Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 8 September to 26 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

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)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Reddish solid below 25°C / liquid above 25°C
Details on test material:
- State of aggregation: N/A
- Particle size distribution: N/A
- Mass median aerodynamic diameter (MMAD): N/A
- Geometric standard deviation (GSD): N/A
- Shape of particles: N/A
- Surface area of particles: N/A
- Crystal structure: N/A
- Coating: N/A
- Surface properties: N/A
- Density: 1.06 g/cm3
- Moisture content: N/A
- Residual solvent: N/A
- Activation: N/A
- Stabilisation: N/A
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: MR92143651
- Expiration date of the lot/batch: 01 January 2017
- Purity: 86.4% (No correction was made for purity of the test substance)
- Purity test date: 18 June 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Yes, maximum temperature: 50°C, maximum duration: unknown (until liquefaction).
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Before use the test substance was warmed in a 45°C water bath to obtain a homogeneous sample..
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands)
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): not specified
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)) and incubated for the minimum of 1 hour at 32 +/- 1°C.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- Indication of any antibiotics used: no

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL, NEGATIVE AND POSITIVE CONTROLS:
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): as such

Duration of treatment / exposure:
10 +/- 1 minutes at 32 +/- 1°C
Duration of post- treatment incubation (in vitro):
120 +/- 10 minutes at 32 +/- 1°C in fresh cMEM medium.
Number of animals or in vitro replicates:
triplicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C.
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations will be performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea will be read against a cMEM filled chamber, and the initial opacity reading thus determined will be recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

QUALITY CHECK OF THE ISOLATED CORNEAS: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: physiological saline (Eurovet Animal Health, Bladel, The Netherlands)

SOLVENT CONTROL USED (if applicable): N/A

POSITIVE CONTROL USED: 10% (w/v) Benzalkonium Chloride (Merck KGaA, Darmstadt, Germany) [CAS Number 63449-41-2] solution prepared in physiological saline.

APPLICATION DOSE AND EXPOSURE TIME: 750 µL for 10 +/- 1 minutes at 32 +/- 1°C

TREATMENT METHOD: not specified

POST-INCUBATION PERIOD: yes. If YES please specify duration: 120 +/- 10 minutes at 32 +/- 1°C

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential
Medium, Life Technologies) and thereafter with cMEM .

- POST-EXPOSURE INCUBATION: see above

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microplate reader (TECAN Infinite® M200 Pro Plate Reader) (OD490)
- Others (e.g, pertinent visual observations, histopathology): Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

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
Value:
>= -0.3 - <= 1.4
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (IVIS ranged from -2.8 to 0.9 so < 3). The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
- Acceptance criteria met for positive control: yes (IVIS ranged from 108 to 143 so > 55). The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 120 and was within two standard deviations of the current historical positive control mean.
- Range of historical values if different from the ones specified in the test guideline: Negative control: IVIS ranged from -3.1 to 2.3; Positive control: IVIS ranged from 80.5 to 186.1.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
1-Phenyldecane-1,3-dione induced an IVIS ≤ 3 in this BCOP test therefore no classification is required for eye irritation or serious eye damage.
Executive summary:

The aim of this study was to evaluate the eye hazard potential of 1-Phenyldecane-1,3-dione using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the potency of 1-Phenyldecane-1,3-dione to induce serious eye damage after topical application for 10 minutes on isolated bovine corneas. . The study procedures described in this report were based on OECD guideline no. 437 and EC guideline B.47 and performed in compliance with Good Laboratory Practices.

Batch MR92143651 of 1-Phenyldecane-1,3-dione was a reddish solid below 25°C / liquid above 25°C with a purity of 86.4%. The test item was applied as it is (750 μl) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 120 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

1-Phenyldecane-1,3-dione did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score (IVIS) of 0.6 after 10 minutes of treatment.

Since 1-Phenyldecane-1,3-dione induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage according to Globally Harmonized System of Classification and Labelling of Chemicals (GHS) and Regulation (EC) No 1272/2008.