Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion:

Key study: In-vitro skin corrosion: Test method OECD 430. GLP study. The substance was determined to be not corrosive to the skin.

Key study: In-vitro skin irritation: Test method OECD 439. GLP study. The substance was determined to be irritating to the skin (false positive)

Key study: In-vivo skin irritation/corrosion: Test method OECD 404. GLP study. The substance was determined to be not irritating to the skin.

Eye irritation/severe damage:

Key study: In-vitro eye damage: Test method OECD Guideline 438. GLP study. The substance was determined not to cause severe eye damage.

Key study: In-vivo eye irritation: Test method OECD Guideline 405. GLP study. The substance was determined to be not irritatint to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 June 2015 - 25 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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 system:
isolated skin discs
Source species:
rat
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Source strain:
Wistar
Details on animal used as source of test system:
SOURCE ANIMAL
- Source:Centre for Experimental Medicine at the Medical University in Katowice
- Sex: Female
- Age at study initiation (in days):At the beginning of the experiment, the animals were 21 days old. The skin discs used in the experiment were obtained from two 29-day-old female rats.
- 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: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20ºC
- Humidity (%): 49-56%
- Air changes (per hr): about 16 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Justification for test system used:
The Transcutaneous Electrical Resistance (TER) test method is a validated and accepted method for the classification of substances as Cat. 1 or non-corrosive.
Vehicle:
unchanged (no vehicle)
Remarks:
Moistened with 150 µL distilled water.
Details on test system:
SKIN DISC PREPARATION
- Procedure used:
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was greater than 10 kΩ.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 21-22 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: One.
- Observable damage in the tissue due to washing: Not rpeorted.
- Modifications to validated SOP: Not reported.


NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: Two.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean TER value is less than or equal to 5 kΩ and the skin disk is obviously damaged.
- The test substance is considered to be non-corrosive to skin if the mean TER value obtained for the test substance is greater than 5 kΩ, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage.

The dye binding procedure was not necessary in this case since all TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A sufficient amount of the solid was applied evenly to the disc to ensure that the whole surface of the epidermis was covered. Then it was moistoned with 150 µL distilled water.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 150 µL distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 150 µL 36 % hydrochloric acid.
- Concentration (if solution): 36 %
Duration of treatment / exposure:
24 hours
Number of replicates:
Three skin discs obtained per animal per run.
Two runs.
Irritation / corrosion parameter:
transcutaneous electrical resistance (in kΩ)
Run / experiment:
1 (mean of three skin discs)
Value:
10.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
transcutaneous electrical resistance (in kΩ)
Run / experiment:
2 (mean of three skin discs)
Value:
12.64
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: 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.

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

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

Animal number

Skin disc number

TER value (kΩ)

1

1

14.41

2

17.28

2

1

17.21

2

16.98

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

0.81

0.81 ± 0.01

2

0.82

3

0.80

Negative control – distilled water

1

19.72

19.19 ± 0.46

2

18.95

3

18.90

Test item

1

10.11

10.30 ± 0.35

2

10.70

3

10.08

2

Positive control –

36% HCl

1

0.87

0.87 ± 0.01

2

0.86

3

0.87

Negative control – distilled water

1

11.93

12.13 ± 0.20

2

12.13

3

12.32

Test item

1

12.35

12.64 ± 0.39 

2

12.49

3

13.09

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 10.30 kΩ (animal no. 1) and 12.64 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:
GHS criteria not met
Remarks:
CLP Implementation.
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 10.30 kΩ (animal no. 1) and 12.64 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:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 October 2015 - 30 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD Guideline 439. GLP study.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
human
Details on test animals and environmental conditions:
In-vitro test: Test system: Human Skin. EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France.
Type of coverage:
other: in-vitro test
Preparation of test site:
other: in-vitro test
Vehicle:
other: in-vitro test
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg
- Concentration (if solution): The test item was applied in its original form, no formulation was required.
Duration of treatment / exposure:
15 min.
Observation period:
42 hours post-incubation.
Number of animals:
In-vitro: EpiSkinTMSM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2).
Details on study design:
Pre-incubation (day [-1]-0):
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of an assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5 % CO2.

