Reaction mass of 1-[rac-(1R,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol isomer 1 and 1-[rac-(1S,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

eye corrosion

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 2018-02-09 to 2018-05-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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 1-[rac-(1R,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol isomer 1 and 1-[rac-(1S,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol

EC Number:

947-711-0

Molecular formula:

C14H28O

IUPAC Name:

Reaction mass of 1-[rac-(1R,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol isomer 1 and 1-[rac-(1S,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol

Test animals / tissue source

Species:

chicken

Strain:

other: Gallus gallus e.g. Ross 308 Broiler

Details on test animals or tissues and environmental conditions:

- Species: Spring chickens ( Gallus gallus e.g. Ross 308 Broiler)
- Number: Multiple chicken heads (three eyes for the test item, three eyes for the positive control item and two eyes for the negative control item)
- Sex: Male or female
- Age of chicken (at slaughter): Approximately 56 days old
- Weight of chicken (at slaughter): Approximately 3 kg
- Storage, temperature and transport conditions of ocular tissue: Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day. Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.
- Time interval prior to initiating testing: The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.03 mL per eye of the test item

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

observations prior to treatment and at 30, 75, 120, 180 and 240 minutes after decontamination with isotonic saline

Number of animals or in vitro replicates:

3 eyes for the test item
3 eyes for the positive control
2 eyes for the negative control

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Eyelids were carefully excised. The integrity of the cornea was measured with a drop of 2 % (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined for damage to the cornea. When the fluorescein retention and corneal opacity scores were ≤ 0.5, the eyes were selected for the test. Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32.0 ± 1.5 °C.

EQUILIBRATION AND BASELINE RECORDINGS
Once all eyes were placed in the superfusion apparatus, the eyes were examined again to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device at the center of each cornea. After the approval process the eyes were incubated for 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES
3 eyes for the test item
3 eyes for the positive control
2 eyes for the negative control

NEGATIVE CONTROL USED
0.03 mL of the negative control item was applied ot the cornea of each negative control eye.

POSITIVE CONTROL USED
0.03 mL of the positive control item, Benzalkonium chloride (5% v/v), was applied and after 10 seconds was rinsed with 20 mL isotonic saline.

APPLICATION DOSE AND EXPOSURE TIME
0.03 mL of the test item were applied to the cornea. The tets item was used in its initial state. The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of 0.9 % (w/v) sodium chloride solution.

OBSERVATION PERIOD
observations prior to treatment and at 30, 75, 120, 180 and 240 minutes after decontamination with isotonic saline

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL of 0.9 % (w/v) sodium chloride solution after 10 seconds exposure

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: calculated with the most densely opacified areas for scoring
- Damage to epithelium based on fluorescein retention: calculated at the 30 minute time interval only
- Swelling: assessed from corneal thickness measurements
- Macroscopic morphological damage to the surface: observations for pitting, sloughing, roughening of the corneal surface, and adhering of test item were made

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

240 mins

Value:

1.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class: III

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

30 mins

Value:

1.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class: II

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

240 mins

Value:

8.96

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class: III

Any other information on results incl. tables

Table 1. Summary of test items results for all endpoints

Mean Corneal Opacity (ICE class)	Mean Fluorescein Retention (ICE class)	Mean Corneal Thickness% Compared to Time Zero (ICE class)					Combination of the 3 Endpoints
		30 mins	75 mins	120 mins	180 mins	240 mins
1.7 (III)	1.5 (II)	8.02 (II)	13.21 (III)	8.49 (II)	8.02 (II)	8.96 (II)
		(III)					1 x II, 2 x III
Classification:		No Prediction Can Be Made

Corneal Opacity Scores

Scattered or diffuse areas; details of the iris are clearly visible was noted in one test item treated eye and Easily discernible translucent area; details of the iris are slightly obscured were noted in two test item treated eyes.

Complete corneal opacity; iris invisible was noted in all positive control treated eyes.

Very faint opacity was noted in the negative control treated eyes.

No morphological effects were noted in the test item or negative control item treated eyes. Sloughing was noted in two of the positive control treated eyes.

Fluorescein Retention Scores

Very minor single cell staining was noted in one test item treated eye. Focal or confluent dense single cell staining was noted in two test item treated eyes. Confluent large areas of the cornea retaining fluorescein were noted in all positive control treated eyes. No fluorescein retention to very minor single cell staining was noted in the negative control treated eyes.

Positive control item

Maximal mean score for corneal opacity:       4.0       ICE Class IV

Mean score of Fluorescein Retention:            3.0       ICE Class IV

Maximal mean corneal swelling compared to time zero:       37.98%       ICE Class IV

Negative Control Item

Maximal mean score for corneal opacity:       0.5       ICE Class I

Mean score of Fluorescein Retention:            0.3       ICE Class I

Maximal mean corneal swelling compared to time zero:       0.00%       ICE Class I

Applicant's summary and conclusion

Interpretation of results:

other: not classified in Category 1

Conclusions:

The substance is not to be classified as severely eye damaging (Cat. 1).

Executive summary:

This ex vivo study was performed to assess the eye irritation potential of the test item by means of the Isolated Chicken Eye Test according to OECD 438 and GLP. The study was performed to evaluate the possible corrosivity or severe irritancy potential of the test item as measured by its ability to induce toxicity in an enucleated chicken eye. 0.03 mL of the test item were applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item (Benzalkonium chloride). A further two enucleated eyes were treated with Sodium chloride for control purposes. Treatment with the test item showed inconclusive results with regard to eye irritation. In conclusion, the substance is not to be classified as severely eye damaging (Cat. 1).