2-hydroxypropan-1-aminium sulfate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Feb 2017 to 28 Feb 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)

Version / remarks:

July 26, 2013

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

CAS/EC/IUPAC not yet assigned

IUPAC Name:

CAS/EC/IUPAC not yet assigned

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Zschimmer and Schwarz GmbH + Co. KG.
- Identification: 0125 SCHWEFELSÄURE, MIPA-SALZ
- Batch: CH 220612
- Purity/Composition: UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Test item storage: At room temperature
- Stable under storage conditions until: 07 December 2017 (expiry date)

OTHER SPECIFICS
- Appearance: Yellow turbid liquid

Test animals / tissue source

Species:

cattle

Strain:

other: bovine

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Test system: Bovine eyes were used as soon as possible after slaughter.
- Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
- 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.
- 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:

TEST MATERIAL AND CONCURRENT CONTROLS
750 µL of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea.

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:

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. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

TEST ITEM PREPARATION
The purity of the test item is 75.1%; the remainder is water. A correction factor is not applicable for the BCOP test, therefore no correction was made for the purity of the test item. The test item was tested neat.

TREATMENT OF CORNEAS AND OPACITY MEASUREMENTS
Due to a technical error, the first experiment was aborted and a repeat assay was performed. 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.
OPACITY MEASUREMENT
- 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=[ I(0) / I - 0.9894] / 0.0251
With I(0) 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.

APPLICATION OF SODIUM FLUORESCEIN
- 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.

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

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.

INTERPRETATION
- 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 the table in ‘Any other information on materials and methods incl. tables’.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Mean opacity

Value:

-1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

fluorescein leakage

Run / experiment:

mean permeability

Value:

0.016

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

mean in vitro irritation score

Value:

-1.3

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:
- The corneas were clear after the 10 minutes of treatment with the test item.
- No pH effect of the test item was observed on the rinsing medium.

ACCEPTANCE OF RESULTS:
- Acceptance criteria for negative control are met.
- Acceptance criteria for positive control are met.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met