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Description of key information

Skin irritation / corrosion: Key study. Method according to US CPSC (16 CFR 1500.41), GLP study. The test item was found to be corrosive to the skin. Supporting study: According to peer reviewed handbook data, the test item is expected to be irritating to skin. Based on the available information, the test item is considered as corrosive to the skin.

Eye irritation: Key study. Method according to OECD 428, GLP study. The test item causes severe eye damage. Based on the available information, it should be classified as Eye Damage, Category 1.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion, other
Type of information:
other: data from handbook
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Principles of method if other than guideline:
Data from peer reviewed handbook. No methods indicated.
GLP compliance:
no
Remarks on result:
not measured/tested
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Based on peer reviewed handbook data, the test item is expected to be a moderate skin irritant.
Executive summary:

As reported in 'Patty's' (peer reviewed handbook data), the test item is a strong base, and would be expected to be a moderate skin irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 26th to February 25th, 2016.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
no
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: the eyeballs were collected from chickens obtain a licensed slaughterhouse, i.e. Zakład Przemysłu Drobiarskiego JAS-DROP in Krzyżowice where they were killed for human consumption.
- Characteristics of donor animals (e.g. age, sex, weight): age approximately 7 weeks, weight 1.5 - 2.5 kg. All eyeballs used in the tests came from the same group of eyes collected on a specific day.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): After electric shock and incision of the neck for bleeding, the chicken heads were transported to the laboratory in a plastic container at ambient temperature. During the transport, the heads were humidified with a physiological salt solution by placing moistened paper towels inside the container.Till the moment of their transfer, the eyeballs were kept in special containers at temperature of -18°C.
- Time interval prior to initiating testing: The time interval between the collection of the chickens’ heads and the use of their eyeballs in the ICE test was 30 minutes.
- indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: no
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
240 ± 5 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES: In order to control the quality of the procedure, the eyeballs used for the purpose of the experiment were assessed for potential damage by corneal opacity profusion and fluorescein retention determination. Result of corneal opacity for all examined eyeballs was less than 0.5. Deviation of corneal thickness for all examined eyeballs was less than 10%.
After a careful excision of the eyelids so as not to damage the cornea, the eyeball surface was treated with a 2% fluorescein solution (w/v) for a few seconds in order to evaluate the corneal integrity. After the removal of the dye by rinsing the corneal surface with a physiological salt solution, fluorescein retention and corneal opacity scores were determined using the slit-lamp microscope BP 900 LED (HAAG STREIT) to ensure that the cornea was undamaged. Only eyeballs without damage were analyzed (fluorescein retention and opacity profusion scores fell below 0.5). The enucleated eyeball was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to the superfusion apparatus so that the entire cornea was supplied with the physiological salt drip.
After the insertion of the eyeballs to the apparatus, another evaluation of corneal opacity and swelling was performed. Result of corneal opacity for all examined eyeballs was less than 0.5. Deviation of corneal thickness for all examined eyeballs was less than 10%. The corneal thickness was determinated using the depth measuring device no. 1 on the slit-lamp microscope and an SP-100 pachymeter (TOMEY).

EQUILIBRATION AND BASELINE RECORDINGS: Before the application of the test item and the control items, all examined and approved eyeballs were incubated at temperature of 32 ± 1.5°C for 45-60 minutes in order to equilibrate them to the test system. After that, a zero reference measurement for corneal thickness and opacity was recorded. The fluorescein score determined at dissection served as the baseline measurement for that endpoint.

NUMBER OF REPLICATES: 3
NEGATIVE CONTROL USED: physiological saline, 0.03 mL
POSITIVE CONTROL USED: 10% acetic acid, 0.03 mL
APPLICATION DOSE AND EXPOSURE TIME: mL, exposure 10s

OBSERVATION PERIOD: The corneas treated with the test item and the control items were evaluated p
retreatment 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: the test item and the control items were rinsed
from the eye with 20 mL of physiological salt solution at ambient temperature. Next, the eyeballs in their
holders were placed in the superfusion apparatus in the original upright position.
- Indicate any deviation from test procedure in the Guideline: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: qualitative assessment by assigning appropiate values to opaque areas, the mean corneal opacity value was calculated for all test eyeballs for all observation time points. Based on the highest mean score corneal opacity the final score was given.
- Damage to epithelium based on fluorescein retention: yes. Qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention) 30 min after end of exposure.
- Swelling: quantitative measurement, using a slit-lamp microscope and an SP-100 pachymeter.The mean percentage of corneal swelling for all test eyeballs was calculated for all observation time points. Based on the highest mean score for corneal swelling, at any time point, the final category score was given.
- Macroscopic morphological damage to the surface: qualitative assessment.
- Others: histopathological evaluations of morphological damage to the surface.

