Registration Dossier

Administrative data

Endpoint:
eye irritation
Remarks:
other: validated "in vitro" test method
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-08-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study reliable without restrictions Deviations from the guideline without an impact on the outcome of this study: According to the OECD guideline, for surfactants (means with a surface tension < 60 mN/m) a different treatment protocol should be used (e.g. tested at concentration of 10% w/v test item solution instead of 20% w/v and reduced exposure time for treatment of cornea are needed). Although the tested substance has surface active properties (41.9 mN/m), the protocol for a non surface active solid substance was used for test performance. However, it is explicitly stated that this has no impact on the outcome of this study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guideline for Testing of Chemicals 437: Bovine Corneal Opacity and Permeability Test method for Identifying Ocular and Severe Irritants (September 2009)
Deviations:
yes
Remarks:
, please refer to the "Rationale for reliability incl. deficiencies" above
Qualifier:
according to
Guideline:
other: Commission Regulation (EU) No 1152/2010: B. 47. Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants (Official Journal of the European Union, L 324, 9.12.2010).
Deviations:
yes
Remarks:
, please refer to the "Rationale for reliability incl. deficiencies" above
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-03-30

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes
Details on test material:
- CAS name: Cyclohexanol, 4-(1,1-dimethylethyl)-, trans-
- Molecular formula: C10H20O
- Molecular weight: 156.3 g/mol
- Physical state: solid

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.

Test system

Vehicle:
physiological saline
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL of the test item was applied
- Concentration (if solution): 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item

Duration of treatment / exposure:
240 minutes
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
not applicable
Details on study design:
PREPARATION OF THE INCUBATION MEDIA
The medium MEM (Minimum Essential Medium), supplemented with sodium bicarbonate and L-glutamine was prepared one day prior to the start of the assay, and stored in a refrigerator (2 - 8 °C).
Immediately before starting the test, MEM was supplemented with 1% foetal calf serum (FCS). Afterwards, it was called cMEM (complete medium).
The OECD foresees the use of EMEM (Eagle’s minimal essential medium) which is in composition and osmolarity equivalent to the cMEM, thus cMEM could be used without restriction.

COLLECTION OF BOVINE EYES
Freshly isolated bovine eyes from at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in Hank’s BSS supplemented with streptomycin / penicillin at ambient temperature. The corneas were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 supplemented with Lglutamine, Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day. Shortly before use, Dextran was added to the medium.

PREPARATION OF CORNEAE
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the experiment were collected in Hank’s BSS supplemented with streptomycin / penicillin and checked finally with a view box for defects listed above.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, annex III, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (Oring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneas in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the complete medium was removed from both compartments and replaced by fresh cMEM, and the basal opacity was determined (t0).

OUTLINE OF STUDY
The anterior compartment received the test item or negative control (saline (produced in-house, lot no. 25.6.11)) or positive control (10% (w/v) Benzalkonium chloride (Sigma, Steinheim, Germany, lot no. 036K0208) in 0.9% (w/v) NaCl in deionised water) at a volume of 0.75 mL each on the surface of the corneas and was incubated at 32 ± 1 °C in the waterbath in a horizontal position. The test item, positive control and negative control were tested in triplicate.
Prior to the application the test item was melted up to 75 °C in the water bath and suspended in saline (20% (w/v)). The positive control was 10% (w/v) Benzalkonium chloride in saline. Saline was used as negative control item.
According to the OECD guideline, for surfactants (means with a surface tension < 60 mN/m) a different treatment protocol should be used (e.g. tested at concentration of 10% w/v test item solution instead of 20% w/v and reduced exposure time for treatment of cornea are needed).
Although the tested substance SymSitive® 1609 pur has surface active properties (< 60 mN/m), the protocol for a non surface active solid substance was used for test performance. However, it is explicitly stated that this has no impact on the outcome of this study.
The incubation time lasted 240 minutes (± 5 minutes).
After the test item or control items, respectively, were rinsed off for three times from the application side with saline. No visual evidence of the test item could be observed afterwards. Fresh cMEM was added into the anterior compartment and opacity was measured (t240). There was no need to add phenol red to the medium as a second marker for successful rinsing since the washing steps were performed very stringently.
In the second step of the assay, permeability of the cornea was determined. 1 mL of a Nafluorescein solution, 0.5 % (w/v) dissolved in HBSS (Hank’s buffered salt solution), was placed in the anterior compartment. Corneas were incubated again in a horizontal position for an additional 90 minutes at 32 ± 1 °C in the water-bath. The optical density of an aliquot of the mixed complete medium from the posterior chamber was measured spectrophotometrically at 490 nm (OD490).

OPACITY MEASUREMENT
The basal opacity of all corneas was recorded using an opacitometer. Each corneas with a value of the basal opacity > 7 was discarded.
After the incubation time of 240 minutes with the test item, positive control and negative control the opacity was measured again (t240).

PERMEABILITY DETERMINATION
The permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) fluorescein solution in HBSS. Corneas were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

CRITERIA FOR DETERMINATION OF A VALID TEST
The test was acceptable if the in vitro irritation score of the positive control was ≥ 30 and the in vitro irritation score of the negative control was ≤ 3.

EVALUATION OF RESUTLS
- Opacity: the change of opacity value of each treated cornea or positive and negative control corneas was calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae was calculated and this value was subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- Permeability: the corrected OD490 value of each cornea treated with positive control and test item was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IN VITRO SCORE CALCULATION
The following formula was used to determine the in vitro irritation score of the negative control:
In vitro Irritation Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro irritation score of the positive control and the test item:
In vitro Irritation Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The in vitro irritation score was calculated for each individual treatment and positive control cornea. The mean in vitro irritation score irritation value of each treated group was calculated from the individual in vitro irritation score values. Depending on the score obtained, the test item was classified into the category according to OECD guideline 437 (table 1 in the field "Any other information on materials and methods incl. tables" below).

Results and discussion

In vivo

Results
Irritation parameter:
other: In vitro score
Basis:
mean
Time point:
other: 240 minutes
Score:
0.33
Irritant / corrosive response data:
Relative to the negative control, the test item did not cause any increase of the corneal opacity or permeability. The calculated mean in vitro irritation score was 0.33. According to OECD 437 the test item is classified as not corrosive / not severe irritant to the eye.

Any other information on results incl. tables

Table 1: Results after 240 minutes incubation time

Test group

Opacity value = Difference (t240 – t0) of Opacity

Permeability at 490 nm (OD490)

In vitro Score

Mean in vitro irritation score

Proposed in vitro Irritation Scale

 

 

Mean

 

Mean

 

 

 

Negative control

1

1.00

0.077

0.075

2.16

2.12

Non corrosive /non severe irritant

1

0.073

2.10

1

0.074

2.11

Positive control

191.00*

0.939*

205.09

287.24

Corrosive /severe irritant

377.00*

2.390*

412.86

230.00*

0.917*

243.76

SymSitive®1609 pur

1.00*

0.039*

1.59

0.33

Non corrosive /non severe irritant

-1.00*

0.029*

-0.56

0.00*

-0.003*

-0.04

* corrected values

- with the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro irritation score 2.12).

-the positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneas (mean in vitro irritation score 287.24) corresponding to a classification as corrosive /severe irritant to the eye (CLP/EPA/GHS (Cat 1)).

Table 2: Historical Data

 

Positive control

Negative control

Meanin vitroIrritation Score

162.65

1.70

Standard Deviation

38.05

0.79

Applicant's summary and conclusion

Interpretation of results:
other: not corrosive/severely eye irritating
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, the test item is not corrosive / not severely irritating to the eye (CLP (Cat 1)).