Application (day 0):
Three replicates / experiment were used for the test item and controls respectively. The epidermal surface was first moistened with 5 μL deionised water in order to improve further contact between powder and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface.
Positive and negative controls: A volume of 10 μL positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface by using a suitable pipette.
Additional controls for dyes and chemicals able to colour the tissue: One additional chemical-treated tissue was used for the non-specific OD evaluation.

Exposure (day 0):
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature. After the incubation time the EpiSkinTMSM units were removed and rinsed thoroughly with PBS 1x solution to remove all of the test material from the epidermal surface.

Post-incubation (day 0-2):
The units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5 % CO2.

MTT test after 42 hours incubation (day 2):
The EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37°C in an incubator with 5 % CO2 protected from light.

Formazan extraction (day 2):
The epidermis was separated with and placed into a tube of 500 μL acidified isopropanol (one tube corresponding to one well of the tissue culture plate). The capped tubes were thoroughly mixed by using a vortex mixer and incubated for at least two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction. At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

Cell viability measurements (day 2):
Following the formazan extraction, 2×200 μL sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at 570 nm using acidified isopropanol solution as the blank (6×200 μL). Individual OD values are corrected with the mean blank OD. The % viability for each replicate (test item and positive control) was calculated relative to the mean negative control.

Data calculation for dyes and chemicals able to colour the tissue: For test items detected as able to stain the tissues the non-specific OD is evaluated due to the residual chemical colour (unrelated to mitochondrial activity) and subtracted before calculation of the “true” viability %.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
19
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
31
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The test item is considered to be irritant to skin, since the mean relative viability after 15 minutes exposure and 42 hours post incubation is less to 50% of the negative control.

Two experiments were performed in order to minimize the possibilities of false results:

No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary.

Cell viability:

First experiment:

Substance

Optical density (OD)

Viability (%)

Negative Control:

1x PBS

1

1.022

117

2

0.847

97

3

0.761

84

Mean

0.877

100

Standard deviation (SD)

15.15

Positive Control:

SDS (5 % aq.)

1

0.178

20

2

0.205

23

3

0.165

19

Mean

0.183

21

Standard deviation (SD)

2.31

Test Item:

1

0.106

12

2

0.178

20

3

0.207

24

Mean

0.164

19

Standard deviation (SD)

5.90

Second experiment:

Substance

Optical density (OD)

Viability (%)

Negative Control:

1x PBS

1

0.962

98

2

0.869

88

3

1.117

114

Mean

0.982

100

Standard deviation (SD)

12.75

Positive Control:

SDS (5 % aq.)

1

0.124

13

2

0.105

11

3

0.073

7

Mean

0.101

10

Standard deviation (SD)

2.61

Test Item:

1

0.351

36

2

0.269

27

3

0.295

30

Mean

0.305

31

Standard deviation (SD)

4.27

As the test item has an intrinsic colour (yellow), one additional chemical-treated tissue was used for the non-specific OD evaluation on both occasions.

OD values and NSC % of additional control:

First experiment:

Additional color control

Optical density (OD)

Non specific color % (NSC %)

Test item treated tissue without MTT incubation

1

0.029

3.3

Second experiment:

Additional color control

Optical density (OD)

Non specific color % (NSC %)

Test item treated tissue without MTT incubation

1

0.016

1.6

Positive and negative controls showed the expected cell viability values within acceptable limits.

Each calculated standard deviation value (SD) for the % viability was below 18.

