Reaction mass of (3R)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]butanal and (3S)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]butanal

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 August - 20 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (3R)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]butanal and (3S)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]butanal

EC Number:

944-855-6

Cas Number:

199445-85-7

Molecular formula:

Not applicable (reaction mass of stereochemical isomers)

IUPAC Name:

Reaction mass of (3R)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]butanal and (3S)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]butanal

Test material form:

liquid

Details on test material:

Test Item Storage: At room temperature

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Assumed stable. Stability at higher temperatures not availbale.
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Recognised supplier
- Number of animals: 9 eyes
- Characteristics of donor animals (e.g. age, sex, weight): Not reported
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: Not reported
- indication of any existing defects or lesions in ocular tissue samples: Eyes exhibiting defects were discarded.
- Indication of any antibiotics used: Not reported.

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

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

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 replicates per test group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS

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. 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 ºC. The corneas were incubated for the minimum of 1 hour at 32 ± 1 ºC.

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 replicates per test group.

NEGATIVE CONTROL USED

Yes, physiological saline.

SOLVENT CONTROL USED (if applicable)

Not applicable.

POSITIVE CONTROL USED

Yes, ethanol.

APPLICATION DOSE AND EXPOSURE TIME

750 µL, for 10 minutes.

TREATMENT METHOD: [closed chamber / open chamber]

Closed chamber.

POST-INCUBATION PERIOD: yes/no. If YES please specify duration

Yes, for 120 ± 10 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.
- POST-EXPOSURE INCUBATION: 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 ºC.

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 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. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany).

- 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 (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 ºC. 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): (please specify: None

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Mean value

Value:

1.3

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 retention score

Run / experiment:

Mean score

Value:

0.022

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 value

Value:

1.6

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: None

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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: Reported

Any other information on results incl. tables

Table 1:       Summary of Opacity, Permeability andin vitroscores

Treatment	Mean Opacity^1	Mean Permeability^1	Meanin vitroIrritation Score^1,2
Negative control	1.7	0.007	1.8
Positive control (ethanol)	18.5	2.113	50
Test item	1.3	0.022	1.6

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

Evaluation of the eye hazard potential of the test substance was conducted in accordance with OECD 437 using the Bovine Corneal Opacity and Permeability test (BCOP test) in accordance GLP.

Undiluted test substance was applied topically (750 µL) to the cornea of bovine eyes for 10 minutes under controlled conditions. 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 50 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.

The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.6 after 10 minutes of contact time.

The test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.