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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Sep 2017 - Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
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:
26 July 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Commission Regulation (EU) 2017/735 amending Regulation (EC) No. 440/2008 adopted 14. Feb. 2017
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline for the Testing of Chemicals, Series on Testing and Assessment No. 160
Version / remarks:
25. Oct. 2011
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: lslaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany
- Characteristics of donor animals (e.g. age, sex, weight): adult cattle (typically 12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled con-tainer within 1 hour 10 minutes.
- Time interval prior to initiating testing: same day of slaughter.
- indication of any existing defects or lesions in ocular tissue samples: After the arrival of the corneas, they were examined and only corneas which were free from damages 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 (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

Experimental Parameters
Date of treatment 19. Oct. 2017
Incubation time 4 h
Negative control HBSS
Positive control Imidazole, 20 % solution in HBSS

Test system

Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Replicate Amount
1 240.3 mg
2 261.8 mg
3 254.7 mg
The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with test item.

Duration of treatment / exposure:
4 hours
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
3 replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
After the arrival of the corneas, they were examined and only corneas which were free from dam-ages 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 (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.
A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

QUALITY CHECK OF THE ISOLATED CORNEAS
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.

NUMBER OF REPLICATES
3 per treatment

NEGATIVE CONTROL USED
HBSS

POSITIVE CONTROL USED
Imidazole, 20 % solution in HBSS

APPLICATION DOSE AND EXPOSURE TIME
ca 240 to 262 mg/cornea, 240 mins

TREATMENT METHOD: open chamber; direct application of the powder to the cornea

POST-INCUBATION PERIOD: for the permeability test, a sodium fluorescein solution with a concentration of 4 mg/mL was used. The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: the final opacity value of each cornea was recorded with the opacitometer. The change of opacity value of each treated cornea with test item, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea.
The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test item and positive control to obtain a corrected opacity.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate photometer at 492 nm. The corrected OD492 value of each cornea treated with test item and positive control was calculat-ed by subtracting the mean negative control cornea value from the original permeability value for each cornea. The mean OD492 value for each treatment group (test item, positive control and negative control) was determined by averaging the final corrected OD492 values of the treated corneas for one treatment group.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The IVIS of each replicate of the negative control was calculated from the following equation:
IVIS = opacity difference + (15 x corrected OD492 value)

The IVIS of each replicate of the positive control and of the test item were calculated from the following equation:
IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492 – mean OD492 of the negative control)]


DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. Yes

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
32.77
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:
According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean.
The negative control has to show an IVIS ≤ 3.
The validity criteria and findings are given in the following table:

Parameter Criterion Found Assessment
IVIS of negative control HBSS ≤ 3 1 .52 ok
IVIS of positive control 20% imidazole solution 70.37 – 159.05 88.32 ok

Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
No prediction of eye irritation can be made.
Executive summary:

This in vitro study was performed to assess corneal damage potential ofBasic Orange 22 by quantitative measurements of changes in opacity and permeability in a bovine cornea following OECD Guideline 437 and EU Method B.47.

Bovine corneas were used. They were collected from slaughtered cattle which were between 12 and 60 months old.

The test itemBasic Orange 22 in its powdered form (ca 240 to 262 mg/cornea) was evenly distributed onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured.

The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. The mean opacity detected with an opacitometer at the end of the test item exposure period was 31.19. After the determination of opacity, the epithelial surface was treated with sodium fluorescein solution for 90 minutes, to investigate alteration in cornea permeability. The calculated mean permeability OD492value of the corneas treated with the test item was 0.1057.

 

HBSS was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (In Vitro Irritancy Score) is 1.52.

20% imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS (In Vitro Irritancy Score) is 88.32.

 

Under the conditions of this study, the test item Basic Orange 22 showed effects on the cornea of the bovine eye. The calculated IVIS (In Vitro Irritancy Score) is 32.77.

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 an UN GHS Category for eye damage with the BCOP test only.