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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Jul 2019 - 27 Nov 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

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:
Version 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Cadmium hydroxide
EC Number:
244-168-5
EC Name:
Cadmium hydroxide
Cas Number:
21041-95-2
Molecular formula:
CdH2O2
IUPAC Name:
cadmium hydroxide
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material:
CP5275
- Expiration date of the lot/batch:
04 June 2020
- Purity test date:
98.92%
- Description:White Solid (in the powder form)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Controlled room temperature (15-25°C, ≤70% relative humidity).
- Safety precautions: Enhanced safety precautions (half mask at least with P3 filter cartridge, nitrile gloves, lab coat) were applied considering the supplied safety datasheet to assure personnel health and safety.

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary), commercial abattoir.
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): 7 wekks old (no info on sex or weight).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): ambient temperature, heads were wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at Charles River Laboratories Hungary Kft.. and processed within 2 hours of collection.
- Time interval prior to initiating testing: not specified
- indication of any existing defects or lesions in ocular tissue samples: none
- Indication of any antibiotics used: not specified

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 30mg


Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
No, however, test item remained stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.
Number of animals or in vitro replicates:
Three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
- Eyes selection: After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
-Preparation of eyes: The eyeball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EQUILIBRATION AND BASELINE RECORDINGS - yes

NUMBER OF REPLICATES - 7

NEGATIVE CONTROL USED - yes: physiological saline

POSITIVE CONTROL USED - yes: imidazole

APPLICATION DOSE AND EXPOSURE TIME - exposure time of 10s before rinsing
-test substance treated chicken eye: treated with 30 mg cadmium hydroxide
-positive control chicken eye: treated with 30 mg imidazole
-negative control eye: treated with 30µL physiological saline (0.9% (w/v) NaCl solution

OBSERVATION PERIOD - The control eye and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.
Minor variations within ±5 minutes were considered acceptable.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: cornea surface was rinsed thoroughly with 20ml physiological saline. Additional gentle rinsing with 3x20 mL (positive control treated eyes) or 5x20 mL (test item treated eyes) saline was performed at each time point when the test item and positive control material remaining on the cornea was observed.
- Time after start of exposure: after 10's of exposure
- Indicate any deviation from test procedure in the Guideline : none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.
- Damage to epithelium based on fluorescein retention: Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Haag-Streit Bern 900 slit-lamp microscope
- Macroscopic morphological damage to the surface: Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.
- Others (e.g, histopathology): via microscopy

SCORING SYSTEM:
- Mean corneal swelling (%) : according to TG, measured at all time points.
- Mean maximum opacity score: according to TG, measured at all time points.
- Mean fluorescein retention score at 30 minutes post-treatment : according to TG, measured on two occasions, at base line (t=0) and 30 minutes after the post-treatment rinse.

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. - yes

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Run / experiment:
up to 75 minutes
Value:
3.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Physiological saline
Positive controls validity:
valid
Remarks:
Imidazole
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Run / experiment:
up to 240 minutes
Value:
4.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Physiological saline
Positive controls validity:
valid
Remarks:
Imidazole
Remarks on result:
positive indication of irritation
Irritation parameter:
cornea opacity score
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Physiological saline
Positive controls validity:
valid
Remarks:
Imidazole
Remarks on result:
positive indication of irritation
Irritation parameter:
fluorescein retention score
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Physiological saline
Positive controls validity:
valid
Remarks:
Imidazole
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not observable (test item remained stuck on cornea despite washing

DEMONSTRATION OF TECHNICAL PROFICIENCY: not indicated

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes - within historical data range, The negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category.
- Acceptance criteria met for positive control: yes - within historical data range, The positive control Imidazole was classified as severely irritating, UN GHS Classification: Category 1.
- Range of historical values if different from the ones specified in the test guideline:
For NEGATIVE control (physiological saline):
-Maximum corneal swelling at up to 75 min: Min. Value: -3.2 % ; Max. Value: 3.4 %
-Maximum corneal swelling at up to 240 min: Min.: -4.8 % ; Max.: 3.4 %
Maximum corneal opacity change: Min.: 0.00 ; Max.: 0.50
Fluorescein retention change: Min.: 0.00 ; Max.: 0.50
Number of eyes: 536
For POSITIVE control (Imidazole):
-Maximum corneal swelling at up to 75 min: Min. Value: -6.6 % ; Max. Value: 25.0 %
-Maximum corneal swelling at up to 240 min: Min.: -15.9 % ; Max.: 36.7 %
Maximum corneal opacity change: Min.: 3.50 ; Max.: 4.00
Fluorescein retention change: Min.: 2.00 ; Max.: 3.00
Number of eyes: 665

Any other information on results incl. tables

The mean values of the treated eyes for maximum corneal thickness change, corneal opacity and fluorescein retention are given below. The conclusion on eye irritancy was based on the OECD guideline quantitative assessments.

 

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

3.2 %

I

Mean maximum corneal swelling at up to 240 min

4.2 %

I

Mean maximum corneal opacity

1.00

II

Mean fluorescein retention

1.00

II

Other Observations

Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.

Overall ICE Class

1xI 2xII

Based on the performed in vitro eye irritation assay in isolated chicken eyes with Cadmium hydroxide, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.The results were very close to the borderline for non-irritant, but as it was just over the limit, the test item can not be classified. If a REACH classification is required, it is recommended that another in vitro test (for example the Epiocular test) is performed.