The experiments were considered to be valid

Interpretation of results:
Category 2 (irritant)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
EpiSkinTM SM test determined that the item was irritant to skin, since the mean relative viability after 15 minutes exposure and 42 hours post incubation was less to 50% of the negative control.
Executive summary:

EpiSkinTM SM test was performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Guideline 439 (GLP test). Disks of EPISKIN (three units / experiment) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. As the test item has an intrinsic colour (yellow), one additional chemical-treated tissue was used for the non-specific OD evaluation. SDS (5% aq.) and 1×PBS treated epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. Two experiments were performed in order to minimize the possibilities of false results. The test item in both experiments showed significantly reduced cell viability in comparison to the negative control (mean value: 19 % and 31 %). All obtained test item viability results were below 50 % when compared to the viability values obtained from the negative control, therefore the test item was considered to be irritating to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiments were considered to be valid. In this in vitro skin irritation test in the EPISKIN model the results indicated that the test item is Irritant [UN GHS: Category 2].

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 January 2016 - 9 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD Guidance 404. GLP study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Experimental Department of the National Research Institute of Animal Production, Balice near Kraków
- Age at study initiation: 9-11 months
- Weight at study initiation: 3.49-4.52 kg
- Housing: Individually in metal cages (73 x 70 x 45 cm).
- Diet (e.g. ad libitum): Ad libitum (LSK Agropol)
- Water (e.g. ad libitum): Ad libitum (tap water)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23 ºC
- Humidity (%): 35-58%
- Air changes (per hr): 16 times/h
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g
Duration of treatment / exposure:
4 hours
Observation period:
7 days
Number of animals:
3 female rabbits
Details on study design:
TEST SITE
- Area of exposure: 6 cm2.
- Type of wrap if used: Gauze patch was covered with PVC foil and fixed with a non-irritating sticking plaster. An elastic bandage and a sticking plaster were used to make a circular protecting band.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, with water.
- Time after start of exposure: 4 hours

SCORING SYSTEM:
According to OECD Guideline 404.
Erythema and edema: 1, 24, 48 and 72 hours and 7 days, after exposure.

OTHER OBSERVATIONS:
Clinical observations: 1, 24, 48 and 72 hours and 7 days, after exposure.
Body weights: Application day (day 0) and at the end of experiment (day 7)
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
other: 24-72 hours
Score:
1.7
Max. score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
other: 24-72 hours
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
other: 24-72 hours
Score:
1.3
Max. score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
animal #1
Time point:
other: 24-72 hours
Score:
0
Max. score:
0
Irritation parameter:
edema score
Basis:
animal #2
Time point:
other: 24-72 hours
Score:
0
Max. score:
0
Irritation parameter:
edema score
Basis:
animal #3
Time point:
other: 24-72 hours
Score:
0
Max. score:
0
Irritant / corrosive response data:
Very slight (barely perfectible) erythema was observed on animal no. 1 after the patch removing, 1 hour and 72 hours, and well defined erythema after 24 and 48 hours. If was fully reversible by day 7. No edema was observed. On animal no. 2, very slight (barely perceptible) erythema was observed after 1 hour and 24 hours. It was fully reversible by 48 hours. No edema was observed. On animal no. 3, well defined erythema was observed after 1 hour and 24 hours and very slight (barely perceptible) erythema after 48 and 72 hours. If was fully reversible by day 7. No edema was observed.
Other effects:
No mortality was observed. No adverse local nor systemic affects were observed.

Animal number

Changes

Evaluation after

Average after 24, 48, and 72 hours

the patch removing

1 hour

24 hours

48 hours

72 hours

7 days

1

erythema

1

1

2

2

1

0

1.7

edema

0

0

0

0

0

0

0.0

other

-

-

-

-

-

-

-

2

erythema

-

1

1

0

0

-

0.3

edema

-

0

0

0

0

-

0.0

other

-

-

-

-

-

-

-

3

erythema

-

2

2

1

1

0

1.3

edema

-

0

0

0

0

0

0.0

other

-

-

-

-

-

-

-

Body weights of the animal:

At the beginning of the experiment: 4.39 kg (animal 1), 3.49 kg (animal 2) and 4.52 kg (animal 3).

