Registration Dossier

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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

Skin irritation


A number of skin irritation studies were included as part of NONS registrations. In vivo dermal irritation studies in rabbits have been conducted on 3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea (PU12/A123; EC 406-690-3), 3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea (R95; EC 406-370-3), N,N''-(methylenedi-4,1-phenylene)bis[N'-octyl]urea (A124; EC 445-760-8), and a mixture of: 3,3'-dicyclohexyl- 1,1'-methylenebis(4,1-phenylene)diurea; 3-cyclohexyl-1-(4-(4-(3-octadecylureido)benzyl)phenyl)urea; 3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea (PU10; A002; PU18;EC 406-530-2),. The results from all tests concluded the substances were non-irritants and no classifiable irritant responses were observed in any of the studies when applied to skin.


 


As part of an updated testing program, in vitro skin irritation tests (OECD 439) were conducted on:


- Reaction mass of 4,4'-methylenediphenyl diisocyanate and Amines, soya alkyl (A003; EC 905-837-3)


- Reaction product of MDI, Octadecylamine and Magnesium Hydroxide (PU05; EC 944-730-6)


- Reaction product of MDI and p-toluidine (PU07; EC 926-809-7)


- Reaction product of MDI, Octylamine and Cyclohexylamine (PU08; EC 926-119-6)


- Reaction product of MDI, Octylamine and Hexamethylenediamine (PU09; EC 924-670-7)


- Polyurea, produced by reacting diphenylmethane diisocyanate with octylamine and dodecyl amine (R03; EC 812-490-0)


- Polyurea, produced by reacting diphenylmethane diisocyanate with octyl amine and stearyl amine (R04; EC 812-491-6)


- Reaction product of 4,4'-methylenediphenyl diisocyanate, ethylenediamine and dodecylamine (R05; EC 924-043-8)


Negative results were observed in all substances tested, indicating the substances were non-irritants to the skin.


 


Under the conditions of the in vitro and in vivo tests, the substances within the MDI category did not demonstrate a skin irritant response.


 


Eye irritation


A number of eye irritation studies were included as part of NONS registrations. In vivo eye irritation studies in rabbits have been conducted on 3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea (PU12/A123; EC 406-690-3), 3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea (R95; EC 406-370-3), N,N''-(methylenedi-4,1-phenylene)bis[N'-octyl]urea (A124; EC 445-760-8), and a mixture of: 3,3'-dicyclohexyl- 1,1'-methylenebis(4,1-phenylene)diurea; 3-cyclohexyl-1-(4-(4-(3-octadecylureido)benzyl)phenyl)urea; 3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea (PU10; A002; PU18;EC 406-530-2). The results for 3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea (PU12/A123; EC 406-690-3), 3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea (R95; EC 406-370-3), and a mixture of: 3,3'-dicyclohexyl- 1,1'-methylenebis(4,1-phenylene)diurea; 3-cyclohexyl-1-(4-(4-(3-octadecylureido)benzyl)phenyl)urea; 3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea (PU10; A002; PU18;EC 406-530-2) demonstrated no classifiable irritant response to the eyes.


 


The substance N,N''-(methylenedi-4,1-phenylene)bis[N'-octyl]urea (EC 445-760-8), which contains two C8 structures, was reported positive for eye irritation based on ocular reactions: irritation of the conjunctivae, seen as redness and chemosis, and a slight corneal opacity in all animals on day 2, which persisted up to day 2 (one animal) or 4 (one animal). In one animal, the effects on the conjunctivae score (redness) and chemosis score were not fully reversed within the observation period of 21 days. In the other animals, the observed effects were fully reversed within 21 days. However, in this study 100 mg of the substance (a white powder with a very poor water solubility of 4.38 µg/L) was put directly onto the eye (without vehicle) and no washing was applied. It was recorded that the material was still present in the eyes >24 h after application. It is therefore most likely that the observed ocular effects have been caused by mechanical irritation rather than inherent irritative properties of the material and this study is not considered to be reliable.


 


As part of an updated testing program, in vitro eye irritation tests (OECD 437) were conducted on:


- Reaction mass of 4,4'-methylenediphenyl diisocyanate and Amines, soya alkyl (A003; EC 905-837-3)


- Reaction product of MDI and p-toluidine (PU07; EC 926-809-7)


- Reaction product of MDI, Octylamine and Cyclohexylamine (PU08; EC 926-119-6)


- Reaction product of MDI, Octylamine and Hexamethylenediamine (PU09; EC 924-670-7)


- Polyurea, produced by reacting diphenylmethane diisocyanate with octyl amine and stearyl amine (R04; EC 812-491-6)


- Reaction product of 4,4'-methylenediphenyl diisocyanate, ethylenediamine and dodecylamine (R05; EC 924-043-8)


 


Negative results were observed for all substances tested, indicating the substances were non-irritants to the eye.


 


In addition, an in vivo ocular irritation study in rabbits was conducted on Polyurea, produced by reacting diphenylmethane diisocyanate with octylamine and dodecyl amine (R03; EC 812-490-0). No classifiable irritant response was observed.


 


Under the conditions of the in vitro and in vivo tests, the substances within the MDI category did not demonstrate an eye irritant response.


 


Respiratory irritation


No data were identified relating to irritation of the respiratory tract. Given the results from the skin and eye irritation studies, it is unlikely that MDI category members would cause respiratory irritation. The members of the MDI category are grease thickeners and although they were tested in the isolated form, they are only manufactured and marketed within the grease base where near stoichiometric quantities of starting materials are used to avoid the presence of excess isocyanate. Additionally, following the substance identification analysis conducted, it is noted in all the study reports that no unreacted MDI starting material is present in the substances to be registered. Based on this information, it is considered that a respiratory classification is not required for any of the substances being registered in the MDI category. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.
Qualifier:
according to guideline
Guideline:
other: Annex V
GLP compliance:
yes
Species:
rabbit
Strain:
other: Japanese White
Type of coverage:
occlusive
Preparation of test site:
not specified
Vehicle:
other: Liquid paraffin
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 500 mg

VEHICLE
- Other: Liquid paraffin
Duration of treatment / exposure:
4 hours
Observation period:
Not reported
Number of animals:
6
Details on study design:
Conducted according to guideline and GLP
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other: Timepoint not specified, standard timepoint assumed
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other: Timepoint not specified, standard timepoint assumed
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other: Timepoint not specified, standard timepoint assumed
Irritation parameter:
erythema score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other: Timepoint not specified, standard timepoint assumed
Irritation parameter:
erythema score
Basis:
animal #5
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other: Timepoint not specified, standard timepoint assumed
Irritation parameter:
erythema score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other: Timepoint not specified, standard timepoint assumed
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other: Timepoint not specified, standard timepoint assumed
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other: Timepoint not specified, standard timepoint assumed
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other: Timepoint not specified, standard timepoint assumed
Irritation parameter:
edema score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other: Timepoint not specified, standard timepoint assumed
Irritation parameter:
edema score
Basis:
animal #5
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other: Timepoint not specified, standard timepoint assumed
Irritation parameter:
edema score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other: Timepoint not specified, standard timepoint assumed
Irritant / corrosive response data:
None reported
Other effects:
None
Interpretation of results:
GHS criteria not met
Conclusions:
Edema and erythema scores were 0 for all animals. Therefore, the test item is not classified for skin irritation.
Executive summary:

The skin irritancy potential of the test item was assessed using 500 mg of the test item. The study was conducted according to a standard guideline using Japanese white rabbits. The mean and max edema and erythema scores were 0 for all animals. No other effects were observed. Therefore, the test item is not classified for skin irritation.

The study is a GLP compliant, guideline study and is acceptable with restrictions for evaluation of this endpoint.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.
Qualifier:
according to guideline
Guideline:
other: Annex V
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Type of coverage:
semiocclusive
Preparation of test site:
not specified
Vehicle:
water
Remarks:
Milli-Q water
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Not reported
- Concentration (if solution): 500 mg
Duration of treatment / exposure:
4 hours
Observation period:
Not reported
Number of animals:
3
Details on study design:
Conducted according to guideline and GLP
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Irritation parameter:
erythema score
Basis:
mean
Time point:
72 h
Score:
0
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
mean
Time point:
72 h
Score:
0
Irritant / corrosive response data:
Any changes observed were fully reversible within 1 day
Other effects:
Slight erythema was noted in one animal after removal of dressing.
Interpretation of results:
GHS criteria not met
Conclusions:
Mean edema and erythema scores after 24, 48 and 72 hours were 0 for all animals. Therefore, the test item is not classified for skin irritation.
Executive summary:

The skin irritancy potential of the test item was assessed with New Zealand White rabbits according to a standard guideline. 500 mg of the test item was applied to 3 rabbits and the exposure period was 4 hours. The edema and erythema scores after 24, 48 and 72 hours were 0 for all animals. Slight erythema was noted in one animal after removal of dressing. Any changes observed were fully reversible within 1 day. Therefore, the test item is not classified for skin irritation.

 

The study is a GLP compliant, guideline study and is acceptable with restrictions for evaluation of this endpoint.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.
Qualifier:
according to guideline
Guideline:
other: Annex V
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Type of coverage:
semiocclusive
Preparation of test site:
not specified
Vehicle:
other:
Remarks:
Substance moistened with water
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 500 mg
Duration of treatment / exposure:
4 hours
Observation period:
Not reported
Number of animals:
3
Irritation parameter:
erythema score
Basis:
animal #1
Remarks:
Mean Score 1
Time point:
24/48/72 h
Score:
0
Irritation parameter:
erythema score
Basis:
animal #2
Remarks:
Mean Score 2
Time point:
24/48/72 h
Score:
0
Irritation parameter:
erythema score
Basis:
animal #3
Remarks:
Mean Score 3
Time point:
24/48/72 h
Score:
0
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
animal #1
Remarks:
Mean Score 1
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
animal #2
Remarks:
Mean Score 2
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
animal #3
Remarks:
Mean Score 3
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Irritant / corrosive response data:
None reported
Other effects:
None
Interpretation of results:
GHS criteria not met
Conclusions:
Mean and maximum edema and erythema scores were 0 for all animals at all timepoints assessed. Therefore, the test item is not classified for skin irritation.
Executive summary:

The skin irritancy potential of the test item was assessed in a study conducted according to EU Annex V guidelines. 500 mg of test item was applied to 3 New Zealand White rabbits. The mean and maximum edema and erythema scores were 0 for all animals. No other effects were observed. Therefore, the test item is not classified for skin irritation.

