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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:
06 June 2017 - 13 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
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)
Version / remarks:
26 July 2013
Deviations:
yes
Remarks:
see table 7.3.2/1 in Any Other Information on Materials and Methods
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-methylheptyl acrylate
EC Number:
256-005-5
EC Name:
1-methylheptyl acrylate
Cas Number:
42928-85-8
Molecular formula:
C11H20O2
IUPAC Name:
octan-2-yl prop-2-enoate
Test material form:
liquid

Test animals / tissue source

Species:
other: bovine cornea
Details on test animals or tissues and environmental conditions:
Species: bovine cattle

Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
Age: as French Authorities avoid the use of any organs from the head of bovines aged more than 12 months, bovine cattle were up to 12 months old (typically, 5 to 8 months old).

Reason for choice: bovine corneas are recommended by Regulatory Authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.

Transport from Supplier to laboratory: the eyes were transported to the laboratory at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas. Upon arrival at the laboratory, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny.
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
Washing of the corneas: the corneas were rinced in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature.
The corneas were used immediately.

(Pre)Incubation T°C: 32°C

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL
- Concentration: undiluted

VEHICLE
- none
Duration of treatment / exposure:
Exposure period of 10 minutes (± 30 seconds) at +32°C, followed by rinsing.
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
2 hours (± 10 minutes) at +32°C
Number of animals or in vitro replicates:
The test item and the negative control were tested on three corneas each.
The positive control was tested on two corneas, instead of three.
Details on study design:
REMOVAL OF THE TEST SUBSTANCE
On completion of the treatment period, the test item was removed from the front opening of the anterior chamber and the epithelium was rinsed as follows:
- the anterior chamber was emptied using a metal gavage tube attached to a vacuum pump,
- residual test item adhering to the walls of the anterior chamber was removed with a cotton bud,
- the corneas were rinsed four times with pre-warmed cMEM containing phenol red (i.e. until the test item had been completely removed from the chamber or until the phenol red was not discoloured). Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.
Some difficulties were encountered since residual amounts of test item were observed on the walls of the anterior chamber, but they were removed at the end of the rinsing step.
- Time after start of exposure: 10 minutes

SCORING SYSTEM / TOOL USED TO ASSESS SCORE:
- Opacity:
Using an opacitometer
The average change in opacity during exposure is determined. It is corrected by subtracting the average negative control value from values of positive control and test item treated corneas.
- Permeability:
Using a spectrophotometer: optical density (OD) at 490 nm wavelength
The optical density is corrected by subtracting the average negative control value from values of positive control and test item treated corneas.
- Scoring:
In vitro irritancy score (IVIS) = Corrected Opacity + (15 x Corrected OD)

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
1
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
53
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
0.3
Negative controls validity:
valid
Remarks:
0.0
Positive controls validity:
valid
Remarks:
18.0
Irritation parameter:
fluorescein leakage
Run / experiment:
mean
Value:
0.062
Negative controls validity:
valid
Remarks:
0.023
Positive controls validity:
valid
Remarks:
2.315
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system:
No notable opaque spots or irregularities were observed on all negative control-treated corneas.
Fluorescein fixation was observed on the three corneas treated with the test item.
Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (Negative control: opacity = 0.0 +/- 0.0; permeability = 0.023 +/- 0.005, Upper limit of the historical mean for opacity and permeability 3 and 0.036 respectively)
- Acceptance criteria met for positive control: yes ( IVIS = 53 +/-1.7 The positive control IVIS was within the range of historical mean +/- 2 SD: 27 - 63).

Any other information on results incl. tables

Table 7.3.2/3: Individual and Mean Corneal Opacity and Permeability Measurements

 

Treatment

Cornea number

 

OPACITY

PERMEABILITY

In Vitro Irritancy Score (IVIS)

Negative control

Pre-Treatment

Post-Treatment

Change Post- Pre-Treatment

Corrected Value

OD490 nm

Corrected Value

71

2

1

-1

0

0.024

0.024

 

77

2

1

-1

0

0.018

0.018

 

74

2

1

-1

0

0.027

0.027

 

Mean

 

 

 

0.0

 

0.023

 

SD

 

 

 

0.0

 

0.005

 

 

Test item

Cornea number

Pre-Treatment

Post-Treatment

Change Post- Pre-Treatment

Corrected Value

OD490 nm

Corrected Value

In Vitro Irritancy Score (IVIS)

85

2

3

1

1

0.010

0.010

1

82

3

2

-1

0

0.155

0.132

2

67

3

3

0

0

0.078

0.055

1

Mean

 

 

 

0.3

 

0.062

1

SD

 

 

 

0.6

 

0.066

0.6

 

Positive

control

Cornea number

Pre-Treatment

Post-Treatment

Change Post- Pre-Treatment

Corrected Value

OD490 nm

Corrected Value

In Vitro Irritancy Score (IVIS)

59

2

23

21

21

2.060

2.037

52

62

2

17

15

15

2.616

2.593

54

 

 

 

 

 

 

 

 

Mean

 

 

 

18.0

 

2.315

53

SD

 

 

 

4.2

 

0.393

1.7

OD= Optical Density

SD: Standard Deviation

Negative control: 0.9% NaCl

Positive control: 100% Ethanol

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
the test substance is considered as not requiring classification for eye irritation or serious eye damage.
Executive summary:

A study was performed to assess the ocular irritancy potential of the test substance to the isolated bovine cornea. The study was conducted according to the OECD guideline No. 437 and in compliance with the principles of Good Laboratory Practice .Negative (Sodium chloride 0.9%) and positive (Ethanol 100%) control items were tested concurrently. 

A single experiment was performed using three corneas for test substance and negative control; and two corneas for positive control.

Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test substance, applied undiluted, the negative and the positive controls were evaluated in a single experiment using a treatment time of 10 minutes and the closed-chamber treatment method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. The corneas were then incubated for 2 hours at +32 °C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated for 90 minutes at +32°C. At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined to generate an In Vitro Irritancy Score (IVIS). 

To consider the study as valid, the positive control should elicit an In Vitro Score that falls within two standard deviations of the historical mean for the laboratory, which was the case; IVIS of ethanol was found to be 53 +/- 1.7. Furthermore, the negative control mean opacity and mean OD490nm should be less than the established upper limit of historical means which was the case as opacity of sodium chloride 0.9% was 0.0 +/- 0.0 and historical value was 3.00 while the permeability of sodium chloride 0.9% was 0.023 +/-0.005 and historical value was 0.036.

 IVIS of the test substance was determined to be 1 +/- 0.6, which was below the cut-off value of 3 and thus indicating that the test substance was not requiring classification for eye irritation or serious eye damage.

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