Registration Dossier

Diss Factsheets

Toxicological information

Eye irritation

Currently viewing:

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between the 03 January 2019 and 03 January 2019.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 2-methoxy-6-methylocta-1,5-diene and 2-methoxy-6-methylocta-2,5-diene
EC Number:
950-170-3
Molecular formula:
C10H18O
IUPAC Name:
Reaction mass of 2-methoxy-6-methylocta-1,5-diene and 2-methoxy-6-methylocta-2,5-diene
Specific details on test material used for the study:
Identification: PG-RAW-0004
Physical state/Appearance: Clear colorless liquid
Storage Conditions: Room temperature in the dark

Test animals / tissue source

Species:
other: Eyes from adult cattle
Strain:
other: Not applicable
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 prepared immediately on arrival.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the test item or control items were applied to the cornea
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
Triplicate
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 ± 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 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 item and three corneas to the positive control item.

Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. 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 10 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 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.

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.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
1.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The positive control In Vitro Irritancy Score was within the acceptance range. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity and permeability values below the established upper limits. The negative control acceptance criteria were therefore satisfied.

Any other information on results incl. tables

Corneal Epithelium Condition

The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment

In Vitro Irritancy Score

Test Item

1.9

Negative Control

1.1

Positive Control

53.5

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD492)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post Incubation

Post-Incubation - Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

2

3

3

4

1

 

0.000

 

 

3

6

6

8

2

 

0.012

 

 

4

6

6

6

0

 

0.000

 

 

 

 

 

 

1.0*

 

0.004¨

 

1.1

Positive Control

5

6

52

50

44

43.0

0.572

0.568

 

6

3

48

45

42

41.0

0.988

0.984

 

9

3

32

34

31

30.0

1.545

1.541

 

 

 

 

 

 

38.0·

 

1.031·

53.5

Test Item

10

4

6

7

3

2.0

0.045

0.041

 

11

3

5

6

3

2.0

0.006

0.002

 

12

3

4

5

2

1.0

0.001

0.000

 

 

 

 

 

 

1.7·

 

0.014·

1.9

OD= Optical density           * = Mean of the post-incubation -pre‑treatment values           ¨= Mean permeability                     ·= Mean corrected value

Corneal Epithelium Condition Post Treatment and Post Incubation

Treatment

Cornea Number

Observation

Post Treatment

Post Incubation

Negative Control

2

Clear

Clear

3

Clear

Clear

4

Clear

Clear

Positive Control

5

Cloudy

Cloudy

6

Cloudy

Cloudy

9

Cloudy

Cloudy

Test Item

10

Clear

Clear

11

Clear

Clear

12

Clear

Clear

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test item was considered not to be an ocular corrosive or severe irritant.
Executive summary:

The eye irritancy potential of the PG-RAW-0004 was assessed according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and is not classified, according to EU CLP criteria.