Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-03 to 2009-06-05
Reliability:
1 (reliable without restriction)
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Environmental Protection Agency. 1996. Label Review Manual: 2nd Edition. EPA737-B-96-001. Washington, DC: U.S. Environmental Protection Agency
Deviations:
no
Qualifier:
according to
Guideline:
other: European Union. 2001. General Classification and Labelling Requirements for Dangerous Substances and Preparations. In Directive 2001/59/EC (28th adaption); Annex 6 of Council Directive 67/548/EEC on the approximation of the laws, regulations and administr
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Aldimine 2
- Physical state: liquid, almost colourless / faint yellow
- Analytical purity: 96.7 %
- Lot/batch No.: ub2.398B/11
- Expiration date of the lot/batch: 04 August 2011
- Stability under test conditions: stable for at least 3 years
- Storage condition of test material: room temperature, closed bottle

Test animals / tissue source

Species:
other: Eyes from 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) containing the antibiotics Penicillin (100 IU/mL) and Streptomycin (100 µg/mL) and transported to the laboratory on ice packs. The eyes were used within 5 hours of slaughter.

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 BCOP holders.
The anterior and posterior chambers of each BCOP holder were filled with fresh complete MEM and plugged. The holders were incubated at 32 ±1ºC for at least 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Test system

Vehicle:
other: sodium chloride
Controls:
not required
Amount / concentration applied:
For the purpose of this study the test material was prepared as a 10% v/v concentration in 0.9% w/v sodium chloride solution.
0.75 mL of the test material or control materials were applied directly to the epithelial surface of the appropriate corneas.
Duration of treatment / exposure:
The test material was applied as a 10% v/v concentration for 10 minutes followed by a post incubation period of 120 minutes with MEM (without the tets substance).
Observation period (in vivo):
The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.
Number of animals or in vitro replicates:
Not applicable
Details on study design:
Application of Sodium Fluorescein:
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from both chambers (anterior chamber first) was removed. The posterior chamber was refilled with fresh complete MEM and 1 mL of 4 mg/mL sodium fluorescein was applied to the anterior chamber. The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1ºC for 90 minutes ± 5 minutes.

Permeability Determinations:
After incubation all the medium in the posterior chamber of each holder was decanted using a syringe with a needle attached and thoroughly mixed in tubes, pre-labelled according to holder number, so that a representative sample was obtained for the OD492 determination.
360 µL of medium representing each cornea was applied to a designated well on a 96 well plate. Two wells were designated as blanks remained empty.These were used for blank subtraction purposes. The optical density at 492nm (OD492) was measured using the Anthos 2001 micro plate reader.
A sodium fluorescein calibration curve was performed to determine the linear range of the testing facilities microplate reader.

Results and discussion

In vivo

Results
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.
Score:
1.6
Reversibility:
other: not applicable
Remarks on result:
other: Regarding the maximal score, a substance that induces an IVIS (in vitro score) >= 55.1 is defined as a corrosive or severe irritant.
Irritant / corrosive response data:
Following assessment of the data for all endpoints the test material was considered to be a mild ocular irritant (see table below) and same as for the negative control.
Other effects:
No other effects

Any other information on results incl. tables

The in vitro Irritancy scores are summarised as follows:

Treatment

In VitroIrritancy Score

Classification

Test Material

1.6

Mild Irritant

Negative Control

0.9

Mild Irritant

Positive Control

77.1

Severe Irritant

As it appears in the table, the test material and the negative control scores are relatively similar and therefore were classified identically in this table. Thus, the test item is practically not irritating.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
Comparison of the in vitro irritancy scores of the test substance, negative control and positive control showed that the test substance is not a skin irritant.
Executive summary:

The study was performed to assess the ocular irritancy potential of the test substance to isolated bovine cornea. The study followed the procedures of the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) recommended protocol for the Bovine Corneal Opacity and Permeability (BCOP) test method. The test material was prepared as a 10% v/v concentration in 0.9% w/v sodium chloride solution. 0.75 mL of the test material or control materials were applied directly to the epithelial surface of the appropriate corneas (ones that were found at the beginning of the study to be free of damage) for 10 minutes, followed by a post incubation period of 120 minutes with MEM (without the test substance). The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. Once the opacity and mean permeability (OD490) values have been corrected for background opacity and the negative control permeability OD490 values, the mean opacity and permeability OD490 values for each treatment group was combined in an empirically-derived formula to calculate an in vitro score (IVIS) for each treatment group. The in vitro irritancy scores for the test material (1.6) and the negative control (0.9% sodium chloride solution) (0.9) were relatively similar while the result of the positive control (ethanol) was as expected high (77.1). A substance that induces an IVIS (in vitro score) ≥ 55.1 is defined as a corrosive or severe irritant, therefore the test material was not considered to be a severe skin irritant. Results of the BCOP test can indicate of positive results - a severe skin irritant or corrosive and when the test results are negative (not skin irritating not corrosive) an in vivo study is required. As under the REACH requirements for the test substance tonnage (REACH section 8.2 column 2) only an in vitro study for eye irritation is required, the study conclusion defined the test substance as a mild irritant although it is practically not irritant (see classification and scores of of test substance and negative control in the table of remarks on results in IUCLID 5 7.3.2)) thereby avoiding the possibility of testing Sika Hardener LG in an in vivo eye irritation study which is anyway not required under REACH.