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Description of key information

Skin corrosion: The substance is not considered corrosive in absence of skin and eye irritating effects.

Skin irritation (OECD TG 439): Not irritating
Eye irritation or serious eye damage (OECD TG 438): Not 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:
Experimental start date 24 May 2016, experimental completion date 30 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Adopted 28 July 2015
GLP compliance:
yes (incl. certificate)
Test system:
other: EPISKIN reconstructed human epidermis model
Source species:
human
Cell type:
other: adult human-derived epidermal keratinocytes
Source strain:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM:
EPISKIN™ Reconstructed Human Epidermis Model Kit:
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 24 May 2016
EpiSkinTM Tissues (0.38cm2) lot number: 16-EKIN-021
Maintenance Medium lot number: 16-MAIN3-035
Assay Medium lot number: 16-ESSC-021

The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13 Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

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 the cellular 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 by using killed tissues to act as controls.

Test for Direct MTT Reduction:
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:

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 (Day 0: TISSUE ARRIVAL):
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes

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.

MAIN TEST:
Application of Test Item and Rinsing (Day 1):
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.

MTT Loading/Formazan Extraction (Day 3):
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 EPISKIN 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.

Absorbance/Optical Density Measurements (Day 6):
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.

DATA EVALUATION:
Quantitative MTT Assessment (Percentage Tissue Viability):
For the test item the mean tissue viability obtained after the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3) to generate the relative mean tissue viability. The relative mean viability was calculated in the following way:
Relative mean viability (%) = ((mean OD562 of test item) / (mean OD562 of negative control)) * 100

Classification of irritation potential is based upon relative mean tissue viability following the 15-Minute exposure period followed by the 42-Hour post exposure incubation period according to the following table:

Criteria for in vitro interpretation: Prediction
Relative mean tissue viability is ≤50%: Irritant
Relative mean tissue viability is >50%: Non-irritant

Quality Criteria:
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Positive Control:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.

Negative Control:
The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%.

Test Item:
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.

Major Computerized Systems:
The following computerized system was used in the study:
Delta Controls – ORCAview (Version 3.4.0)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 µL
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
other: Relative mean viability (%)
Value:
82.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
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 (10 µL of test item in 90 µL of sterile water) containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

Test Item, Positive Control Item and Negative Control Item:
The individual and mean OD562 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given under “Any other information on results incl. tables”. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given under “Any other information on results incl. tables”.

The relative mean viability of the test item treated tissues was 82.8% after a 15 Minute exposure period and 42 Hour post exposure incubation period.
It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

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 18.0%. 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

OD562of tissues

Mean OD562of triplicate tissues

±SDof OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

1.265

1.293

0.045

97.8

100*

3.5

1.345

104.0

1.269

98.1

Positive Control Item

0.076

0.063

0.011

5.9

4.9

0.9

0.058

4.5

0.055

4.3

Test Item

0.934

1.071

0.232

72.2

82.8

18.0

1.339

103.6

0.940

72.7


OD= Optical Density

SD = Standard deviation

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

Interpretation of results:
other: Not a skin irritant
Remarks:
according to EU CLP criteria and its amendments (1272/2008)
Conclusions:
The relative mean tissue viability after 15 minutes treatment with the substance compared to the negative control tissue was 82.8%. Since the mean relative tissue viability for the substance was above 50%, the substance is considered to be a non-irritant to the skin.
Executive summary:

The possible skin irritation potential of the substance was evaluated using the EPISKIN reconstructed human epidermis model in compliance with OECD TG 439 and GLP principles. Triplicate skin tissues were treated by application of 10 µL undiluted test substance for an exposure period of 15 minutes. After 42 hours post-exposure incubation period, determination of the cytotoxic (irritancy) effect was performed.Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test substance treated tissues relative to the negative controls.The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 82.8%. Since the mean relative tissue viability for the substance was higher than 50% after 15 minutes treatment the substance is considered to be non-irritant. The positive control had a relative mean tissue viability of 4.9% (< 40%) after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than or equal to 18% (<=18%), for the substance, the positive and the negative control, indicating that the test system functioned properly.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16-03-2016 to 15-04-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
Adopted on 26 July 2013
GLP compliance:
yes (incl. certificate)
Remarks:
Triskelion, Utrechtseweg 48, 3700 AV, Zeist
Species:
other: eyes of male or female chickens (ROSS, spring chickens)
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: approximately 1.5 - 2.5 kg
-Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- The preparation and validation of the eyes prior to the ICE-test were all according to OECD guideline 438.
Vehicle:
unchanged (no vehicle)
Controls:
other: Positive controls: Benzalkonium Chloride. Negative control: Physiological saline.
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 30 µL
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
240 minutes
Number of animals or in vitro replicates:
3 eyes
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: The eyes were rinsed with 20 mL saline
- Time after start of exposure: 10 seconds

SCORING SYSTEM: According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes

TOOL USED TO ASSESS SCORE: All examinations were carried out with the slit-lamp microscope. Fluorescein retention was only scored at approximately 30 minutes after treatment.

CONTROLS: A negative control (30 µL physiological saline) and 3 positive controls (30 µL Benzalkonium Chloride 5%) were included.
Irritation parameter:
percent corneal swelling
Run / experiment:
Slit-lamp examination
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Maximum mean score
Irritation parameter:
cornea opacity score
Run / experiment:
Slit-lamp examination
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Maximum mean score
Irritation parameter:
fluorescein retention score
Run / experiment:
Slit-lamp examination
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Slit-lamp examination: The test substance did not cause any corneal effects. The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Microscopic examination: Microscopic examination of the corneas treated with the test substance did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane, and endothelial necrosis (two corneas).
Interpretation of results:
other: Not an eye irritant.
Remarks:
according to EU CLP 1272/2008 and its amendments
Conclusions:
Under the test conditions (OECD 438 and GLP) the test substance is not considered to be an eye irritant.
Executive summary:

In accordance to OECD guideline 438 and GLP the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane, and endothelial necrosis (two corneas). This information on the negative and positive controls shows that the test is valid. The test substance did not cause any corneal effects. Microscopic examination of the corneas treated with the test substance did not reveal any abnormalities. Based on these results, the test substance is considered to be not severely eye irritating.

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

Additional information

Skin corrosion: The substance is not considered to be corrosive in absence of skin and eye irritation.

Skin irritation: The possible skin irritation potential of the substance was evaluated using the EPISKIN reconstructed human epidermis model in compliance with OECD TG 439 and GLP principles. Triplicate skin tissues were treated by application of 10 µL undiluted test substance for an exposure period of 15 minutes. After 42 hours post-exposure incubation period, determination of the cytotoxic (irritancy) effect was performed.Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test substance treated tissues relative to the negative controls.The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 82.8%. Since the mean relative tissue viability for the substance was higher than 50% after 15 minutes treatment the substance is considered to be non-irritant. The positive control had a relative mean tissue viability of 4.9% (< 40%) after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than or equal to 18% (<=18%), for the substance, the positive and the negative control, indicating that the test system functioned properly.

 

The above test was preferred above an in vivo skin irritation study (somewhat similar to OECDTG 404) with concentrations of 2.5% and considered not fulfilling the REACH requirements. 

 

Eye irritation: In accordance to OECD guideline 438 and GLP the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane, and endothelial necrosis (two corneas). This information on the negative and positive controls shows that the test is valid. The test substance did not cause any corneal effects. Microscopic examination of the corneas treated with the test substance did not reveal any abnormalities. Based on these results, the test substance is considered to be not severely eye irritating.

Justification for classification or non-classification

Skin irritation:

Based on the negative result in the skin irritation test the substance does not need to be classified as a skin irritant according to EU CLP (EC 1272/2008 and its amendments).

Eye irritation:

Based on the negative result in the eye irritation test the substance does not need to be classified for this endpoint according to EU CLP (EC 1272/2008 and its amendments).