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Diss Factsheets

Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 August 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes (incl. QA statement)

Test material

1
Chemical structure
Reference substance name:
(8S)-7,7,8,9,9-pentamethyl-5H,6H,7H,8H,9H-cyclopenta[h]quinazoline
EC Number:
801-093-8
Cas Number:
1315251-11-6
Molecular formula:
C16H22N2
IUPAC Name:
(8S)-7,7,8,9,9-pentamethyl-5H,6H,7H,8H,9H-cyclopenta[h]quinazoline
Test material form:
solid

Test animals / tissue source

Species:
other: Eyes from adult cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee and placed in Hanks’ Balanced Salt Solution (HBSS), supplemented with Penicillin/Streptomycin, and transported to the laboratory on ice packs. The corneas were prepared immediately on arrival.

Test system

Vehicle:
other: 0.9 % w/v sodium chloride solution.
Controls:
yes
Amount / concentration applied:
The substance was applied at a concentration of 20 % w/v in 0.9 % w/v sodium chloride solution.
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
Not applicable
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 (epithelial side uppermost) 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. (MEM) and plugged. The holders were incubated at 32 ± 1 ºC 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 MEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the substance and three corneas to the positive control item.

Treatment of Corneas
The MEM was removed from the anterior chamber of the BCOP holder and the substance preparation or control items were applied to the cornea. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes. At the end of the exposure period the substance preparation and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior and posterior chambers were refilled with fresh complete MEM. A post treatment opacity reading was taken and each cornea was visually observed.

Application of Sodium Fluorescein
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 sodium fluorescein solution (5 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 medium representing each cornea was applied to a designated well on a 96 well plate and the optical density at 492 nm (OD492) was measured. If values greater than 2.500 OD492 were obtained a 1 in 5 dilution of the medium in complete MEM was performed and the measurement repeated. The modified value was multiplied by 5 to reflect the 1 in 5 dilution.

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

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Remarks:
mean
Value:
1.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
No. 6
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
No. 7
Value:
3.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
No. 9
Value:
1.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: Permeability
Run / experiment:
No. 6
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: Permeability
Run / experiment:
No. 7
Value:
0.001
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: Permeability
Run / experiment:
No. 9
Value:
0.026
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Table 1 Individual and Mean Corneal Opacity and Permeability Measurements:

Treatment

Cornea No.

Opacity

Permeability (OD)

In vitro irritancy score

Pre-treatment

Post-treatment

Post-treatment – pre-treatment

Corrected value

 

Corrected value

Negative control

1

0

2

2

 

0.042

 

 

4

1

4

2

 

0.044

 

 

8

1

4

2

 

0.026

 

 

 

 

 

2.7*

 

0.037***

 

3.2

Positive control

2

0

57

57

54.3

1.725

1.688

 

3

0

63

63

60.3

1.541

1.504

 

5

2

46

44

41.3

1.165

1.128

 

 

 

 

 

52.0**

 

1.440**

73.6

Substance

6

2

4

2

0.0

0.032

0.000

 

7

2

8

6

3.3

0.038

0.001

 

9

0

4

4

1.3

0.063

0.026

 

 

 

 

 

1.6**

 

0.009**

1.7

OD = Optical density

* = Mean of the post-treatment-pre-treatment values

***= Mean permeability

**= Mean corrected value

Table 2 Corneal Epithelium Condition Post Incubation

Treatment

Cornea no.

Observation

Post treatment

Negative control

1

Clear

4

Clear

8

Clear

Positive control

2

Cloudy

3

Cloudy

5

Cloudy

Substance

6

Clear

7

Clear

9

Clear

Applicant's summary and conclusion

Interpretation of results:
other:
Remarks:
Not a eye irritant in accordance with EU CLP (EC no 1272/2008 and its amendments)
Conclusions:
The substance was considered not to be an ocular corrosive or severe irritant based on the OECD TG 437
Executive summary:

A study was performed to assess the ocular irritancy potential of the substance to the isolated bovine cornea. The method was designed to be compatible with OECD TG 437 and GLP principles. The substance was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive control items were tested concurrently. 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). A substance that induces an In Vitro Irritancy Score ≥ 55.1 is defined as an ocular corrosive or severe irritant.

Results showed that the in vitro irritant score were 1.7, 3.2 and 73.6 for the substance, negative control and positive control, respectively.

In conclusion, the substance was considered not to be an ocular corrosive or severe irritant.