SCORING SYSTEM:
- Mean corneal swelling (%): The degree of corneal swelling was determined by measuring corneal thickness using a slit-lamp microscope and an SP-100 pachymeter.The mean percentage of corneal swelling for all test eyeballs was calculated for all observation time points. Based on the highest mean score for corneal swelling, at any time point, the final category score was given.

- Mean maximum opacity score:
0 No opacity
0.5 Very faint opacity
1 Scattered or diffuse areas; details of the iris are clearly visible
2 Easily discernible translucent areas; details of the iris 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
Corneal swelling was calculated as % as follows:corneal swelling = [corneal thickness at time t – corneal
thickness at time t = 0/ corneal thickness at time t = 0] x100

- 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: as indicated in the TG.
Irritation parameter:
fluorescein retention score
Run / experiment:
mean
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
ICE class IV.
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
ICE class IV.
Irritation parameter:
percent corneal swelling
Run / experiment:
mean
Value:
>= 40.5 - <= 70.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
ICE class IV.
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: yes
- Gross examinations of the eyeballs treated with the test item revealed roughening of the corneal surface.
- Histopathological examinations of the corneas treated with the test item revealed: wrinkling, karyolysis, karyopyknosis, cell swelling, coagulation, necrosis (eyeballs no. 1, no. 2, no. 3), detachment (eyeball no. 2), slight erosions (eyeball no. 1) of the anterior corneal epithelium, local vacuolation and dissection of the anterior corneal epithelium (eyeball no. 3)

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 1. Fluorescein retention

 

observation after

time t

(minutes)

 

 

test item

 

positive control

imidazole

 

 

negative control

physiological saline

eyeball no.

eyeball no.

eyeball no.

1

2

3

4

5

6

7

8

9

0

 

0

0

0

0

0

0

0

0

0

30

 

3.0

3.0

3.0

3.0

3.0

3.0

0.0

0.0

0.0

Table 2. Corneal opacity

 

observation after

time t

(minutes)

 

 

test item

 

positive control

imidazole

 

 

negative control

physiological saline

eyeball no.

eyeball no.

eyeball no.

1

2

3

4

5

6

7

8

9

0

 

0

0

0

0

0

0

0

0

0

30

 

4.0

4.0

4.0

4.0

4.0

4.0

0.0

0.0

0.0

75

4.0

4.0

4.0

4.0

4.0

4.0

0.0

0.0

0.0

120

4.0

4.0

4.0

4.0

4.0

4.0

0.0

0.0

0.0

180

4.0

4.0

4.0

4.0

4.0

4.0

0.0

0.0

0.0

240

4.0

4.0

4.0

4.0

4.0

4.0

0.0

0.0

0.0

 

Table 3. Corneal swelling (%)

 

observation after

time t

(minutes)

 

 

test item

 

positive control

imidazole

 

 

negative control

physiological saline

eyeball no.

eyeball no.

eyeball no.

1

2

3

4

5

6

7

8

9

0

 

0

0

0

0

0

0

0

0

0

30

 

35.0

56.7

29.9

50.4

70.0

41.2

-6.7

-7.4

-5.3

75

55.5

75.3

32.5

61.6

77.4

50.7

-4.0

-6.3

-4.6

120

74.7

51.7

49.4

70.5

106.1

73.9

-6.5

-8.4

-8.9

180

83.9

59.1

55.7

74.8

118.1

91.1

-6.5

-10.1

-7.2

240

96.4

56.5

58.8

82.7

122.5

109.1

-10.3

-4.1

-2.0

“-”= decrease in corneal thickness, no swelling

 

Table 4. Gross evaluation of the treated corneas

 

observation after

time t

(minutes)

 

 

test item

 

positive control

imidazole

 

 

negative control

physiological saline

eyeball no.

eyeball no.

eyeball no.

1

2

3

4

5

6

7

8

9

30

 

SIGNS

SIGNS

SIGNS

SIGNS

SIGNS

SIGNS

NC

NC

NC

75

SIGNS

SIGNS

SIGNS

SIGNS

SIGNS

SIGNS

NC

NC

NC

120

SIGNS

SIGNS

SIGNS

SIGNS

SIGNS

SIGNS

NC

NC

NC

180

SIGNS

SIGNS

SIGNS

SIGNS

SIGNS

SIGNS

NC

NC

NC

240

SIGNS

SIGNS

SIGNS

SIGNS

SIGNS

SIGNS

NC

NC

NC

NC = no changes; SIGNS = roughening of the corneal surface

Table 5. Evaluation of fluorescein retention

 

observation after

time t

(minutes)

 

