Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was according to OECD guideline 437.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Principles of method if other than guideline:
Test System:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2 - 4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hank’s balanced salt solution (supplemented with 0.01 % streptomycin and 0.01 % penicillin). Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 hour.

Preparations:
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 °C. On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1 % fetal calf serum (= complete MEM) and stored in a water bath at 32 °C ± 1 °C. The same was performed with the MEM with phenol red. After the arrival of the corneas they were examined and only corneas which were free from defects were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM without phenol red was filled. The holders were then incubated for one hour in the incubation chamber at 32 °C.

Method Description:
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. The baseline opacity was measured by placing the holder with the cornea in a spectral photometer and recording the absorption at 570 nm. Opacity is calculated from the measured absorption. For each treatment group (negative control solution, test item suspension and positive control solution), three replicates were used. After removal of the pre-incubation medium, 750 μL negative control solution resp. test item suspension resp. positive control solution were applied to each replicate

According to the characteristics of the test item, the following treatment procedure was performed: Open Chamber Method:
The “open chamber-method” is used for solid substances. In order to apply the test item, the nut was unscrewed to remove the glass disc. The test item could be applied directly on the cornea now. 750 μL of the test item were tested as suspension at 20 % concentration in 0.9 % sodium chloride solution. The test item suspension was given on the epithelium in such a manner that as much as possible of the cornea was covered with test item suspension. Exposition time on the corneas was 4 h ± 5 min. at 32 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded at once (again by measurement at 570 nm). The cMEM without phenol red was then removed from the front chamber, and 1 mL sodium fluorescein solution (concentration
5 mg/mL) was added to the front chamber. The chambers were then closed again and incubated for 90 ± 5 min at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability of the cornea was measured as optical density of the liquid at 490 nm.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Quinine Base Anhydrous
- Molecular formula (if other than submission substance): C20H24N2O2
- Molecular weight (if other than submission substance): 324.43 g/mol
- Substance type: white powder
- Physical state: solid
- Analytical purity:98.6% Quinine,
- Impurities (identity and concentrations): 1.3% Dihydroquinine, 0.1% Other alcaloids (HPLC)
- Composition of test material, percentage of components: 100.0 % Alcaloid base (Titration)
- Lot/batch No.: Lot 7974
- Stability under test conditions: H2O; EtOH; acetone: 96h; CH3CN; DMSO: unknown
- Storage condition of test material: does not require any special temperature storage conditions, keep away from light and humidity

Test animals / tissue source

Species:
other: Bos primigenius Taurus (Fresh bovine corneas)

Test system

Vehicle:
physiological saline
Remarks:
0.9 % sodium chloride solution
Amount / concentration applied:
TEST MATERIAL
- Concentration (if solution): 20% in 0.9 % sodium chloride solution
Duration of treatment / exposure:
Incubation time: 4 hours at 32 ± 1 °C
Details on study design:
Negative Control: Sodium chloride solution: 0.9 % NaCl (CAS-No. 7647-14-5), dissolved in demin. water.
Positive Control: Imidazole solution: 20 % C3H4N2 (CAS-No. 288-32-4), dissolved in 0.9 % NaCl.
The test item is a non-surfactant solid substance. It was tested as a suspension at a concentration of 20% in 0.9 % sodium chloride solution.

Results and discussion

Any other information on results incl. tables

Opacity and Permeability Values

The absorption (570 nm) and opacity values which were measured before and after exposition are given in the following table:

A) Absorption and Opacity Values Negative Control:

 Parameter  Negative Control       
 Absorption before exposition  0.1534  0.1456  0.1523
 Absorption after exposition  0.2548  0.1948  0.2557
 Opacity before exposition  1.4236  1.3983  1.4200
 Opacity after exposition  1.7980  1.5660  1.8018
 Opacity Difference  0.3744  0.1677  0.3817

Mean opacity difference of the negative control is 0.3080.

B) Absorption and Opacity Values Test Item and Positive Control:

 Parameter  Test Item Quinine Base Anhydrous         Positive Control       
 Absorption before exposition  0.1916   0.2158  0.1423  0.1539  0.1389  0.1598
 Absorption after exposition  0.6452  0.5839 0.5777   1.5211  1.7040  1.5634
 Opacity before exposition  1.5545  1.6436  1.3877  1.4253  1.3769  1.4448
 Opacity after exposition  4.4177  3.8362  3.7818  33.1971  50.5825  36.5932
 Opacity Difference  2.8632  2.1926  2.3941  31.7718  49.2056 35.1484 

For the permeability measurement, three replicates for each treatment group were measured. The optical density values at 490 nm are given in the following table:

C) Optical density at 490 nm:

 Repl.  Negative Control  

 Test Item Quinine Base Anhydrous

 Positive Control       
 Meas.  0.0074 0.0059  0.0099   0.2255  0.2459  0.1908  0.5096 0.3197   0.3642
 Corr.  0.0370  0.0295  0.0495  1.1275  1.2295  0.9540  2.5480  1.5985  1.8210
 Mean 0.0387                 --

Note: In order to correct the path length, a factor of 5 was taken into account when calculating the IVIS.

IVIS Values

IVIS was calculated using the values in tables A, B and C and the equation IVIS = (opacity difference – mean opacity difference of the negative controls) + [15 * (corr.OD490 – mean corr. OD490 of the negative controls)]

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

D) IVIS:

 Test Group IVIS   Mean IVIS  Relative Standard Deviation IVIS
 Negative Control 0.9% NaCl  0.929    
   0.610  0.888  29.2 %
   1.124    
Test Item Quinine Base Anhydrous  18.887    
   19.747  18.150  11.4 %
   15.816    
 Positive Control 20% imidazole  69.103    
   72.295  67.658  8.1 %
  61.575     

Applicant's summary and conclusion

Interpretation of results:
Category 2B (mildly irritating to eyes)
Remarks:
Migrated information Criteria used for interpretation of results: other: UN GHS
Conclusions:
According to the study report, the read across substance quinine base anhydrous showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 18.15. Therefore, quinine base anhydrous is mildly irritating to eyes and we assume, that quinine dihydrochloride is also mildly irritating to eyes.
Executive summary:

This in vitro study was performed to assess serious eye damage of the read across substance quinine base anhydrous by quantitative measurements of changes in opacity and permeability in a bovine cornea. Two experiments were performed. The first experiment was cancelled, because the value of the positive control was not within the range of the historical data. The second experiment was valid. The test item quinine base anhydrous was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for one hour and whose opacity had been determined. A 20% quinine base anhydrous solution was incubated on the cornea for four hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. Physiological sodium chloride solution was used as negative control and imidazole (20 % solution in 0.9 % sodium chloride solution) was used as positive control. The negative control showed no irritation effects and no serious eye damage, mean IVIS was 0.888. The positive control induced serious eye damage on the cornea, mean IVIS was 67.658. Under this test condition, quinine base anhydrous showed effects on the cornea of the bovine eye. A mean IVIS of 18.150 was calculated.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in a UN GHS Category. But according to the Background Review Document Current Status of In Vitro Test Methods for Identifying Mild/Moderate Ocular Irritants: Bovine Corneal Opacity and Permeability Test Method, 2010 a substance with an IVIS of 3.1 - 25 is mild irritant. Therefore, we assume that the read across substance quinine base anhydrous is mildly irritating to eyes and conclude that quinine dihydrochloride is also mildly irritating to eyes.