Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
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
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)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
TEST TISSUES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
750ul
Duration of treatment / exposure:
240 minutes (+/-10 minutes)
Observation period (in vivo):
Not appliable
Duration of post- treatment incubation (in vitro):
Not applicable
Number of animals or in vitro replicates:
Study performed in triplicate
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32°C +/-1°C The corneas were incubated for the minimum of 1 hour at 32°C +/-1°C.

After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany).

QUALITY CHECK OF THE ISOLATED CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
Yes, physiological saline

POSITIVE CONTROL USED
Yes, 20% (w/v) Imidazole (Merck Schuchardt OHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline.

APPLICATION DOSE AND EXPOSURE TIME
750ul, 240 minutes (+/-10 minutes)

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
The medium from the anterior compartment was removed and 750 µl of either the negative control, positive control (20% (w/v) Imidazole solution) or 20% (w/v) solution of the test item was introduced onto the epithelium of the cornea. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 +/- 10 minutes at 32 ° +/- 1°C. After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) was calculated

With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. Yes

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Value:
98
Vehicle controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Value:
93
Vehicle controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Value:
0.313
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: No

Any other information on results incl. tables

Table 1: Summary of Opacity, Permeability and In Vitro Scores

Treatment

Mean

Opacity

Mean

Permeability

Mean In vitro Irritation Score1, 2

Negative control

0.7

-0.001

0.7

Positive control

104

1.390

125

The test item

93

0.313

98

1  Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

2  In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Table 2:            In Vitro Irritancy Score

Treatment

Final Opacity2

Final OD4902

In vitroIrritancy Score1

 

Negative control

-0.2

0.002

-0.2

2.8

-0.003

2.8

-0.6

-0.002

-0.6

 

Positive control

122

1.667

147

118

1.308

138

73

1.195

91

 

The test item

92

0.349

97

96

0.210

99

92

0.381

98

1  In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

2  Positive control and test item are corrected for the negative control.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In conclusion, since Mercaptamine induced an IVIS ≥ 55, it is concluded that Mercaptamine induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report