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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:
weight of evidence
Study period:
March - December 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 oxide
EC Number:
215-146-2
EC Name:
Cadmium oxide
Cas Number:
1306-19-0
Molecular formula:
CdO
IUPAC Name:
oxocadmium
Test material form:
solid: particulate/powder
Details on test material:
- Product name: Cadmium oxide
- Physical state: solid (in the powder form), Red-brown
- Purity: 99.8% CdO
i.e. 87.4% Cd
- Lot No.:CDOF78170174
- Expiration date: 22 October 2020
- Storage condition of test material: Controlled room temperature (15-25°C, ≤70% relative humidity)
Specific details on test material used for the study:
Name: Cadmium oxide
Batch/Lot number: CDOF78170174
CAS number: 1306-19-0
Description: Red-brown solid (in the powdered form)
Purity: 99.8%
Expiry date: 22 October 2020
Storage conditions: 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) will be applied considering the supplied safety datasheet to assure personnel health and safety.
As agreed with the Sponsor, no correction for purity of the test item will be applied.

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: commercial abattoir - TARAVIS KFT. (Address: H-9600 Sárvár, Rábasömjéni utca 129., Hungary)
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): 7 weeks old, age and sex not specified
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box) and transported at ambient temperature. 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):
None, however test material remained stuck to the cornea even after the 240minute post-treatment rinse.
Number of animals or in vitro replicates:
Three test item treated eyes, three positive control treated eyes and one negative control eye were examined during the study.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED 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 (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit
-preparation: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 - At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No significant corneal thickness changes (1.6%) were observed in one eye and no corneal thickness changes were observed in the other eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED - yes

POSITIVE CONTROL USED - yes

APPLICATION DOSE AND EXPOSURE TIME - exposure time of 10s with the following:
-test substance treated chicken eye: treated with 30 mg cadmium oxide
-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 eyes 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 approximately ±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 solution at ambient temperature. Additional gentle rinsing with 3x20 mL saline was performed at each time point when the test material or the positive control material remaining on the cornea was observed.
- Indicate any deviation from test procedure in the Guideline

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The cornea thickness and cornea opacity were measured at all time points (approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse, minor variations within ±5 minutes considered acceptable)
- Damage to epithelium based on fluorescein retention: Fluorescein retention was measured on two occasions, at base line (t=0) and 30 minutes after the post-treatment rinse.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: not specified
- Macroscopic morphological damage to the surface: not observable
- Others (e.g, histopathology): performed, see results for report

SCORING SYSTEM:
- Mean corneal swelling (%) : according to ICE classification criteria for corneal thickness
- Mean maximum opacity score: according to ICE classification criteria
- Mean fluorescein retention score at 30 minutes post-treatment : according to ICE classification criteria

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
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Physiological saline
Positive controls validity:
valid
Remarks:
Imidazole
Remarks on result:
not determinable because of methodological limitations
Irritation parameter:
percent corneal swelling
Run / experiment:
up to 240 minutes
Value:
14.5
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:
1.33
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:
1.83
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: Difficult to evaluate because the test item fully stuck the all cornea surfaces. The cornea swelling was not detected until 180 minutes observation and the cornea opacity until 120 minutes observation because of this reason.
HISTOPATH: The negative control (0.9 % Sodium chloride, Salsol solution) cornea showed no abnormalities. Positive control, 30 mg Imidazole was associated with severe epithelial erosion in 3/3 cases. Moderate pyknotic nuclei were observed in outer region of stroma in 3/3 cases. No endothelial changes were recorded. The test item, cadmium carbonate produced slight erosion of the corneal epithelium in 6/6 sections. Very slight vacuolation of the low part of the epithelium in 3/6 sections and slight vacuolation of the low part of the epithelium was noted in 2/6 sections. No stromal or endothelial changes were observed. Based on the published criteria for histopathological changes, the test item, cadmium oxide was classified as Category 1.

DEMONSTRATION OF TECHNICAL PROFICIENCY: not specified

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, classified as non-irritating, according to the EU regulations. UN GHS Classification: No Category.
- Acceptance criteria met for positive control: Yes, classified as severe irritant according to the EU regulations. UN GHS Classification: Category 1.
- Range of historical values if different from the ones specified in the test guideline: see historical data table in additional results section

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.

The mean maximum corneal swelling up to 240 min, the mean maximum corneal opacity and the mean fluorescein retention ICE classes are used for EC and GHS classification.

