Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 September 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and well described study in accordance with GLP and OECD Guideline 437 without any deviation.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: MEXORYL SCK
- Physical state: Beige powder
- Analytical purity: saponines content (determined by HPLC assay) 55.8 % w/w
- Lot/batch No.: R0069579A 003 P 001
- Shelf life/Retest date: April 2011
- Storage condition of test material: Stored at room temperature and away from light and moisture

Test animals / tissue source

Species:
other: bovine eye
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
- Eyes selection: from reception of eyes: detection of defects of the cornea, elimination of eyes presenting defects, elimination of the too big eyes, all stages were performed by avoiding touching corneas, in order not to damage them.
- Preservation of the corneas: eyes were placed in buffered Hanks medium enriched with antibiotics and preserved at 5 ± 3 °C for maximum duration of 24 h.

Test system

Vehicle:
other: distilled water
Controls:
other: negative control: 0.9 % w/v sodium chloride solution
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL ± 8 µL per cornea
- Concentration (if solution): Diluted to 20 % (w/w) in distilled water
- pH of the preparation : 5.5


Duration of treatment / exposure:
- Contact timepoint: 4 h ± 10 minutes at 32 ± 1 °C.
Observation period (in vivo):
- Corneal opacity was measured pre-treatment, post-treatment and post-incubation (after 4 h ± 10 minutes of incubation at 32 ± 1 °C).
- Application of Fluorescein (5 mg/mL) and corneal permeability was measured after 90 ± 5 min of incubation at 32 ± 1 °C.
Number of animals or in vitro replicates:
Total: 9 corneas - 3 corneas/group for test item, negative and positive controls
Details on study design:
Details of test procedure: Eyes were placed in buffered Hanks medium enriched with antibiotics and preserved at 5 ± 3 °C for maximum duration of 24 h. Corneas were mounted in corneal holders and preincubated in nutritive medium in water-bath at 32 ± 1 °C for at least one hour. Before the treatment, opacity measurement was performed using an OP KIT opacitometer. The test item (750 µL ± 8 µL) was applied on each cornea and incubated for 4 h ± 10 minutes at 32 ± 1 °C. At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with nutritive medium containing phenol red at 32 ± 1 °C before a final rinse with nutritive medium. The anterior chamber was refilled with nutritive medium. Measurement of the opacity OPT2 (OPT “2 hours”) of each cornea vs an empty holder (containing nutritive medium only). Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of fluorescein solution (5 mg/mL) and the holders were incubated at 32 ± 1 °C for 90 ± 5 minutes. After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 µL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 490 nm (OD490) was measured.

Results and discussion

In vivo

Results
Irritation parameter:
other: In Vitro Irritancy Score (IVIS)
Basis:
mean
Time point:
other: 4 h
Score:
82.2
Reversibility:
not reversible
Remarks on result:
other: score: 82.2 ± 4.9
Irritant / corrosive response data:
- In Vitro Irritancy Score (IVIS) were 82.2 ± 4.9 and 193.0 ± 2.4 for test item and positive controls, respectively.
Other effects:
None

Any other information on results incl. tables

Table 7.3.2/1: In vitro eye irritation – results

Holder No

Treatment

OPT0

OPT2

OPT2 – OPT0

Corrected opacity

DO

Corrected OD

Score OPc + 150DOc

97

Negative control

0

0

0

 

0.070

 

 

440

3

3

0

 

0.091

 

 

86

0

0

0

 

0.071

 

 

Comments: Nil

Mean

0.0

0.077

 

SD

0.0

0.012

 

29

Imidazole 20 %

1

146

145

145.0

3.256

3.179

192.7

300

0

147

147

147.0

3.312

3.235

195.5

210

0

142

142

142.0

3.332

3.255

190.8

Comments: Detachment of epithelium and edema

Mean

144.7

3.223

193.0

SD

2.5

0.039

2.4

2

MEXORYL SCK

2

27

25

25.0

4.092

4.015

85.2

12

I.E.C: 101382 0000B455

4

24

20

20.0

3.848

3.771

76.6

58

Diluted to 20 %

0

28

28

28.0

3.866

3.779

84.7

Comments: Detachment of epithelium and edema

Mean

24.3

3.855

82.2

SD

4.0

0.139

4.9

Applicant's summary and conclusion

Interpretation of results:
corrosive
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions, MEXORYL SCK applied diluted to 20 % (w/w) in distilled water, is classified as corrosive or severe irritant for the isolated bovine cornea, after 4 h of contact.
Executive summary:

In an in vitro eye irritation study performed according to the OECD Guideline 437 and in compliance with GLP, test item, MEXORYL SCK diluted to 20 % (w/w) in distilled water (750 µL ± 8 µL per cornea) was applied to isolated bovine corneas mounted in corneal holders, and incubated for 4 h ± 10 minutes at 32 ± 1 °C. Three corneas were used for each treated series (test item; negative control; positive control: Imidazole 20 %). The two endpoints, corneal opacity and corneal permeability were measured and In Vitro Irritancy Score (IVIS) was calculated. 

In Vitro Irritancy Score (IVIS) were 82.2 ± 4.9 and 193.0 ± 2.4 for test item and positive controls, respectively.

Under the test conditions, MEXORYL SCK applied diluted to 20 % (w/w) in distilled water, is classified as corrosive or severe irritant for the isolated bovine cornea, after 4 h of contact.