The end time for the 2-hour incubation period was not recorded in the raw data
Principles of method if other than guideline:
The end time for the 2-hour incubation period was not recorded in the raw data. However, the subsequent procedure was noted as completed 2 hours and 10 minutes after the start of the incubation period. This deviation from protocol was believed not to have affected the integrity or outcome of the study as the negative and positive controls gave satisfactory results. In addition, the study was considered to be GLP-compliant but the expiry date for the test article stability was not based on known stability data.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Corneas from bovine eyes were obtained from a local abattoir. The eyes were removed after slaughter, completely immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL) in a suitably sized container and transported on the same day to the testing facility. On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
A volume of 750 µL of the test article was applied to each of three corneas followed by a ten minute incubation at 32 °C. The test article was administered without dilution. A volume of 750 µL of the negative or positive control was similarly applied to further groups of three corneas
Duration of treatment / exposure:
Ten minute incubation period
Duration of post- treatment incubation (in vitro):
Corneas were incubated (horizontally) for approximately 2 hours prior to the measurement of corneal opacity. For the permeability endpoint (optical density), corneas were incubated in the vertical position for 1 hour and 25 minutes. The negative and positive control groups were subject to the same procedures
Number of animals or in vitro replicates:
Three corneas (triplicate)
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined for defects on arrival, including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used. The selected corneas were excised from the eyes and loaded onto a holder that contained chambers (posterior and anterior) filled with pre-warmed Minimal Essential Medium (MEM). To avoid bubbles forming, the posterior chamber was filled first. The holders were incubated at 32 ± 1 °C for at least 1 hour. After this point, the media in both chambers was exchanged for fresh MEM in the order of posterior chamber followed by anterior chamber, therein ensuring that the corneas retained their natural curvature

QUALITY CHECK OF THE ISOLATED CORNEAS
Following preparation in the MEM, the opacity of each cornea was measured using an opacitometer. Any corneas found to have scratches or increased neovascularization or an opacity of >7 opacity units when examined prior to treatment were discarded

NUMBER OF REPLICATES
Three (triplicate)

NEGATIVE CONTROL USED
Yes, the negative control substance was 0.9% sodium chloride solution

POSITIVE CONTROL USED
Yes, the positive control substance was dimethylformamide (DMF)

APPLICATION DOSE AND EXPOSURE TIME
The test article was not diluted prior to administration. A volume of 750 µL of the test article was applied to each of three corneas followed by a 10 minute incubation at 32 °C

POST-INCUBATION PERIOD: Yes

REMOVAL OF TEST SUBSTANCE
After the 10 minute incubation period, the corneas were individually washed with media containing phenol red (as a pH indicator) until this indicator showed no pH effect occurring and demonstrating that the test article had been removed successfully. The corneas were then washed once in media without phenol red

POST-EXPOSURE INCUBATION
Yes. After washing, the anterior chamber was filled with fresh MEM and the corneas were incubated (horizontally) for approximately 2 hours. Corneal opacity was subsequently measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the corneas were incubated in the vertical position for 1 hour and 25 minutes. Following this period, the media in the posterior chamber was removed and held in a labelled tube for later analysis

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: Opacitometer
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of spectrophotometry (OD490)
- Others: It was apparent from visual observation that the corneas treated with the test article appeared cloudy post treatment and the corneas treated with the positive control were cloudy and wrinkled following treatment

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA
The test article is concluded as inducing serious eye damage if the IVIS is >55 or as not requiring classification for eye irritation if the IVIS is ≤3. No prediction can be made if the IVIS is >3 but ≤55

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
Mean corrected
Value:
18.69
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The corneas treated with 1-[(2-hydroxyethyl)thio]propan-2-ol appeared cloudy post-treatment and the corneas treated with the positive control were noted to be cloudy and wrinkled following treatment

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the negative control yielded opacity and permeability values that were less than the established upper limits for the endpoints for bovine corneas as treated at the testing facility
- Acceptance criteria met for positive control: yes, the positive control yielded an IVIS within 2 standard deviations of the historical control mean

Any other information on results incl. tables

Corneal Opacity

Test Substance Cornea Number Initial Opacity Post Incubation Opacity Change in Opacity Mean Change in Opacity Corrected Opacity Mean Corrected Opacity
Negative 23 0 -1 -1 -0.67 -0.3 0
Negative 24 0 -1 -1 -0.3
Negative 25 0 0 0 0.7
Test article 18 0 16 16 N/A 16.7 16
Test article 19 0 15 15 15.7
Test article 20 0 15 15 15.7
Positive 14 -1 60 61 N/A 61.7 62.7
Positive 15 -1 69 70 70.7
Positive 16 -2 53 55 55.7

Corneal Permeability (Density)

Test Substance Cornea Number Mean Blank OD490 OD490 Corrected OD490 Mean Corrected OD490 Final Corrected OD490 Mean Group Corrected OD490
Negative 23 0.04 0.041 0.001 0.002 0 0
Negative 24 0.042 0.003 0.001
Negative 25   0.04 0.001 -0.001
Test article 18 0.423 0.383 N/A 0.381 0.18
Test article 19 0.173 0.133 0.132
Test article 20   0.067 0.027 0.026
Positive 14 0.416 0.376 N/A 0.375 0.243
Positive 15 0.13 0.091 0.089
Positive 16   0.307 0.276 0.266

Calculated IVIS

Test Chemical Mean Opacity Mean Permeability IVIS (Mean Opacity + (15 x Mean Permeability))
Test article 16 0.18 18.69
Negative control 0 0 0
Positive control 62.7 0.243 66.31

N/A Not applicable.

The data in these tables was computer generated. In this system individual and derived figures are rounded. Thereby recalculation of derived values from the individual data will, in some instances, yield minor variations

Applicant's summary and conclusion

Interpretation of results:
other: inconclusive
Conclusions:
The results of this in vitro Bovine Corneal Opacity and Permeability (BCOP) assay suggests that a conclusive prediction cannot be made on the eye irritation potential, or for the hazard classification, of 1-[(2-hydroxyethyl)thio]propan-2-ol as the IVIS was >3 but < 55.1 (IVIS = 18.69).
Executive summary:

An in vitro Bovine Corneal Opacity and Permeability (BCOP) assay was performed in line with OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method).

The undiluted test material was applied to three cattle corneas obtained from an abattoir at a volume of 750 µL, after which each cornea was incubated at 32 ± 1 °C for 10 minutes and then washed with phenol red-containing media followed by a media without phenol red. Corneal opacity was measured after a 2 hour period of horizontal incubation. For permeability, the corneas were incubated in the vertical position for 1 hour and 25 minutes at 32 ± 1°C within a sodium fluorescein solution. Thereafter, three 350 μL aliquots of this media (per cornea) were analysed for optical density at 490 nanometers (OD490). A positive (dimethylformamide) and negative (0.9% sodium chloride solution) control was applied using the same procedure to additional groups of corneas.

The mean corrected opacity reading for the test article, positive control, and negative control was 16.0, 62.7, and 0.0, respectively. The mean group corrected optical density for the test article was 0.180 and the mean group corrected optical density for the positive and negative control was 0.243 and 0.000, respectively. An In Vitro Irritation Score (IVIS) of 18.69 was calculated for 1-[(2-hydroxyethyl)thio]propan-2-ol from corneal opacity and permeability measurements. No prediction can be made if the IVIS is >3 but ≤ 55 and, therefore, the study is inconclusive.