On the last day of the experiment: 4.39 kg (animal 1), 3.52 kg (animal 2) and 4.56 kg (animal 3).

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was determined to be non irritant to the skin of rabbits.
Executive summary:

The acute skin irritation/corrosion study was performed on the test item according to OECD Guideline 404 (GLP study). The study commenced with a sighting study on one animal. Transcutaneous Electrical Resistance Test (TER) of the test item was performed and it was determined that the substance does not lead to skin corrosion/severe irritation, and therefore in the sighting study the test item could be applied to one animal for four hours. The test item in amount of 0.5 ml was applied to the shaved skin of one animal (rabbit no. 1) and covered with a protecting band. The exposure lasted 4 hours. After the examination of the treated skin, the test item was applied to the skin of the next two animals (rabbits no. 2 and 3) in order to confirm the irritant or negative response. General clinical observations of the animals for morbidity and mortality were performed daily during the entire experiment. Detailed clinical observations of the treated skin were performed 1, 24, 48, 72 hours and 7 days after the end of the exposure. Body weights of the animals were determined on the application day (day 0), i.e. directly before the application, and on the last day of the experiment. The mean 24 -72 hours erythema scores were 1.7, 0.3 and 1.3 for each animal respectively being the effects fully reversible by day 7. Since no edema was observed, the mean 24 -72 hours edema scores were 0.0 for three animals. No other adverse effects were observed. Based on these results (erythema and edema scores <2.3 and fully reversible), the test item was determined to be non irritating to the skin.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 August 2015 - 19 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Experimental Department of the National Research Institute of Animal Production in Balice near Kraków.
- Age at study initiation: 6-month-old
- Weight at study initiation: 2.9-3.6 kg
- Housing: Individually housed in metal cages (73 x 70 x 45 cm).
- Diet (e.g. ad libitum): ad libitum (LSK)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25 ºC
- Humidity (%): 50-90%
- Air changes (per hr): 16 times per hour
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL (0.0680 g)
Duration of treatment / exposure:
1 hour
Observation period (in vivo):
14 days
Number of animals or in vitro replicates:
3 male rabbits
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): The test item was not removed from the eye by physiological mechanism, so the eye was rinsed with saline.
- Time after start of exposure: 1 hour.

SCORING SYSTEM:
Ocular lesions were graded according to OECD Guideline 405/EU Method B5.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: Mean 24-72 h
Score:
0
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
other: Mean 24-72 h
Score:
0
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
other: Mean 24-72 h
Score:
0
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: Mean 24-72 h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: 24 h
Irritation parameter:
iris score
Basis:
animal #2
Time point:
other: Mean 24-72 h
Score:
0
Irritation parameter:
iris score
Basis:
animal #3
Time point:
other: Mean 24-72 h
Score:
0.7
Max. score:
1
Reversibility:
fully reversible within: 72 h
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
other: Mean 24-72 h
Score:
1.3
Max. score:
2
Reversibility:
fully reversible within: 7 d
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
other: Mean 24-72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 72 h
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
other: Mean 24-72 h
Score:
1.7
Max. score:
2
Reversibility:
fully reversible within: 7 d
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: Mean 24-72 h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: Mean 24-72 h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
other: Mean 24-72 h
Score:
1.3
Max. score:
2
Reversibility:
fully reversible within: 7 d
Other effects:
No other adverse effects were observed.

Evaluation of the animals’ ocular lesions:

Animal No.