Positive Control

 

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

9.6 %

II

Mean maximum corneal swelling at up to 240 min

27.8 %

III

Mean maximum corneal opacity

4.00

IV

Mean fluorescein retention

3.00

IV

Other Observations

Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.

Overall ICE Class

1xIII 2xIV

The positive control Imidazole was classified as severely irritating, UN GHS Classification: Category 1.


 

NEGATIVE Control

 

 

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0.0 %

I

Mean maximum corneal swelling at up to 240 min

0.0 %

I

Mean maximum corneal opacity

0.00

I

Mean fluorescein retention

0.00

I

Other Observations

None

Overall ICE Class

3xI

 

The negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category.

Pathology report:

Semi-quantitative microscopic evaluation was performed on the cornea in the ICET. The classification of histopathology findings was performed based on the publications: M.K. Prinsen et al./Toxicology in Vitro 25 (2011), 1475-1479), Elodie Cazelle et al./ Toxicology in Vitro 28 (2014), 657-666 and Atlas of histopathological lesions of Isolated Chicken Eyes, M.V.W. Wijnands and M.K. Prinsen, June 2015. The negative control, 30 μL of 0.9 % Sodium chloride (Salsol solution) cornea showed no abnormalities. Positive control, 30 mg Imidazole caused severe epithelial erosion of corneal epithelium in 3/3 cases and detachment form the basement membrane in 2/3 cases. No compromised Bowman’s or basement membrane as well as no endothelial changes were recorded.

Cadmium hydroxide produced very slight erosion of the corneal epithelium in 6/6 sections. Very slight and/or slight vacuolation of the top of the epithelium was seen in 6/6 sections. No stromal or endothelial changes were observed as well as no effects on integrity on basement, Bowman's and Descemet's membranes. Based on the published criteria for histopathological changes, no predictions can be made for Cadmium hydroxide.

Microscopic Findings:

Test Material

 

Eye number

 

Epithelium

Notes

Stroma

Endothelium

Erosion

Necrosis

Vacuolation

 

 

Disorder of fibers

 

 

Pyknotic nuclei

Necrosis

 

outer region (adjacent to epithelium)

inner region (adjacent to endothelium)

 

Top

Mid

Low

 

Test item

11A

1/2

-

-

-

1

-

-

-

-

-

 

11B

1/2

-

-

-

1

-

-

-

-

-

 

12A

1/2

-

-

-

½

-

-

-

-

-

 

12B

1/2

-

-

-

½

-

-

-

-

-

 

13A

1/2

-

-

-

-

-

-

-

-

-

 

13B

1/2

-

-

-

½

-

-

-

-

-

 

Imidazole

14

3

-

-

-

-

x

-

1

-

-

 

15

3

-

-

-

-

-

-

1

-

-

 

16

3

-

-

-

-

x

-

1

-

-

 

Physiological saline solution (Salsol solution,NaCl 0.9% (w/v)

Solution

17

-

-

-

-

-

-

-

-

-

-

 

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

x- detachment from basement membrane

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
Based on the performed in vitro eye irritation assay in isolated chicken eyes with Cadmium hydroxide, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification. Further histopathological observations were performed by the study's pathologist and did not reveal evidence of severe irritant effects.
Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (25 June 2018).

 

After the zero reference measurements, the eye was held in horizontal position and 30 mg test itemwas applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds exposure time, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 µL ofphysiological saline(0.9% (w/v) NaCl solution). Three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

 

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in this experiment. Thus, the study was considered to be valid.

No significant cornea swelling (mean swelling ≤5%) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (severity 1) was observed on any eyes. Slight fluorescein retention change (severity 1) was observed on any eyes.Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.

The results were very close to the borderline for non-irritant, but as it was just over the limit, the test item can not be classified. If a REACH classification is required, it is recommended that another in vitro test (for example the Epiocular test) is performed.

Based on the performed in vitro eye irritation assay in isolated chicken eyes with Cadmium hydroxide, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

Histopathological observations were made on two sections of each of the 3 corneas treated with test item (6 sections). Microscopic evaluation showed very slight erosion of the corneal epithelium in 6/6 sections. Very slight and/or slight vacuolation of the top epithelium were seen in 6/6 cases. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, no prediction can be made for Cadmium hydroxide.

SUMMARY TABLE FOR UN GHS CLASSIFICATION

Criteria for “No category” (all true)

 

3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II or 1 endpoint classed as I and 2 endpoints classed as II:

True

No severe corneal morphological changes:

True

Test item was not stuck to the cornea at 240 minutes after the post-treatment rinse:

False

 

Criteria for “Category 1” (one or more true)

 

2 or more endpoints classed as IV:

False

Corneal opacity ≥ 3 at 30 min (in at least 2 eyes):

False

Corneal opacity = 4 at any time point (in at least 2 eyes):

False

Severe loosening of epithelium (in at least 1 eye):

False

 

Criteria for “No prediction can be made” (one or two true)

 

Based on the endpoints not classifiable for No Category, or for Category 1:

False

Particles of test item were stuck to the cornea and could not be washed off during the study:

True