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Administrative data

Description of key information

Skin corrosion (a.o. based on absence of skin irritation up to 100% application in the LLNA (OECD TG 429) data): not corrosive

Skin irritation (OECD TG 439): irritating

Eye irritation (OECD TG 438): irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 May - 30 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
In accordance with GLP
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Adopted 28 July 2015
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Test system:
human skin model
Source species:
other: 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 TM
- Tissue batch number(s): 16-EKIN-021
- Production date/ Shipping date: no data
- Delivery date: 24 May 2016
- Date of initiation of testing: 24 May 2016

PRE-TEST PROCEDURE
- Pre-incubation: On day of receipt the tissues were transferred into a 12-wells plate filled with maintenance medium. A different 12-wells plate was used for the test item and each control item. The tissue were incubated at 37°C, 5% CO2 in air overnight.
- Test item colour interference: To assess colour interference, 10 μL of test substance was added to 90 μL sterile water. The mixture was mixed for approximately 15 minutes. At the end of the shaking period a colour check was performed.
- Test item MTT reduction: To assess the ability of the test item to directly reduce MTT, 10 μL of the test item was added to 2 mL MTT solution (0.3 mg/mL in assay medium). The mixture was incubated in the dark for 3 hours at 37°C, 5% CO2 in air. Untreated MTT solution was used as a control. At the end of the incubation period a colour check was performed.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment: room temperature for 15 minutes, post-treatment incubation: 37 °C for 42 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
-Tissues were washed with DPBS with Ca++ and Mg++ to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in assay medium
- Incubation time: 3 hours at 37 °C, 5% CO2 in air
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm (without reference filter)

NUMBER OF REPLICATE TISSUES: 3

DECISION CRITERIA
- A test substance is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post-exposure incubation is ≤ 50% of the mean viability of the negative controls.
- A test substance is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post-exposure incubation is > 50% of the mean viability of the negative controls.

ACCEPTANCE CRITERIA
- 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%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 μL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): 5% (w/v)
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Relative mean tissue viability compared to the negative control tissues (100%)
Value:
5.9
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive 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 solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

ACCEPTANCE OF RESULTS:
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 1.3%. The test item acceptance criterion was therefore satisfied.

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

Item

OD562of tissues

Mean OD562 of triplicate tissues

± SD of 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

0.062

0.077

0.017

4.8

5.9

1.3

0.096

7.4

0.072

5.6


OD = Optical Density

SD = Standard deviation

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

⊕ = The control groups were shared with Envigo - Shardlow study number TW67BS, MS48XJ, FC59GT, YB24MH, RS46VL, JD61DM and NL15CD.

Interpretation of results:
other: Skin irritant, Category 2
Remarks:
according to EU CLP criteria (1272/2008) and its amendments.
Conclusions:
Under the conditions of this test, the relative mean tissue viability for the test item treated tissues was determined to be 5.9%. This value is below the threshold for irritancy of ≤50%. Based on the results obtained, the test substance is considered to be irritating to the skin, Category 2 according to GHS.

Executive summary:

The possible skin irritation potential of the substance was tested in vitro, using a reconstructed human epidermis model, according to OECD TG 439 guideline and GLP principles. Skin tissues were treated by topical application of 10 μL undiluted test substance for 15 minutes. After a 42 hours post-exposure 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 relative mean tissue viability of 4.9% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18% for test item, positive control and negative control treated tissues, indicating that the test system functioned properly. Under the conditions of this test, the relative mean tissue viability for the test item treated tissues is determined to be 5.9%. This value is below the threshold for irritancy of ≤50%. Based on the results obtained, the test substance is considered to be irritating to the skin.

Endpoint:
skin corrosion: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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
Remarks:
In accordance with GLP conditions
Reference:
Composition 1
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)
Test material information:
Composition 1
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:
6
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Maximum mean values
Irritation parameter:
cornea opacity score
Run / experiment:
Slit-lamp examination
Value:
1
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Maximum mean values
Irritation parameter:
fluorescein retention score
Run / experiment:
Slit-lamp examination
Value:
1.3
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
Slit-lamp examination: The test substance caused corneal effects consisting of slight corneal swelling (mean of 6%), slight opacity (mean score of 1.0) and slight or moderate fluorescein retention (mean score of 1.3). 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 revealed very slight or slight erosion, very slight or slight necrosis (two corneas) and moderate vacuolation of the epithelium (one cornea), and the epithelium partly detached from the basement membrane (one cornea). 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: Category 2 (irritating to eyes)
Remarks:
in accordance with EU CLP criteria (1272/2008) and its amendments.
Conclusions:
Under the test conditions (OECD 438 and GLP) the test substance is considered to be an eye irritant. Therefore, the substance should be classified Category 2 irritating to eyes in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC). In accordance with GHS criteria, the substance should be classified as Category 2B (mildly irritating to eyes).
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 test substance caused corneal effects consisting of slight corneal swelling (mean of 6%), slight opacity (mean score of 1.0) and slight or moderate fluorescein retention (mean score of 1.3). 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 of the corneas treated with the test substance revealed very slight or slight erosion, very slight or slight necrosis (two corneas) and moderate vacuolation of the epithelium (one cornea), and the epithelium partly detached from the basement membrane (one cornea). 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). Based on these results, the test substance is considered to be eye irritating.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

In vitro skin corrosion

The substance is not considered to be corrosive based on the absence of skin irritation in the LLNA in which the test substance was tested up to 100%. In addition, the substance is a hydrocarbon with an acetal attached, which is not considered to be very reactive in a way that it could cause corrosion.

In vitro skin irritation

The skin irritation potential of the substance was tested in vitro, using a reconstructed human epidermis model, according to OECD TG 439 guideline and GLP principles. Skin tissues were treated by topical application of 10 μL undiluted test substance for 15 minutes. After a 42 hours post-exposure 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 relative mean tissue viability of 4.9% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18% for test item, positive control and negative control treated tissues, indicating that the test system functioned properly. Under the conditions of this test, the relative mean tissue viability for the test item treated tissues is determined to be 5.9%. This value is below the threshold for irritancy of ≤50%. Based on the results obtained, the test substance is considered to be irritating to skin.

In vitro 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 test substance caused corneal effects consisting of slight corneal swelling (mean of 6%), slight opacity (mean score of 1.0) and slight or moderate fluorescein retention (mean score of 1.3). 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 of the corneas treated with the test substance revealed very slight or slight erosion, very slight or slight necrosis (two corneas) and moderate vacuolation of the epithelium (one cornea), and the epithelium partly detached from the basement membrane (one cornea). Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Based on these results, the test substance is considered to be eye irritating.

Justification for classification or non-classification

Skin corrosion: The substance is not considered corrosive based on absence of skin irritation in a 100% application in an LLNA test.

Skin irritation: Based on the positive results found in the in-vitro skin irritation test, the test substance is considered to cause skin irritation. In accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC) and its amendments, this results in skin irritation Category 2 classification for this endpoint (H315 / Causes skin irritation).

Eye irritation: Based on the positive results found in the in vitro eye irritation test, the test substance is considered to cause eye irritation. In accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC) and its amendments, this results in a Category 2 irritating to eyes classification for this endpoint (H319 / Causes serious eye irritation).