Registration Dossier

Administrative data

Description of key information

Skin irritation: OECD 430, EU B.40. GLP study. The test item do not lead to skin corrosion/severe irritation.
Eye irritation: OECD 438, EU B.48. GLP study. The test item did not cause eye damage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 November 2013 - 2 November 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD Guideline 430. GLP study.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
SOURCE OF THE BIOLOGICAL MATERIAL:
TEST ANIMALS:
- Source: Centre for Experimental Medicine at the Medical University in Katowice
- Age at study initiation: 29 days old (hair follicles in dormant phase)
- Housing: Plastic cage covered with a wire bar lid. Dimensions: 58 x 37 x 21 cm. Bedding: UV-sterilized wood shavings.
- Diet (e.g. ad libitum): ad libitum, "Murigran” standard granulated laboratory fodder
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20ºC
- Humidity (%): 49-52%
- Air changes (per hr): about 16 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Type of coverage:
other: Not applicable: in-vitro test
Preparation of test site:
other: Not applicable: in-vitro test
Vehicle:
water
Controls:
yes, concurrent vehicle
Amount / concentration applied:
VEHICLE
- Amount(s) applied (volume or weight with unit): 150 ul of distilled water
Duration of treatment / exposure:
24 hours
Number of animals:
2
Irritant / corrosive response data:
On the grounds of the study, it may be stated that the test ite belongs to a group of substances which do not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs

Results of the control transcutaneous electrical resistance test (TER):

Animal number

Skin disc number

TER value (kΩ)

1

1

17.28

2

18.79

2

1

14.91

2

15.07

The skin discs gave the resistance values greater than 10 kΩ; therefore, the remainder of the skin discs of the animals could have been used in the experiment.

Results of the transcutaneous electrical resistance test (TER):

Animal number

Tested substance

Skin disc number

TER value (kΩ)

Mean TER value ± SD (kΩ)

1

Positive control –

36% HCl

1

0.88

0.88 ± 0.02

2

0.87

3

0.90

Negative control – distilled water

1

17.03

16.35 ± 0.51

 

2

15.29

3

16.74

Test item

1

5.72

6.18 ± 0.51

 

2

6.08

3

6.73

2

Positive control –

36% HCl

1

0.92

0.90 ± 0.02

2

0.88

3

0.90

Negative control – distilled water

1

17.17

17.68 ± 1.11

 

2

16.92

3

18.95

Test item

1

6.75

6.65 ± 0.52

 

2

6.08

3

7.11

The concurrent mean values for the positive and negative controls were within the acceptable ranges for the method:

Positive control: 0.5-1.0 kΩ

Negative control: 10 -25 kΩ

The mean TER results for the skin discs treated with the test item were equal to 6.18 kΩ (animal no. 1) and 6.65 kΩ (animal no. 2).

Gross changes on the surface of the treated skin discs:

Animal number

Tested substance

Skin disc number

Gross changes

1

Positive control –

36% HCl

1

perforation

2

perforation

3

perforation

Negative control – distilled water

1

no changes

2

no changes

3

no changes

Test item

1

no changes

2

no changes

3

no changes

2

Positive control –

36% HCl

1

perforation

2

perforation

3

perforation

Negative control – distilled water

1

no changes

2

no changes

3

no changes

Test item

1

no changes

2

no changes

3

no changes

The gross examination showed that the positive control skin discs exhibited skin perforation, whereas the negative control skin discs and the ones treated with the test item did not reveal any changes.