Test item

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

not detected

no data

Mean maximum corneal swelling at up to 240 min

14.5 %

II

Mean maximum corneal opacity

1.33

II

Mean fluorescein retention

1.83

III

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

2xII 1xIII

The cornea swelling was not detected until 180 minutes observation and the cornea opacity until 120 minutes observation because the test item fully stuck the all cornea surfaces. Based on this in vitro eye irritation study in isolated chicken eyes withCadmium oxide, 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.

Positive Control

 

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

9.2 %

II

Mean maximum corneal swelling at up to 240 min

20.0 %

III

Mean maximum corneal opacity

3.83

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

Based on these observations, the positive control substance Imidazole was classified as severe irritant according to the EU regulations. UN GHS Classification: Category 1.


 

Negative Control

 

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

1.6 %

I

Mean maximum corneal swelling at up to 240 min

1.6 %

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,according to the EU regulations. UN GHS Classification:No Category.

Histopathology: 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

 

Cadmium oxide

14A

1

-

-

-

1

-

-

-

-

-

 

14B

1

-

-

-

1

-

-

-

-

-

 

15A

1

-

-

-

½

-

-

-

-

-

 

15B

1

-

-

-

½

-

-

-

-

-

 

16A

1

-

-

-

-

-

-

-

-

-

 

16B

1

-

-

-

½

-

-

-

-

-

 

Imidazole

17

3

-

-

-

-

-

-

2

-

-

 

18

3

-

-

-

-

-

-

2

-

-

 

19

3

-

-

-

-

-

-

2

-

-

 

NaCl 0.9% (w/v)

Solution

20

-

-

-

-

-

-

-

-

-

-

 

Summary table for 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:

False

No severe corneal morphological changes:

False

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:

True

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

True

Pathology report:

Score form histopathology chicken eyes

Test Material

Chamber ID

Epithelium

Notes

Stroma

Endothelium

Category

Erosion

Necrosis

Vacuolation

Disorder of fibers

Pyknotic nuclei

Necrosis

outer region (adjacent to epithelium)

inner region (adjacent to endothelium)

Top

Mid

Low

Cadmium oxide

14A

1

-

-

-

1

 

-

-

-

-

Category 1

14B

1

-

-

-

1

 

-

-

-

-

Cadmium oxide

15A

1

-

-

-

½

 

-

-

-

-

15B

1

-

-

-

½

 

-

-

-

-

Cadmium oxide

16A

1

-

-

-

-

 

-

-

-

-

16B

1

-

-

-

½

 

-

-

-

-

 

Positive Control

17

3

-

-

 

-

-

-

2

-

-

Positive Control

18

3

-

-

-

-

-

-

2

-

-

Positive Control

19

3

-

-

-

-

-

-

2

-

-

Negative Control

20

-

-

-

-

-

-

-

-

-

-

Historical data:

 

Maximum corneal swelling at up to 75 min

Maximum corneal swelling at up to 240 min

Maximum corneal opacity change

Fluoresccein retention change

Number
of eyes

 

Min.

Value

Max.

Value

Mean

SD

Min.

Value

Max. Value

Mean

SD

Min.

Value

Max. Value

Min.

Value

Max. Value

 

