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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 9 JULY 2019 to 19 JULY 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

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:
yes
Remarks:
The eyes were incubated between 45 and 64 minutes instead of between 45 and 60 minutes, as initially scheduled. As the results obtained with the negative control, the deviation is considered as without impact on the conclusion of the study.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile
EC Number:
274-126-1
EC Name:
1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile
Cas Number:
69808-32-8
Molecular formula:
C17H17ClN4O2
IUPAC Name:
1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: powder
- Particle size distribution: not determined
- Mass median aerodynamic diameter (MMAD): not determined
- Geometric standard deviation (GSD): not determined
- Shape of particles: not determined
- Surface area of particles: not determined
- Crystal structure: not determined
- Coating: none
- Surface properties: not determined
- Density: 1.3526 ± 0.0010 g/cm3 at 20.0 ± 0.2°C
- Moisture content: <0.5%
- Residual solvent: not detected
- Activation: none
- Stabilisation: none
- Other:

Test animals / tissue source

Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: slaughterhouse (Etablissement Brun, 33820 Etauliers, France)
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight): 7 weeks old, 1.5-2.5 Kg
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transport time 75 minutes, at ambient temperature in plastic boxes humidified with towels moistened with physiological saline
- Time interval prior to initiating testing:
- indication of any existing defects or lesions in ocular tissue samples: none
- Indication of any antibiotics used:

Test system

Vehicle:
unchanged (no vehicle)
Controls:
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):

VEHICLE
- Amount(s) applied (volume or weight with unit): none
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:
Duration of treatment / exposure:
10 seconds
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. when the eye is removed from the orbit, a visible portion of the optic nerve should be attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.

The enucleated eye was mounted in a stainless steel clamp with he cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. Then clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.0ºC and 32.4ºC.

After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or (iii) any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.

EQUILIBRATION AND BASELINE RECORDINGS
Once all eyes had been examined and approved (see table in appendix 4), the eyes were incubated between 45 and 64 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES
3 replicates for test item test.

NEGATIVE CONTROL USED
Concurrent negative control (physiological saline - Dutscher Batch No. 3013132). One eye was treated with the negative control, rinsed twice with 10 mL of physiological saline and a humidified cotton swab at ambient temperature (as for the eye treated with the test item).

SOLVENT CONTROL USED (if applicable)
None

POSITIVE CONTROL USED
Positive control (sodium hydroxide - Fisher Scientific, Batch No. 1550248) were included in this experiment. Three eyes were treated with the positive control.

APPLICATION DOSE AND EXPOSURE TIME
Three eyes (in their holder) were removed from the superfusion apparatus, placed in a horitzontal position, and 30 mg of the test item was applied, after being reduced in fine powder, during 10 seconds to the cornea such that the entire surface of the cornea was evenly covered with the test item.

OBSERVATION PERIOD

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The eyes were rinssed twice with 10 mL of physiological saline and humidified cotton swab at ambient temperature.
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Haag-Streit BP900 split-lamp microscope with depth-measuring device no. I.
- Damage to epithelium based on fluorescein retention: Haag-Streit BP900 split-lamp microscope with depth-measuring device no. I.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Haag-Streit BP900 split-lamp microscope with depth-measuring device no. I.
- Macroscopic morphological damage to the surface:Haag-Streit BP900 split-lamp microscope with depth-measuring device no. I.
- Others (e.g, histopathology):

SCORING SYSTEM:
- Mean corneal swelling (%): 0%, corresponding to ICE class I
- Mean maximum opacity score: 0.0, corresponding to ICE class I
- Mean fluorescein retention score at 30 minutes post-treatment: 1.5, corresponding to ICE class II

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
The combination of the three endopoints for the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile was 2 x I, 1 x II.

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihidro-6-hydroxy-4-methyl-2-oxonicotinonitrile does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No Category).
No signal word and hazard statement are required.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Run / experiment:
Run 1 (10 seconds)
Value:
ca. 0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as "No Category", as expected.
Positive controls validity:
valid
Remarks:
The combination of the three endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe Irritant", as expected.
Irritation parameter:
fluorescein retention score
Run / experiment:
Run 1 (exposure 10 seconds)
Value:
ca. 1.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as "No Category", as expected.
Positive controls validity:
valid
Remarks:
The combination of the three endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe Irritant", as expected.
Irritation parameter:
cornea opacity score
Run / experiment:
Run 1 (duration of exposure: 10 seconds)
Value:
ca. 0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as "No Category", as expected.
Positive controls validity:
valid
Remarks:
The combination of the three endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe Irritant", as expected.
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: he combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as "No Category", as expected.
- Acceptance criteria met for positive control:The combination of the three endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe Irritant", as expected.
- Range of historical values if different from the ones specified in the test guideline:

In vivo

Irritant / corrosive response data:
See Table 9 (overall remarks, attachments)

Any other information on results incl. tables























































































































































































Endpoint measured



Eye No.



Time (min)



0



30



75



120



180



240



Corneal opacity



4



0



0



0



0



0



0



5



0



0



0



0



0



0



6



0



0



0



0



0



0



mean



0.0



0.0



0.0



0.0



0.0



0.0



ICE class



 



I



Fluorescein retention



4



0.5



1



-



-



-



-



5



0.5



3



-



-



-



-



6



0.5



0.5



-



-



-



-



Mean



0.5



1.5



-



-



-



-



ICE class



 



II



-



-



-



-



Corneal thickness



4



0.58



0.58



0.58



0.58



0.58



0.58



5



0.57



0.57



0.57



0.57



0.57



0.57



6



0.56



0.56



0.56



0.56



0.56



0.56



Corneal swelling



4



-



0



0



0



0



0



5



-



0



0



0



0



0



6



-



0



0



0



0



0



Mean



-



0



0



0



0



0



ICE class



I



Combination of the 3 Endpoints



2 x I, 1 x II



CLASSIFICATION



No category



TEST ITEM:


1-butyl-5-[4-chlorophenyl]azo]-1,2-dihidro-6-hidroxy-4-methyl-2-oxonicotinonitrile


 


Application: 30 mg of pure test item


Application date: 10 September 2018


Table 9


 


Note:


No morphological effects were noted, whatever the examination time.


 


 


 


 


 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In ccordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitriledoes not require classification for eye irritation and serious eye damage as defined by the UN GHS (No Category).
No signal word and hazard statement are required.

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