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Description of key information

Based on the in vitro and ex vivo study results, the test substance is considered to be non-irritating to both skin and eyes.

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:
From June 01, 2011 to June 21, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Cell type:
other: three-dimensional reconstructed human epidermis keratinocytes
Cell source:
other: EPISKIN model - reconstructed human epidermis
Vehicle:
unchanged (no vehicle)
Details on test system:
EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier: SkinEthic Laboratories, France
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
The test substance was used as supplied. First 5 µL of the distilled water was topically applied to ensure contact between test substance and tissue, and then 10 mg of the test substance was applied on epidermal surface
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
Triplicates for the test substance, negative and positive control.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 minutes exposure
Value:
103
Negative controls validity:
valid
Remarks:
100 % tissue viability
Positive controls validity:
valid
Remarks:
10.9 % tissue viability
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Direct MTT Reduction test:
The MTT solution containing the test substance did not turn blue which indicated that the test substance did not directly reduce MTT.

Test substance, Positive Control substance and Negative Control substance:
The relative mean viabillity of the test substance treated tissues was 103.0 % after 15 minutes exposure period.

Quality Criteria:
The relative mean tissue viability for the positive control treated tissues was <40 % relative to the negative control treated tissues and the standard deviation value of the percentage viability was <18 %. The positive control acceptance criterion was therefore satisfied.
The mean OD540 for the negative control treated tissues was >0.6 and the standard deviation value of the percentage viability was >18 %. The negative control acceptance criterion was therefore satisfied.
The standard deviation calculated from individual percentage tissue viabilities of the three identically test substance treated tissues was 18 %. The test substance acceptance criterion was therefore satisfied.
Interpretation of results:
other: not classified based on EU CLP criteria
Conclusions:
Under the study conditions, the test substance was determined to be non-irritating to the skin.
Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate', using Episkin method, according to EU Method B.46, in compliance with GLP. Skin irritation potential was examined using the EpiSkin reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 h. To identify possible interference of the test substance with MTT endpoint, prior to the main test the test substance was checked for the ability to directly reduce MTT. On Day 1 of the main test, triplicate tissues were treated with the undiluted test substance for an exposure period of 15 minutes. First 5 µL of the distilled water was topically applied to ensure contact between test substance and tissue, and then 10 mg of the test substance was applied on epidermal 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. At the end of the exposure period each tissue was rinsed before incubating for 42 h at 37 °C, 5 % CO2 in air. Formazan extraction was performed at Day 3. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. Absorbance optical density measurements were conducted at Day 6. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 540 nm. The relative mean viability of the test substance treated tissues was 103% (which is well above the threshold for classification) after the 15-minute exposure period and 42 h post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied. Under the study conditions, the test substance was determined to be non-irritating to the skin (Harlan, 2012).

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 07, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Eyes from adult cattle were obtained from local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee and placed in Hanks’s Balanced Salt Solution (HBSS), supplemented with Penicillin/Streptomycin, and transported to the laboratory on ice packs. The eyes were refrigerated on arrival and used within 24 of receipt.


Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Neat test substance. For the purpose of this study the test substance was pulverised and chopped to a suitable consistency before use.
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
First incubation, with the test substance for 240 min. Subsequently a post treatment opacity reading was taken and each cornea was visually observed.
Second incubation: 1 mL of sodium fluorescein solution (5 mg/mL) at 32 ± 1 ºC for 90 minutes.
Number of animals or in vitro replicates:
Three corneas were allocated to the negative, positive control and the test substance.
Details on study design:
Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. 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 cornea were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete minimum essential medium (MEM) and plugged. The holders were incubated at 32 ± 1 °C for at least 60 minutes. At the end of incubation period each cornea was examined fr defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete MEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. Three corneas were also numerically allocated to the negative control substance and three corneas to the positive control test substance.

Treatment of Corneas
The MEM was removed from the anterior chamber of the BCOP holder and the neat test substance or control substances were applied to the cornea. The holders were gently tilted back and forth to ensure a uniform application of the substance over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 °C for 240 minutes. At the end of exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior and posterior chambers were refilled with fresh complete MEM. A post-treatment opacity reading was taken and each cornea were visually observed.

Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured. If values greater than 1.500 OD492 were obtined a 1 in 5 dilution of the medium in complete MEM was performed and the measurement repeated. The modified value was multiplied by 5 to reflect dilution.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10 % neutral buffered formalin.
Irritation parameter:
in vitro irritation score
Run / experiment:
240 min
Value:
1
Negative controls validity:
valid
Remarks:
3.3
Positive controls validity:
valid
Remarks:
99.6
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Criteria:
- A test substance that induces an In Vitro Irritancy Score >55.1 is defined as an ocular corrosive or severe irritant.
Result: the test substance was considered not to be an occular corrosive or severe irritant.

