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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:
key study
Study period:
2017-01-17 to 2017-03-02
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:
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:
July 26, 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:
December 09, 2010
GLP compliance:
yes (incl. certificate)
Remarks:
Behörde für Gesundheit und Verbraucherschutz

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES

- Source: obtained from a slaughterhouse
- Storage, temperature and transport conditions of ocular tissue : To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution2 (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL
- Time interval prior to initiating testing: not reported
- indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: yes

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 μL
- Concentration: 20% suspension in 0.9% sodium chloride solution (w/v)
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
90 ± 5 minutes
Number of animals or in vitro replicates:
3 replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Bovine eyes from cattle were completely submerged in Hanks’ Balanced Salt Solution containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL. The eyes were examined for defects (e.g., increased opacity, scratches, and neovascularisation), and only corneas from eyes free of defects were used. The closed-chamber method was then used where the dissected corneas (2 to 3 mm rim of sclera) were mounted to 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. After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
0.9% sodium chloride solution

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

APPLICATION DOSE AND EXPOSURE TIME
750 μL for 240 minutes

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes; 90 ± 5 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After exposure, 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.

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: 1 mL sodium fluorescein9 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. 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.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) ; IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA: A test item that induces an IVIS ≤ 3 is defined as no category; > 3 and ≤ 55; no prediction can be made; and > 55 as a corrosive or severe irritant.

A test was considered acceptable if the positive control gave an IVIS that fell within two standard deviations of the current historical mean. The negative or solvent/vehicle control responses were less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control. In cases of borderline results in the first testing run, a second testing run should be considered. A borderline result is defined as:

• 2 of the 3 corneas gave discordant predictions from the mean of all 3 corneas, OR,
• 1 of the 3 corneas gave discordant prediction from the mean of all 3 corneas AND the discordant result was > 10 IVIS units from the cut-off threshold of 55.
• If the repeat testing run corroborates the prediction of the initial testing run (based upon the mean IVIS value), then a final decision can be taken without further testing. If the repeat testing run results in a non-concordant prediction from the initial testing run (based upon the mean IVIS value), then a third and final testing run should be conducted to resolve equivocal predictions, and to classify the test chemical. It may be permissible to waive further testing for classification and labelling in the event any testing run results in a UN GHS Category 1 prediction.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: mean opacity score
Value:
ca. 2.536
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation parameter:
other: mean permeability value
Value:
ca. 0.18
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Value:
ca. 5.241
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

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:
other: non corrosive according to Regulations (EC) No 1272/2008.
Conclusions:
Under the present test conditions Dimelamine pyrophosphate tested in the in vitro BCOP test method, had an IVIS value of 5.241, which 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.