Registration Dossier

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:
2011-07-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with OECD guideline 437 (2009) : Bovine Corneal Opacity and Permeability Assay.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 437 : Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants (September 2009)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name : Jarocol Blue 2
Batch number : JBT03110003
Purity : 99.4%
CAS number : 33229-34-4
EC number : 251-410-3
Name : 2-[(4-N,N-bis-(2'-hydroxyethyl)amino-2-nitrophenyl)amino]ethanol

Test animals / tissue source

Species:
other: In-vitro assessment using fresh bovine corneae
Strain:
other: In-vitro assessment using fresh bovine corneae

Test system

Vehicle:
other: 0.9% (w/v) NaCl in deionised water)
Controls:
other: In-vitro assessment using fresh bovine corneae
Amount / concentration applied:
20% (w/v) preparation in 0.9% (w/v) sodium chloride solution of the test item
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
Not applicable
Details on study design:
This study should provide a rational basis for risk assessment in humans. An ocular contact is one of the probable routes of human exposure. This test is designed to measure the opacity of the cornea by quantifying the ability of light to pass through it. The permeability, as a result of the irritation potential of the test item, is determined using Na-fluorescein solution. Comparison of the opacity before and afterexposure to test item and determination of permeability after treatment provide an indication of the irritant of the test item. For this purpose the inflammatory in vitro changes of the bovine cornea after application of the test item is measured with regard to the opacity and permeability of dye. The results of both criteria are combined. The resulting in vitro irritation factor is compared with a criterion scale, produced prior to the experiment. The changes are classified into four grades : mild, moderate, severe, very severe.

Results and discussion

In vivo

Results
Irritation parameter:
other: In-vitro irritation score
Basis:
other: Test item corneae
Time point:
other: 240 minutes
Score:
5.4

Any other information on results incl. tables

Evaluation of Results

Opacity

The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneae. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

Permeability

The corrected OD490 was calculated by subtracting the mean OD490 of the negative control corneae from the OD490 value of each treated cornea. The OD 490 value of each treatment group was calculated by averaging the corrected OD490 values of the treated corneae for the treatment group.

In-Vitro Score Calculation

The following formula was used to determine the in-vitro irritancy score :

In-vitro Irritancy Score = mean opacity value + (15 X mean OD490 value)

Results

Corneal Opacity and Permeability Measurements

 Test Group  Cornea Number  Opacity Value = Difference (t240 -t0) of Opacity  Permeability at 490 nm(OD490nm)  In-Vitro Irritancy Score
 Negative Control  1  0  0.055  
 Negative Control  2  2  0.033  
 Negative Control  3  3  0.063  
 Negative Control    1.7*  0.050**  2.4
 Positive Control  4  64.3  2.745  
 Positive Control  5  71.3  2.260  
 Positive Control  6  54.3  2.670  
 Positive Control    63.3***  2.558***  101.7
 Test Item  7  7.3  0.024  
 Test Item  8  3.3  0.041  
 Test Item  4.3 0.019   
 Test Item    5.0*** 0.028***   5.4

* : Mean of the post treatment

** : Mean permeability

*** : Mean corrected value

Corneal Epithelium Condition Post Treatment

 Treatment  Cornea Number  Observation Post Treatment
 Negative Control  1  clear
Negative Control   clear
 Negative Control  clear
 Positive Control  4  cloudy
 Positive Control  5  cloudy
 Positive Control  6  cloudy
 Test Item  7  clear
 Test Item  8  clear
 Test Item  9  clear

Applicant's summary and conclusion

Interpretation of results:
other: Not an ocular corrosive or severe irritant
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 in-vitro study was performed to assess the corneal irritation and damage potential of 2-[(4-N,N-bis-(2'-hydroxyethyl)amino-2-nitrophenyl)amino]ethanol (HC Blue 2) by means of the BCOP assay using fresh bovine corneae. Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an in-vitro irritancy score. Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints. The positive control (20% w/v imidazole) showed clear opacity and distinctive permeability of the corneae and therefore is classified as very severe eye irritant. The test item 2-[(4-N,N-bis-(2'-hydroxyethyl)amino-2-nitrophenyl)amino]ethanol (HC Blue 2) is considered not to be an ocular corrosive or severe irritant.