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 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:
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:
Adopted : 26 July 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by HFC Prestige Service, Batch No. DRDGCNP110705W
- Expiration date of the lot/batch: 12 April 2019
- Purity test date: 2 May 2016

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was used neat
- Final dilution of a dissolved solid, stock liquid or gel: no dilution was used
- Final preparation of a solid:not specified

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS, spring chickens
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Bor poultry slaughterhouse (Netherlands)
- Age at study initiation: 6 weeks old
- Weight at study initiation: 1.5 - 2.5 kg

ENVIRONMENTAL 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.

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 ; 30 µL of positive control
- 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:
Number of animals or 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, 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 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 take n 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). The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32 deg Celsius (water pump set at 36.4 deg Celsius). 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 Depth Measuring Attachment No. I for the Haag Streit slit lamp microscope, set at 0.095mm. 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 unacceptably 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 re
tention. 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

POSITIVE CONTROL USED
Benzalkonium Chloride (BAC)

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 treatment 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 observa
tion 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: please specify if the decision criteria as indicated in the TG was used.

On the basis of the severity of the observed findings for corneal swelling, corneal opacity and fluoresc
ein retention, the effects are divided into four categories, viz. I = no effect; II = slight effect; III = m
oderate 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

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Run / experiment:
Test item
Value:
4
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
Test item
Value:
1
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
Irritation parameter:
in vitro irritation score
Run / experiment:
Test item
Value:
65
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: NaOH was used as positive control and provided informations about suitability of the test system

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: Not specified

After the exposure procedure, a considerable orange/red staining of the sclera, cornea and iris was observed. The staining persisted during the 4-hr observation period. At sampling of the corneas for histopathological examination, an orange/red staining of the vitreous body was observed. 2-Amino-6-Chloro-4-Nitrophenol (B099; GTS119166) caused corneal effects consisting of very slight or slight corneal swelling (mean swelling 4%), slight corneal opacity (mean score 1.0) and moderate fluorescein retention (mean score 2.0).
Microscopic examination of the corneas treated with 2-Amino-6-Chloro-4-Nitrophenol (B099; GTS119166) revealed very slight (2/3 corneas) erosion and very slight (1/3 corneas, top region) vacuolation of the epithelium.

Any other information on results incl. tables

Table 1 - Summary results of the slit-lamp examination

Test material

 

Maximum mean score for:

 

 

Irritation

catego­ries1

Irritation

Index2

Classifi­ca­tions

(EU-CLP3/UN-GHS4)

 

Swelling %

Opaci­ty

Fluores­cein

retention

2-Amino-6-Chloro-4-Nitrophenol (B099; GTS119166)

4

1.05

2.0

I;II;III

65

2/2B

NaOH(positive control)

44

4.06

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 Considerable orange/red staining of the sclera, cornea and iris which was persistent throughout the 4-hr observation period; at sampling the cornea for histopathology an orange/red staining of the vitreous body was observed.

6 Immediate iris constriction and severe loosening of epithelium


 

 

Table 2 - Individual histopathological findings

 

Test Material

 

Eye no.

 

Epithelium

Notes

Stroma

Endothelium

Erosion

Necrosis

Vacuolation

 

 

Disorder of fibers

 

 

Pyknoticnuclei

Necrosis

top

mid

low

outer region (adjacent to epithelium)

inner region (adjacent to endothelium)

2-Amino-6-Chloro-4-Nitrophenol (B099; GTS119166)

7

-

-

-

-

-

 

-

-

-

-

8

½

-

½

-

-

 

-

-

-

-

9

½

-

-

-

-

 

-

-

-

-

NaOH

(positive control)

13

3

-

-

-

-

-

-

-

P

14

3

-

-

-

-

-

-

-

P

15

3

-

-

-

-

-

-

-

P

Saline

(negative control)

16

-

-

-

-

-

 

-

-

-

-

 

- = not observed; P = present; ½ = very slight; 1 = slight; 2 = moderate; 3 = severe;

= scored in the top/mid/low section of the epithelium;‡ = stromal necrosis


 

 

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Under experimental conditions of the study, the registered substance 2-Amino-6-Chloro-4-Nitrophenol (B099; GTS119166) caused corneal effects consisting of very slight or slight corneal swelling (mean swelling 4%), slight corneal opacity (mean score 1.0) and moderate fluorescein retention (mean score 2.0). Microscopic examination of the corneas did revealed very slight (2/3 corneas) erosion and very slight (1/3 corneas, top region) vacuolation of the epithelium.According to the CLP regulation and OECD, the ICE test cannot be used for classify Eye irritation Category 2 due to false negative rate of the test (including alcohol). However, microscopic observation was performed on histopathologic slide of the chicken eye. Hence, by caution, the substance was considered as irritant for thee eyes due to erosion, vacuolation of the epithelium in this test, and was classified as Category 2.
Executive summary:

This GLP-compliant study was performed to assess the potential irritation/corrosion property of the registered substance 2-Amino-6-Chloro-4-Nitrophenol in a Isolated Chicken Eye test (ICE test) according to OECD guideline 438 method.

2-Amino-6-Chloro-4-Nitrophenol (B099; GTS119166) 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.

Under experimental conditions of the study, the registered substance 2-Amino-6-Chloro-4-Nitrophenol (B099; GTS119166) caused corneal effects consisting of very slight or slight corneal swelling (mean swelling 4%), slight corneal opacity (mean score 1.0) and moderate fluorescein retention (mean score 2.0). Microscopic examination of the corneas did revealed very slight (2/3 corneas) erosion and very slight (1/3 corneas, top region) vacuolation of the epithelium.According to the CLP regulation and OECD, the ICE test cannot be used for classify Eye irritation Category 2 due to false negative rate of the test (including alcohol). However, microscopic observation was performed on histopathologic slide of the chicken eye. Hence, by caution, the substance was considered as irritant for thee eyes  due to erosion, vacuolation of the epithelium in this test, and was classified as Category 2.