Reaction mass of ethyl 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxylate and ethyl 2,6,6-trimethylcyclohexa-2,4-diene-1-carboxylate and ethyl 6,6-dimethyl-2-methylenecyclohex-3-enecarboxylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD Guideline No.437 and under GLP compliance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

Adopted July 26, 2013.

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 2,6,6-trimethylcyclohexa-1,3-ene-1-carboxylate

EC Number:

252-335-9

EC Name:

Ethyl 2,6,6-trimethylcyclohexa-1,3-ene-1-carboxylate

Cas Number:

35044-59-8

Molecular formula:

C12H18O2

IUPAC Name:

ethyl 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxylate

Test material form:

liquid

Details on test material:

SOURCE OF TEST MATERIAL
- Physical appearance: colourless to pale yellow liquid
- Stored in a dry, well ventilated and dark location at room
temperature (i.e. 10–30°C)

Test animals / tissue source

Species:

cattle

Strain:

other: bovine cattle (Bos taurus)

Details on test animals or tissues and environmental conditions:

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. 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:

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 at 32 ± 1°C

Duration of post- treatment incubation (in vitro):

120 ± 10 minutes at 32 ± 1°C

Number of animals or in vitro replicates:

Three corneas for each treated series (test item formulation, positive control and vehicle control) were used.

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

TREATMENT METHOD
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= [((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 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.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

One of the negative control eyes was excluded since it was slightly translucent resulting in an opacity value which was outside the normal range.

Positive controls validity:

valid

Other effects / acceptance of results:

ETHYL SAFRANATE was tested neat.

The individual in vitro irritancy scores for the negative controls ranged from -1.3 to -0.4 (study plan deviation). 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 individual positive control in vitro irritancy scores ranged from 53 to 73 for Ethanol. The corneas treated with the positive control item were turbid after the 10 minutes of treatment. The mean in vitro irritancy score of the positive control (Ethanol) was 62 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 corneas treated with ETHYL SAFRANATE showed opacity values ranging from -0.1 to 2.9 and permeability values ranging from -0.013 to 0.057. The corneas were clear after the 10 minutes of treatment with ETHYL SAFRANATE. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 0.1 to 2.7 after 10 minutes of treatment with ETHYL SAFRANATE.
ETHYL SAFRANATE did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.5 after 10 minutes of treatment.
Since ETHYL SAFRANATE induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Any other information on results incl. tables

Table 7.3.2/1: Summary of opacity, permeability and in vitro scores

 

Treatment	Mean opacity^1	Mean permeability^1	Mean in vitro irritation score^1,2
Negative control	-1.0	0.010	-0.8
Positive control (Ethanol)	22.3	2.636	61.8
ETHYL SAFRANATE	1.2	0.019	1.5

¹ Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.
² In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

 

Individual values for opacity, permeability and in vitro scores were available in the report attached in the section 'Attached full study report'.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of the test item was predicted and, ETHYL SAFRANATE induced an IVIS ≤ 3. Therefore, the test item is not corrosive/not severely irritating to the eye according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Executive summary:

An ex vivo eye irritation study was performed according to the OECD Guideline 437 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on bovine corneas.

 

Corneas obtained from young slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for the minimum of 1 hour at 32 ± 1°C.

A single experiment was performed using three corneas for each treated series (test item, positive control and negative control). Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer.

The test item was applied undiluted, in a single experiment using a treatment time of 10 minutes onto the epithelium of the cornea. Negative and positive controls were applied using the same treatment time. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. The corneas were then incubated for 120 ± 10 minutes at 32 ± 1°C before a second opacity measurement was performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes(± 5 minutes) at 32 ± 1°C. At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities. Opacity, residual test item on corneas and fluorescein fixation were observed on each cornea treated with the test item.

 

The individual in vitro irritancy scores for the negative controls ranged from -1.3 to -0.4 (study plan deviation). 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 individual positive control in vitro irritancy scores ranged from 53 to 73 for Ethanol. The corneas treated with the positive control item were turbid after the 10 minutes of treatment. The mean in vitro irritancy score of the positive control (Ethanol) was 62 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 corneas treated with ETHYL SAFRANATE showed opacity values ranging from -0.1 to 2.9 and permeability values ranging from -0.013 to 0.057. The corneas were clear after the 10 minutes of treatment with ETHYL SAFRANATE. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 0.1 to 2.7 after 10 minutes of treatment with ETHYL SAFRANATE. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.5 after 10 minutes of treatment.
Since ETHYL SAFRANATE induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

 

The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 1.5. Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of the test item was predicted and, ETHYL SAFRANATE induced an IVIS ≤ 3. Therefore, the test item is not corrosive/not severely irritating to the eye according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).