Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
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
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Trideca-1,1,1,2,2,3,3,4,4,5,5,6,6-fluorohexane
EC Number:
206-581-9
EC Name:
Trideca-1,1,1,2,2,3,3,4,4,5,5,6,6-fluorohexane
Cas Number:
355-37-3
Molecular formula:
C6HF13
IUPAC Name:
Trideca-1,1,1,2,2,3,3,4,4,5,5,6,6-fluorohexane
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AGC Chemicals Europe Ltd.(UK); Batch no. 41251
- Expiration date of the lot/batch: 01 March 2017 (nominal expiry date) (taken from label)
- Purity test date: Not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: until 01 March 2017 (nominal expiry date) (taken from label)

FORM AS APPLIED IN THE TEST (if different from that of starting material): Colourless liquid

OTHER SPECIFICS:

Purity/Composition: 100.00%
Molecular weight: 320

Test animals / tissue source

Species:
cattle
Strain:
other: 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), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Number of animals: 3 corneas each for replicate of the test material; negative control; and positive control
- 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): Bovine eyes from young cattle were obtained from the where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: Not specified
- 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
- Amount(s) applied (volume or weight with unit): 750 µL undiluted

Duration of treatment / exposure:
10 ± 1 minutes
Duration of post- treatment incubation (in vitro):
120 ± 10 minutes at 32 ±1°C
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Preparation of 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. The isolated corneas were stored in a petri dish with cMEM (Earle’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.

Cornea selection and Opacity reading
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group

NEGATIVE CONTROL USED
A negative control, physiological saline (Eurovet Animal Health, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.

POSITIVE CONTROL USED
Ethanol

APPLICATION DOSE AND EXPOSURE TIME
750 µl of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea for 10 ± 1 minutes at 32 ± 1°C

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes
After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM.

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 (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.

- POST-EXPOSURE INCUBATION:
corneas were incubated for 120 ± 10 minutes at 32 ±1°C

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.The opacity value (measured with the device OP-KIT) was calculated according to:

Opacity = ((I0/I) – 0.9894)/0.0251

Where: I0 = the empirically determined illuminance through a cornea holder but with windows and medium

I = the measured illuminance through a holder with cornea.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader] (OD490): Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/ml cMEM solution. The holders were slightly
rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ±1°C.

After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS): The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints. The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS
Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given in Table 1.

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

he assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Test Material
Value:
ca. -0.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: corneas were clear after the 10 minutes of treatment
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The individual in vitro irritancy scores for the negative controls ranged from -2.1 to -0.1.
- Acceptance criteria met for positive control: The individual positive control in vitro irritancy scores ranged from 34 to 39 for Ethanol. The corneas treated with the positive control item were turbid after the 10 minutes of treatment.OTHER EFFECTS:
- Range of historical values if different from the ones specified in the test guideline: 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 (Ethanol) was 35 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.

Any other information on results incl. tables

Table 2. Summary of opacity, permeability andin vitroscores

Treatment

Mean Opacity1

Mean Permeability1

Mean In vitro

Irritation Score1,2

Negative control

-1.2

0.002

-1.1

Positive control

(Ethanol)

19.7

1.039

35.3

Asahiklin™ AC-2000

-0.1

-0.001

-0.2

1  Calculated using the negative control mean opacity and mean permeability values for the positive  control and test item.

2  In vitro irritancy score (IVIS) = mean opacity value

Applicant's summary and conclusion

Interpretation of results:
other: Not classified
Remarks:
GHS Criteria
Conclusions:
The test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.2 after 10 minutes of treatment.
 
Since 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

In a key guideline (OECD 437) in vitro eye irritation study, the eye hazard potential of test material (1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane) was evaluated using the Bovine Corneal Opacity and Permeability test (BCOP test).

 

The test material was topically applied undiluted on the epithelium of the bovine cornea for 10 minutes. Post exposure the cornea was thoroughly rinsed to remove the test item and incubated for 2 hours with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein. A negative control, physiological saline was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints while ethanol was used as the positive control in this study.

 

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 was 35 and was within two standard deviations of the current historical positive control mean.

 

The test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.2 after 10 minutes of treatment.

 

Since 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.