The study is a GLP compliant, guideline study and is acceptable with restrictions for evaluation of this endpoint.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Type of coverage:
semiocclusive
Preparation of test site:
not specified
Vehicle:
unchanged (no vehicle)
Remarks:
Substance wetted with reverse osmosis purified water
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 500 mg
Duration of treatment / exposure:
4 hours
Observation period:
Not reported
Number of animals:
3
Details on study design:
Conducted according to guideline and GLP
Irritation parameter:
erythema score
Basis:
animal #1
Remarks:
Mean Score 1
Time point:
24/48/72 h
Score:
0
Irritation parameter:
erythema score
Basis:
animal #2
Remarks:
Mean Score 2
Time point:
24/48/72 h
Score:
0
Irritation parameter:
erythema score
Basis:
animal #3
Remarks:
Mean Score 3
Time point:
24/48/72 h
Score:
0
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: Not reported
Score:
0
Irritation parameter:
edema score
Basis:
animal #1
Remarks:
Mean Score 1
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
animal #2
Remarks:
Mean Score 2
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
animal #3
Remarks:
Mean Score 3
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
mean
Time point:
other: Not reported
Score:
0
Irritant / corrosive response data:
None reported
Other effects:
None
Interpretation of results:
GHS criteria not met
Conclusions:
Mean and maximum edema and erythema scores were 0 for all animals at all timepoints assessed. Therefore, the test item is not classified for skin irritation.
Executive summary:

The skin irritancy potential of the test item was assessed with a 500 mg application to New Zealand White rabbits in a study conducted according to according to a OECD 404 and EU B4 guidelines. The mean and maximum edema and erythema scores were 0 for all animals. No other effects were observed. Therefore, the test item is not classified for skin irritation.

The study is a GLP compliant, guideline study and is acceptable with restrictions for evaluation of this endpoint.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 June 2021 - 18 June 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2020
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Adult human-derived
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPIDERM™ Reconstructed Human Epidermis Model
- Tissue batch number(s): 34165
- Date of initiation of testing: 15 June 2021
- Source: MatTek In Vitro Life Sciences Laboratories

REMOVAL OF TEST MATERIAL AND CONTROLS:
- Washing: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS without Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert approximately 15 times using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were then submerged 3 times in 150 ml of DPBS without Ca++ and Mg++ before being transferred to the upper wells of a new 6-well post exposure incubation plate pre-filled with 0.9 ml assay medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: No filter

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
25 mg of the test item was added to 1 mL of a 1.0 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 1 hour. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and non-viable, freeze killed, tissues for quantitative correction of the results. An assessment of colour interference of the test item was also made. 25 mg of test item was added to 300 µL of sterile water and 300 µL of isopropanol. After incubating at 37 °C, 5% CO2 in air for 1 hour a visual assessment of the color was made.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-irritating to skin if the viability after 60 minutes exposure is greater than or equal to 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
102.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
115.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
103.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
106.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Colour interference with MTT: The solutions containing the test item were colorless. It was therefore unnecessary to run color correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was ≥0.8 and ≤2.8 (observed: 2.076). The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was ≤20% relative to the negative control treated tissues (observed: 3.2%). The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for standard deviation: The standard deviation between the three tissue replicates of each treatment group did not exceed 18% (observed: 7.1 %). The acceptance criterion was therefore satisfied.

Table: Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item  OD570of tissues  Mean OD570of triplicate tissues  ± SD of OD570  Relative individual tissue viability (%)  Relative mean viability (%)  ± SD of Relative mean viability (%)
Negative Control Item  

 1.979

 2.076

 0.085

 95.3

 100*      

 4.1

 2.114

 101.8

 2.136

 102.9

Positive Control Item

 0.059

 0.066

 0.008

2.8

 3.2

 0.4

 0.064

3.1

 0.075

3.6

 Test item

 2.122

 2.219

 0.148

 102.2

 106.9

 7.1

 2.389

 115.1

 2.146

 103.4

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

     

Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro skin irritation potential of A003 was assessed in this study. Under the experimental conditions reported, the test item was classified as a non-irritant.
Executive summary:

The in vitro skin irritation potential of A003 was tested in the reconstructed human epidermis test model (OECD 439). The mean viability of the test item was 106.9 % relative to the negative control and therefore under the experimental conditions reported, the test item was classified as a non-irritant.

The study is a GLP compliant guideline experiment study acceptable without restrictions for assessment of this endpoint.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 October 2021 - 15 October 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2021
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Adult human-derived
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPIDERM™ Reconstructed Human Epidermis Model
- Tissue batch number(s): 34198
- Delivery date: 12 October 2021
- Date of initiation of testing: 12 October 2021
- Source: MatTek In Vitro Life Sciences Laboratories

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS without Ca(2+) and Mg(2+). Rinsing was achieved by filling and emptying each tissue insert approx. 15 times using a constant soft stream of DPBS. The rinsed tissues were submerged 3 times in 150 mL of DPBS without Ca(2+) and Mg(2+) before a final rinse inside and outside of the tissue insert and drying with a cotton swab. The tissues were then transferred to the upper wells of a new 6-well post exposure incubation plate pre-filled with 0.9 mL assay medium.
- Observable damage in the tissue due to washing: None reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: no filter

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
25 mg of the test item was added to 1 mL of a 1.0 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C, 5% CO2 in air for 1 hour. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and non-viable, freeze-killed, tissues for quantitative correction of the results. An assessment of colour interference of the test item was also made. 25 mg of test item was added to 300 μL of sterile water and 300 μL of isopropanol. After incubating at 37 °C, 5% CO2 in air for 1 hour a visual assessment of the colour was made.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-irritating to skin if the viability after 60 minutes exposure is greater than 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
97
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
95.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
97.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
97.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Colour interference with MTT: The solutions containing the test item were colourless, therefore it was not necessary to run colour correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was ≥0.8 and ≤2.8 (observed: 2.077). The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was ≤20% relative to the negative control treated tissues (observed: 3.3%). The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation between the three tissue replicates of each treatment group did not exceed 18% (observed standard deviation: 0.9 %). The acceptance criterion was therefore satisfied.

Table 1: Mean OD570 Values and Viabilities for the Negative control item, Positive control item, and Test item

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control is set at 100%

Item

OD570 of tissues Mean OD570 of triplicate tissues

± SD of OD570

Relative individual tissues viability (%) Relative mean viability (%) ± SD of Relative mean viability (%)
Negative control item 2.312 2.077 0.260 111.3 100* 12.5
2.120 102.1
1.798 86.6
Positive control item 0.078 0.067 0.009 3.8 3.3 0.5
0.064 3.1
0.060 2.9
Test item 2.030 2.014 0.020 97.7 97.0 0.9
2.021 97.3
1.992 95.9
Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro skin irritation potential of PU05 was assessed in this study. Under the experimental conditions reported, the test item was classified as a non-irritant.
Executive summary:

The in vitro skin irritation potential of the test item was tested in the reconstructed human epidermis test model (OECD 439 and EU Method B.46). The mean viability of the test item was 97.0 % relative to the negative control and therefore, under the experimental conditions reported, the test item was classified as a non-irritant.

The study is a GLP compliant guideline experiment study acceptable without restrictions for assessment of this endpoint.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 November 2021 to 06 November 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2019, corrected 2020
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Adult human-derived
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPIDERM™ Reconstructed Human Epidermis Model
- Tissue batch number(s): 36102
- Delivery date: 03 November 2021
- Date of initiation of testing: 03 November 2021
- Source: MatTek In Vitro Life Sciences Laboratories

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS without Ca(2+) and Mg(2+). Rinsing was achieved by filling and emptying each tissue insert approx. 15 times using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were then submerged 3 times in 150 ml of DPBS without Ca(2+) and Mg(2+) before a final rinse inside and outside of the tissue insert and drying with a cotton swab. After the rinsing process, the tissues were transferred to the upper wells of a new 6-well post exposure incubation plate pre-filled with 0.9 ml assay medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: no filter

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
25 mg of the test item was added to 1 mL of a 1.0 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C, 5% CO2 in air for 1 hour. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and non-viable, freeze-killed, tissues for quantitative correction of the results. A colour check was also performed. 25 mg of test item was added to 300 μL of sterile water and 300 μL of isopropanol. After incubating at 37°C, 5% CO2 in air for 1 hour a visual assessment of the colour was made.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-irritating to skin if the viability after 60 minutes exposure is greater than 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
98.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
94.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
98.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
102
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Colour interference with MTT: The solutions containing the test item were colourless, therefore it was not necessary to run colour correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was ≥0.8 and ≤2.8 (observed: 2.010). The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was ≤20% relative to the negative control treated tissues (observed: 4.3 %). The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation between the three tissue replicates of each treatment group did not exceed 18% (observed: 3.7 %). The acceptance criterion was therefore satisfied.

Table 1: Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item, and Test Item

OD = Optical Density

SD = Standard Deviation

* = The mean viability of the negative control tissues is set at 100%

Item OD570 of tissues Mean OD570 of triplicate tissues

± SD of OD570

Relative individual tissue viability (%) Relative mean viability (%) ± SD of Relative mean viability (%)
Negative control item 2.100 2.010 0.085 104.5 100* 4.3
1.930 96.0
2.000 99.5
Positive control item 0.087 0.086 0.001 4.3 4.3 0.1
0.085 4.2
0.086 4.3
Test item 2.051 1.979 0.074 102.0 98.5 3.7
1.983 98.7
1.904 94.7
Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro skin irritation potential of the test item was assessed in this study. Under the experimental conditions reported, the test item was classified as a non-irritant.
Executive summary:

The in vitro skin irritation potential of the test item was tested in the reconstructed human epidermis test model (OECD 439 and EU Method B.46). The mean viability of the test item was 98.5 % relative to the negative control and therefore, under the experimental conditions reported, the test item was classified as a non-irritant.

