Leuco polysulfided 4-[(2,4-dinitrophenyl)amino]phenol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

23 August - 22 September, 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)

Version / remarks:

26 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:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Leuco polysulfided 4-[(2,4-dinitrophenyl)amino]phenol

EC Number:

310-132-3

EC Name:

Leuco polysulfided 4-[(2,4-dinitrophenyl)amino]phenol

Cas Number:

61902-31-6

Molecular formula:

not applicable

IUPAC Name:

Reaction product of 4-[(2,4-dinitrophenyl)amino]phenol with sodium polysulfide, leuco derivatives

Test material form:

solid: particulate/powder

Details on test material:

Test item: Leuco Sulfur Blue 11
Appearance: dark black powder
CAS No. 61902-31-6
EC No. 310-132-3
Storage: room temperature

Specific details on test material used for the study:

Expiration date: 29 April 2020
Storage: Room temperature (15-25°C)

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Characteristics of donor animals: All eyes used in the assay were from the same groups of eyes collected on one specific day.
- Temperature and transport conditions of ocular tissue: 2 h at ambient temperature (19.4 ºC to 20.3 ºC)
- indication of any existing defects or lesions in ocular tissue samples: 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.
- Indication of any antibiotics used: not specified

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 mg/eye

Duration of treatment / exposure:

10 sec

Duration of post- treatment incubation (in vitro):

30, 75, 120, 180 and 240 minutes

Number of animals or in vitro replicates:

3 test item treated eyes and 3 positive control eyes and one negative control eye were used in the 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 .

Tool used to assess score: hand-slit lamp/biomicroscope/fluorescein
The appropriate number of eyes was selected and after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic 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 using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm.
Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current).

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. 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.


SELECTION AND PREPARATION OF ISOLATED EYES
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.

EQUILIBRATION AND BASELINE RECORDINGS
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 avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied 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 saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 5 drops/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, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic 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 using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm. 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 was conducted for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods. 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 ±5-7 % within approximately 45 to 60 minutes before the start of application. Slight changes in thickness (0% to 2%) were observed in the eyes, finding considered as normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

NUMBER OF REPLICATES
3 test item treated eyes and 3 positive control eyes and one negative control eye were used in the study.

NEGATIVE CONTROL USED
30 µL NaCl (9 g/L saline)

POSITIVE CONTROL USED
30 mg Imidazole

APPLICATION DOSE AND EXPOSURE TIME
30 mg test item, 10 s

OBSERVATION PERIOD
30, 75, 120, 180 and 240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 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.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Yes
- Damage to epithelium based on fluorescein retention: Yes
- Swelling: measured with optical pachymeter on a slit-lamp microscope, yes

SCORING SYSTEM:
- Mean corneal swelling (%) : Yes
- Mean maximum opacity score : Yes
- Mean fluorescein retention score at 30 minutes post-treatment : Yes

DECISION CRITERIA: Tthe decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: over all ICE score: 1xI; 1xII; 1xIII

Remarks:

Overall in vitro classification is neither UN GHS Classification Category I nor No Category; test item has been categorized as “No prediction can be made”.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

Table 2: Summary of the results test item)

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	3%	I
Mean maximum corneal swelling at up to 240 min	4%	I
Mean maximum corneal opacity	1.7	III
Mean fluorescein retention	1.3	II
Other Observations	None
Overall ICE Class^1	1xI, 1xII, 1xIII

Table 3: Positive Control (Imidazole)

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	25%	III
Mean maximum corneal swelling at up to 240 min	33%	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, UNGHS Classification: Category 1.

 

Table 4: Negative Control: (NaCl, 9 g/L saline))

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	2%	I
Mean maximum corneal swelling at up to 240 min	3%	I
Mean maximum corneal opacity	0.5	I
Mean fluorescein retention	0.0	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 range.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In an ex vivo assay in isolated chicken eyes (ICE) according to OECD guideline 438, the overall ICE class was 1xI, 1xII, 1xIII. The overall in vitro classification is neither UN GHS Classification Category I nor No Category. Thus, the test item has been categorized as “No prediction can be made”.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICE Test) was to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test was performed according to OECD guideline 438. 30 mg/ of the test item or positive control or 30 µL of the negative control (NaCl, 9 mg/L saline), repectively, were applied to the cornea. Three test item treated eyes, three positive eyes and one negative control eye were used in this study.

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. Tested corneas were evaluated pre-treatment and at 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 time points above. The overall ICE class for the test item was 1xI, 1xII, 1xIII. Positive and negative controls showed the expected results. The experiment was considered to be valid.

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