6,6'-di-tert-butyl-4,4'-diethyl-2,2'-methylenediphenol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 07 to December 07, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

September 22, 2015

Test material

Test material information

Constituent 1

Reference substance name:

6,6'-di-tert-butyl-4,4'-diethyl-2,2'-methylenediphenol

EC Number:

201-814-0

EC Name:

6,6'-di-tert-butyl-4,4'-diethyl-2,2'-methylenediphenol

Cas Number:

88-24-4

Molecular formula:

C25H36O2

IUPAC Name:

2-tert-butyl-6-[(3-tert-butyl-5-ethyl-2-hydroxyphenyl)methyl]-4-ethylphenol

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

chicken

Strain:

other: COBB 500 (Experiment I) and Ross 308 (Experiment II)

Details on test animals or tissues and environmental conditions:

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 paper moistened with physiological 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.

SELECTION AND PREPARATION OF EYES FOR THE TEST
* 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. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

* Preparation of eyes
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 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). 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 the acclimatization and treatment periods.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

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.

Number of animals or in vitro replicates:

Three replicates for the test item and the positive control
One replicate for the negative control

Details on study design:

TREATMENT
After the zero reference measurements, the eye (in its retainer) was removed from the chamber, held in a horizontal position and the test item was applied onto the centre of the cornea, taking care not to damage or touch the cornea. 30 mg of the test item was applied to the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance.
Negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 mg powdered imidazole.
Three test item treated eyes, three positive control treated eyes and one negative control eye were examined during the study.

TEST ITEM REMOVAL
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 the test item if possible. Additional gentle rinsing with 20 mL saline was performed after treatment and at each time point when the test item or positive control material remaining on the cornea was observed. The test item treated eyes (where the test item was stuck on the cornea
surfaces) were rinsed additional gentle rinsing at least 2x20 mL saline after treatment in each experiment.

OBSERVATION AND ASSESSMENT OF CORNEAL EFFECTS
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.
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.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Remarks:

at up to 75 min

Run / experiment:

Experiment I

Value:

0

Remarks on result:

other: ICE Class I

Results 2

Irritation parameter:

percent corneal swelling

Remarks:

at up to 240 min

Run / experiment:

Experiment I

Value:

1.1

Remarks on result:

other: ICE Class I

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment I

Value:

0.5

Remarks on result:

other: ICE Class I

Results 4

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment I

Value:

0.33

Remarks on result:

other: ICE Class I

Results 5

Irritation parameter:

percent corneal swelling

Remarks:

at up to 75 min

Run / experiment:

Experiment II

Value:

0.5

Remarks on result:

other: ICE Class I

Results 6

Irritation parameter:

percent corneal swelling

Remarks:

at up to 240 min

Run / experiment:

Experiment II

Value:

1.1

Remarks on result:

other: ICE Class I

Results 7

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment II

Value:

0

Remarks on result:

other: ICE Class I

Results 8

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment II

Value:

0.17

Remarks on result:

other: ICE Class I

Other effects / acceptance of results:

TEST ITEM
In Experiment I and II, mnimal amount of test item was stuck on three and two cornea surfaces, resp. after the post-treatment rinse. The cornea surfaces were cleared at 120 minutes after the post-treatment rinse. The test item showed no significant corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results. Based on these results, the test item was non-irritant, UN GHS Classification: No Category.

POSITIVE CONTROL
The positive control (Imidazole) was classified as severely irritating, UN GHS Classification: Category 1.

NEGATIVE CONTROL
The negative control (Physiological saline) was classified as non-irritating, UN GHS Classification: Non-classified.

VALIDITY OF THE TEST
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range. Therefore this experiment was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the in vitro eye irritation assay in isolated chicken eyes, 6,6'-di-tert-butyl-4,4'-diethyl-2,2'-methylenediphenol is non-irritant.

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 No. 438 guideline. After the zero reference measurements, the eye was held in horizontal position and 30 mg of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (9% (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

The test item showed no significant corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results. Therefore, based on these in vitro eye irritation tests in isolated chicken eyes with 6,6'-di-tert-butyl-4,4'-diethyl-2,2'-methylenediphenol, the test item was non-irritant, UN GHS Classification: No Category.