Interpretation of results:
other: non-corrosive
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The substance do not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.
Executive summary:

The in vitro skin corrosion: transcutaneous electrical resistance test (TER) was performed according to OECD Guideline 430 Guideline and EU Method B.40. (GLP study). Skin discs used in the experiment were obtained from two 29-day-old rats. The test item (ground to a powder) was uniformly applied to the epidermal surface of the skin disc placed inside a tube. Positive (36% hydrochloric acid) and negative (distilled water) controls were conducted concurrently. Three skin discs obtained from each animal were used for the test item and three for each control item. The test item and the control items were evenly applied to the discs for 24 hours and kept at 21-22°C. Then, they were removed by washing with a jet of tap water and the surface tension of the skin was reduced by adding 70% ethanol. After removing the ethanol the tissue was hydrated by the addition of 3 mL of a solution of MgSO4 (154 mM). A LCR 6401 low-voltage, alternating current databridge was used to measure the electrical resistance of the skin in kΩ by placing the databridge electrodes on either side of the skin disc. The skin discs were subjected to a gross examination. The mean TER results were equal to 6.18 kΩ (animal no. 1) and 6.65 kΩ (animal no. 2). The concurrent positive and negative control values fell within the acceptable ranges for the method. Gross examinations of the skin discs did not reveal any pathological changes. On the grounds of the study, it may be stated that the test item do not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
10 April 2014 - 12 May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD Guideline 438. GLP study.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
other: chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Zakład Przemysłu Drobiarskiego JAS-DROP in Krzyżowice
- Age at study initiation: 7-week-old
- Weight at study initiation: 1.5 - 2.5 kg

BIOLOGICAL MATERIAL
After sedation of the chickens by electric shock and incision of the neck for bleeding, their 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. The time interval between the collection of the chickens’ heads and the use of their eyeballs in the ICE test was 30 minutes.
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 g

Application and removal of the test item and the control items:
There were three groups, i.e. one group treated with the test item and two control groups, including a positive one (imidazole) and a negative one (physiological salt) used concurrently in the study to ensure its quality. The test item and the control items were tested on three eyeballs each. Immediately following the zero reference measurements, the eyeballs in their holders were removed from the superfusion apparatus and placed in a horizontal position in order to apply the test item and the control items. The test item (ground to a powder) and the item used in the positive control (imidazole) were applied in the amount of 0.03 g, whereas the item used in the negative control (physiological salt) was applied in a volume of 0.03 mL. The test item and the control items were uniformly applied to the corneal surface for 10 seconds. Then, they were rinsed from the eye with 20 mL of physiological salt at ambient temperature.
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
4 hours after post-treatment rinse.
Number of animals or in vitro replicates:
9 eyebalss (3 eyeballs per treatment: test item, negative control, positive control)
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes (physiological salt)
- Time after start of exposure: After 10 seconds of exposure.

MEASURED PARAMETERS:
Pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.
At all observation times points, corneal opacity and swelling were evaluated, whereas morphological changes of the corneal surface were recorded. The quantitative determination of fluorescein retention was performed only once, i.e. 30 minutes after the end of the exposure.

Scoring system
Florescein retention:
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

Corneal opacity:
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:
The degree of corneal swelling was determined by measuring corneal thickness with a SP-100 pachymeter.

Gross evaluation:
To determine whether any morphological effects, e.g. pitting of corneal epithelial cells, roughening of the corneal surface, and sticking of the test item to the cornea were visible.

Histopathological evaluation of the treated corneas:
Following the final evaluation of the treated eyeballs (240 minutes after the application of the test item and the control items), the eyeballs were fixed in a 4% solution of formaldehyde. Next, specimens were collected (one specimen in the plane including the cornea, lens, and optic nerve). The tissue material was dehydrated and prepared using a paraffin technique. Paraffin blocks were cut into smaller parts, whose thickness was 5 μm, with a microtome and stained using Hematoxylin and Eosin. The following layers of the cornea were evaluated: anterior epithelium, anterior elastic lamina (Bowman’s membrane), corneal stroma, posterior elastic lamina (Descemet’s membrane), and posterior epithelium. All treated eyeballs were subject to this evaluation.
Irritant / corrosive response data:
On the grounds of the study and the overall in vitro Irritancy Classification, it may be stated that the test item did not cause eye damage. According to UN GHS classification criteria, no prediction can be made, since the ICE Class combination of the 3 endpoints was 2xII and 1xI.

