1,3-Dioxole, 2,2,4-trifluoro-5-(trifluoromethoxy)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07-Apr-2017 to 01-Sep-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 437. Furthermore, functional model conditions and references to historical control data are included in the report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Version / remarks:

Adopted 26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Assessed on 07-11, 14 and 16 September 2015. Dated on the 03 November 2015.

Test material

Test material information

Constituent 1

Reference substance name:

605-263-0

EC Number:

605-263-0

Cas Number:

161611-74-1

Molecular formula:

C4F6O3

IUPAC Name:

605-263-0

Test material form:

liquid

Details on test material:

- Physical state: Colourless liquid; odorless
- Storage condition of test material: At room temperature

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: as soon as possible after slaughter

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
- Concentration (if solution): undiluted

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

Number of animals or in vitro replicates:

3 corneas were selected at random for each treatment group

Details on study design:

SELECTION AND 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.

QUALITY CHECK OF THE ISOLATED CORNEAS
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. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

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

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

POSITIVE CONTROL USED
Ethanol (Purity >= 99.9%)

APPLICATION DOSE AND EXPOSURE TIME
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 +/- 1 minutes at 32 +/- 1°C.

TREATMENT METHOD: closed chamber

POST-INCUBATION 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. 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. Subsequently the corneas were incubated for 120 +/- 10 minutes at 32 +/- 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

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

With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

- Corneal permeability:
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.

- Others (e.g, pertinent visual observations, histopathology): Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

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

DECISION CRITERIA:
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 hereafter:

In vitro score range: UN GHS:
=< 3 No Category
> 3 ; =< 55 No prediction can be made
> 55 Category 1

ACCEPTABILITY CRITERIA
The 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

Value:

-0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No, the corneas were clear after the 10 minutes of treatment with Perfluoro methoxy dioxole.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline:
The positive control should elicit an In Vitro Irritancy Score that falls within two standard deviations of the historical mean (2 x SD 12.64 to 56.68) for the laboratory.
The negative control mean opacity change value should be ≤3.0 and the permeability mean value ≤0.042 (assays performed from February 2014 to February 2017).

In vivo

Other effects:

No pH effect of the test item was observed on the rinsing medium.

Any other information on results incl. tables

Table 7.3.2/1 - Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity ^1	Mean Permeability ^1	Mean In Vitro Score ^1, 2
Negative control	-0.5	0.003	-0.4
Positive control (Ethanol)	19.6	2.737	60.7
Test item	-1.2	0.027	-0.8

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 + (15 x mean OD490 value).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, Perfluoro methoxy dioxole induced an IVIS =< 3, therefore, the test substance is not classified for eye irritation or serious eye damage, according to Regulation (EC) No. 1272/2008 (CLP) and to the UN GHS Regulation.

Executive summary:

The eye hazard potential of the Perfluoro methoxy dioxole was evaluated using the Bovine Corneal Opacity and Permeability test (BCOP test), under GLP compliance, according to the OECD Guideline No. 437.

The corneal damage potential of test substance was assessed using fresh bovine corneae. 750 µL of test item was applied to cornea for 10 minutes followed by an incubation period of 2 hours at 32 ± 1 °C and corneal opacity was measured. Three corneas were used for each treated series (undiluted test item; negative control: physiological saline; positive control: ethanol). Following the opacity measurement, the permeability of the corneas to sodium fluorescein was evaluated. Two endpoints, corneal opacity and permeability, were measured and combined to give an In Vitro Irritancy Score.

 

The individual in vitro irritancy scores for the negative controls ranged from -1.2 to 0.3. The individual positive control in vitro irritancy scores ranged from 47 to 72. The corneas treated with the positive control item were turbid after the 10 minutes of treatment. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range (3.0 and 0.042, respectively) indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 61 and within two standard deviations of the current historical positive control mean (34.7 - 78.2). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The corneas treated with Perfluoro methoxy dioxole were clear after the 10 minutes of treatment with Perfluoro methoxy dioxole. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -2.1 to 0.7 after 10 minutes of treatment with Perfluoro methoxy dioxole and, the mean IVIS was lower than or equal to 3.