Reaction product of 4-aminophenol with 2-ethyl-6-methylbenzenamine and sodium polysulfide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 December 2016 - 30 January 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:

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:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of 4-​aminophenol with 2-​ethyl-​6-​methylbenzenamine and sodium polysulfide

EC Number:

600-519-8

Cas Number:

1040873-93-5

Molecular formula:

not applicable

IUPAC Name:

Reaction product of 4-​aminophenol with 2-​ethyl-​6-​methylbenzenamine and sodium polysulfide

Test material form:

solid: particulate/powder

Details on test material:

Test item: Blue Sema
Appearance: Black to brownish black, solid
CAS No: 1040873-93-5
EC No: 600-519-8

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal
(19.4ºC to 20.3ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4 5 heads/box).
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 fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with 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 eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item and positive control applied in an amount of 0.03 g/eye.

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

Three test item treated eyes, three positive control and one negative control eyes were used in this study.

Details on study design:

isolated chicken eye test (ICET)

Removal of test item:
The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum. The Imidazole and test item were stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole treated eyes after the 30, 75, 120 and 180 minutes of observation, but cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all (three eyes) test item treated eyes after the 30 and 75 minutes of observation. All test item treated eyes were totally cleared at 120 minutes after the post-treatment rinse.

Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an ICE (Isolated Chicken Eye) class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for each test substance.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

1-3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The overall ICE class was 3xII.

Remarks:

The overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, the test item has been categorised as “No prediction can be made”.

Any other information on results incl. tables

Test Item:

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	10%	II
Mean maximum corneal swelling at up to 240 min	18%	II
Mean maximum corneal opacity	1.3	II
Mean fluorescein retention	1.3	II
Other Observations	None
Overall ICE Class^1	3xII

Positive Control: Imidazole 

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	28%	IV
Mean maximum corneal swelling at up to 240 min	37%	IV
Mean maximum corneal opacity	4.0	IV
Mean fluorescein retention	3.0	IV
Other Observations	Cornea opacity score 4 was observed in two eyes at 30 minutes after the post-treatment rinse.
Overall ICE Class^1	3xIV

The positive control Imidazole was classed as corrosive/severely irritating, UN GHS Classification: Category 1.

 

Negative Control: NaCl (9 g/L saline) 

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	0%	I
Mean maximum corneal swelling at up to 240 min	0%	I
Mean maximum corneal opacity	0.0	I
Mean fluorescein retention	0.5	I
Other Observations	None
Overall ICE Class^1	3xI

The negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.

Positive and negative control values were within the corresponding historical control data ranges.

Applicant's summary and conclusion

Interpretation of results:

other: not Category 1 (irreversible effects on the eye) based on GHS criteria

Remarks:

The test item has been categorized as “No prediction can be made”.

Conclusions:

In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes (not UN GHS Cat. 1).

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) according to OECD guideline 438 was to evaluate the potential ocular corrosivity of the test item by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points. The test item and Imidazole (positive control) was ground before use in the study and applied in an amount of 30 mg/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 µL saline solution. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with about 20 mL saline solution at ambient temperature and this procedure was repeated for each eye.

Adherence of the test item and the positive control Imidazole was observed on the cornea surfaces at 240 min after the post-treatment rinse. Positive and negative controls showed the expected results. The experiment was considered to be valid. The overall ICE class of the test item was 3xII. The test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes (not UN GHS Cat. 1).