 

The study is a GLP compliant guideline experiment study acceptable without restrictions for assessment of this endpoint.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 June 2021 - 18 June 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2020
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Adult human-derived
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPIDERM™ Reconstructed Human Epidermis Model
- Tissue batch number(s): 34165
- Date of initiation of testing: 15 June 2021
- Source: MatTek In Vitro Life Sciences Laboratories

REMOVAL OF TEST MATERIAL AND CONTROLS:
- Washing: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS without Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert approximately 15 times using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were then submerged 3 times in 150 ml of DPBS without Ca++ and Mg++ before being transferred to the upper wells of a new 6-well post exposure incubation plate pre-filled with 0.9 ml assay medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: No filter

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
25 mg of the test item was added to 1 mL of a 1.0 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37° C, 5% CO2 in air for 1 hour. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and non-viable, freeze killed, tissues for quantitative correction of the results. An assessment of colour interference of the test item was also made. 25 mg of test item was added to 300 µL of sterile water and 300 µL of isopropanol. After incubating at 37 °C, 5% CO2 in air for 1 hour a visual assessment of the color was made.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-irritating to skin if the viability after 60 minutes exposure is greater than or equal to 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
105.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
98.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
107.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
104
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Colour interference with MTT: The solutions containing the test item were colorless. It was therefore unnecessary to run color correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was ≥0.8 and ≤2.8 (observed: 2.076). The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was ≤20% relative to the negative control treated tissues (observed: 3.2%). The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for standard deviation: The standard deviation between the three tissue replicates of each treatment group did not exceed 18% (observed: 4.8 %). The acceptance criterion was therefore satisfied.

Table: Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item  OD570of tissues  Mean OD570of triplicate tissues  ± SD of OD570  Relative individual tissue viability (%)  Relative mean viability (%)  ± SD of Relative mean viability (%)
Negative Control Item  

 1.979

 2.076

 0.085

 95.3

 100*      

 4.1

 2.114

 101.8

 2.136

 102.9

Positive Control Item

 0.059

 0.066

 0.008

2.8

 3.2

 0.4

 0.064

3.1

 0.075

3.6

 Test item

 2.194

 2.159

 0.100

 105.7

 104.0

 4.8

 2.047

 98.6

 2.237

 107.8

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro skin irritation potential of the test item was assessed. Under the experimental conditions reported, the test item was classified as a non-irritant.
Executive summary:

The in vitro skin irritation potential of the test item was tested in the reconstructed human epidermis test model (OECD 439 and EU Method B.46). The mean viability of the test item was 104.0 % relative to the negative control and therefore, under the experimental conditions reported, the test item was classified as a non-irritant.

 

The study is a GLP compliant guideline experiment study acceptable without restrictions for assessment of this endpoint.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 October 2021 to 15 October 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2021
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Adult human-derived
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPIDERM™ Reconstructed Human Epidermis Model
- Tissue batch number(s): 34198
- Delivery date: 12 October 2021
- Date of initiation of testing: 12 October 2021
- Source: MatTek In Vitro Life Sciences Laboratories

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS without Ca(2+) and Mg(2+). Rinsing was achieved by filling and emptying each tissue insert approximately 15 times using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were then submerged 3 three times in 150 ml of DPBS without Ca(2+) and Mg(2+) before a final rinse inside and outside of the tissue insert and drying with a cotton swab. After the rinsing process, the tissues were transferred to the upper wells of a new 6-well post exposure incubation plate pre-filled with 0.9 ml assay medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: no filter

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
25 mg of the test item was added to 1 mL of a 1.0 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C, 5% CO2 in air for 1 hour. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and non-viable, freeze-killed, tissues for quantitative correction of the results. An assessment of colour interference was also performed. 25 mg of test item was added to 300 μL of sterile water and 300 μL of isopropanol. After incubating at 37°C, 5% CO2 in air for 1 hour a visual assessment of the colour was made.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-irritating to skin if the viability after 60 minutes exposure is greater than 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
98.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
102.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
93.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
98.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Colour interference with MTT: The solutions containing the test item were colourless, therefore it was not necessary to run colour correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was ≥0.8 and ≤2.8 (observed: 2.077). The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was ≤20% relative to the negative control treated tissues (observed: 3.3 %). The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation between the three tissue replicates of each treatment group did not exceed 18% (observed: 4.5 %). The acceptance criterion was therefore satisfied.

Table 1: Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item, and Test Item

OD = Optical Density

SD = Standard Deviation

* = The mean viability of the negative control tissues is set at 100%

Item OD570 of tissues Mean OD570 of triplicate tissues

± SD of OD570

Relative individual tissue viability (%) Relative mean viability (%) ± SD of Relative mean viability (%)
Negative Control Item 2.312 2.077 0.260 111.3 100* 12.5
2.120 102.1
1.798 86.6
Positive Control Item 0.078 0.067 0.009 3.8 3.3 0.5
0.064 3.1
0.060 2.9
Test Item 2.050 2.040 0.094 98.7 98.2 4.5
1.941 93.5
2.128 102.5
Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro skin irritation potential of the test item was assessed in this study. Under the experimental conditions reported, the test item was classified as a non-irritant.
Executive summary:

Thein vitroskin irritation potential of the test item was tested in the reconstructed human epidermis test model (OECD 439 and EU Method B.46). The mean viability of the test item was 98.2 % relative to the negative control and therefore, under the experimental conditions reported, the test item was classified as a non-irritant.

 

The study is a GLP compliant guideline experiment study acceptable without restrictions for assessment of this endpoint.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 June 2021 - 18 June 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2020
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Adult human-derived
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPIDERM™ Reconstructed Human Epidermis Model
- Tissue batch number(s): 34165
- Date of initiation of testing: 15 June 2021
- Source: MatTek In Vitro Life Sciences Laboratories

REMOVAL OF TEST MATERIAL AND CONTROLS:
- Washing: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS without Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert approximately 15 times using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were then submerged 3 times in 150 ml of DPBS without Ca++ and Mg++ before being transferred to the upper wells of a new 6-well post exposure incubation plate pre-filled with 0.9 ml assay medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: No filter

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
25 mg of the test item was added to 1 mL of a 1.0 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37° C, 5% CO2 in air for 1 hour. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and non-viable, freeze killed, tissues for quantitative correction of the results. An assessment of colour interference of the test item was also made. 25 mg of test item was added to 300 µL of sterile water and 300 µL of isopropanol. After incubating at 37 °C, 5% CO2 in air for 1 hour a visual assessment of the color was made.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-irritating to skin if the viability after 60 minutes exposure is greater than or equal to 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
105.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
97.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
103.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
102.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Colour interference with MTT: The solutions containing the test item were colorless. It was therefore unnecessary to run color correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was ≥0.8 and ≤2.8 (observed: 2.076). The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was ≤20% relative to the negative control treated tissues (observed: 3.2 %). The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for standard deviation: The standard deviation between the three tissue replicates of each treatment group did not exceed 18% (observed: 4.2 %). The acceptance criterion was therefore satisfied.

Table: Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item  OD570of tissues  Mean OD570of triplicate tissues  ± SD of OD570  Relative individual tissue viability (%)  Relative mean viability (%)  ± SD of Relative mean viability (%)
Negative Control Item  

 1.979

 2.076

 0.085

 95.3

 100*      

 4.1

 2.114

 101.8

 2.136

 102.9

Positive Control Item

 0.059

 0.066

 0.008

2.8

 3.2

 0.4

 0.064

3.1

 0.075

3.6

 Test item

 2.191

 2.119

 0.087

 105.5

 102.1

 4.2

 2.022

 97.4

 2.143

 103.2

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro skin irritation potential of the test item was assessed in this study. Under the experimental conditions reported, the test item was classified as a non-irritant.
Executive summary:

The in vitro skin irritation potential of the test item was tested in the reconstructed human epidermis test model (OECD 439 and EU Method B.46). The mean viability of the test item was 102.1 % relative to the negative control and therefore, under the experimental conditions reported, the test item was classified as a non-irritant.

 

The study is a GLP compliant guideline experiment study acceptable without restrictions for assessment of this endpoint.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 October 2021 to 15 October 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2021
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Adult human-derived
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPIDERM™ Reconstructed Human Epidermis Model
- Tissue batch number(s): 34198
- Delivery date: 12 October 2021
- Date of initiation of testing: 12 October 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS without Ca(2+) and Mg(2+). Rinsing was achieved by filling and emptying each tissue insert approximately 15 times using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were then submerged 3 times in 150 ml of DPBS without Ca(2+) and Mg(2+) before being transferred to the upper wells of a new 6-well post exposure incubation plate pre-filled with 0.9 ml assay medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: no filter

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
25 mg of the test item was added to 1 mL of a 1.0 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C, 5% CO2 in air for 1 hour. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and non-viable, freeze killed, tissues for quantitative correction of the results. An assessment of colour interference was also performed. 25 mg of test item was added to 300 µL of sterile water and 300 µL of isopropanol. After incubating at 37°C, 5% CO2 in air for 1 hour a visual assessment of the colour was made.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-irritating to skin if the viability after 60 minutes exposure is greater than 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
103.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
109.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
102.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
97.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Colour interference with MTT: The solutions containing the test item were colourless, therefore it was not necessary to run colour correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was ≥0.8 and ≤2.8 (observed: 2.077). The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was ≤20% relative to the negative control treated tissues (observed: 3.3 %). The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for standard deviation: The standard deviation between the three tissue replicates of each treatment group did not exceed 18% (observed: 5.7 %). The acceptance criterion was therefore satisfied.