Part of the eye

Readings after

Average 24-72 h

1 h

24 h

48 h

72 h

7 d

1

Cornea

0

0

0

0

0

0.0

Iris

1

1

0

0

0

0.3

Conjuctiva

Erythema

3

2

1

1

0

1.3

Swelling

2

1

0

0

0

0.3

2

Cornea

0

0

0

0

-

0.0

Iris

0

0

0

0

-

0.0

Conjuctiva

Erythema

2

2

1

0

-

1.0

Swelling

2

1

0

0

-

0.3

3

Cornea

0

0

0

0

0

0.0

Iris

1

1

1

0

0

0.7

Conjuctiva

Erythema

3

2

2

1

0

1.7

Swelling

3

2

1

1

0

1.3

Interpretation of results:
GHS criteria not met
Remarks:
CLP Implementation.
Conclusions:
The test item was determined to be not irritating to the eye in rabbits.
Executive summary:

The acute eye irritation/corrosion study was performed in albino New Zealand rabbits according to OECD Guideline 405 (GLP study). The test item in an amount of 0.068 g was applied to the conjunctival sac of one eye of one animal. The other eye, which remained untreated, served as a control. The animal was observed for 7 days. Following the examination of the treated eye, the test item was applied to the eyes of rabbits no. 2 and 3 to confirm the irritant or negative response. General clinical observations of the animals for morbidity and mortality were performed several times a day during the first 3 days, and twice a day on the remaining days of the experiment. Detailed clinical observations for changes in the cornea, iris, and conjunctiva were performed 1, 24, 48, and 72 hours and 7 days after the application. Body weights of the animals were determined on the application day (day 0), i.e. directly before the application, and on the last day of the experiment. After the observation period, the animals were euthanized. After application of the test item, changes in the iris of two rabbits and changes in the conjunctiva of three rabbits were stated (cornea scores = 0; iris scores <1, conjuctiva scores <2, chemosis scores <2). These changes were transient and fully reversible by day 7. The substance was determined to be not irritating to the eye.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 June 2015 - 10 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
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 eyeballs (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
Fluorescein 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.
Irritation parameter:
fluorescein retention score
Run / experiment:
1
Value:
1.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Reported value correspond to ICE Class II
Irritation parameter:
fluorescein retention score
Run / experiment:
2
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Reported value corresponds to ICE class II.
Irritation parameter:
cornea opacity score
Run / experiment:
1
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Reported value is the maximum mean score observed at any time, it corresponds to ICE class II.
Irritation parameter:
cornea opacity score
Run / experiment:
2
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Reported value is the maximum mean score observed at any time, it corresponds to ICE class II.
Irritation parameter:
percent corneal swelling
Run / experiment:
1
Value:
3.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Reported value is the maximum mean score observed at any time, it corresponds to ICE class I.
Irritation parameter:
percent corneal swelling
Run / experiment:
2
Value:
10.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Reported value is the maximum mean score observed at any time, it corresponds to ICE class I.
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None reported.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, it was classified as GHS non-classified.
- Acceptance criteria met for positive control: Yes, it was classified as GHS Category I.

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

1.3

II

3.0

IV

0.0

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

1.0

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

0.0

I

75

1.0

II

4.0

IV

0.0

I

120

1.0

II

4.0

IV

0.0

I

180

1.0

II

4.0

IV

0.0

I

240

1.0

II

4.0

IV

0.0

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

2.7

IV

4.0

IV

0.0

I

75

2.7

IV

4.0

IV

0.0

I

120

2.7

IV

4.0

IV

0.0

I

180

2.7

IV

4.0

IV

0.0

I

240

2.7

IV

4.0

IV

0.0

I

 

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

3.4*

I

18.8

III

0.0

I

75

0.7*

I

37.3

IV

0.2

I

120

0.8*

I

45.1

IV

0.2

I

180

0.4*

I

49.8

IV

0.2*

I

240

3.7*

I

51.9

IV

0.2*

I

* - percentage of corneal thickness decrease, no swelling

 

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

4.8*

I

16.7

III

0.8*

I

75

7.8*

I

29.0

III

1.1*

I

120

10.2*

I

38.2

IV

0.4*

I

180

8.6*

I

43.7

IV

1.5*

I

240

10.2*

I

54.9

IV

3.1*

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 examination of the corneas treated with the test item - the first run.

