6,6'-di-tert-butyl-2,2'-thiodi-p-cresol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 April 2017 to 20 April 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

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)

Version / remarks:

OECD Guidelines for the Testing of Chemicals 438 (26th July 2013)

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)

Version / remarks:

EU Commission Regulation (EC) No 1152/2010 (8th December 2010) amending, Regulation (EC) No 440/2008: Method B 48.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

OPPTS 870.2400 (EPA 712-C-98-195) August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

6,6'-di-tert-butyl-2,2'-thiodi-p-cresol

EC Number:

202-009-7

EC Name:

6,6'-di-tert-butyl-2,2'-thiodi-p-cresol

Cas Number:

90-66-4

Molecular formula:

C22H30O2S

IUPAC Name:

6,6'-di-tert-butyl-2,2'-thiodi-p-cresol

Test material form:

solid: particulate/powder

Details on test material:

Name: 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol
Other name: LOWINOX® TBP-6
CAS number: 90-66-4
Batch/Lot Number: C034J0059 / C036K0111
Description: White powder
Purity*: 99.8%
Expiry date: 12 May 2017
Storage condition: Controlled room temperature (15-25 ºC, below 70 RH%)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
* No correction for purity of the test item was applied.

Specific details on test material used for the study:

No further details specified in the study report.

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

CHICKEN HEADS COLLECTION AND TRANSPORT
Strain of chicken: ROSS 308
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 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 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 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.

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.

Identification
The eyes were identified by chamber number, marked on the door of the chamber.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

In each experiment, 30 mg of the test item was applied onto the entire surface of the cornea

Duration of treatment / exposure:

exposure period of 10 seconds from the end of the application the cornea surface

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. Minor variations within approximately ±5 minutes were considered acceptable.

Number of animals or in vitro replicates:

One eye was treated with physiological saline, three eyes with the test item and another three with powdered Imidazole in each experiment.

Details on study design:

THE BASELINE ASSESSMENTS
The 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 changes in thickness (0.0%) were observed in the eyes in each 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.

TEST PROCEDURE
Treatment
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 each experiment, 30 mg 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 each experiment negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 mg powdered Imidazole.
One eye was treated with physiological saline, three eyes with the test item and another three with powdered Imidazole in each experiment.

Test item removal
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.
Additional gentle rinsing with 20 mL saline was performed at each time point when the test item or positive control material remaining on the cornea was observed. The test item treated eyes were rinsed additional gentle rinsing with 20 mL saline after treatment in each experiment.

OBSERVATION
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 Bern 900 slit-lamp microscope was used for the measurements.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

mean maximum

Run / experiment:

Experiment I

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

fluorescein retention score

Remarks:

mean

Run / experiment:

Experiment I

Value:

0.17

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

cornea opacity score

Remarks:

mean maximum

Run / experiment:

Experiment II

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

fluorescein retention score

Remarks:

mean

Run / experiment:

Experiment II

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test item 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol 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.
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in experiment. This experiment was considered to be valid.

Any other information on results incl. tables

TEST ITEM

Experiment I
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.50	I
Mean fluorescein retention	0.17	I
Other observations	Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) was cleared at 30 minutes after the post-treatment rinse.
Overall OCE Class	3xI

 

Experiment II
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.6%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other observations	Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) was cleared at 30 minutes after the post-treatment rinse.
Overall OCE Class	3xI

 

POSITIVE CONTROL

Experiment I
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	9.7%	II
Mean maximum corneal swelling at up to 240 min	28.1%	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other observations	Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse
Overall OCE Class	1xIII 2xIV

 

Experiment II
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	9.7%	II
Mean maximum corneal swelling at up to 240 min	25.8%	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other observations	Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Overall OCE Class	1xIII 2xIV

 

NEGATIVE CONTROL

Experiment I
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other observations	None
Overall OCE Class	3xI

 

Experiment II
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other observations	None
Overall OCE Class	3xI

 

VALIDITY OF THE TEST

Historical Control data

 

Negative Control: Physiological Saline

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	-3.2%	3.4%
Maximum corneal swelling at up to 240 min	-4.8%	3.4%
Maximum corneal opacity change	0.00	0.50
Fluorescein retention	0.00	0.50
Number of studies	254

 

Positive Control: Imidazole

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	-6.6%	25.0%
Maximum corneal swelling at up to 240 min	-15.9	36.7%
Maximum corneal opacity change	3.50	4.00
Fluorescein retention	2.00	3.00
Number of studies	117

 

 

SUMMARY TABLE FOR UN GHS CLASSIFICATION

EXPERIMENT I AND EXPERIMENT II

Criteria for No Category	True/False
3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint class as II:	True
Test item was not stuck to the cornea:	True
Criteria for Category 1	True/False
2 or more endpoints classed as IV:	False
Corneal opacity≥3 at 30 min (in at least 2 eyes):	False
Corneal opacity = 4 at any time point (in at least 2 eyes):	False
Severe loosening of epithelium (in at least 1 eye):	False
Criteria for No predication can be made	True/False
Based on the endpoints not classifiable for No Category and for Category 1:	False
Particles of test item were stuck to the cornea and could not be washed off during the study	False

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the in vitro eye irritation assays in isolated chicken eyes with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol, the test item was non-irritant, UN GHS Classification: No Category.

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 (26 July 2013).

 

In each experiment after the zero reference measurements, the eye was held in horizontal position and 30 mg test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% (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 within the historical control data range in experiment. Thus, the experiment was considered to be valid.

 

Experiment I: No corneal swelling was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity change (severity 0.5 on all three eyes) was observed on three eyes. No significant fluorescein retention change (severity 0.5 on one eye) was noted on all three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

 

Experiment II: No significant corneal swelling (mean ≤5%) was observed during the four hour observation period on test item treated eyes. No cornea opacity change and no fluorescein retention change were observed on three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

 

Based on these in vitro eye irritation assays in isolated chicken eyes with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol, the test item was non-irritant, UN GHS Classification: No Category.