Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19.07.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2013
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Purity: 99.9%

Test animals / tissue source

Species:
cattle
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
- Source: isolated corneas obtained on the test day as a by-product from animals freshly slaughtered (abattoir A. Moksel AG, Buchloe, Germany)
- Transport: in HBSS containing Pen/Strep on ice. Immediately after arrival of the eyes, cornea preparation was initiated
- Inspection: carefully examined for defects and any defective eyes were discarded
- Preparation of the tissue: the tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS
- Preparation of the corneas: corneas were mounted in corneal holders (endothelial side against the O-ring of the posterior chamber). The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI) for 1 hour at 32 ± 1 °C (equilibration period)

Test system

Vehicle:
physiological saline
Remarks:
20% concentration
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Volume: 750 μL
Duration of treatment / exposure:
4 hours ± 5 minutes
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
3 corneas for the test item, the positive control and the negative control, respectively
Details on study design:
Treatment of the corneas
- An initial measurement was performed on each of the corneas (opacitometer). Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay
- The medium was removed from the anterior chamber and replaced with the test item or control
- 750 μL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method) for 4 hours ± 5 minutes incubation at 32 ± 1 °C. Then test substance or control substance was removed and the epithelium washed at least three times with MEM (containing phenol red) till the medium was free of test substance, finally the cornea was rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed. Corneas were observed visually and pertinent observations were recorded.

- After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Acceptance criteria
- The BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
- The negative control responses resulted should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Evaluation of results
- The following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer: Opacity = ( I0/I-b)/a with a = 0.025 and b = 0.9894.
The value I0 is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated periodically.

- The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.

- The mean OD490 for the blank cuvettes was calculated. The mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490

- The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

-The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)

- The IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are: IVIS ≤ 3: no category; > 3 ≤ 55: no prediction can be made; > 55: category 1


Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
3 corneas
Value:
>= 1.45
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The acceptability criteria were fullfilled and the validity of the test system was confirmed

Any other information on results incl. tables

 In Vitro Irritation Score

Cornea No. Test Item Corrected Opacity Corrected OD490 Value IVIS
1 Negative Control 0.64 0.005  
2 1.18 0.019  
3 0.9 0.014  
MV 0.9 0.013 1.09
4 Positive Control 112.97 1.757  
5 110.15 1.837  
6 98.06 2.977  
MV 107.06 2.191 139.92
7 Test Item 1.06 -0.002  
8 0.88 -0.006  
9 2.7 -0.013  
MV 1.55 -0.007 1.45

MV: Mean Value

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
According to the evaluation criteria the test item L-Tyrosine is classified into 'UN GHS No Category'.
Executive summary:

In the current study the eye irritation potential of the test item L-Tyrosine was investigated in the bovine corneal opacity and permeability assay. The test was performed according to OECD 437 and in compliance to GLP.

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.

Thus the acceptability criteria were fullfilled and the validity of the test system was confirmed.

The test item was suspended with physiological saline 0.9% NaCl to obtain a 20% concentration.

All 3 corneas treated with L-Tyrosine showed no opacity of the tissue but the test item bequeathed few white spots.

The following mean in vitro irritation score was calculated:

1.45

Therefore the test item was classified into UN GHS No Category.