2-methyl-4-phenylbutan-2-ol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 May 2017 - 23 August 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 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:

2-methyl-4-phenylbutan-2-ol

EC Number:

203-074-4

EC Name:

2-methyl-4-phenylbutan-2-ol

Cas Number:

103-05-9

Molecular formula:

C11H16O

IUPAC Name:

2-methyl-4-phenylbutan-2-ol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Yinghai (Cangzhou) Aroma Chemical Company Ltd.,; CP008-170101
- Expiration date of the lot/batch: 30 June 2018
- Purity:99.7%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25 ºC, below 70 RH%). Protected from light and humidity

Test animals / tissue source

Species:

chicken

Strain:

other: COBB 500

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út 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 heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.

After collection, the heads were inspected for appropriate quality and wrapped with tissue aper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within approximately 2 hours of collection.

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): 30 μL

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

Three eyes per group

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
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 fluoresceintreated 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. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

The eye ball 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 to 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.

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). Again avoid too much pressure on the eye by the clamp. 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 thenacclimatization and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
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 changes in thickness (0.0%) were observed in the eyes of this study. 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
3

NEGATIVE CONTROL USED
Physiological saline (0.9% (w/v) NaCl

POSITIVE CONTROL USED
5% (w/v) Benzalkonium chloride solution

APPLICATION DOSE AND EXPOSURE TIME
10 seconds

OBSERVATION PERIOD
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period:
The time of application was observed, 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 at ambient temperature, taking care not to damage the cornea but attempting to remove all residual of the test item if possible.

METHODS FOR MEASURED ENDPOINTS:
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit BP 900® slit-lamp microscope was used for the measurements.

- Macroscopic morphological damage to the surface:
Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the Investigator.

SCORING SYSTEM:
- Mean corneal swelling (%):Refer to Section 3.8.1 of the report
- Mean maximum opacity score: Refer to Section 3.8.2 of the report
- Mean fluorescein retention score at 30 minutes post-treatment: Refer to Section 3.8.3 of the report

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Remarks:

75 mins

Run / experiment:

DMPEC

Value:

2.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class

Remarks:

I

Results 2

Irritation parameter:

percent corneal swelling

Remarks:

240 mins

Run / experiment:

DMPEC

Value:

3.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class

Remarks:

I

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

DMPEC

Value:

1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class

Remarks:

II

Results 4

Irritation parameter:

fluorescein retention score

Run / experiment:

DMPEC

Value:

3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class

Remarks:

IV

Results 5

Irritation parameter:

percent corneal swelling

Remarks:

75 mins

Run / experiment:

Positive Control

Value:

12.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class

Remarks:

III

Results 6

Irritation parameter:

percent corneal swelling

Remarks:

240 mins

Run / experiment:

Positive control

Value:

28

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class

Remarks:

III

Results 7

Irritation parameter:

cornea opacity score

Run / experiment:

Positive control

Value:

4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class

Remarks:

IV

Results 8

Irritation parameter:

fluorescein retention score

Run / experiment:

Positive control

Value:

3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class

Remarks:

IV

Results 9

Irritation parameter:

other: All parameters

Run / experiment:

Negative Control

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class

Remarks:

I

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:
Positive control only: Severe loosening of epithelium was observed on one eye (1/3) at 180 minutes and on two eyes (2/3) at 240 minutes after the post-treatment rinse.

ACCEPTANCE OF RESULTS:
The results from all eyes used met the quality control standards. The negative control and positive control results were in line with historic data. This experiment was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on this in vitro eye irritation in the isolated chicken eyes test with DMPEC, the test item is not classified as a severe irritant and not classified as non-irritant.

Executive summary:

In an in vitro eye irritation test in isolated chicken eyes (ICE) assay (17_094-038CS), isolated chicken eyes were exposed to DMPEC (99.7%) for 10 seconds. Physiological saline (0.9% (w/v) NaCl) was used for the negative control and 5% (w/v) Benzalkonium chloride solution was used for the positive control. The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse.

No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. Slight corneal opacity change (severity 0.5

or 1) was noted on three eyes. Severe fluorescein retention change (severity 3) was observed. No other corneal effect was observed. The positive and negative controls gave the appropriate responses. The ICE classes for DMPEC were: 1xI (mean corneal swelling) 1xII (mean corneal opacity) 1xIV (mean fluorescein retention). DMPEC did not meet any of the criteria for Eye damage – Category 1. DMPEC did not meet all the criteria for Not classified (i.e. it was false that 3 endpoints were classed as I or 2 endpoints were classed as I and 1 endpoint classed as II. Based on this in vitro eye irritation in the isolated chicken eyes test with DMPEC, the test item is not classified as a severe irritant and not classified as non-irritant.

This in vitro eye irritation test in isolated chicken eyes (ICE) assay is acceptable and satisfies the guideline requirement for an OECD 438 study.