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Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From February 07, 2017 to February 28, 2017
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 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:
yes
Remarks:
see "Principles of method if other than guideline"
Principles of method if other than guideline:
Corneas were incubated for 10 ± 1 minutes at 25 °C in experiment 1 and at 28.5 °C in experiment II.
Evaluation: Since the test compound is not stable at higher temperatures (maximum temperature 30 ºC), the corneas were incubated with the test compound for 10 minutes at a temperature below 30 ºC. Positive and negative controls were in experiment 1 also incubated at the lower temperature. Ten minutes incubation at a slightly lower temperature has no effect on the results. The controls in experiment I were within the historical data control range and therefore this deviation has no effect on the study integrity.
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
(Z)-N-[2-(2-hydroxyethoxy)ethyl]-9-octadecenamide
Cas Number:
20429-33-8
Molecular formula:
C22H43NO3
IUPAC Name:
(Z)-N-[2-(2-hydroxyethoxy)ethyl]-9-octadecenamide
Test material form:
solid
Specific details on test material used for the study:
Batch: LAB 4417; Appearance: light yellow paste
- No correction factor for purity was applied
- Stability at higher temperatures: yes, maximum temperature: 30°C

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Test System: bovine eyes were used as soon as possible after slaughter.
Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
No correction was made for the purity/composition of the test substance. An excessive amount of test substance that completely covered the cornea was applied.
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
First experiment: 10 ± 1 minutes at 25.0 °C (study plan deviation),
Second sexperiment: 10 ± 1 min at 28.5 °C (study plan deviation).
Number of animals or in vitro replicates:
3 replicates
Details on study design:
Preparation of Corneas:

The eyes were checked for unacceptable defects. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM. The isolated corneas were mounted in a corneal holder (one cornea per holder) (BASF) 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, 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.

Treatment of Corneas and Opacity Measurements: Two experiments were performed.

- All experiments were performed at a temperature slightly below 30 °C since the test substance was not considered stable above 30 °C.
- The medium from the anterior compartment was removed and 750 uL of either the negative control, positive control (Ethanol) or an excessive amount of test substance 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 substance over the entire cornea.

- Experiment I:
Corneas treated with the test substance, negative and positive control were in the first experiment incubated in a horizontal position for 10 ± 1 minutes at 25.0 °C (study plan deviation). A second experiment was performed since the IVIS values were spread over two categories.

- Experiment II:
The second experiment was aborted since the controls and test substance were incubated on the corneas for more than 10 min. The second experiment was repeated. Corneas treated with negative and positive control were in the repeat of the second experiment incubated in a horizontal position for 10 ± 1 minutes at 32.0 °C ± 1 °C. The test substance was incubated in a horizontal position for 10 ± 1 min at 28.5 °C (study plan deviation). After the incubation the solutions and the test substance were removed and the epithelium was washed with MEM with phenol red and thereafter with cMEM.
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.

Application of Sodium Fluorescein:

Following the final opacity measurement, permeability of the cornea to Na-fluorescein 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/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.

Permeability Determinations:

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 substance was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
Experiment I
Value:
1.37
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Experiment II
Value:
-1.53
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
mean of two experiments
Value:
-0.1
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- 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 53 and 64. In experiment I and II, respectively and 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

Results Experiment I:

 

- The IVIS for the negative controls ranged from 1.0 to 2.9. The individual positive control IVIS ranged from 48 to 62.

- The corneas treated with the positive control substance were translucent after the 10 min of treatment. The cornea treated with the negative control were slightly translucent (but all IVIS values were within the historical control data range).

- The corneas treated with the test substance showed opacity values ranging from -0.8 to 2.3 and permeability values ranging from 0.023 to 0.060. The corneas were slightly translucent after the 10 min of treatment with the test substance. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 0.0 to 3.2 after 10 min of treatment with the test substance. Since the individual in vitro irritancy scores were spread over 2 categories (0.0, 0.9 and 3.2 respectively), the test was inconclusive and a repeat experiment was performed.

Results Experiment II:

 

- The IVIS for the negative controls ranged from -1.2 to -0.5. The individual positive control IVIS ranged from 48 to 82. The corneas treated with the positive control were turbid after the 10 min of treatment.

- The corneas treated with the test substance showed opacity values ranging from -2.2 to -1.0 and permeability values ranging from 0.003 to 0.040. The corneas were clear after the 10 min of treatment with the test substance. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -2.2 to -1.0 after 10 minutes of treatment with the test substance.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the study conditions, over two experiments, the test substance did not induce ocular irritation through both endpoints with an IVIS <3 in 5 out of 6 eyes, resulting in a mean in vitro irritancy score of -0.1 (-2.2 to 3.2) after 10 minutes of treatment. Since the test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

A study was conducted to determine the eye irritation potential of the test substance as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea according to OECD Guideline 437 (Bovine Corneal Opacity and Permeability test). Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, The Netherlands). Two experiments were performed. The cornea of a bovine eye was pre-incubated with cMEM without phenol red at 32 ± 1°C for 1 h and its opacity had been determined. The test substance (pure) was applied on cornea in a way that as much as possible of the cornea surface was covered. Subsequently cornea was incubated for 10 min at 32 ± 1°C. After removal of the test substance and 2 h post-incubation, opacity and permeability values were measured. Physiological saline was used as negative control and ethanol as positive control. The meanin vitroirritancy score was 1.4 after 10 min of treatment with the test substance. Since the individualin vitroirritancy scores were spread over 2 categories (0.0, 0.9 and 3.2 respectively) the test was inconclusive and a repeat experiment was performed. In the second experiment, the test substance did not induce ocular irritation through both endpoints, resulting in a meanin vitroirritancy score of -1.5 after 10 min of treatment. In both experiments, the negative controls responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative controls did not induce irritancy on the corneas. The meanin vitroirritancy score of the positive controls were within standard deviations of the current historical positive control means. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Under the study conditions, over two experiments, the test substance did not induce ocular irritation through both endpoints with an IVIS <3 in 5 out of 6 eyes, resulting in a meanin vitroirritancy score of -0.1 (-2.2 to 3.2) after 10 minutes of treatment. Since the test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage (Westernik, 2017).

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