2-(4-amino-2-nitroanilino)ethanol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 August 2016 to 28 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

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. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-amino-2-nitroanilino)ethanol

EC Number:

220-701-7

EC Name:

2-(4-amino-2-nitroanilino)ethanol

Cas Number:

2871-01-4

Molecular formula:

C8H11N3O3

IUPAC Name:

2-(4-amino-2-nitroanilino)ethanol

Test material form:

solid: crystalline

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch No. 7213003567
- Expiration date of the lot/batch: 18/09/2016
- Purity test date: no certificate of analysis was available in the report

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (15-25 °C)
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: no vehicle used

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no treatment, the test item was used as supplied
- Final dilution of a dissolved solid, stock liquid or gel: no dilution performed
- Final preparation of a solid: no preparation performed

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: Bor poultry slaughterhouse (Netherlands)
- Number of animals: Not specified, 7 eyes were used
- Characteristics of donor animals (e.g. age, sex, weight): 6 weeks old, 1.5 kg- 2.5 kg
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): 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: 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): 30mg of test item
- Concentration (if solution): the positive control was used neat (ground)

VEHICLE
The test item was used pure

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit):30 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 30 mg
- Concentration (if solution): pure

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

0, 30, 75, 120, 240 minutes

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

in vitro replicates
1 for Negative Control
3 for Positive Control
3 for Test group

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES

Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium 2.0% w/v (Minims, Chauvin, England) was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein treated cornea was examined with a slit lamp microscope (Slit lamp 900 BP, Haag Streit AG, Liebefeld Bern, Switzerland) to ensure that the cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye ball from the orbit without cutting off the optical nerve too short.
The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and 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 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, Watson-Marlow 205CA). 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 superfu¬sion apparatus, the eyes were examined again with the slit lamp microscope to ensure that they were not damaged. Corneal thickness was measured using the Depth Measuring Attachment No. I for the Haag Streit 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 fluorescein (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 equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.

NUMBER OF REPLICATES
1 for Negative Control ; 3 for Positive Control ; 3 for Test group

NEGATIVE CONTROL USED
Physiological Saline NaCl 0.9% w/v

POSITIVE CONTROL USED
Sodium Hydroxide NaOH

APPLICATION DOSE AND EXPOSURE TIME
30 μL of negative control or 30 mg of postive control and test item were applied for 10 seconds.

OBSERVATION PERIOD
0, 30, 75, 120, 180 and 240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The eye was rinsed 10 seconds after t
reatment with test item or control. They were rinsed with 20 mL saline solution.
- Indicate any deviation from test procedure in the Guideline : no deviation

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: slit lamp microscope
- Damage to epithelium based on fluorescein retention: Slit lamp microscope
- Swelling: measured with optical pachymeter on a slit-lamp microscope
- Macroscopic morphological damage to the surface: No
- Others (e.g, histopathology): Histopathological examination

SCORING SYSTEM:
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”.
The mean percentage of swelling for the three test eyes will be calcula¬ted for each of the observation time points of 30, 75, 120, 180, and 240 minutes. The maximum mean percentage (can be at any of the time points) will be used for classification into one of four categories

- 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)
The mean corneal opacity value for all test eyes is calculated for the observation time points of 30, 75 , 120, 180, and 240 minutes. The maximum mean opacity score (can be at any of the time points) will be used for classification into one of four categories

- 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)

DECISION CRITERIA: On the basis of the severity of the observed findings for corneal swelling, corneal opacity and fluorescein retention, the effects are divided into four categories, viz. I = no effect; II = slight effect; III = moderate effect; IV = severe effect.

