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Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 August 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

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)
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Ammonium manganese(3+) diphosphate
EC Number:
233-257-4
EC Name:
Ammonium manganese(3+) diphosphate
Cas Number:
10101-66-3
Molecular formula:
MnKx (NH4)1-x P2O7 (0 =< x =< 0.5)
IUPAC Name:
Ammonium manganese diphosphate, monoclinic and triclinic
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
chicken
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: licensed slaughterhouse, Etablissement Brun, 33820 Etauliers, France.
- Characteristics of donor animals (e.g. age, sex, weight): healthy approximately 7-week-old chickens used for human consumption; their body weights ranging from 1.5 - 2.5 kg.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): intact heads were transported to the laboratory at ambient temperature, in plastic boxes humidified with towels moistened with physiological saline.
- Time interval prior to initiating testing: The heads were collected on 09 August 2021 at 8:14 am and they were enucleated at Laboratoire ICARE – Site de Martillac on 09 August 2021 at 10:36 am.
- Indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: no
- Selection and preparation of corneas: 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 > 0.5; (ii) corneal opacity > 0.5; or, (iii) any additional signs of damage were replaced. For eyes that were 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 were rejected.

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 of test item
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
No post-treatment incubation was performed.
Number of animals or in vitro replicates:
3
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 left 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 the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The 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 31.8°C and 31.9°C.
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 in the superfusion apparatus between 45 and 65 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

NEGATIVE CONTROL USED: 30 μL physiological saline (Dutscher Batch No. C0543A01).

POSITIVE CONTROL USED: 30 mg sodium hydroxide (Fisher Scientific, Batch No. 0000080257).

APPLICATION DOSE AND EXPOSURE TIME: 30 mg test item were applied for 10 seconds.

OBSERVATION PERIOD: Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: eyes were rinsed twice with 10 mL of physiological saline at ambient temperature.
- Indicate any deviation from test procedure in the Guideline: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was then given for each test or control item.
- Damage to epithelium based on fluorescein retention: mean fluorescein retention value for all test eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item.
- Swelling: measured with optical pachymeter on a slit-lamp microscope (Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I); slit-width setting: at 9½, equaling 0.095 mm.
- Macroscopic morphological damage to the surface: The aim of this evaluation was to determine whether any “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea were visible.These findings can vary in severity and may occur simultaneously.

SCORING SYSTEM:
- Mean corneal swelling (%) = [(corneal thickness measurement at time t - corneal thickness at time=0) / corneal thickness at time =0] x 100

- Mean maximum opacity score:
0 - No opacity,
0.5 - Very faint opacity
1 - Scattered or diffuse areas; details of the iris clearly visible
2 - Easily discernible translucent area; details of the ris are slightly obscured,
3 - Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely
discernible
4 - Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment
0 - No fluorescein retention,
0.5 - Very minor single cell staining,
1 - Single cell staining scattered throughout the treated area of the cornea,
2 - Focal or confluent dense single cell staining,
3 - Confluent large areas of the cornea retaining fluorescein

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

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
mean
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
maximum
Value:
5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
morphological effects
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no. No morphological effects were noted, whatever the examination time.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. 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.
- Acceptance criteria met for positive control: yes. The combination of the three endpoints for the positive control, Sodium Hydroxyde, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

Any other information on results incl. tables

Table 1. Results for test item


























































































































































































Endpoint measured



Eye No.



Time (min)



-45



30



75



120



180



240



Corneal opacity



13



0



0



0.5



1



1



1



14



0



0



0



0



0



0



15



0



0



0



0.5



0.5



0.5



Mean



0.0



0.0



  0.2



0.5



0.5



0.5



ICE class



I



Fluorescein retention



13



0.5



0.5



-



-



-



-



14



0.5



0.5



-



-



-



-



15



0.5



0.5



-



-



-



-



Mean



0.5



0.5



-



-



-



-



ICE class



 



I



-



-



-



-



Corneal thickness



13



0.52



0.54



0.54



0.54



0.54



0.54



14



0.53



0.54



0.54



0.55



0.55



0.55



15



0.54



0.56



0.58



0.58



0.58



0.58



Corneal swelling (%)



13



-



4



4



4



4



4



14



-



2



2



4



4



4



15



-



4



7



7



7



7



Mean



-



3



4


5

5



5



ICE class



 



I



Combination of the 3 Endpoints



 



3 x I



CLASSIFICATION



 



No category



 


Table 2. Results for the positive control. 






























































































































































































Endpoint measured



Eye No.



Time (min)



 



 



-45



30



75



120



180



240



Corneal opacity



1



0



4



4



4



4



4



2



0



4



4



4



4



4



3



0



4



4



4



4



4



Mean



0.0


4.04.04.04.0

4.0



ICE class



-



IV



 



Fluorescein retention



1



0.5



3



-



-



-



-



2



0.5



3



-



-



-



-



3



0.5



3



-



-



-



-



Mean



0.5



3.0



-



-



-



-



ICE class



-



IV



-



-



-



-



Corneal thickness



1



0.49



0.70



0.72



0.82



0.85



0.85



2



0.52



0.66



0.84



0.96



0.96



0.96



3



0.50



0.72



0.82



0.96



0.97



0.97



Corneal swelling (%)



1



( - )



43



47



67



73



73



2



( - )



27



62



85



85



85



3



( - )



44



64



92



94



94



Mean



-


38578184

84



ICE class



-



IV



Combination of the 3 Endpoints



 



3 x IV



CLASSIFICATION



 



Category 1 : "Corrosive/Severe Irritant"



Table 3. Results for the negative control.


















































































































Endpoint measured



Eye No.



Time (min)



-45



30



75 



120



180



240



Corneal opacity



16



0



0



0



0



0



0



Mean



0.0



0.0



 0.0



0.0



0.0



0.0



ICE class



I



Fluorescein retention



16



0.5



0.5



 



-



-



-



Mean



0.5



0.5



-



-



-



-



ICE class



 



I



-



-



-



-



Corneal thickness



16



0.52



0.52



0.52



0.52



0.52



0.52



Corneal swelling (%)



16



-



0



0



0



0



0



Mean



-



0



  0 



0



0



0



ICE class



 



I



Combination of the 3 Endpoints



 



3 x I



CLASSIFICATION



 



No Category


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Not irritant (CLP Regulation EC no. 1272/2008)
Conclusions:
Based on this in vitro eye irritation study, the combination of the 3 endpoints was 3 x I. Therefore, the test item is not eye-irritant.
Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye-damaging effects of the test item according to the OECD 438 (Isolated Chicken Eye), under GLP conditions.


Eyeballs were isolated from chickens killed for human consumption, the test system was equilibrated and zero reference measurements were taken. Three groups of three eyeballs each were exposed to either 30 mg test item, 30 mg sodium hydroxide (positive control), or 30 μL physiological saline (negative control). After 10 seconds of exposure, all eyeballs were rinsed with 20 mL of physiological saline at ambient temperature and examined. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 45, 30, 75, 120, 180, and 240 minutes post-dose to assign an ICE class.


The results for the test item treated eyes showed mean fluorescein retention of 0.5 (ICE class I), maximum mean corneal opacity of 0.5 (ICE class I), and a maximal mean corneal swelling of 5.0 % (ICE class I). Concurrent positive and negative control values fell within the acceptable ranges for the method. Gross examinations did not exhibit any changes of the treated test item corneal surface.


Based on these results, the combination of the 3 endpoints was 3 x I. Therefore, the test item does not have any negative effects on the chicken cornea and is deemed not irritating.