Evaluation of fluorescein retention– the first run.

 

Observation after (minutes)

Test item

Positive control

Imidazole

Negative control

Physiological saline

Average

ICE class

Average

ICE class

Average

ICE class

30

1.0

II

3.0

IV

0.3

I

 

Evaluation of fluorescein retention – the second run.

 

Observation after (minutes)

Test item

Positive control

Imidazole

Negative control

Physiological saline

Average

ICE class

Average

ICE class

Average

ICE class

30

1.3

II

3.0

IV

0.0

I

 

Evaluation of corneal opacity– the first run.

 

Observation after (minutes)

Test item

Positive control

Imidazole

Negative control

Physiological saline

Average

ICE class

Average

ICE class

Average

ICE class

30

1.0

II

4.0

IV

0.3

I

75

1.0

II

4.0

IV

0.3

I

120

1.0

II

4.0

IV

0.3

I

180

1.0

II

4.0

IV

0.3

I

240

1.0

II

4.0

IV

0.3

I

 

Evaluation of corneal opacity – the second run.

 

Observation after (minutes)

Test item

Positive control

Imidazole

Negative control

Physiological saline

Average

ICE class

Average

ICE class

Average

ICE class

30

1.3

II

4.0

IV

0.0

I

75

1.3

II

4.0

IV

0.0

I

120

1.3

II

4.0

IV

0.0

I

180

1.3

II

4.0

IV

0.0

I

240

1.3

II

4.0

IV

0.0

I

 

Table 7a.Evaluation of corneal swelling (%)– the first run.

 

Observation after (minutes)

Test item

Positive control

Imidazole

Negative control

Physiological saline

Average

ICE class

Average

ICE class

Average

ICE class

30

4.6*

I

70.9

IV

2.3*

I

75

5.5*

I

78.2

IV

4.5*

I

120

6.8*

I

82,8

IV

6.2*

I

180

10.1*

I

86.1

IV

7.6*

I

240

11.9*

I

88.1

IV

10.2*

I

* - percentage of corneal thickness decrease, no swelling

 

Table 7b. Evaluation of corneal swelling (%) – the second run.

 

Observation after (minutes)

Test item

Positive control

Imidazole

Negative control

Physiological saline

Average

ICE class

Average

ICE class

Average

ICE class

30

2.7*

I

73.6

IV

1.3*

I

75

3.9*

I

74.9

IV

3.6*

I

120

4.8*

I

77.7

IV

5.1*

I

180

6.3*

I

80.5

IV

6.6*

I

240

6.5*

I

81.6

IV

7.5*

I

* - percentage of corneal thickness decrease, no swelling

Gross evaluation of the treated corneas - the first run.

 

 

Observation after (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

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

75

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

120

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

180

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

240

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

NC = no changes

SIGNS = roughening of the corneal surface

 

Gross evaluation of the treated corneas - the second run.

 

 

Observation after (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

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

75

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

120

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

180

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

240

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

NC = no changes

SIGNS = roughening of the corneal surface

Histological examinaion of the corneas treated with the test item - the first run.

Exfoliation of the anterior corneal epithelium (eyeballs no. 1, 2, and 3) and dissection of the corneal stroma (eyeballs no. 1, 2 and 3) were observed.

Histopathological examination of the corneas treated with the test item - the second run.

Exfoliation of the anterior corneal epithelium (eyeballs no. 1, 2, and 3), dissection of the corneal stroma (eyeballs no. 1 and 2) and detachment of the posterior corneal epithelium (eyeball no. 2) were observed.

