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:
weight of evidence
Study period:
From September 20, 2017 to September 22, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
BCOP test was performed to identify test substance that can induce serious eye damage and to identify test substance not requiring classification for eye irritation or serious eye damage.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
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:
Deviation was considered to have not affected the integrity or validity of the study.
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
yes
Remarks:
Deviation was considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: paste

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were refrigerated on arrival and used within 24 h of receipt.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL test (undiluted) or reference substance
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
32 ± 1ºC for 120 minutes
Number of animals or in vitro replicates:
Triplicates

Details on study design:
Preparation of corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of corneas and opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test substance and three corneas to the positive control substance.

Treatment of corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test substance or control substances were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the substance over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed. The negative and positive control data was shared with Envigo study number HN60FW and PB95SY.

Application of sodium fluorescein
Following the final 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 sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 µL of media representing each cornea was dispensed into the appropriate wells of a pre labeled 96 well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Data evaluation
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

Opacity measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

Permeability measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

In Vitro Irritancy Score
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test substance induced a response through only one of the two endpoints.

Visual observation
The condition of the cornea was visually assessed post treatment.

Major Computerized Systems
The following computerized systems were used in the study:
• Delta Building Monitoring System.
• Labtech LT-4500 microplate reader and LT-com software

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
ca. 32.7
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No prediction could be made about eye irritation potential
Other effects / acceptance of results:
The positive control In Vitro Irritancy Score was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied. The study has met validity criteria.

Any other information on results incl. tables

Results

Individual and mean corneal opacity and permeability measurement


Treatment

Cornea Number

Opacity

Permeability (OD)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post Incubation

Post-Incubation - Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control*

1

3

3

3

0

 

0.000

 

 

2

5

6

6

1

 

0.002

 

 

3

3

3

4

1

 

0.001

 

 

Mean

 

 

 

0.7

 

0.001

 

0.7

Positive Control*

4

4

36

34

30

29.3

0.664

0.663

 

5

4

35

35

31

30.3

1.280

1.279

 

6

2

35

35

33

32.3

1.067

1.066

 

Mean

 

 

 

 

30.7

 

1.003

45.7

Test substance

7

4

14

20

16

15.3

1.241

1.240

 

8

4

20

20

16

15.3

0.745

0.744

 

9

3

12

15

12

11.3

1.765

1.764

 

Mean

 

 

 

 

14.0

 

1.249

32.7

OD= Optical density            

* = Control data shared with Envigo - Shardlow study number HN60FW and PB95SY

Corneal epithelium condition post treatment

Treatment

Cornea Number

Observation

Post Treatment

Post Incubation

Negative Control

1

Clear

Clear

2

Clear

Clear

3

Clear

Clear

Positive Control

4

Cloudy

Cloudy

5

Cloudy

Cloudy

6

Cloudy

Cloudy

Test substance

7

Partly Cloudy

Cloudy

8

Partly Cloudy

Cloudy

9

Partly Cloudy

Cloudy

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

 

Treatment

In Vitro Irritancy Score

Test substance

32.7

Negative Control

0.7

Positive Control

45.7

 

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
other: inconclusive results based on CLP criteria
Conclusions:
Under the study conditions, no prediction of eye irritation potential for the test substance could be made.




Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the test substance, 'Cocodimonium hydroxypropyl hydrolysed wool' (active: 62.8%), using the Bovine corneal Opacity Test (BCOP) method, according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. Appropriately prepared bovine cornea, in triplicates were exposed to 0.75 mL of the test substance (undiluted) and reference substances (Negative control: sodium chloride 0.9% w/v, Positive control: Ethanol). The holders were gently tilted back and forth to ensure a uniform application of the substance over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1ºC for 10 minutes. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete Eagle’s Minimum Essential Medium (EMEM) containing phenol red before a final rinse with complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed. The negative and positive control data was shared with Envigo study number HN60FW and PB95SY. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The IVIS for test substance, negative control and positive control were reported to be 32.7, 0.7 and 45.7 respectively. The study had met all validity criteria, except for negative control, however, the deviation was considered to have not affected the integrity or validity of the study. Under the study conditions, based on the IVIS score of the test substance, which is >3 and <55%, no prediction of eye irritation potential could be made (Envigo, 2018).