Interpretation of corneal swelling, corneal opacity, and fluorescein retention and categorisation into the four categories is done according the following methodology:

Corneal swelling:
Mean corneal swelling (%) Category
0 5 I
>5 12 II
>12 - 18 (>75 min. after treatment) II
(≤75 min. after treatment) III
>18 26 III
>26 - 32 (>75 min. after treatment) III
(≤75 min. after treatment) IV
>32 IV

Corneal opacity:
Max. mean opacity score Category
0.0 0.5 I
0.6 1.5 II
1.6 2.5 III
2.6 4.0 IV

Fluorescein retention:
mean fluorescein retention score Category
at 30 min after treatment:
0.0 0.5 I
0.6 1.5 II
1.6 2.5 III
2.6 3.0 IV

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

Test Item

Value:

4

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Test Item

Value:

0.2

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

fluorescein retention score

Run / experiment:

Test Item

Value:

0.2

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

After the exposure procedure, a faint, red/purple staining of the sclera was observed. The intensity of the staining decreased over time and had cleared at 120 min after administration. At sampling of the corneas for histopathological examination, a red staining of the vitreous body was observed.
HC Red No. 3 (B050; GTS119151) caused corneal effects consisting of very slight corneal swelling (mean swelling 4%), no or very slight corneal opacity (mean score 0.2) and no or very slight fluorescein retention (mean score 0.2).

- Visible damage on test system: Not specified

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes, Sodium Hydroxide showed positive response when used as control

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate

- Acceptance criteria met for positive control: The positive control NaOH caused severe swelling (mean score 44%), 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

- Range of historical values if different from the ones specified in the test guideline: not specified

Any other information on results incl. tables

Table 1 - Summary results of the slit-lamp examination

Test material	Maximum mean score for:			Irritation catego­ries^1	Irritation Index^2	Classifi­ca­tions (EU-CLP^3/UN-GHS^4)
	Swelling %	Opaci­ty	Fluores­cein retention
HC Red No. 3 (B050; GTS119151)	4	0.2^5	0.2	I;I;I	12	NC/NC
NaOH(positive control)	44	4.0^6	3.0	IV;IV;IV	184	1/1

1 I = no effect; II = slight effect; III = moderate effect; IV = severe effect.

2 Irritation Index = maximum mean corneal swelling + maximum mean opacity (x 20) + mean fluorescein score (x 20)

3 EU-CLP: NC = not classified; Category 2 = Irritating to eyes; Category 1 = irreversible effects on the eye/serious damage to the eye. Regulation (EC) No 1272/2808 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2806.

4 UN-GHS: NC = not classified; Category 2B = mild irritant, causes eye irritation; Category 2A = irritant, causes eye irritation; Category 1 = irreversible effects on the eye/serious damage to the eye. United Nations-Economic Commission for Europe (UN/ECE) (2003). Globally Harmonized System of Classification and Labelling of Chemicals (GHS). UN, New York and Geneva, 2007.

5 Faintly red/purple staining of the sclera; during the first 120 min the staining cleared; at sampling the cornea for histopathology a red staining of the vitreous body was observed.

6 Immediate iris constriction and severe loosening of epithelium

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental condition of the study, the Test Article did not showed eye irritation effect.HC Red No. 3 (B050; GTS119151) caused corneal effects consisting of very slight corneal swelling (mean swelling 4%), no or very slight corneal opacity (mean score 0.2) and no or very slight fluorescein retention (mean score 0.2). Microscopic examination of the corneas did not reveal any abnormalities. Based on this report and according to the CLP regulation, the test article HC Red No.3 was Not Classified for Eye Irritation.

Executive summary:

This GLP compliant study was performed in order to evaluate the eye irritation potential of the test article HC Red No.3 in an Isolated Chicken Eye test, performed with OECD 438 guideline method.

HC Red No. 3 (B050; GTS119151) was evaluated neat for eye irritation potential in the Isolated Chicken Eye (ICE) test. In addition, the test included a negative control (saline) and a positive control (NaOH). 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.

After the exposure procedure, a faint, red/purple staining of the sclera was observed. The intensity of the staining decreased over time and had cleared at 120 min after administration. At sampling of the corneas for histopathological examination, a red staining of the vitreous body was observed.

HC Red No. 3 (B050; GTS119151) caused corneal effects consisting of very slight corneal swelling (mean swelling 4%), no or very slight corneal opacity (mean score 0.2) and no or very slight fluorescein retention (mean score 0.2). Microscopic examination of the corneas did not reveal any abnormalities, other than very slight erosion of the epithelium in one cornea.

Under the experimental condition of the study, the Test Article did not showed eye irritation effect. Based on this report and according to the CLP regulation, the test article HC Red No.3 was Not Classified for Eye Irritation.