Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-05-05 - 2017-05-18
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 guidelineopen allclose all
Qualifier:
according to guideline
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
dated December 08, 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
N-butyltriphenylphosphonium bromide
EC Number:
918-205-7
Cas Number:
1779-51-7
Molecular formula:
C22H24P.Br
IUPAC Name:
N-butyltriphenylphosphonium bromide

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: slaughterhouse
- Characteristics of donor animals (e.g. age, sex, weight): Bovine eyes from cattle in the age range of 6 to 12 months; no data on sex and weight
- indication of any existing defects or lesions in ocular tissue samples: Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used. The quality of each cornea was also evaluated at later steps in the assay. Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded.
- Indication of any antibiotics used: To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution, (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL; no further data

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% suspension in 0.9% sodium chloride solution (w/v)

VEHICLE
- 0.9% sodium chloride solution
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
n.a.
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse. The corneas were dissected with a 2 to 3 mm rim of sclera.

QUALITY CHECK OF THE ISOLATED CORNEAS
Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used. The quality of each cornea was also evaluated at later steps in the assay. Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
Solvent control was used as negative control

SOLVENT CONTROL USED: yes; 0.9% sodium chloride solution

POSITIVE CONTROL USED: yes ; 20% Imidazole (CAS no. 288-32-4) in 0.9% sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME: 750 µL, 240 minutes

TREATMENT METHOD: The corneas were mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM), while preventing bubble formation. The corneal holder was equilibrated at 32 ± 1°C for at least one hour.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the exposure period of 240 minutes the test item, the negative and positive controls, were removed from each chamber. Subsequently, the epithelium was washed with EMEM containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.

- POST-EXPOSURE INCUBATION: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
- Corneal permeability: To determine the corneal permeability 1 mL sodium fluorescein solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM. The holder was incubated in a horizontal position at 32 ± 1°C for 90 ± 5 minutes. The amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader (Tecan Sunrise Magellan Version 6.410). Measurements at 490 nm were recorded as optical density (OD490). The fluorescein permeability values were determined using OD490 values based upon a visible light spectrophotometer (Tecan Sunrise) using a standard 1 cm path length.

- Others: After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer. A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment and positive control groups.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS):
After correcting the opacity and mean permeability (OD490) values for background opacity and the negative control permeability OD490 values, the mean opacity, and permeability OD490 values for each treatment group were combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows: IVIS = mean opacity value + (15 x mean permeability OD490 value)
> 3 and <= 55: No prediction can be made
> 55: corrosive or severe irritant

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
Remarks:
opacity
Run / experiment:
mean of three corneas
Value:
10.491
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other:
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent (negative) control: The corneas treated with the negative control item 0.9% sodium chloride solution revealed a mean opacity value of 0.531 ± 0.161 and a mean permeability value of 0.017 ± 0.019. The calculated IVIS value of 0.791 ± 0.328 was well below the cut-off value of 3 (UN GHS no category).
- Acceptance criteria met for positive control: The corneas treated with the positive control item 20% Imidazole in 0.9% NaCl solution revealed a mean opacity value of 66.840 ± 5.811 and a mean permeability value of 2.381 ± 0.389 compared to the solvent control. The calculated IVIS value of 102.560 ± 11.271 was within two standard deviations of the current historical mean and well above the cut-off value of 55. Hence, the acceptance criteria for the test were fulfilled.

Any other information on results incl. tables

Following treatment with n-Butyltriphenylphosphonium bromide a mean opacity of 7.131 ± 2.342 and a mean permeability value of 0.224 ± 0.091 compared to the negative control were determined. The calculated IVIS of 10.491 ± 2.268 is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55, identifying test substances as inducing serious eye damage (UN GHS Category 1).

Consequently no prediction concerning irritant or severely irritant potential of the test item can be made.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The test of n-Butyltriphenylphosphonium bromide in a Bovine Corneal Opacity and Permeability Test resulted in an in vitro irritancy score (IVIS) of 10.491 ± 2.268 indicating serious effects to the eyes. However, the method is not validated to discriminate between Serious Eye Damage Cat. 1 and Serious Eye Irritation Cat. 2. In accordance with the ECHA Guidance document Chapter R7.a, Version 6.0 –July 2017, R.7.2.8.1, Figure R. 7.2-5 5, n-Butyltriphenylphosphonium bromide is classified in Category 1 (irreversible effects on the eye).