Table 1: Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item, and Test Item

Item OD570 of tissues Mean OD570 of triplicate tissues

± SD of OD570

Relative individual tissue viability (%) Relative mean viability (%) ± SD of Relative mean viability (%)
Negative Control Item 2.312 2.077 0.260 111.3 100* 12.5
2.120 102.1
1.798 86.6
Positive Control Item 0.078 0.067 0.009 3.8 3.3 0.5
0.064 3.1
0.060 2.9
Test Item 2.033 2.141 0.119 97.9 103.1 5.7
2.121 102.1
2.269 109.2

OD = Optical Density

SD = Standard Deviation

* = The mean viability of the negative control tissues is set at 100%

Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro skin irritation potential of the test item was assessed in this study. Under the experimental conditions reported, the test item was classified as a non-irritant.
Executive summary:

The in vitro skin irritation potential of the test item was tested in the reconstructed human epidermis test model (OECD 439 and EU Method B.46). The mean viability of the test item was 103.1 % relative to the negative control and therefore, under the experimental conditions reported, the test item was classified as a non-irritant.

 

The study is a GLP compliant guideline experiment study acceptable without restrictions for assessment of this endpoint.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 October 2021 to 24 October 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2021
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Adult human-derived
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPIDERM™ Reconstructed Human Epidermis Model
- Tissue batch number(s): 34194
- Delivery date: 21 October 2021
- Date of initiation of testing: 21 October 2021
- Source: MatTek In Vitro Life Sciences Laboratories

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS without Ca(2+) and Mg(2+). Rinsing was achieved by filling and emptying each tissue insert approximately 15 times using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were then submerged 3 times in 150 ml of DPBS without Ca(2+) and Mg(2+) before a final rinse inside and outside of the tissue insert and drying with a cotton swab. After the rinsing process, the tissues were transferred to the upper wells of a new 6-well post exposure incubation plate pre-filled with 0.9 ml assay medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: no filter

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
25 mg of the test item was added to 1 mL of a 1.0 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C, 5% CO2 in air for 1 hour. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and non-viable, freeze killed, tissues for quantitative correction of the results. An assessment of colour interference was also performed. 25 mg of test item was added to 300 µL of sterile water and 300 µL of isopropanol. After incubating at 37°C, 5% CO2 in air for 1 hour a visual assessment of the color was made.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-irritating to skin if viability after 60 minutes exposure is greater than 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
84.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
86.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
83
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
82.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Colour interference with MTT: The solutions containing the test item were colourless, therefore it was not necessary to run colour correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was ≥0.8 and ≤2.8 (observed: 2.047). The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was ≤20% relative to the negative control treated tissues (observed: 3.3 %). The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for standard deviation: The standard deviation between the three tissue replicates of each treatment group did not exceed 18% (observed: 2.2 %). The acceptance criterion was therefore satisfied.

Table 1: Mean OD570 Values and Viabilites for the Negative Control Item, Positive Control Item, and Test Item

Item

OD570 of tissues

Mean OD570 of triplicate tissues

± SDof OD570

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

1.956

2.047

0.255

95.6

100*

12.5

1.850

90.4

2.335

114.1

Positive Control Item

0.088

0.069

0.017

4.3

3.3

0.8

0.058

2.8

0.060

2.9

Test Item

1.697

1.724

0.044

82.9

84.2

2.2

1.700

83.0

1.774

86.7

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro skin irritation potential of the test item was assessed in this study. Under the experimental conditions reported, the test item was classified as a non-irritant.
Executive summary:

The in vitro skin irritation potential of the test item was tested in the reconstructed human epidermis test model (OECD 439 and EU Method B.46). The mean viability of the test item was 84.2 % relative to the negative control and therefore, under the experimental conditions reported, the test item was classified as a non-irritant.

 

The study is a GLP compliant guideline experiment study acceptable without restrictions for assessment of this endpoint.

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.
Qualifier:
according to guideline
Guideline:
other: Annex V
GLP compliance:
yes
Specific details on test material used for the study:
Nature of substance: Powder
Species:
rabbit
Strain:
New Zealand White
Vehicle:
not specified
Controls:
not specified
Amount / concentration applied:
46 mg
Duration of treatment / exposure:
Not reported
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
3
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.7
Remarks on result:
other: mean
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.3
Remarks on result:
other: Mean
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.7
Remarks on result:
other: Mean
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
mean
Max. score:
2
Remarks on result:
other: Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Remarks on result:
other: Mean
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.7
Remarks on result:
other: Mean
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.3
Remarks on result:
other: Mean
Irritation parameter:
chemosis score
Basis:
mean
Max. score:
2
Remarks on result:
other: Max. duration: 48 h; Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: Mean
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: Mean
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: Mean
Irritation parameter:
cornea opacity score
Basis:
mean
Max. score:
0
Remarks on result:
other: Max. duration: h; Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: Mean
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: Mean
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: Mean
Irritation parameter:
iris score
Basis:
mean
Max. score:
0
Remarks on result:
other: Max. duration: h; Max. value at end of observation period: 0 (related to all animals)
Irritant / corrosive response data:
All response observed were fully reversible within 7 days. Maximun duration of conjunctival redness was 7 days.
Other effects:
No signs of systemic toxicity were observed.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item was observed to have conjunctivae redness scores of 0.7, 1.3 and 1.7, and chemosis scores of 0.3, 0.7 and 1 1 for the 3 test animals at 24, 48 and 72 hours. A maximum score of 2 was observed for both conjunctivae and chemosis. The cornea and iris scores for all animals were 0. Any changes observed were fully reversible within 7 days and no signs of clinical toxicity were observed. Therefore, the test item is not classified for eye irritation
Executive summary:

The eye irritancy potential of the test item was investigated at a concentration of 46 mg with New Zealand White rabbits according to a standard guideline. The test item was observed to have conjunctivae redness scores of 0.7, 1.3 and 1.7, and chemosis scores of 0.3, 0.7 and 1 1 for the 3 test animals at 24, 48 and 72 hours. A maximum score of 2 was observed for both conjunctivae and chemosis. The cornea and iris scores for all animals were 0. Any changes observed were fully reversible within 7 days and no signs of clinical toxicity were observed. Therefore, the test item is not classified for eye irritation

The study is a GLP compliant, guideline study and is acceptable with restrictions for evaluation of this endpoint.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.
Qualifier:
according to guideline
Guideline:
other: Annex V
GLP compliance:
yes
Specific details on test material used for the study:
Powder
Species:
rabbit
Strain:
New Zealand White
Vehicle:
not specified
Controls:
not specified
Amount / concentration applied:
63 mg
Duration of treatment / exposure:
Not reported
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
3
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: Mean
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.7
Remarks on result:
other: Mean
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.7
Remarks on result:
other: Mean
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
mean
Remarks:
Max. Score
Time point:
other: See remarks
Score:
1
Remarks on result:
other: Max. duration: 48 h; Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: Mean
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: Mean
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: Mean
Irritation parameter:
chemosis score
Basis:
mean
Time point:
other: Not reported
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: Mean
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: Mean
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: Mean
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: Not reported
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: Mean
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: Mean
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: Mean
Irritation parameter:
iris score
Basis:
mean
Time point:
other: Not reported
Score:
0
Max. score:
0
Irritant / corrosive response data:
All response observed were fully reversible within 3 days
Other effects:
No signs of systemic toxicity were observed.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item was observed to have conjuctivae redness scores of 0 to 0.7 for the 3 animals tested at 24, 48 and 72 hours. A maximum score of 2 was was observed at 48 hours. The chemosis, cornea and iris scores for all animals were 0. Any changes observed were fully reversible within 3 days and no signs of clinical toxicity were observed. Therefore, the test item is not classified for eye irritation
Executive summary:

The eye irritancy potential of the test item was investigated at a concentration of 63 mg with New Zealand White rabbits according to a standard guideline over 72 hours. The test item was observed to have conjuctivae redness scores of 0 to 0.7 for the 3 animals tested at 24, 48 and 72 hours. A maximum score of 2 was was observed at 48 hours. The chemosis, cornea and iris scores for all animals were 0. Any changes observed were fully reversible within 3 days and no signs of clinical toxicity were observed. Therefore, the test item is not classified for eye irritation

The study is a GLP compliant, guideline study and is acceptable with restrictions for evaluation of this endpoint.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.
Qualifier:
according to guideline
Guideline:
other: Annex V
GLP compliance:
yes
Specific details on test material used for the study:
Nature of substance: Powder
Species:
rabbit
Strain:
New Zealand White
Vehicle:
not specified
Controls:
not specified
Amount / concentration applied:
66 mg (equivalent to 0.1 ml of bulk substance)
Duration of treatment / exposure:
72 hours
Observation period (in vivo):
7 days
Number of animals or in vitro replicates:
3
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #1
Remarks:
Mean score 1
Time point:
24/48/72 h
Score:
0
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #2
Remarks:
Mean score 1
Time point:
24/48/72 h
Score:
1.7
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #3
Remarks:
Mean score 3
Time point:
24/48/72 h
Score:
0.3
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
mean
Time point:
72 h
Max. score:
2
Remarks on result:
other: Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
Mean score 1
Time point:
24/48/72 h
Score:
0
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
Mean score 2
Time point:
24/48/72 h
Score:
0.3
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
Mean score 3
Time point:
24/48/72 h
Score:
0
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24 h
Max. score:
1
Remarks on result:
other: Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
Mean score 1
Time point:
24/48/72 h
Score:
0
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
Mean score 2
Time point:
24/48/72 h
Score:
0
Irritation parameter:
cornea opacity score
Basis:
animal #3
Remarks:
Mean score 3
Time point:
24/48/72 h
Score:
0
Irritation parameter:
cornea opacity score
Basis:
mean
Max. score:
0
Remarks on result:
other: Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
mean score 1
Time point:
24/48/72 h
Score:
0
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
mean score 2
Time point:
24/48/72 h
Score:
0.3
Irritation parameter:
iris score
Basis:
animal #3
Remarks:
mean score 3
Time point:
24/48/72 h
Score:
0
Irritation parameter:
iris score
Basis:
mean
Time point:
24 h
Max. score:
1
Remarks on result:
other: Max. value at end of observation period: 0 (related to all animals)
Irritant / corrosive response data:
Reversibility of any observed effect: Changes fully reversible within 7 days
Other effects:
Following fluorescein application to the eyes of all three animals at 24 hours, 30% of the total cornea area in animal number 2 was stained indicating epithelial damage. A repeat of fluorescein treatment at 72 hours yielded negative findings. Immediately after fluorescein examination at 24 hours, both eyes of all three animals were rinsed with water. Residual test substance remained in the eye of animal no. 3.
Interpretation of results:
GHS criteria not met
Conclusions:
The overall scores at 24, 48 and 72 hours for the three animals were 0, 1.7 and 0.3 for conjunctivae (redness), 0, 0.3 and 0 for chemosis, 0 for all animals for cornea and 0, 0.3 and 0 for iris. A maximum score of 2 was observed at 72 hours for conjunctivae (redness) and a maximum score of 1 was observed at 24 hours for chemosis and iris.
Executive summary:

The test item was analysed for eye irritation effects on New Zealand White rabbits according to EU Annex V guidelines. Three rabbits were exposed to 66 mg of the test item in powder form (corresponding to 0.1 mL of bulk substance). The overall scores at 24, 48 and 72 hours for the three animals were 0, 1.7 and 0.3 for conjunctivae (redness), 0, 0.3 and 0 for chemosis, 0 for all animals for cornea and 0, 0.3 and 0 for iris. A maximum score of 2 was observed at 72 hours for conjunctivae (redness) and a maximum score of 1 was observed at 24 hours for chemosis and iris. Following fluorescein application to the eyes of all three animals at 24 hours, 30% of the total cornea area in animal number 2 was stained indicating epithelial damage. A repeat of fluorescein treatment at 72 hours yielded negative findings. Changes were fully reversible within 7 days. The test item is not classified for eye irritation.

The study is a GLP compliant, guideline experimental study and is acceptable for assessment with restrictions.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
The substance (a white powder with a very poor water solubility of 4.38 µg/L) was put directly onto the eye (without vehicle) and no washing was applied. It was recorded that the material was still present in the eyes >24 h after application. It is therefore most likely that the observed ocular effects have been caused by mechanical irritation rather than inherent irritative properties of the material and the study is not considered to be reliable
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
GLP compliance:
yes
Specific details on test material used for the study:
Nature of substance: Powder
Species:
rabbit
Strain:
New Zealand White
Vehicle:
not specified
Controls:
not specified
Amount / concentration applied:
100 mg
Duration of treatment / exposure:
72 hours
Observation period (in vivo):
22 days
Number of animals or in vitro replicates:
3
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #1
Remarks:
Mean score 1
Time point:
24/48/72 h
Score:
2.3
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #2
Remarks:
Mean score 1
Time point:
24/48/72 h
Score:
2.3
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #3
Remarks:
Mean score 3
Time point:
24/48/72 h
Score:
1.3
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
mean
Remarks:
Max score
Time point:
other: Not reported
Score:
3
Reversibility:
other:
Remarks:
Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
Mean score 1
Time point:
24/48/72 h
Score:
2.7
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
Mean score 2
Time point:
24/48/72 h
Score:
2.3
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
Mean score 3
Time point:
24/48/72 h
Score:
0.7
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
Max score
Time point:
other: Not reported
Score:
4
Remarks on result:
other: Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
Mean score 1
Time point:
24/48/72 h
Score:
1.3
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
Mean score 2
Time point:
24/48/72 h
Score:
1
Irritation parameter:
cornea opacity score
Basis:
animal #3
Remarks:
Mean score 3
Time point:
24/48/72 h
Score:
0.7
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
Max score
Time point:
other: Not reported
Score:
2
Remarks on result:
other: Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
mean score 1
Time point:
24/48/72 h
Score:
0.7
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
mean score 2
Time point:
24/48/72 h
Score:
0
Irritation parameter:
iris score
Basis:
animal #3
Remarks:
mean score 3
Time point:
24/48/72 h
Score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
Max score
Time point:
other: Not reported
Score:
1
Remarks on result:
other: Max. value at end of observation period: 0 (related to all animals)
Irritant / corrosive response data:
Reversibility of any observed effect: Changes not fully reversible within 22 days
Other effects:
Persistent tearing at the end of the observation period

At day 21 there is a degree of conjunctival irritation in an animal; this is not observed any more at the 22nd day but it remains tearing. These persistent effects justify the classification of the substance as "severely irritating to the eyes" (Xi R41).

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The conjunctivae redness scores for the test item were 1.3, 2.3 and 2.3 for the 3 animals and the maximum score was 3 at 24, 48 and 72 hours. The chemosis scores were 0.7, 2.3 and 2.7 for the 3 animals and the maximum score was 4. The cornea and iris scores were 0.7, 1 and 1.3 and 0, 0 and 0.7, respectively with a maximum cornea score of 2 and a maximum iris score of 1. At day 21 there was a degree of conjunctival irritation in an animal; that is not observed at day 22 but it remained tearing. These persistent effects justify the classification of the substance.
Executive summary:

The eye irritancy potential of the test item was investigated at a concentration of 100 mg with New Zealand White rabbits according to EU method C5 and OECD 405 guidelines.

The conjunctivae redness scores for the test item were 1.3, 2.3 and 2.3 for the 3 animals and the maximum score was 3 at 24, 48 and 72 hours. The chemosis scores were 0.7, 2.3 and 2.7 for the 3 animals and the maximum score was 4. The cornea and iris scores were 0.7, 1 and 1.3 and 0, 0 and 0.7, respectively with a maximum cornea score of 2 and a maximum iris score of 1. At day 21 there was a degree of conjunctival irritation in an animal; that is not observed at day 22 but it remained tearing. These persistent effects justify the classification of the substance.

The substance (a white powder with a very poor water solubility of 4.38 µg/L) was put directly onto the eye (without vehicle) and no washing was applied. It was recorded that the material was still present in the eyes >24 h after application. It is therefore most likely that the observed ocular effects have been caused by mechanical irritation rather than inherent irritative properties of the material and the study is not considered to be reliable.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03/08/2021 - 03/08/2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Local abattoir
- Characteristics of donor animals: Adult cattle (typically 12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue: Eyes were transported to the test facility over ice packs on the same day of slaughter and prepared immediately on arrival at the test facility.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- Indication of any antibiotics used: Eyes were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
- Selection and preparation of corneas: The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
- Quality check of the isolated corneas: The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 70 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.5276 grams

CONTROLS
- Amount applied: 0.75 mL
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
Three
Details on study design:
NUMBER OF REPLICATES : Three

NEGATIVE CONTROL USED : Sodium chloride 0.9% w/v (Baxter Healthcare SA)

POSITIVE CONTROL USED : Imidazole (Fisher Bioreagents)

APPLICATION DOSE AND EXPOSURE TIME : Approximately 0.5276 g of the solid test item was found to adequately cover the corneal surface. The BCOP holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity readings were taken using a calibrated opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a Labtech LT-4500 microplate reader (OD492)
- Others: Histopathology and visual observation post treatment.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DATA INTERPRETATION: The decision criteria using the following prediction model:
- IVIS = ≤ 3 No category for UN GHS criteria
- IVIS = > 3 and ≤ 55 No stand-alone prediction can be made for UN GHS
- IVIS = > 55 Category 1 UN GHS

CRITERIA FOR ACCEPTANCE:
- Positive control: 20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during the previous 12 months for this testing facility.
- Negative control: Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values, or an overall IVIS value of < 3 calculated from the previous 12 months data for this testing facility.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 17
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 18
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 19
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were clear post treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control values were within accepted limits (IVIS = 2.4). The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the acceptance range (IVIS = 82.0). The positive control acceptance criterion was therefore satisfied.

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

 Treatment  Cornea Number  Opacity  Permeability (OD492)  In vitro Irritancy Score
 Pre-Treatment  Post-Treatment  Post-Treatment - Pre-Treatment  Corrected Value    Corrected Value
 Negative Control  2  3  4  1    0.002    
 3  3  5  2    0.003    
 6  3  7  4    0.010    
       2.3*    0.005**    2.4
 Positive control  9  4  62  58  55.7  1.138  1.133  
 11  2  67  65  62.7  1.840  1.835  
 12  3  63  60  57.7  1.710  1.705  
         58.7***    1.558***  82.0
 Test Item  17  3  5  2  0.0  0.003  0.000  
 18  3  3  0  0.0  0.006  0.001  
 19  3  5  2  0.0  0.004  0.000  
         0.0***    0.000***  0.0

OD = Optical density

* = Mean of the post-treatment minus pre-treatment values

** = Mean permeability

*** = Mean corrected value

Table 2: Corneal Epithelium Condition Post Treatment

 Treatment  Cornea Number  Observation Post Treatment
 Negative Control  2  Clear
 3  Clear
 6  Clear
 Positive Control  9  Cloudy
 11  Cloudy
 12  Cloudy
 Test Item  17  Clear
 18  Clear
 19  Clear
Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro eye irritation potential of the test item was assessed in this study. Under the experimental conditions reported, the test item was classified as a non-irritant.
Executive summary:

The eye irritation potential of the test item was assessed in a OECD 437 and EU B.47 method guideline study. Approximately 0.5276 g of the test item was applied to the corneal surface of prepared cattle cornea, obtained on the day of extraction, and incubated for 240 minutes followed by rinsing to remove the test item. Additionally, sodium chloride (0.9 % w/v) and Imidazole (20 % w/v solution in 0.9 % w/v sodium chloride) were tested as negative and positive controls alongside the test item analysis. The opacity of treated corneas was assessed using a opacitometer and the permeability was determined at OD492 by microplate reader. The In vitro irritancy score (IVIS), based on the mean opacity and permeability measurement, was calculated for the test item, positive and negative controls; and the classification requirement was assessed based on the IVIS.

