Registration Dossier

Toxicological information

Eye irritation

Currently viewing:

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 3 August 2016 To 26 September 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:
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
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
White to light grey powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Analytical purity: 100%
- Purity test date: not specified
- Lot/batch No.: DRDGAHE0412001
- Expiration date of the lot/batch: 28 Jult 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: not specified
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was sued neat, as supplied

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS, spring chickens
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: poultry slaughterhouse v.d. Bor, Nijkerkerveen
- Number of animals: 7 Eyes were used
- Characteristics of donor animals (e.g. age, sex, weight): Approximately 7 weeks old), body weight range approximately 1.5-2.5 kg
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- Time interval prior to initiating testing: within 2 hours
- indication of any existing defects or lesions in ocular tissue samples: not specified
- Indication of any antibiotics used:not specified

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg
- Concentration (if solution): neat

VEHICLE
- Amount(s) applied (volume or weight with unit): 30mg or 30µL
- Lot: B0988898 347 (positive control ) ; H1123-02 (negative control)
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
The eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment
Number of animals or in vitro replicates:
triplicates were used for test item and positive control condition.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus (Triskelion, Zeist, the Netherlands; see Figure 1). The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate of 0.10 0.15 mL/min (peristaltic pump set at speed 5.00). The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32°C (water pump set at 36.4°C).
After placing in the superfusion apparatus, the eyes were examined again with the slit lamp microscope to ensure that they were not damaged. Corneal thickness was measured using the slit lamp microscope, set at 0.095 mm. Corneal thickness was expressed in instrument units. An accurate measurement was taken at the corneal apex of each eye.
Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unaccep¬tably stained with fluores¬cein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced.

EQUILIBRATION AND BASELINE RECORDINGS
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibrati¬on period of 45 60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculati¬ons.
At time t = 0 (i.e. immedia¬tely after the zero reference measure¬ment), the following procedure was applied for each test eye: The clamp holding the test eye was placed on paper tissues outside the chamber with the cornea facing upwards.

NUMBER OF REPLICATES
triplicates were used for test item and positive control condition.

NEGATIVE CONTROL USED
physiological saline

POSITIVE CONTROL USED
NaOH

APPLICATION DOSE AND EXPOSURE TIME
30 µL or 30 mg

OBSERVATION PERIOD
240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL saline
- Indicate any deviation from test procedure in the Guideline not specified

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: slit lamp microscope
- Damage to epithelium based on fluorescein retention: slit amp microscope
- Swelling: measured with slit-lamp microscope
- Macroscopic morphological damage to the surface:
- Others : histopathology

SCORING SYSTEM:
- Mean corneal swelling (%)
Corneal swelling, expressed as a percentage, is calculated according to the following formula:
“Corneal thickness at time t minus corneal thickness at time t = 0, divided by corneal thickness at time t = 0 and multiplied by 100”.
A negative swelling up to -5% (not unusual for control eyes) will be presented as 0 % swelling in the tables/appendices.

- Mean maximum opacity score
Opacity degree of density (area most dense taken for scoring)
0 = no opacity
0.5 = very faint opacity (= very slight)
1 = scattered or diffuse areas, details of iris clearly visible (= slight)
2 = easily discernible translucent area, details of iris slightly obscured (= moderate)
3 = severe corneal opacity, no specific details of iris visible, size of pupil barely discernible (= severe)
4 = complete corneal opacity, iris invisible (= very severe)

- Mean fluorescein retention score at 30 minutes post-treatment
0 = no fluorescein retention
0.5 = very minor single cell staining (= very slight)
1 = single cell staining scattered throughout the treated area of the cornea (= slight)
2 = focal or confluent dense single cell staining (= moderate)
3 = confluent large areas of the cornea retaining fluorescein (= severe)
Intermediate scores can also be assigned. The mean fluorescein retention value for all test eyes is calculated for the observation time point of 30 minutes only. If desired or in case of test substances that have adhered to the cornea, fluorescein retention can be determined at t=240 min or whenever the test compound is removed.

NOTE. Intermediate scores can also be assigned, e.g. slight-to-moderate (1.5) and moderate-to-severe (2.5).

The value for all test eyes is calculated for the observation time points of 30, 75, 120, 180, and 240 minutes.

DECISION CRITERIA: In the ICE test, the eyes were examined at several time intervals after treatment to determine ocular effects using the parameters of corneal thickness (swelling), corneal opacity and fluorescein retention. Defined scoring scales were used for each parameter to define the severity of effects into four categories (I-IV). Four classes of eye irritancy (not irritating; slightly irritating; moderately irritating; severely irritating) were identified in the ICE test by combination of the categories defined for each of the evaluation parameters using the Prediction Model.(Attadched Table 1 ). Classification was performed accoprding to the combination of the categories.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Run / experiment:
Test Item
Value:
9
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
Test Item
Value:
2
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
Test Item
Value:
2
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not specifed

DEMONSTRATION OF TECHNICAL PROFICIENCY: NaOH indiced a positive response and proved the system performance.

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

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate (Appendix 2).
The positive control NaOH caused severe swelling (mean score 45%), very severe corneal opacity (mean score 4.0), severe fluorescein retention (mean score 3.0), and severe loosening of epithelium and demonstrated the ICE test as meeting the acceptance criteria to be considered a valid study

2-Amino-4-Hydroxyethylaminoanisole Sulfate (A084; GTS119130) caused slight corneal swelling (mean swelling 9%), moderate opacity (mean score 2.0) and moderate fluorescein retention (mean score 2.0).
Microscopic examination of the corneas treated with 2-Amino-4-Hydroxyethylaminoanisole Sulfate (A084; GTS119130) revealed very slight (1/3 corneas) erosion of the epithelium.

Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control NaOH revealed severe erosion (3/3 corneas) and severe necrosis (3/3 corneas) of the epithelium, stromal necrosis (3/3 corneas), and necrosis of the endothelium (3/3 corneas).

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the experimental condition of the study, the test item induced irritation to the chicken eyes when tested neat in this ICE assay. Additionally, the report concluded to the classification of Category 2A. However, according to the CLP regulation, an ICE can only classfied Category 1 or Not classified. Hence, the registered item was considered to have irritant potential, but Category 2A for the classification cannot be applied according to CLP criteria. Further studies (as OECD TG 492) study are necessary to define the eye irritation classification.
Executive summary:

The purpose of this GLP compliant study was to evaluate the potential irritation to eye property of the test item A084 when applied neat in chicken eye, performed in a Isolated Chicken Eye Assay according to OECD 439 Guideline .

Chicken eyes were obtained from slaughter animals used for human consumption. The isolated chicken eyes were exposed to a single application of 30 mg for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology of the corneas was performed.

2-Amino-4-Hydroxyethylaminoanisole Sulfate (A084; GTS119130) caused slight corneal swelling (mean swelling 9%), moderate opacity (mean score 2.0) and moderate fluorescein retention (mean score 2.0). Microscopic examination of the corneas revealed very slight (1/3 corneas) erosion of the epithelium.

Under the experimental condition of the study, the test item induced irritatation to the chicken eyes when tested neat in this ICE assay. Additionally, the report concluded to the classification of Category 2A. However, according to the CLP regulation, an ICE can only classfied Category 1 and Not classified. Hence, the registered item was considered to have irritant potential, but Category 2A for the classification cannot be applied according to CLP criteria. Further studies (as OECD TG 492 study) are necessary to define the eye irritation classification.