 

test item

 

positive control

imidazole

 

 

negative control

physiological saline

average

ICE class

average

ICE class

average

ICE class

30

 

3.0

IV

3.0

IV

0.0

I

 

Table 6. Evaluation of corneal opacity

 

observation after

time t

(minutes)

 

 

test item

 

positive control

imidazole

 

 

negative control

physiological saline

average

ICE class

average

ICE class

average

ICE class

30

 

4.0

IV

4.0

IV

0.0

I

75

 

4.0

IV

4.0

IV

0.0

I

120

4.0

IV

4.0

IV

0.0

I

180

4.0

IV

4.0

IV

0.0

I

240

4.0

IV

4.0

IV

0.0

I

 

Table7. Evaluation of corneal swelling (%)

 

observation after

time t

(minutes)

 

 

test item

 

positive control

imidazole

 

 

negative control

physiological saline

average

ICE class

average

ICE class

average

ICE class

30

 

40.5

IV

53.8

IV

-6.5

I

75

 

54.4

IV

63.2

IV

-5.0

I

120

58.6

IV

83.5

IV

-7.9

I

180

66.2

IV

94.7

IV

-7.9

I

240

70.6

IV

104.8

IV

-5.4

I

“-”           - percentage of corneal thickness decrease, no swelling

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Remarks:
EU criteria.
Conclusions:
The test item causes serious eye damage (combination of the 3 endpoints is 3xIV). Therefore, it can be classified as Eye Damage Category 1.
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 guideline 438 under GLP conditions. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to either the test item, 10% acetic acid (positive control) or physiological saline (negative control). Three eyeballs were used for the test item and for each control. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438 recomendations, histopathological evaluation of the corneal layers was conducted. The average fluorescein retention was 3.0, resulting in an ICE class IV interpretation, the mean corneal opacity value for the test item was 4.0, resulting in an ICE class IV interpretation, and the mean corneal swelling values for the test item were 40.5 -70.6, resulting in a ICE class IV interpretation. Histopathological examinations of the corneas treated with the test item revealed: wrinkling, karyolysis, karyopyknosis, cell swelling, coagulation, necrosis (eyeballs no. 1, no. 2, no. 3), detachment (eyeball no. 2), slight erosions (eyeball no. 1) of the anterior corneal epithelium, local vacuolation and dissection of the anterior corneal epithelium (eyeball no. 3). According to the overall in vitro classification (UN GHS), and taking into account the results from the histopathological examination, the combination of the 3 endpoints was 3xIV, leading to an unequivocal result. Therefore, the test item leads to serious eye damage, and can be classified as Eye Damage Category 1.


Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion: Key study. The potential of the test item to produce dermal irritation after a single topical 4h application was studied in 3 male and 3 female New Zealand White rabbits, according to US CPSC (16 CFR 1500.41) - Method of testing primary irritant substances, similar to OECD 404 (GLP study). All animals died during the 4 hour application period. Blood was observed around the nose and mouth, and the position of the animals indicated convulsions prior to death. The gross necropsy indicated signs of hemorrhaging in the lungs and pericardial sac and the blood appeared a dark blue color. Severe necrosis was observed at all test sites, indicating irreversible corrosive damage to the skin. Based on the test results, the test item was found to be corrosive to the skin. Supporting study: According to peer reviewed handbook data, the test item is expected to be irritating to skin. Based on the available information, the test item is considered as corrosive to the skin.

Eye irritation: An in vitro (ex vivo) study was conducted with the test item according to the OECD guideline 438 (GLP study). Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to either the test item, 10% acetic acid (positive control) or physiological saline (negative control). Three eyeballs were used for the test item and for each control. The average fluorescein retention was 3.0 (ICE class IV), the mean corneal opacity value for the test item was 4.0 (ICE class IV), and the mean corneal swelling values for the test item were 40.5 -70.6 (ICE class IV). Histopathological examinations of the corneas treated with the test item revealed: wrinkling, karyolysis, karyopyknosis, cell swelling, coagulation, necrosis (eyeballs no. 1, no. 2, no. 3), detachment (eyeball no. 2), slight erosions (eyeball no. 1) of the anterior corneal epithelium, local vacuolation and dissection of the anterior corneal epithelium (eyeball no. 3).The combination of the 3 endpoints (3xIV) and the histopathological observations led to an unequivocal result. Therefore, the test item leads to serious eye damage, and can be classified as Eye Damage Category 1.

Justification for classification or non-classification

Based on the available information, the test item is classified as Skin Corrosive, Category 1, H314, according to CLP Regulation (EU) 1272/2008.

Based on the available information, the test item is classified as Eye Damage Category 1, H318, according to CLP Regulation (EU) 1272/2008.

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