 

The mean IVIS for the item is 0.0, and based on the assessment criteria the test item is assigned no category under the UN GHS criteria; though it is noted that the test item could not be formulated to a concentration of 20% w/v solution in sodium chloride 0.9% w/v. This did not have an effect on the study results. The mean IVIS for the negative and positive controls were 2.4 and 82.0, respectively, and therefore the acceptability criteria of the study were met.

 

The study is a GLP compliant guideline experimental study, and is deemed acceptable without restrictions for assessment of this endpoint.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21/10/2021 - 21/10/2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Local abattoir
- Characteristics of donor animals: Adult cattle (typically 12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue: Eyes were transported to the test facility over ice packs on the same day of slaughter and prepared immediately on arrival at the test facility.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- Indication of any antibiotics used: Eyes were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
- Selection and preparation of corneas: The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
- Quality check of the isolated corneas: The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 70 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.5402 grams

CONTROLS
- Amount applied: 0.75 mL
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
Three
Details on study design:
NUMBER OF REPLICATES : Three

NEGATIVE CONTROL USED : Sodium chloride 0.9% w/v (Baxter Healthcare SA)

POSITIVE CONTROL USED : Imidazole (Acros Organics)

APPLICATION DOSE AND EXPOSURE TIME : Approximately 0.5402 g of the solid test item was found to adequately cover the corneal surface. The BCOP holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity readings were taken using a calibrated opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a Labtech LT-4500 microplate reader (OD492)
- Others: Histopathology and visual observation post treatment

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

CRITERIA FOR ACCEPTANCE:
- Positive control: 20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during the previous 12 months for this testing facility.
- Negative control: Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values, or an overall IVIS value of < 3 calculated from the previous 12 months data for this testing facility.

DATA INTERPRETATION: The decision criteria using the following prediction model:
- IVIS = ≤ 3 No category for UN GHS criteria
- IVIS = > 3 and ≤ 55 No stand-alone prediction can be made for UN GHS
- IVIS = > 55 Category 1 UN GHS
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
2.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 14
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 15
Value:
4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 16
Value:
5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were clear post treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control values were within accepted limits (IVIS = 1.7). The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the acceptance range (IVIS = 110.0). The positive control acceptance criterion was therefore satisfied.

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

 Treatment  Cornea Number  Opacity  Permeability (OD492)  In vitroIrritancy Score
 Pre-Treatment  Post-Treatment  Post-Treatment - Pre-Treatment  Corrected Value    Corrected Value
 Negative Control  2  3  4  1    0.005    
 3  3  5  2    0.004    
 6  3  5  2    0.003    
       1.7*    0.004**    1.7
 Positive control  9  2  75  73  71.3  1.790  1.786  
 11  4  101  97  95.3  1.825  1.821  
 12  3  91  88  86.3  1.535  1.531  
         84.3***    1.713***  110.0
 Test Item  14  3  5  2  0.3  0.011  0.007  
 15  3  7  4  2.3  0.050  0.046  
 16  2  7  5  3.3  0.017  0.013  
         2.0***    0.022***  2.3

OD = Optical density

* = Mean of the post-treatment minus pre-treatment values

** = Mean permeability

*** = Mean corrected value

Table 2: Corneal Epithelium Condition Post Treatment

 Treatment  Cornea Number  Observation Post Treatment
 Negative Control  2  Clear
 3  Clear
 6  Clear
 Positive Control  9  Cloudy
 11  Cloudy
 12  Cloudy
 Test Item  14  Clear
 15  Clear
 16  Clear
Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro eye irritation potential of the test item was assessed in this study. Under the experimental conditions reported, the test item was classified as a non-irritant.
Executive summary:

The eye irritation potential of the test item was assessed in an OECD 437 and EU B.47 method guideline study. Approximately 0.5402 g of the test item was applied to the corneal surface of prepared cattle cornea, obtained on the day of extraction, and incubated for 240 minutes followed by rinsing to remove the test item. Additionally, sodium chloride (0.9 % w/v) and Imidazole (20 % w/v solution in 0.9 % w/v sodium chloride) were tested as negative and positive controls alongside the test item analysis. The opacity of treated corneas was assessed using an opacitometer and the permeability was determined at OD492 by microplate reader. The In vitro irritancy score (IVIS), based on the mean opacity and permeability measurement, was calculated for the test item, positive and negative controls; and the classification requirement was assessed based on the IVIS.

 

The mean IVIS for the item is 2.3, and based on the assessment criteria the test item is assigned no category under the UN GHS criteria; though it is noted that the test item could not be formulated to a concentration of 20% w/v solution in sodium chloride 0.9% w/v. This did not have an effect on the study results. The mean IVIS for the negative and positive controls were 1.7 and 110.0, respectively, and therefore the acceptability criteria of the study were met.

The study is a GLP compliant guideline experimental study, and is deemed acceptable without restrictions for assessment of this endpoint.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03/08/2021 - 03/08/2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Local abattoir
- Characteristics of donor animals: Adult cattle (typically 12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue: Eyes were transported to the test facility over ice packs on the same day of slaughter and prepared immediately on arrival at the test facility.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- Indication of any antibiotics used: Eyes were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
- Selection and preparation of corneas: The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
- Quality check of the isolated corneas: The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 70 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.6142 grams

CONTROLS
- Amount applied: 0.75 mL
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
Three
Details on study design:
NUMBER OF REPLICATES : Three

NEGATIVE CONTROL USED : Sodium chloride 0.9% w/v (Baxter Healthcare SA)

POSITIVE CONTROL USED : Imidazole (Fisher Bioreagents)

APPLICATION DOSE AND EXPOSURE TIME : Approximately 0.6142 g of the solid test item was found to adequately cover the corneal surface. The BCOP holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity readings were taken using a calibrated opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a Labtech LT-4500 microplate reader (OD492)
- Others: Histopathology and visual observation post treatment

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DATA INTERPRETATION: The decision criteria using the following prediction model:
- IVIS = ≤ 3 No category for UN GHS criteria
- IVIS = > 3 and ≤ 55 No stand-alone prediction can be made for UN GHS
- IVIS = > 55 Category 1 UN GHS

CRITERIA FOR ACCEPTANCE:
- Positive control: 20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during the previous 12 months for this testing facility.
- Negative control: Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values, or an overall IVIS value of < 3 calculated from the previous 12 months data for this testing facility.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 14
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 15
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 16
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were clear post treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control values were within accepted limits (IVIS = 2.4). The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the acceptance range (IVIS = 82.0). The positive control acceptance criterion was therefore satisfied.

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

 Treatment  Cornea Number  Opacity  Permeability (OD492)  In vitro Irritancy Score
 Pre-Treatment  Post-Treatment  Post-Treatment - Pre-Treatment  Corrected Value    Corrected Value
 Negative Control  2  3  4  1    0.002    
 3  3  5  2    0.003    
 6  3  7  4    0.010    
       2.3*    0.005**    2.4
 Positive control  9  4  62  58  55.7  1.138  1.133  
 11  2  67  65  62.7  1.840  1.835  
 12  3  63  60  57.7  1.710  1.705  
         58.7***    1.558***  82.0
 Test Item  14  2  2  0  0.0

 0.003

 0.000

 

 15

 2

 3

 1

 0.0

 0.006

 0.001

 

 16

 2

 3

 1

 0.0

 0.004

 0.000

 

 

 

 

 

 0.0***

 

 0.000***

 0.0

OD = Optical density

* = Mean of the post-treatment minus pre-treatment values

** = Mean permeability

*** = Mean corrected value

Table 2: Corneal Epithelium Condition Post Treatment

 Treatment

 Cornea Number

 Observation Post Treatment

 Negative Control

 2

 Clear

 3

 Clear

 6

 Clear

 Positive Control

 9

 Cloudy

 11

 Cloudy

 12

 Cloudy

 Test Item

 14

 Clear

 15

 Clear

 16

 Clear

Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro eye irritation potential of the test item was assessed in this study. Under the experimental conditions reported, the test item was classified as a non-irritant.
Executive summary:

The eye irritation potential of the test item was assessed in an OECD 437 and EU B.47 method guideline study. Approximately 0.6142 g of the test item was applied to the corneal surface of prepared cattle cornea, obtained on the day of extraction, and incubated for 240 minutes followed by rinsing to remove the test item. Additionally, sodium chloride (0.9 % w/v) and Imidazole (20 % w/v solution in 0.9 % w/v sodium chloride) were tested as negative and positive controls alongside the test item analysis. The opacity of treated corneas was assessed using an opacitometer and the permeability was determined at OD492 by microplate reader. The In vitro irritancy score (IVIS), based on the mean opacity and permeability measurement, was calculated for the test item, positive and negative controls; and the classification requirement was assessed based on the IVIS.

 

The mean IVIS for the item is 0.0, and based on the assessment criteria the test item is assigned no category under the UN GHS criteria; though it is noted that the test item could not be formulated to a concentration of 20% w/v solution in sodium chloride 0.9% w/v. This did not have an effect on the study results. The mean IVIS for the negative and positive controls were 2.4 and 82.0, respectively, and therefore the acceptability criteria of the study were met.

