complex mixture from biological origin

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-03-04 to 2022-06-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

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:

26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

(8Z)-16-{[(2R,4S,5S)-6-[(acetyloxy)methyl]-3-{[(2S,4S,5S)-6-[(acetyloxy)methyl]-3,4,5-trihydroxyoxan-2-yl]oxy}-4,5-dihydroxyoxan-2-yl]oxy}heptadec-8-enoic acid; [(1S,4S,5S,8R,17Z,27S,31R)-28-[(acetyloxy)methyl]-4,5,30,31-tetrahydroxy-10-methyl-25-oxo-2,7,9,26,29-pentaoxatricyclo[25.2.2.0³,⁸]hentriacont-17-en-6-yl]methyl acetate

IUPAC Name:

(8Z)-16-{[(2R,4S,5S)-6-[(acetyloxy)methyl]-3-{[(2S,4S,5S)-6-[(acetyloxy)methyl]-3,4,5-trihydroxyoxan-2-yl]oxy}-4,5-dihydroxyoxan-2-yl]oxy}heptadec-8-enoic acid; [(1S,4S,5S,8R,17Z,27S,31R)-28-[(acetyloxy)methyl]-4,5,30,31-tetrahydroxy-10-methyl-25-oxo-2,7,9,26,29-pentaoxatricyclo[25.2.2.0³,⁸]hentriacont-17-en-6-yl]methyl acetate

Test material form:

other: liquid or pasty, solidified melt

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Batch number: 210810 FSL1
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 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: slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Characteristics of donor animals (e.g. age, sex, weight): Bovine eyes from young cattle were used
- 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 and tested the day of arrival in the laboratory.
- Time interval prior to initiating testing: Bovine eyes were used as soon as possible after slaughter.
- 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.

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): The test material was tested neat

Duration of treatment / exposure:

10 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 (+/-) 10 minutes

Number of animals or in vitro replicates:

triplicate

Details on study design:

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: physiological saline

POSITIVE CONTROL USED: ethanol

APPLICATION DOSE AND EXPOSURE TIME: 750 µL; the test item was tested neat; exposure time: 10 ± 1 min

TREATMENT METHOD: The isolated
corneas were mounted in a corneal holder (one cornea per holder) 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 were performed on each of the corneas using an opacitometer. 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. The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test material 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 material over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1 °C.

POST-INCUBATION PERIOD: yes, 120 ± 10 min

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. Possible pH effects of the test material 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 cornea treated with the test material 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 material 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: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490): 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 (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. 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 material was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

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 materials as inducing serious eye damage (UN GHS Category 1) and test materials not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
≤ 3 = No category
> 3; ≤ 55 = No prediction can be made
> 55 = Category 1
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

Run / experiment:

mean of 3

Value:

10

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
A pH effect of the test material was observed on the rinsing medium, the corneas were rinsed until no color change of the medium was observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, 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. Altough the negative control treated corneas were translucent, all results are within the acceptability range therefore this has no impact on the study result.
- Acceptance criteria met for positive control: yes, The mean in vitro irritancy score of the positive control (Ethanol) was 51 and within two standard deviations of the current historical positive control mean

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

further information is required for classification according to IATA guidance

Conclusions:

The test material induced ocular irritation through one endpoint (permeability), resulting in a mean in vitro irritancy score of 10 after 10 minutes of treatment. In conclusion, since PC-2021-999 induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Executive summary:

In an in vitro eye irritation guideline study (bovine corneal opacity and permeability assay) according to OECD 437 under GLP conditions, 0.75 mL of a of “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, lactone intermediate” was applied on corneas for 10 min at 32 °C. The test was performed in triplicates.

Physiological saline was used as negative control, ethanol as positive control. Both controls confirmed the validity of the study.

The test material induced ocular irritation through one endpoint (permeability), resulting in a mean in vitro irritancy score of 10 after 10 minutes of treatment.

In conclusion, no prediction on the classification can be made.