Storax (balsam)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Apr 2017 - 25 Apr 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)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Storax (balsam)

EC Number:

232-458-4

EC Name:

Storax (balsam)

Cas Number:

8046-19-3

Molecular formula:

Not applicable, UVCB - NCS

IUPAC Name:

Storax (balsam)

Test material form:

other: liquid

Details on test material:

CAS : 8046-19-3 EINECS : 232-458-4
- Name of test material (as cited in study report): STYRAX RESINOID OIL
- Test Facility test item number: 208399/A
- Appearance: Orange viscous liquid
- Substance type: pure substance
- Physical state: liquid
- Analytical purity: 100%

Specific details on test material used for the study:

Identification: Styrax resinoid oil
Appearance: Orange viscous liquid

Test item storage: At room temperature
Stable under storage conditions until: 02 February 2019

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- 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. Bovine eyes were used as soon as possible after slaughter. 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:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: 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:

3

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) 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. Three corneas were selected at random for each treatment group.

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.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
physiological saline

POSITIVE CONTROL USED
Ethanol

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. 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 was measured with the OP-KIT device.
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.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: According to guideline.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

-2.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

- 0.7

Positive controls validity:

valid

Remarks:

17.2

Remarks on result:

other: values ranging from -3.7 to -2.0

Results 2

Irritation parameter:

fluorescein leakage

Run / experiment:

mean

Value:

0.004

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.004

Positive controls validity:

valid

Remarks:

0.937

Remarks on result:

other: values ranging from -0.003 to 0.018.

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

-2.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

-0.6

Positive controls validity:

valid

Remarks:

31.3

Remarks on result:

other: ranging from -3.4 to -2.0

Other effects / acceptance of results:

The individual in vitro irritancy scores for the negative controls ranged from -1.8 to 1.1. The individual positive control in vitro irritancy scores ranged from 24 to 40. The corneas treated with the positive control item were turbid after the 10 minutes of treatment. The corneas treated with Styrax resinoid oil showed opacity values ranging from -3.7 to -2.0 and permeability values ranging from -0.003 to 0.018. The corneas were clear after the 10 minutes of treatment with Styrax resinoid oil. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -3.4 to -2.0 after 10 minutes of treatment with Styrax resinoid oil.

Styrax resinoid oil did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -2.8 after 10 minutes of treatment.

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 31 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.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since Styrax resinoid oil induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

To evaluate the eye hazard potential of Styrax resinoid oil an Bovine Corneal Opacity and Permeability test (BCOP test) was performed according to OECDTG 437. The eye damage of Styrax resinoid oil was tested through topical application for 10 minutes.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 (Ethanol) was 31 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.Styrax resinoid oil did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of –2.8 after 10 minutes of treatment. In conclusion, since Styrax resinoid oil induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.and according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).