Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 - 19 Aug 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

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)
Version / remarks:
(2013)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
Triethoxyhexadecylsilane
EC Number:
240-465-9
EC Name:
Triethoxyhexadecylsilane
Cas Number:
16415-13-7
Molecular formula:
C22H48O3Si
IUPAC Name:
triethoxy(hexadecyl)silane
Test material form:
liquid

Test animals / tissue source

Species:
cattle
Strain:
other: not provided
Details on test animals or tissues and environmental conditions:
TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in vitro test method used for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The opacity and permeability assessments of the cornea are combined to derive an in vitro
irritancy score (IVIS), which is used to classify the irritancy level of the test substance.

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: A. Moksel AG, Buchloe, Germany
- Transport medium and temperature conditions: HBSS containing Pen/Strep on ice

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: Isolated corneas were mounted in cornea holders
- Type of cornea holder used: not specified, Duratec GmbH, Germany
- Description of the cornea holder: The cornea holders consist of an anterior and a posterior compartment, which interface with the epithelial and endothelial sides of the cornea
- Test medium and temperature conditions used in the cornea holder: RPMI 1640 (without phenol red) supplemented with 1% [v/v] fetal bovine serum and 2 mM [v/v] L-glutamine; prior to use: pre-warmed to 32 ± 1 °C
- Equilibration time: 1 h at 32 ± 1°C

DETERMINATION OF THE INITIAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer
- Specification of the device: opacitometer (BASF-OP3.0, Duratec GmbH, Germany)

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: number of eyes for the negative control: 3; number of eyes for the positive control: 3
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied in the test: 750 μL
- Concentration (if solution): liquids: 100%

VEHICLE
- Substance: physiol. saline
- Concentration (if solution): 0.9% NaCl solution
- Amount(s) applied in the test: 750 μL

POSITIVE SUBSTANCE
- Substance: ethanol
- Concentration (if solution): 100%
- Amount(s) applied in the test: 750 μL
Duration of treatment / exposure:
10 min
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
number of eyes for the test item: 3
Details on study design:
TEST CONDITIONS
- Open-Chamber method: Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The controls or test substance were applied directly to the epithelial surface of the cornea.

POST-EXPOSURE TREATMENT
- Removal of the test substance: After 10 minutes incubation at 32 ± 1°C either the test substance or the control substance was removed and the epithelium washed at least three times.
- Medium for washing the corneas: MEM containing phenol red
- Medium for final rinsing: complete RPMI (without phenol red)

DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer
- Time of determination: Directly after refilling fresh RPMI without phenol red in the anterior chamber the final opacity was measured
- Specification of the device: BASF-OP3.0, Duratec GmbH, Germany

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: Sodium fluorescein solution was added to the anterior chamber of the cornea holder while the posterior chamber was filled with RPMI fresh medium. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured via spectrophotometry at 490 nm recorded as optical density (OD490)
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (4 mg/mL)
- Incubation time: 90 min at 32 ± 1°C

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
10 min
Value:
0.16
Vehicle controls validity:
valid
Remarks:
0.95
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
18.16
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
10 min
Value:
0.25
Vehicle controls validity:
valid
Remarks:
1.16
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
40.34
Remarks on result:
no indication of irritation
Irritation parameter:
other: permeability
Run / experiment:
10 min
Value:
0.006
Vehicle controls validity:
valid
Remarks:
0.014
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
1.479
Other effects / acceptance of results:
The eye irritancy potential of the test item was investigated in the bovine corneal opacity and permeability assay resulting in an in vitro irritation score (IVIS) of 0.25. Therefore, the test item was considered as not requiring classification for eye irritation or serious eye damage. The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Any other information on results incl. tables

Table 1: Corneal opacity

Parameter

OD490

(Initial opacity)

OD490

(final opacity)

OD490

(change of opacity value)

 

Corrected opacity value

Mean

 Negative control

1.14

2.53

1.39

 

-

 

0.95

1.06

2.03

0.96

1.21

1.69

0.48

 Positive control

2.14

22.78

20.64

19.69

 

18.16

2.26

19.69

17.43

16.49

2.03

21.28

19.25

18.30

 

Test item

0.35

1.80

1.45

0.50

 

0.16

1.28

2.37

1.09

0.15

0.92

1.69

0.77

-0.18

Table 2: Permeability data

Parameter

OD490

 

Corrected OD490

 

Mean

 

Negative control

0.018

 

-

 

0.014

0.010

0.014

 

Positive control

1.890

1.876

 

1.479

1.208

1.194

1.380

1.366

 

Test item

0.008

-0.006

 

0.006

0.034

0.020

0.018

0.004

Table 3: In Vitro Irritation Score

 

Mean IVIS

Negative control

1.16

Positive control

40.34

Test item

0.25

Table 4: Historical Mean In Vitro Irritation Score of the Negative and Positive Control

 

Mean IVIS

Standard Deviation

Negative control

0.73

0.57

Positive control

48.80

10.37

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria are not met, no classification required according to Regulations (EC) No 1272/2008
Conclusions:
According to the bovine corneal opacity and permeability assay, the test item can be considered as not requiring classification for eye irritation or serious eye damage.