Interpretation of results:
other: no prediction can be made
Remarks:
Criteria used for interpretation of results: OECD GHS
Conclusions:
The test item did not cause eye damage. According to UN GHS classification criteria, no prediction can be made.
Executive summary:

The isolated chicken eye test (in vitro) was performed according to OECD Guideline 438 and EU Method B.48 (GLP study). The study was conducted in two runs. The first study led to a GHS NC outcome, so a second run of nine eyeballs was conducted to confirm or discard the negative outcome. The test item (ground to a powder) and the item used in the positive control (imidazole) were applied in the amount of 0.03 g, whereas the item used in the negative control (physiological salt) was applied in a volume of 0.03 mL. Three eyeballs were used for the test item and three for each control item. Every time, the test item and the control items were applied to the corneal surface for 10 seconds and kept at temperature between 20 – 23º C. Then, they were rinsed from the eye with 20 mL of physiological salt at ambient temperature. The corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse. At all observation time points, corneal opacity and swelling were evaluated, whereas morphological changes of the corneal surface were recorded. The quantitative determination of fluorescein retention was performed only once, i.e. 30 minutes after the end of the exposure. Following the final evaluation of the treated eyeballs, i.e. 240 minutes after the application of the test item and the control items, the eyeballs were fixed in a 4% solution of formaldehyde in order to allow histopathological examinations to be conducted. The test item did not cause eye damage. The obtain mean fluorescein retention scores for the eyeball treated with test item were 1.0 (ICE class II) and 1.3 (ICE class II) in the first and second round respectively. The mean corneal opacity scores were 1.0 (ICE class II) and 1.3 (ICE class II). No swelling was observed in both runds (ICE class I). Gross examination did not reveal any changes of the corneal surface. In the first run, the histopathological examinations of the corneas showed exfoliation of the anterior corneal epithelium and dissection of the corneal stroma. Moreover, in the second run, the detachment of the posterior corneal epithelium was observed in one treated eyeball. These results were accepted since the concurrent positive and negative control values fell within the acceptable ranges for the method. On the grounds of the study results and the overall in vitro Irritancy Classification, it may be stated that the test item did not cause eye damage. According to UN GHS classification criteria, no prediction can be made, since the ICE Class combination of the 3 endpoints was 2xII and 1xI.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Skin irritation/corrosion: Key study: The in vitro skin corrosion: transcutaneous electrical resistance test (TER) was performed according to OECD Guideline 430 Guideline and EU Method B.40. (GLP study). The mean TER results for the skin discs (rat) treated with the test item for 24 hours were equal to 6.18 kΩ (animal no. 1) and 6.65 kΩ (animal no. 2). The concurrent positive and negative control values fell within the acceptable ranges for the method. Gross examinations of the skin discs did not reveal any pathological changes. On the grounds of the study, it may be stated that the test item do not lead to skin corrosion/severe irritation.

Eye irritation: Key study: The isolated chicken eye test (in vitro) was performed according to OECD Guideline 438 and EU Method B.48 (GLP study). The obtain mean fluorescein retention scores for the eyeball treated with test item were 1.0 (ICE class II) and 1.3 (ICE class II) in the first and second run respectively. The mean corneal opacity scores were 1.0 (ICE class II) and 1.3 (ICE class II). No swelling was observed in both runs (ICE class I). Gross examination did not reveal any changes of the corneal surface. In the first run, the histopathological examinations of the corneas showed exfoliation of the anterior corneal epithelium and dissection of the corneal stroma. Moreover, in the second run, the detachment of the posterior corneal epithelium was observed in one treated eyeball. On the grounds of the study results and the overall in vitro Irritancy Classification, the test item did not cause eye damage. According to UN GHS classification criteria, no prediction can be made, since the ICE Class combination of the 3 endpoints was 2xII and 1xI.


Justification for selection of skin irritation / corrosion endpoint:
Only one study is available.

Justification for selection of eye irritation endpoint:
Only one study is available.

Justification for classification or non-classification

Based on the available information, the substance is not classified for irritation/corrosion according to CLP Regulation (EC) no. 1272/2008.