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Administrative data

Description of key information

Skin corrosion: In view of the substance not being an irritant it is not corrosive either.

Skin irritation (OECD TG 439): Not irritating
Eye irritation and severe damage (OECD TG 405): Irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 May 2016 - 30 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France.
Source strain:
other: Not applicable.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM, 0.38 cm^2
- Tissue batch number: 16-EKIN-021
- Twenty five μL of the undiluted test substance was added into 12-well plates on top of the skin tissues.
- The test item was applied topically to the corresponding tissues ensuring uniform covering.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

PRE-TEST PROCEDURE:
Assessment of Direct Test Item Reduction of MTT:
MTT Salt Metabolism, Cell Viability Assay:
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of thecellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction:
10 μL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue or 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 water-killed tissues for quantitative correction of the results.
Assessment of Color Interference with the MTT endpoint:
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. 10 μL of test item was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.

PRE-INCUBATION:
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

APPLICATION/TREATMENT OF TEST SUBSTANCE:
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

CELL VIABILITY MEASUREMENT:
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

DECISION CRITERIA:
The test item may be considered non-irritant to skin when the tissue viability after exposure and post-treatment incubation is more than > 50% and an irritant if the viability is ≤ 50% in accordance with OECD TG 439.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Test material
- Applied volume: 10 μL
Duration of treatment / exposure:
15-Minute exposure period and 42 hours post-exposure incubation period.
Number of replicates:
A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.
Vehicle:
unchanged (no vehicle)
Irritation / corrosion parameter:
other: relative mean viability
Value:
85
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: The relative mean tissue viability compared to the negative control tissues (100%).
Other effects / acceptance of results:
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.
Assessment of Color Interference with the MTT endpoint:
The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

Test Item:
The relative mean viability of the test item treated tissues was 85.0% after a 15-Minute exposure period and 42-Hour post-exposure incubation period.

Quality Criteria:
The relative mean tissue viability for the positive control treated tissues was 4.9% relative to the negative control treated tissues and the standard deviation value of the viability was 0.9%.
The positive control acceptance criteria were therefore satisfied.
The mean OD562 for the negative control treated tissues was 1.293 and the standard deviation value of the viability was 3.5%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 9.5%. The test item acceptance criterion was therefore satisfied.

Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item

OD562 of

tissues

Mean OD562

of triplicate

tissues

± SD of

OD562

Relative

individual

tissue

viability (%)

Relative

mean

viability (%)

Negative

Control Item

1.265

1.293

0.045

97.8

100

1.345

104.0

1.269

98.1

Positive Control Item

0.076

0.063

0.011

5.9

4.9

0.096

4.5

0.055

4.3

Test Item

0.961

1.100

0.122

74.3

85.0

1.149

88.9

1.189

92.0

OD = Optical Density

SD = Standard deviation

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

Interpretation of results:
other: Not skin irritating.
Remarks:
According to Regulation (EC) No. 1272/2008 and its updates.
Conclusions:
Since the mean relative tissue viability for the substance was above 50% the substance is considered to be not skin irritating.
Executive summary:

The possible skin irritation potential of the substance was tested in two in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 10 μL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 4.9%. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 85%.

Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment the substance is considered to be not skin irritating.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 14, 1964 - February 19, 1964
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Reliability has been presented as 2 because similar to OECD Guideline protocol has been followed but not GLP.
Reference:
Composition 1
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
1981
Deviations:
yes
Remarks:
No details on animal housing and environmental conditions
GLP compliance:
no
Test material information:
Composition 1
Species:
rabbit
Strain:
other: Albino
Vehicle:
unchanged (no vehicle)
Controls:
other: One eye of each animal remained untreated and served as the reference control.
Amount / concentration applied:
Amount applied: 0.1 mL
Duration of treatment / exposure:
Single instillation on Day 1
Observation period (in vivo):
7 days
Number of animals or in vitro replicates:
3
Details on study design:
STUDY DESIGN
The test material was applied undiluted in 3 animals.

TREATMENT
In accordance with OECD 405 (1981). The test substance is placed in the conjunctival sac of right eye of each animal after gently pulling the lower lid away from the eyeball. The lids are then gently held together for about one second. The other eye serves at control.

REMOVAL OF TEST SUBSTANCE
-Washing: No

OBSERVATIONS
- Irritation:
The eyes of each animal were examined approximately 24, 48, 72, 96 hours and 7 days after instillation of the test substance.
The irritation scores and a description of all other (local) effects were recorded. The irritation was assessed according to OECD 405 (1981).
Irritation parameter:
cornea opacity score
Remarks:
(opacity)
Basis:
animal: #1, #2 and #3 (mean)
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal: #1, #2 and #3 (mean)
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal: #1 and #2
Remarks:
(mean)
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal #3
Remarks:
(mean)
Time point:
24/48/72 h
Score:
1.33
Max. score:
3
Reversibility:
fully reversible within: 4 days
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
(mean)
Time point:
24/48/72 h
Score:
1.67
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
(mean)
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
fully reversible within: 4 days
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
(mean)
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 3 days
Irritant / corrosive response data:
The substance caused a mild conjunctival (redness) and a slight/mild chemosis in all three animals. The effects were fully reversible within 7 days.

Ocular Irritant response data (for the undiluted test substance)

 

                                           Time after administration

 

       1 day

       2 days

       3 days

       4 days

       7 days

 Animal:  1  3  1  2  3  1  2  3
 Cornea score (opacity)  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0
 Iris score  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0
 Conjunctivae score (redness)  2  3  2  2  2  1  2  1  1  1  0  0  0  0
 Chemosis score  2  2  2  2  1  1  1  1  0  1  0  0  0  0  0
Interpretation of results:
other: Eye irritant Cat 2
Remarks:
According to Regulation (EC) No. 1272/2008 and its amendments.
Conclusions:
In an eye irritation study with three rabbits, performed equivalent to OECD 405 guideline, irritation was observed. Based on the results of this study, the substance is considered an eye irritant.
Executive summary:

The substance was tested in an eye irritation test in three rabbits equivalent to OECD 405 guideline. The substance did not cause effects on cornea or iris. In 2/3 animals conjunctivae score 2 during the first two days meeting the criteria for considering the substance a severe eye irritant. Effects on chemosis are mild. The effects were fully reversible within 7 days. The results show that the substance is considered to be severe irritating to eyes.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Skin corrosion: In view of the substance not being an irritant it is not corrosive either.

Skin irritation, in vitro:The possible skin irritation potential of the substance was tested in two in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 10 μL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 4.9%. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 85%. Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment the substance is considered to be not skin irritating.

Eye irritation, in vivo:

The substance was tested in an eye irritation test in three rabbits equivalent to OECD 405 guideline. The substance did not cause effects on cornea or iris. In 2/3 animals conjunctivae score 2 during the first two days meeting the criteria for considering the substance a severe eye irritant. Effects on chemosis are mild. The effects were fully reversible within 7 days. The results showed that the substance is considered to be severe irritating to eyes (Cat 2).

Justification for classification or non-classification

Based on the results the substance does not need to be classified for skin corrosion and skin irritation according to EU CLP (1272/2008 and its amendments.

Based on the results, the substance should be classified as irritating to eyes cat.2 and labelled with H319: Causes serious eye irritation according to Regulation (EC) No. 1272/2008 and its amendments.