Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 December 2012 to 05 August 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted according to current test guidelines and GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: Oxypyrion-Acetate
CAS Number: 1742-79-6
Batch number: 11085913
Purity: Not stated
Date of arrival: 20 November 2012
Storage conditions: Refrigerated (2 to 8°C)

Test animals / tissue source

Species:
other:
Strain:
other:
Details on test animals or tissues and environmental conditions:
Bovine corneas supplied by 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.
Upon arrival at the test facility, the corneas were excised from the eyes and loaded onto the specifically designed holders. Both chambers of each holder were filled with pre-warmed Eagle’s Minimal Essential Medium (EMEM) (posterior chamber first), ensuring that no bubbles were formed. The holders were incubated at 32°C ± 1°C for at least 1 hour. After the incubation, the media was removed from both the anterior and posterior chambers. Fresh media was added to the posterior chamber first and then the anterior chamber (this media replacement order ensured the cornea retained its natural curvature as much as possible). The opacity of each cornea was measured using an opacitometer (model OP-KIT-3). Any corneas found to have scratches or increased neovascularization or an opacity of >7 opacity units when examined prior to treatment were discarded.

Test system

Vehicle:
other: The test item was tested as a 20% w/v solution in 0.9% sodium chloride solution.
Controls:
no
Amount / concentration applied:
The test item was tested as a 20% w/v solution in 0.9% sodium chloride solution.
The formulation was prepared on the day of use and was used approximately 3.5 hours after preparation, not within 2 hours of preparation as specified in the protocol. This deviation from protocol was considered not to have affected the integrity or outcome of the study as the OECD test guideline dose not specify a time period between preparation and use.

A volume of 750 µl of the test item formulation was applied to each of three corneas.
Duration of treatment / exposure:
A volume of 750 µl of the test item formulation was applied to each of three corneas followed by four hour vertical position incubation at 32°C, with shaking. After this incubation, each cornea was washed and the anterior chambers filled with pre-warmed EMEM (without phenol red) the opacities measured and the anterior chamber emptied. For the permeability endpoint, 1 ml of sodium fluorescein (5 mg/ml solution) was added into the anterior chamber and the corneas were incubated in the vertical position for 1.5 hours ± 5 minutes. Following this period, the media in the posterior chamber was removed and held in a labelled tube. Three 350 µl aliquots of this media (per cornea) were analysed for optical density at 490 nanometers (OD490).
A volume of 750 µl of the negative or positive control was similarly applied to further groups of three corneas. These groups were subject to the procedures detailed above.
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
Not applicable
Details on study design:
The study was conducted to meet the known requirements of OECD Guidelines for Testing of Chemicals Method 437 (adopted 7 September 2009).

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
151.27
Negative controls validity:
valid
Remarks:
in vitro irritation score 0
Positive controls validity:
valid
Remarks:
in vitro irritation score 66.87
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
Corneal Opacity:
The corneas treated with the test item were noted to be slightly cloudy. The corneas treated with the positive control were noted to be cloudy.
The mean opacity reading for the test item was 151.3
The mean opacity reading for the negative control was 0.
The mean opacity reading for the positive control was 40.7.

Corneal Permeability:
The mean group corrected optical density for the test item was -0.004.
The mean group corrected optical density for the negative control was 0.
The mean group corrected optical density for the positive control was 1.747.

In Vitro Irritation Score:
The calculated in vitro irritation score for the test item was 151.27
The calculated in vitro irritation score for the negative control was 0.
The calculated in vitro irritation score for the positive control was 66.87.

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

Test item

24

1

160

159

N/A

154.3

151.3

Test item

30

1

158

157

152.3

Test item

31

1

153

152

147.3

Negative

21

0

11

11

4.7

6.3

0.0

Negative

22

0

1

1

-3.7

Negative

23

0

2

2

-2.7

Positive

33

1

43

42

N/A

37.3

40.7

Positive

34

0

45

45

40.3

Positive

35

1

50

49

44.3

 

 

Corneal Permeability

 

Test substance

Cornea number

Mean blank OD490

OD490

Corrected OD490

Mean Corrected OD490

Final Corrected OD490

Mean group Corrected OD490

Test item

24

N/A

0.003

0.003

N/A

-0.003

-0.004

Test item

30

-0.001

-0.001

-0.006

Test item

31

0.002

0.002

-0.003

Negative

21

0.000

0.004

0.004

0.005

-0.002

0.00

Negative

22

0.005

0.005

0.000

Negative

23

0.008

0.008

0.002

Positive

33

N/A

1.816

1.816

N/A

1.810

1.747

Positive

34

1.492

1.492

1.487

Positive

35

1.949

1.949

1.944

 

 

Calculated IVIS

 

Test substance

Mean opacity

Mean permeability

IVIS (mean opacity + (15 x mean permeability))

Negative control

0

0

0

Positive control

40.7

1.747

66.87

Test item

151.3

-0.004

151.27

 

 

Applicant's summary and conclusion

Interpretation of results:
corrosive
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
The test item, Oxypyrion-Acetate, produced an IVIS score of 151.27 and was considered to be corrosive or severely irritating to the eye.
Executive summary:

This study was conducted to determine whether the test item, Oxypyrion-Acetate (CAS Nr 1742-79-6), causes corrosion or severe irritation to excised bovine corneas using the bovine corneal opacity and permeability (BCOP) assay (OECD guideline 437).

A volume of 750 µl of the test item formulation was applied to each of three corneas followed by a four hour incubation at 32°C. After this incubation, each cornea was washed with media containing phenol red followed by media without phenol red, the opacities measured and then the anterior chamber emptied. For the permeability endpoint, sodium fluorescein solution was added into the anterior chamber and the corneas were incubated in the vertical position for 1.5 hours ± 5 minutes. Following this period, the media in the posterior chamber was removed and three 350 µl aliquots of this media (per cornea) were analysed for optical density at 490 nanometers (OD490).

A volume of 750 µl of the negative or positive control was similarly applied to further groups of three corneas. These corneas were subject to the procedures detailed above.

The mean opacity reading for the test article was 151.3, for the negative control was 0 and for the positive control was 40.7.

The mean group corrected optical density for the test article was -0.004, for the negative control was 0 and for the positive control was 1.747.

The test item, Oxypyrion-Acetate, produced an IVIS score of 151.27 and was considered to be corrosive or severely irritating to the eye.