 

The study is a GLP compliant guideline experimental study, and is deemed acceptable without restrictions for assessment of this endpoint.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05/10/2021 - 05/10/2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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:
2020
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Local abattoir
- Characteristics of donor animals: Adult cattle (typically 12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue: Eyes were transported to the test facility over ice packs on the same day of slaughter and prepared immediately on arrival at the test facility.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- Indication of any antibiotics used: Upon excision, eyes were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
- Selection and preparation of corneas: The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
- Quality check of the isolated corneas: The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1ºC for 60 minutes. At the end of the incubation period, each cornea was examined for defects. Only corneas free of damage were used.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.4682 grams as a solid disc covering the cornea surface

CONTROLS
- Amount applied: 0.75 mL
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
Three
Details on study design:
- Number of replicates: Three

- Negative control: Sodium chloride 0.9% w/v (Baxter Healthcare SA)

- Positive control: Imidazole (Acros Organics)

APPLICATION DOSE AND EXPOSURE TIME : 0.4682 g of the solid test item was found to adequately cover the corneal surface. The BCOP holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity readings were taken using a calibrated opacitometer.
- Corneal permeability: Passage of sodium fluorescein dye from the anterior to posterior chambers was determined by measuring optical density (OD 492) with a Labtech LT-4500 microplate reader
- Others: Histopathology and visual observation post treatment

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DATA INTERPRETATION: The decision criteria using the following prediction model:
- IVIS = ≤ 3 No category for UN GHS criteria
- IVIS = > 3 and ≤ 55 No stand-alone prediction can be made for UN GHS
- IVIS = > 55 Category 1 UN GHS

CRITERIA FOR ACCEPTANCE:
- Positive control: 20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during the previous 12 months for this testing facility.
- Negative control: Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values, or an overall IVIS value of < 3 calculated from the previous 12 months data for this testing facility.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
0.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 17
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 18
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 20
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were clear post treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control values (IVIS = 1.8) were within accepted limits (i.e. =<2.8 for opacity, =<0.015 for permeability, or IVIS =<3). The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control value (IVIS = 99.7) was within the acceptance range (IVIS 61.9 - 113.9). The positive control acceptance criterion was therefore satisfied.

Table 1. Individual and mean corneal opacity and permeability measurements

Treatment Cornea number Opacity Observation post-treatment Permeablity (OD 492) In Vitro Irritancy Score
Pre-treatment Post-treatment Post-treatment - pre-treatment Corrected value Original Corrected
Negative control 2 2 3 1 - Clear 0.006 - -
3 1 3 2 - Clear 0.025 - -
6 2 4 2 - Clear 0.000 - -
- - - 1.7* - - 0.010** - 1.8
Positive control 9 3 70 67 65.3 Cloudy 2.360 2.350 -
11 3 65 62 60.3 Cloudy 2.665 2.655 -
12 1 71 70 68.3 Cloudy 2.005 1.995 -
- - - - 64.7*** - - 2.333*** 99.7
Test item 17 2 4 2 0.3 Clear 0.000 0.000 -
18 3 5 2 0.3 Clear 0.006 0.000 -
20 2 4 2 0.3 Clear 0.003 0.000 -
- - - - 0.3*** - - 0.000*** 0.3

OD = optical density, * = mean of post-treatment - pre-treatment values, ** = mean permeability, *** = mean corrected value

Table 2       Corneal Epithelium Condition Post Treatment

 Treatment  Cornea Number  Observation Post Treatment
 Negative Control  2  Clear
 3  Clear
 6  Clear
 Positive Control  9  Cloudy
 11  Cloudy
 12  Cloudy
 Test Item  17  Clear
 18  Clear
 20  Clear
Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro eye irritation potential of the test item was assessed in this study. Under the experimental conditions reported, the test item was classified as a non-irritant.
Executive summary:

The eye irritation potential of the test item was assessed in an OECD 437 and EU B.47 method guideline study. Approximately 0.4682 g of the test item was applied to the corneal surface of prepared cattle cornea, obtained on the day of extraction, and incubated for 240 minutes followed by rinsing to remove the test item. Additionally, sodium chloride (0.9 % w/v) and Imidazole (20 % w/v solution in 0.9 % w/v sodium chloride) were tested as negative and positive controls alongside the test item analysis. The opacity of treated corneas was assessed using an opacitometer and the permeability was determined at OD492 by microplate reader. The In vitro irritancy score (IVIS), based on the mean opacity and permeability measurement, was calculated for the test item, positive and negative controls; and the classification requirement was assessed based on the IVIS.

 

The mean IVIS for the item is 0.3, and based on the assessment criteria the test item is assigned no category under the UN GHS criteria; though it is noted that the test item could not be formulated to a concentration of 20% w/v solution in sodium chloride 0.9% w/v. This did not have an effect on the study results. The mean IVIS for the negative and positive controls were 1.8 and 99.7, respectively, and therefore the acceptability criteria of the study were met.

 

The study is a GLP compliant guideline experimental study, and is deemed acceptable without restrictions for assessment of this endpoint.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16/12/2019 - 26/12/2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
An in vitro study was not conducted on the substance prior to conducting the in vivo eye irritation study as the study was performed for the substance to be registered in Australia.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L’Arbresle, France
- Age at study initiation: 15 - 26 weeks old
- Weight at study initiation: 2945 to 4262 grams
- Housing: Animals were housed individually in labelled cages with perforated floors (67 x 62 x 55 cm)
- Diet: Pelleted diet for rabbits provided once per day during the study period
- Water: Municipal tap water was freely available to animals via water bottles
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 20
- Humidity (%): 46 - 56
- Air changes (per hr): 10 or greater with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Average of 50.6 mg (50.3 - 51.2 mg), approximately 0.1 mL
Duration of treatment / exposure:
1 hour
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
Three animals
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Eye was rinsed with approximately 50 mL tepid tap water
- Time after start of exposure: 1 hour

SCORING SYSTEM: Numerical scoring system

TOOL USED TO ASSESS SCORE: Fluorescein
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: 1 / 24 / 48 / 72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
other: 1 / 24 / 48 / 72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
other: 1 / 24 / 48 / 72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: 1 h
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
other: 1 / 24 / 48 / 72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
other: 1 h
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #1
Time point:
other: 1 / 24 h
Score:
1
Reversibility:
fully reversible within: 48 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #1
Time point:
other: 48 / 72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #2
Time point:
other: 1 h
Score:
2
Reversibility:
fully reversible within: 48 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #2
Time point:
24 h
Score:
1
Reversibility:
fully reversible within: 48 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #2
Time point:
other: 48 / 72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #3
Time point:
other: 1 hour
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: 1 h
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: 1 h
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
other: 1 h
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #1
Time point:
other: 1 h
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #2
Time point:
other: 1 h
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #3
Time point:
other: 1 h
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Instillation of the test item (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in effects on the iris and conjunctivae. Iridial irritation was noted for two animals after 1 hour only. The irritation of the conjunctivae consisted of redness, chemosis and discharge which completely resolved within 48 hours in all animals. Treatment of the eyes with 2% fluorescein 24 hours after test item installation revealed no corneal epithelial damage.
Other effects:
No signs of systemic toxicity or mortality were observed during the test period.
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the study, the test item should not be classified and has no obligatory labelling requirement for eye irritation according to the GHS criteria.
Executive summary:

The test item was assessed for eye irritation using standard guidelines (OECD 405, EU 440/2008 Part B and OPPTS 870.2400). Approximately 50.6 mg of test item was instilled into one eye each of three rabbits. The rabbits were then observed for eye irritation after 1, 24, 48 and 72 hours. Effects were observed on the iris and conjunctivae, with iridial irritation noted for two animals after 1 hour only. The conjunctivae irritation resulted in redness, chemosis and discharge, which were all completely resolved within 48 hours of treatment. Therefore, the test item does not need to be classified under GHS guidelines.

Endpoint:
eye irritation: in vivo
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
16/12/2019 - 26/12/2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
PU MDI substances all contain the same core structure, with either linear alkyl chains and/or cyclic groups attached. PU MDI structures are therefore similar between all category members, and organisms will be exposed to very similar compounds, only differing by the length of the alkyl chain or whether cyclic groups are present. In the body, there may be metabolism of the PU MDI structures; however, due to the structural similarity of the parent compounds any metabolites are also likely to be similar. See section 13 of IUCLID for further information of the read-across assessment.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L’Arbresle, France
- Age at study initiation: 15 - 26 weeks old
- Weight at study initiation: 2945 to 4262 grams
- Housing: Animals were housed individually in labelled cages with perforated floors (67 x 62 x 55 cm)
- Diet: Pelleted diet for rabbits provided once per day during the study period
- Water: Municipal tap water was freely available to animals via water bottles
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 20
- Humidity (%): 46 - 56
- Air changes (per hr): 10 or greater with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Average of 50.6 mg (50.3 - 51.2 mg), approximately 0.1 mL
Duration of treatment / exposure:
1 hour
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
Three animals
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Eye was rinsed with approximately 50 mL tepid tap water
- Time after start of exposure: 1 hour

SCORING SYSTEM: Numerical scoring system

TOOL USED TO ASSESS SCORE: Fluorescein
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: 1 / 24 / 48 / 72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
other: 1 / 24 / 48 / 72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
other: 1 / 24 / 48 / 72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: 1 h
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
other: 1 / 24 / 48 / 72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
other: 1 h
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #1
Time point:
other: 1 / 24 h
Score:
1
Reversibility:
fully reversible within: 48 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #1
Time point:
other: 48 / 72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #2
Time point:
other: 1 h
Score:
2
Reversibility:
fully reversible within: 48 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #2
Time point:
24 h
Score:
1
Reversibility:
fully reversible within: 48 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #2
Time point:
other: 48 / 72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #3
Time point:
other: 1 hour
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: 1 h
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: 1 h
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
other: 1 h
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #1
Time point:
other: 1 h
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #2
Time point:
other: 1 h
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #3
Time point:
other: 1 h
Score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Instillation of the test item (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in effects on the iris and conjunctivae. Iridial irritation was noted for two animals after 1 hour only. The irritation of the conjunctivae consisted of redness, chemosis and discharge which completely resolved within 48 hours in all animals. Treatment of the eyes with 2% fluorescein 24 hours after test item installation revealed no corneal epithelial damage.
Other effects:
No signs of systemic toxicity or mortality were observed during the test period.
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the study, the test item should not be classified and has no obligatory labelling requirement for eye irritation according to the GHS criteria.
Executive summary:

The test item was assessed for eye irritation using standard guidelines (OECD 405, EU 440/2008 Part B and OPPTS 870.2400). Approximately 50.6 mg of test item was instilled into one eye each of three rabbits. The rabbits were then observed for eye irritation after 1, 24, 48 and 72 hours. Effects were observed on the iris and conjunctivae, with iridial irritation noted for two animals after 1 hour only. The conjunctivae irritation resulted in redness, chemosis and discharge, which were all completely resolved within 48 hours of treatment. Therefore, the test item does not need to be classified under GHS guidelines.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30/09/2021 - 30/09/2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Local abattoir
- Characteristics of donor animals: Adult cattle (typically 12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue: Eyes were transported to the test facility over ice packs on the same day of slaughter and prepared immediately on arrival at the test facility.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- Indication of any antibiotics used: Eyes were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
- Selection and preparation of corneas: The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
- Quality check of the isolated corneas: The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 70 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.7455 grams

CONTROLS
- Amount applied: 0.75 mL
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
Three
Details on study design:
NUMBER OF REPLICATES : Three

NEGATIVE CONTROL USED : Sodium chloride 0.9% w/v (Baxter Healthcare SA)

POSITIVE CONTROL USED : Imidazole (Acros Organics)

APPLICATION DOSE AND EXPOSURE TIME : Approximately 0.7455 g of the solid test item was found to adequately cover the corneal surface. The BCOP holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity readings were taken using a calibrated opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a Labtech LT-4500 microplate reader (OD492)
- Others: Histopathology and visual observation post treatment

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DATA INTERPRETATION: The decision criteria using the following prediction model:
- IVIS = ≤ 3 No category for UN GHS criteria
- IVIS = > 3 and ≤ 55 No stand-alone prediction can be made for UN GHS
- IVIS = > 55 Category 1 UN GHS

CRITERIA FOR ACCEPTANCE:
- Positive control: 20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during the previous 12 months for this testing facility.
- Negative control: Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values, or an overall IVIS value of < 3 calculated from the previous 12 months data for this testing facility.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
0.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 17
Value:
-2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 20
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 21
Value:
-1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were clear post treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control values were within accepted limits (IVIS = 0.7). The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the acceptance range (IVIS = 95.8). The positive control acceptance criterion was therefore satisfied.

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

 Treatment  Cornea Number  Opacity  Permeability (OD492)  In vitro Irritancy Score
 Pre-Treatment  Post-Treatment  Post-Treatment - Pre-Treatment  Corrected Value    Corrected Value
 Negative Control  2  3  5  2    0.005    
 3  3  3  0    0.000    
 6  3  2  0    0.000    
       0.7*    0.002**    0.7
 Positive control  9  4  75  71  70.3  1.890  1.888  
 11  4  75  71  70.3  1.655  1.653  
 12  3  79  76  75.3  1.214  1.212  
         72.0***    1.585***  95.8
 Test Item  17  4  2  -2  0.0  0.004  0.002  
 20  2  2  0  0.0  0.005  0.003  
 21  4  3  -1  0.0  0.011  0.009  
         0.0***    0.005***  0.1

OD = Optical density

* = Mean of the post-treatment minus pre-treatment values

** = Mean permeability

*** = Mean corrected value

Table 2: Corneal Epithelium Condition Post Treatment

 Treatment  Cornea Number  Observation Post Treatment
 Negative Control  2  Clear
 3  Clear
 6  Clear
 Positive Control  9  Cloudy
 11  Cloudy
 12  Cloudy
 Test Item  17  Clear
 20  Clear
 21  Clear
Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro eye irritation potential of the test item was assessed in this study. Under the experimental conditions reported, the test item was classified as a non-irritant.
Executive summary:

The eye irritation potential of the test item was assessed in an OECD 437 and EU B.47 method guideline study. Approximately 0.7455 g of the test item was applied to the corneal surface of prepared cattle cornea, obtained on the day of extraction, and incubated for 240 minutes followed by rinsing to remove the test item. Additionally, sodium chloride (0.9 % w/v) and Imidazole (20 % w/v solution in 0.9 % w/v sodium chloride) were tested as negative and positive controls alongside the test item analysis. The opacity of treated corneas was assessed using an opacitometer and the permeability was determined at OD492 by microplate reader. The In vitro irritancy score (IVIS), based on the mean opacity and permeability measurement, was calculated for the test item, positive and negative controls; and the classification requirement was assessed based on the IVIS.

 

The mean IVIS for the item is 0.1, and based on the assessment criteria the test item is assigned no category under the UN GHS criteria; though it is noted that the test item could not be formulated to a concentration of 20% w/v solution in sodium chloride 0.9% w/v. This did not have an effect on the study results. The mean IVIS for the negative and positive controls were 0.7 and 95.8, respectively, and therefore the acceptability criteria of the study were met.

 

The study is a GLP compliant guideline experimental study, and is deemed acceptable without restrictions for assessment of this endpoint.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30/09/2021 - 30/09/2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Local abattoir
- Characteristics of donor animals: Adult cattle (typically 12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue: Eyes were transported to the test facility over ice packs on the same day of slaughter and prepared immediately on arrival at the test facility.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- Indication of any antibiotics used: Eyes were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
- Selection and preparation of corneas: The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
- Quality check of the isolated corneas: The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 70 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.7123 grams

CONTROLS
- Amount applied: 0.75 mL
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
Three
Details on study design:
NUMBER OF REPLICATES : Three

NEGATIVE CONTROL USED : Sodium chloride 0.9% w/v (Baxter Healthcare SA)

POSITIVE CONTROL USED : Imidazole (Acros Organics)

APPLICATION DOSE AND EXPOSURE TIME : Approximately 0.7123 g of the solid test item was found to adequately cover the corneal surface. The BCOP holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity readings were taken using a calibrated opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a Labtech LT-4500 microplate reader (OD492)
- Others: Histopathology and visual observation post treatment

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DATA INTERPRETATION: The decision criteria using the following prediction model:
- IVIS = ≤ 3 No category for UN GHS criteria
- IVIS = > 3 and ≤ 55 No stand-alone prediction can be made for UN GHS
- IVIS = > 55 Category 1 UN GHS

CRITERIA FOR ACCEPTANCE:
- Positive control: 20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during the previous 12 months for this testing facility.
- Negative control: Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values, or an overall IVIS value of < 3 calculated from the previous 12 months data for this testing facility.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 14
Value:
-1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 15
Value:
-1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Cornea number 16
Value:
-2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were clear post treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control values were within accepted limits (IVIS = 0.7). The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the acceptance range (IVIS = 95.8). The positive control acceptance criterion was therefore satisfied.

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

 Treatment  Cornea Number  Opacity  Permeability (OD492)  In vitro Irritancy Score
 Pre-Treatment  Post-Treatment  Post-Treatment - Pre-Treatment  Corrected Value    Corrected Value
 Negative Control  2  3  5  2    0.005    
 3  3 3  0    0.000    
 6  3  2  0    0.000    
       0.7*    0.002**    0.7
 Positive control  9  4  75  71  70.3  1.890

 1.888

 

 11

 4

 75

 71

 70.3

 1.655

 1.653

 

 12

 3

 79

 76

 75.3

 1.214

 1.212

 

 

 

 

 

 72.0***

 

 1.585***

 95.8

 Test Item

 14

 3

 2

 -1

 0.0

 0.002

 0.000

 

 15

 3

 2

 -1

 0.0

 0.002

 0.000

 

 16

 3

 1

 -2

 0.0

 0.003

 0.001

 

 

 

 

 

 0.0***

 

 0.001***

 0.0

OD = Optical density

* = Mean of the post-treatment minus pre-treatment values

** = Mean permeability

*** = Mean corrected value

Table 2: Corneal Epithelium Condition Post Treatment

 Treatment  Cornea Number  Observation Post Treatment
 Negative Control  2  Clear
 3  Clear
 6  Clear
 Positive Control  9  Cloudy
 11  Cloudy
 12  Cloudy
 Test Item  14  Clear
 15  Clear
 16  Clear
Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro eye irritation potential of the test item was assessed in this study. Under the experimental conditions reported, the test item was classified as a non-irritant.
Executive summary:

The eye irritation potential of the test item was assessed in an OECD 437 and EU B.47 method guideline study. Approximately 0.7123 g of the test item was applied to the corneal surface of prepared cattle cornea, obtained on the day of extraction, and incubated for 240 minutes followed by rinsing to remove the test item. Additionally, sodium chloride (0.9 % w/v) and Imidazole (20 % w/v solution in 0.9 % w/v sodium chloride) were tested as negative and positive controls alongside the test item analysis. The opacity of treated corneas was assessed using an opacitometer and the permeability was determined at OD492 by microplate reader. The In vitro irritancy score (IVIS), based on the mean opacity and permeability measurement, was calculated for the test item, positive and negative controls; and the classification requirement was assessed based on the IVIS.

 

The mean IVIS for the item is 0.0, and based on the assessment criteria the test item is assigned no category under the UN GHS criteria; though it is noted that the test item could not be formulated to a concentration of 20% w/v solution in sodium chloride 0.9% w/v. This did not have an effect on the study results. The mean IVIS for the negative and positive controls were 0.7 and 95.8, respectively, and therefore the acceptability criteria of the study were met.

 

The study is a GLP compliant guideline experimental study, and is deemed acceptable without restrictions for assessment of this endpoint.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The category members are not classified as irritant to skin as all the in vivo rabbit studies on MDI category members were negative.

Category members are also not classified for eye irritation. Although the study conducted with N,N''-(methylenedi-4,1-phenylene) bis[N'-octyl]urea (EC 445-760-8), which contains C8 structures, indicated irreversible effects at the end of the observation period, this study is not considered to be reliable as the test item was administered as a powder, without vehicle, and no rinsing was conducted. All other studies for category members concluded that the substances are not eye irritants, and further testing is ongoing with category members to confirm this.