Histopathological examinations of the corneas treated with the test item showed very slight exfoliation of the anterior corneal epithelium (eyeballs no. 1, no. 2 and no. 3) and vacuolation of the basal cells layer of the anterior corneal epithelium (eyeball no. 2).

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

Histopathological examinations of the corneas treated with the test item showed very slight exfoliation of the anterior corneal epithelium (eyeball no. 1), exfoliation of the anterior corneal epithelium (eyeball no. 3), slight exfoliation of the anterior corneal epithelium and vacuolation of the basal cells layer of the anterior corneal epithelium (eyeball no. 2).

Interpretation of results:
study cannot be used for classification
Remarks:
CLP Implementation.
Conclusions:
According to UN GHS classification criteria, no prediction can be made, since the ICE Class combination of the 3 endpoints were 2xII and 1xI (the first run and the second run).
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 obtain mean fluorescein retention scores for the eyeball treated with test item were 1.3 and 1.0 (ICE class II) in the first and second round respectively. The mean corneal opacity scores were 1.0 (ICE class II) in both runs. The mean corneal swelling value (%) for the eyeballs treated with the test item were from 0.4 to 3.7 (ICE class I) in the first run and from 4.8 to 10.2 (ICE class I) in the second run. Gross examinations of the eyeballs treated with the test item did not exhibit any changes of the corneal surface. Histopathological examinations of the corneas treated with the test item showed very slight or slight exfoliation of the anterior corneal epithelium and vacuolation of the basal cells layer of the anterior corneal epithelium. 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 were 2xII and 1xI (the first run and the second run).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion:

Key study: In-vitro skin corrosion study:

The in vitro skin corrosion: transcutaneous electrical resistance test (TER) was performed according to OECD Guideline 430 (GLP study). The substance did 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.

Key study: In-vitro skin irritation study:

Since the substance was determined to be not corrosive to the skin in the previous in-vitro test, an in-vitro EpiSkinTM SM test was performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Guideline 439 (GLP test). Taking into account that the test item was determined to be not irritating to the eyes (in-vivo test according to OECD 405), the experiment was perfomed twice in order to minimize false results. Nevertheless, all obtained test item viability results were below 50 % when compared to the viability values obtained from the negative control,

therefore the test item was considered to be irritating to skin. The substance resulted to be irritating to the skin according to OECD Guideline 439. Taking into accoun that the test item was not irritating to the eyes (OECD Guideline 405), it was questioned whether the obtained results were related to a false positive result.

Key study: In-vivo skin irritation study:

Finally, the acute skin irritation/corrosion study was performed on the test item with albino New Zealand female rabbits according to OECD Guideline 404 (GLP study). The mean 24-72 hours erythema scores were 1.7, 0.3 and 1.3 for each animal respectively being the effects fully reversible by day 7. Since no edema was observed, the mean 24 -72 hours edema scores were 0.0 for the three animals. No other adverse effects were observed. Based on these results (erythema and edema scores <2.3 and fully reversible), the test item was determined to be non irritating to the skin.

Eye irritation/severe damage:

Key study: In-vitro eye irritation:

The isolated chicken eye test (in vitro) was performed according to OECD Guideline 438 (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. 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 were 2xII and 1xI (the first run and the second run).

Key study: In-vivo eye irritation:

The acute eye irritation/corrosion study was performed in albino New Zealand rabbits according to OECD Guideline 405 (GLP study). The test item was applied to the conjunctival sac of one three animals. The other eye, which remained untreated, served as a control. Animals were observed for 7 days. Changes in the iris of two rabbits and changes in the conjunctiva of three rabbits were stated (cornea scores = 0; iris scores <1, conjuctiva scores <2, chemosis scores <2). These changes were transient and fully reversible by day 7. The substance was determined to be not irritating to the eyes.

Justification for classification or non-classification

Based on available data, the substance is not classified for skin nor eye irritation according to CLP Regulation (EC) No. 1272/2008.