Negative control/ Physiological Saline

Year 2014

-3.2%

3.4%

-0.3%

1.1%

-4.8%

3.4%

-0.6%

1.5%

0.0

0.5

0.0

0.5

115

Year 2015

-1.6%

1.7%

0.0%

0.5%

-1.6%

3.2%

0.0%

0.7%

0.0

0.5

0.0

0.0

126

Year 2016

-1.6%

1.6%

0.1%

0.4%

-3.2%

1.7%

0.1%

0.7%

0.0

0.0

0.0

0.0

76

Year 2017

0.0%

0.0%

0.0%

0.0%

-1.6%

0.0%

0.0%

0.2%

0.0

0.0

0.0

0.0

55

Year 2018

0.0%

0.0%

0.0%

0.0%

-1.6%

1.6%

0.0%

0.4%

0.0

0.0

0.0

0.0

58

Year 2019

0.0%

0.0%

0.0%

0.0%

0.0%

1.7%

0.0%

0.2%

0.0

0.0

0.0

0.0

49

Year 2020

-1.6%

1.6%

-0.2%

0.9%

-3.2%

1.6%

-0.5%

1.3%

0.0

0.0

0.0

0.0

10

Period 2014-2020

-3.2%

3.4%

0.0%

0.6%

%

-4.8%

3.4%

-0.1%

0.9%

0.0

0.5

0.0

0.5

489

Positive control/Benzalkonium Chloride

Year 2014

-7.7%

27.0%

5.9%

8.9%

-10.7%

34.8%

12.3%

13.8%

2.5

4.0

1.5

3.0

186

Year 2015

-7.9%

20.0%

9.7%

4.5%

15.9%

38.3%

27.7%

3.6%

3.0

4.0

2.5

3.0

246

Year 2016

-4.8%

16.7%

10.3%

2.4%

19.4%

33.3%

27.2%

2.6%

4.0

4.0

3.0

3.0

141

Year 2017

-4.8%

15.0%

9.5%

2.7%

20.6%

31.7%

25.7%

2.6%

4.0

4.0

3.0

3.0

78

Year 2018

6.3%

14.5%

10.0%

1.9%

20.6%

35.5%

27.2%

2.9%

3.5

4.0

2.5

3.0

99

Year 2019

4.8 %

16.4%

9.8%

2.2%

17.7%

36.1%

27.5%

3.6%

4.0

4.0

2.5

3.0

72

Year 2020

6.6%             11.7%             9.0%             1.5%             17.7%             36.1%             27.9%             3.5%         4             4.0             2.5             3.0            6

11.7%             9.0%             1.5%             17.7%             36.1%             27.9%             3.5%         4             4.0             2.5             3.0            6

9.2%

1.6%

17.7%

25.4%

21.6%

2.3%

3.5

4.0

2.5

3.0

21

Period 2014-2020

-7.9%

27.0%

9.0%

5.3%

-10.7%

38.3%

23.8%

9.4%

2.5

4.0

1.5

3.0

843

Positive control/Imidazole

Year 2014

-6.6%

25.0%

7.1%

6.1%

-15.9%

35.4%

14.6%

8.7%

3.5

4.0

2.0

3.0

160

Year 2015

6.3%

19.7%

11.5%

2.8%

20.0%

36.7%

28.7%

3.5%

3.5

4.0

2.5

3.0

130

Year 2016

4.8%

15.0%

10.3%

1.9%

18.8%

33.3%

26.9%

2.7%

4.0

4.0

3.0

3.0

90

Year 2017

4.8%

16.7%

10.4%

2.2%

20.6%

33.3%

26.1%

2.6%

4.0

4.0

3.0

3.0

87

Year 2018

6.3%

13.3%

10.6%

1.8%

21.9%

31.7%

26.9%

2.5%

4.0

4.0

3.0

3.0

78

Year 2019

6.3%

16.4%

10.7%

2.1%

23.8%

34.4%

28.1%

2.3%

4.0

4.0

2.5

3.0

81

Year 2020

8.1%

13.3%

9.6%

1.7%

17.7%

25.0%

21.1%

2.1%

3.5

4.0

2.5

3.0

9

Period 2014-2020

-6.6%

25.0%

9.8%

4.0%

-15.9%

36.7%

24.1%

7.5%

3.5

4.0

2.0

3.0

635

CONCLUSION

 

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 of0.9 % Sodium chloride (Salsol solution)cornea showed no abnormalities.Positive control, 30 mg of Imidazole caused severe epithelial erosion of corneal epithelium in 3/3 cases. Moderate presence of pyknotic nuclei in top region of stroma in 3/3 cases.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Based on the performed in vitro eye irritation assay in isolated chicken eyes with Cadmium oxide, 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 slight erosion of the corneal epithelium in 6/6 cases. Very slight vacuolation of the low part of the epithelium in 3/6 sections and slight vacuolation of the low part of the epithelium was noted in 2/6 sections. 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, Cadmium oxide was classified as Category 1.
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 eyes were held in a horizontal position and the test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds exposure time, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 mg test item. The three positive control eyes were treated in a similar way with 30 mg of Imidazole. The negative control eye was treated with 30 µL of physiological saline(0.9% (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated.

 

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 experiment. Thus, the study was considered to be valid.

Slight corneal swelling change (mean = 14.5%) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (severity 1 on two eyes and severity 2 on one eye) was observed on three eyes. Moderate fluorescein retention change (severity 2 on two eyes and severity 1.5 on one eye) was noted on three eyes. The cornea swelling was not detected until 180 minutes observation and the cornea opacity until 120 minutes observation because the test item fully stuck the all cornea surfaces. Test item was stuck on all cornea surfacesafter the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.

Based on this in vitro eye irritation assay in isolated chicken eyes with
Cadmium oxide, 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 slight erosion of the corneal epithelium in 6/6 cases.Very slight vacuolation of the low part of the epithelium in 3/6 sections and slight vacuolation of the low part of the epithelium was noted in 2/6 sections. 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, Cadmium oxide was classified as Category 1.