1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol

EC Number:

255-256-8

EC Name:

1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol

Cas Number:

41199-19-3

Molecular formula:

C13H22O

IUPAC Name:

2,5,5-trimethyl-1,2,3,4,4a,5,6,7-octahydronaphthalen-2-ol

Test material form:

liquid

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

- Source: TARAVIS KFT. (Address: H-9600 Sárvár, Rábasömjéni utca 129., Hungary)
- Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and transported to Charles River Laboratories Hungary Kft. at ambient temperature at the earliest convenience.
- After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at Charles River Laboratories Hungary Kft. and processed within 2 hours of collection.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 μL

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
- Eyes selection: After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit.
- Preparation of eyes and Identification: The eyeball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.
- Eyes examination and acclimatization time: The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Too much pressure on the eye by the clamp was avoided. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity. The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
- Solubility checking: The solubility of the test item in physiological saline was tested prior to the experiment (approximately 30 μL test material in 1 mL physiological saline (Manufacturer: B. Braun Pharmaceuticals SA, Lot number: 91134Y05-1, Expiry date: 28 February 2022)). The test item did not dissolve in physiological saline.
- Baseline assessments: At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No significant corneal thickness changes (1.6% was observed in one eye in Experiment II and -1.6% was observed in one eye, 1.6% was observed in one eye and 1.7% was observed in two eyes in Experiment I) and no corneal thickness changes were observed in the other eyes in both experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
- Three test item treated eyes, three positive control treated eyes and one negative control eye were examined during the study.

CONTROLS, APPLICATION DOSE AND EXPOSURE TIME
- Administration of Test and Control Items: After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea. In both experiments 30 μL of the test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea. In both experiments the positive control eyes were treated in a similar way with 30 μL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution).

APPLICATION DOSE AND EXPOSURE TIME
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.

MEASUREMENTS
The negative and positive control eyes and all test item treated eyes were evaluated pre-treatment (as described in Section 11.2. Baseline assessments) and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable. Haag-Streit BP 900® slit lamp microscope was used for the measurements. Corneal thickness and corneal opacity were measured at each time point indicated above. Fluorescein retention was measured on two occasions, at base line (t=0) and approximately 30 minutes after the post-treatment rinse.
- Corneal thickness determination: For thickness measurements, the slit lamp microscope was focused such that the physiological saline solution appeared as a visible, clear (sharp) image as it moved across the cornea surface.
- Corneal opacity determination: For opacity determination, the slit lamp microscope was focused such that the physiological saline solution appeared as a visible, clear (sharp) image as it moved across the cornea surface.
- Fluorescein retention determination: The fluorescein retention determination the settings of the slit lamp microscope was the same as for opacity assessment, but the green light filter was used.

HISTOPATHOLOGY AND MICROSCOPIC EVALUATION
At the end of the procedure, the corneas were carefully removed from the eyes and placed individually into labelled containers of preservative fluid (10% neutral buffered formalin. The corneas, available for potential histopathology, were stored at room temperature.

DECISION CRITERIA
The conclusion on eye irritancy was based on the relevant OECD guideline quantitative assessments and according to Regulatory Toxicology and Pharmacology 54, 2009, 272-281 (See "Any other information on materials and methods).

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

Exp 1. 75 min

Value:

8.2

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Results 2

Irritation parameter:

percent corneal swelling

Run / experiment:

Exp 2. 75 min

Value:

9.4

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

Exp 1. 240 min

Value:

12.6

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Results 4

Irritation parameter:

percent corneal swelling

Run / experiment:

Exp 2. 240 min

Value:

16.6

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Results 5

Irritation parameter:

cornea opacity score

Run / experiment:

Exp 1.

Value:

2.33

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class III

Results 6

Irritation parameter:

cornea opacity score

Run / experiment:

Exp 2.

Value:

2

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class III

Results 7

Irritation parameter:

fluorescein retention score

Run / experiment:

Exp 1.

Value:

0.33

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Results 8

Irritation parameter:

fluorescein retention score

Run / experiment:

Exp 2.

Value:

1.67

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class III

Other effects / acceptance of results:

MORPHOLOGICAL EFFECT
Severe loosening of epithelium was observed in the test item treated eyes in one eye at 75 minutes after the post-treatment rinse in both experiments. Severe loosening of epithelium was observed in the positive control treated eyes in one eye at 120 minutes after the post-treatment rinse in Experiment I and in one eye at 75 minutes after the post-treatment rinse in Experiment II. No other morphological effect was observed in the study.

POSITIVE CONTROL
EXPERIMENT 1
- Mean maximum corneal swelling at up to 75 min: 12.4%; ICE Class III
- Mean maximum corneal swelling at up to 240 min: 28.6%; ICE Class III
- Mean maximum corneal opacity change: 4.00; ICE Class IV
- Mean fluorescein retention change: 3.00; ICE Class IV
- Other Observations: Loosening of epithelium was observed in one eye at 120 minutes after the post-treatment rinse.
EXPERIMENT 2
- Mean maximum corneal swelling at up to 75 min: 9.2%; ICE Class II
- Mean maximum corneal swelling at up to 240 min: 21.1%; ICE Class III
- Mean maximum corneal opacity change: 4.00; ICE Class IV
- Mean fluorescein retention change: 2.83; ICE Class IV
- Other Observations: Severe loosening of epithelium was observed in one eye at 75 minutes after the post-treatment rinse.

NEGATIVE CONTROL
EXPERIMENT 1
- Mean maximum corneal swelling at up to 75 min: 0.0%; ICE Class I
- Mean maximum corneal swelling at up to 240 min: 0.0%; ICE Class I
- Mean maximum corneal opacity change: 0.00; ICE Class I
- Mean fluorescein retention change: 0.00; ICE Class I
- Other Observations: None
EXPERIMENT 2
- Mean maximum corneal swelling at up to 75 min: 0.0%; ICE Class I
- Mean maximum corneal swelling at up to 240 min: -1.6%; ICE Class I
- Mean maximum corneal opacity change: 0.00; ICE Class I
- Mean fluorescein retention change: 0.00; ICE Class I
- Other Observations: None

VALIDITY CRITERIA
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range. This study was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

other: Serious eye damage

Remarks:

in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

The substance causes serious eye damage in the ICE test (OECD guideline 438)

Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD TG 438 guideline and the classification scheme of Schutte et al (2009). After the zero reference measurements, the eyes were held in a horizontal position and the test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds exposure time, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 μL test item. The three positive control eyes were treated in a similar way with 30 μL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated. The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in experiment. Thus, the study was considered to be valid. Slight corneal swelling change (mean = 12.6%) was observed during the four-hour observation period on test item treated eyes. Moderate cornea opacity change (severity 2 on two eyes and severity 3 on one eye) was observed. No significant fluorescein retention change (severity 0.5 on two eyes and no fluorescein retention change on one eye) was noted. Severe loosening of epithelium was observed in one eye at 75 minutes after the post treatment rinse. Due to the equivocal results of the Experiment I, an additional experiment was performed for clarification. In Experiment II slight corneal swelling change (mean = 16.6%) was observed during the four-hour observation period on test item treated eyes. Moderate cornea opacity change (severity 2 on all three eyes) was observed. Moderate fluorescein retention change (severity 0.5 on two eyes and no fluorescein retention change on one eye) was noted. Severe loosening of epithelium was observed in one eye at 75 minutes after the post treatment rinse. Based on this in vitro eye irritation assay in isolated chicken eyes with Ambrinol, according UN GHS Classification and to Schutte et al (2009), the test item is classed as Category 1.