- 20 % w/v Imidazole was used for positive control purposes. The test is acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean for this testing facility. In this case should be within: 55.8 - 126.1.
Result: passed
Interpretation of results:
other: not classified based on EU CLP criteria
Conclusions:
Under the study conditions, the test substance was determined to be non-corrosive or non-irritating to the eye.
Executive summary:

An in vitro study was conducted to determine the corneal damage potential by the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate' (100%), using the Bovine Corneal Opacity and Permeability (BCOP) method according to OECD Guideline 437, in compliance with GLP. One valid experiment was performed with three replicates for negative control, positive control and the test substance. The undiluted test substance was applied in a way that as much as possible of the corneas surface was covered. Subsequently corneas were incubated for 240 minutes at 32 ± 1°C in BCOP corneal holder. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior and posterior chambers were refilled with fresh complete MEM. A post treatment opacity reading was taken and each cornea was visually observed. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium was replaced with 1 mL of sodium fluorescein solution (5 mg/mL) and corneas were incubated again at 32 ± 1°C for 90 minutes. After incubation 360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured. The two endpoints opacity and permeability were combined in an empirically derived formula to generate an in vitro irritancy score (IVIS). The obtained in vitro irritancy scores for the test substance, negative control and positive control were 1 (which is well below the threshold for no classification for eye), 3.3, and 99.6, respectively. The study was considered to have met all the validity criteria. Under the study conditions, the test substance was determined to be non-corrosive or non-irritating to the eye (Harlan, 2012).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From August 30, 2011 to September 01, 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Species:
other: human skin model
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30 mg
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
3 h
Number of animals or in vitro replicates:
3
Details on study design:
Pre test:
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt to a purple formazan dye by the mitochondrial succinate dehydrogenase in viable cells. The possible interference of the test substance with MTT was checked in pre experiment. 30 mg of the test substance was added to 1 mL of a 0.5 mL MTT solution and incubated at room temperature in the dark for 3 hours. Untreated MTT solution was used as a control. If the MTT solution turned blue, the test substance was presumed to have reduced MTT.

Preparation of tissues:
Using sterile techniques, 1 mL of maintenance medium at room temperature, was dispensed into the appropriate number of wells of 6-well plate designated “treatment plates”. Separate treatment plates were used for the test substance, positive and negative controls to avid cross-contamination occurring. Before treatment, the 7 day old tissues were transferred from the arrival plates into the wells of “treatment plates” containing the maintenance medium.

Main test:
Triplicate tissues were treated with 30 mg of the test substance for 10 minutes. The tissues were dosed at regular timed intervals to allow for the period taken to rinse each insert following exposure and to ensure each tissue received an equal exposure time. Triplicate tissues were treated with 30 uL of DPBS with Ca2+ and Mg2+ to serve as negative controls and triplicate tissues were treated with 30 uL of 2% w/v Dodecyl Sulphate to serve as the positive control. The plates were incubates at 37°C, 5% CO2 in air during the exposure time. At the end of the relevant exposure period, each tissue insert was removed from the well using forceps and rinsed using a wash bottle containing DPBS without Ca2+ and Mg2+. Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test substance. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a prelabeled 24-well plate designated ‘holding plate’ containing 300 uL of maintenance medium until all tissues were rinsed. Following rinsing, the two tissues (two per group) were were transferred into a prelabeled 24-well plate designated “MTT loading plate containing 300 uL” of 0.5/mL MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into incubator for approximately 3 hours at 37°C, 5% CO2 in air. At the end of the incubation period the tissues were visually examined and the degree of MTT staining evaluated (qualitative evaluation of the tissue viability). The inserts were rinsed twice with phosphate buffered saline and blotted on absorbent paper to remove residual MTT solution and transferred to a pre-labelled 24-well plate designated “MTT extraction plate” containing 0.75 mL isopropanol in each of a sufficient number of wells. An extra 0.75 mL of isopropanol was added onto each tissue and plate was sealed. The plate was wrapped in aluminum foil and allowed to stay overnight at room temperature to extract the formazan crystals out of the tissue. At the end of extraction period, each tissue insert was pierced with a pipette fitted with a 1000 uL tip and extraction solution forced vigorously up and down through the tissue insert until a homogenous solution was obtained. The empty inserts were discarded. For each tissue triplicate 200 uL samples were transferred to appropriate wells of the pre-labeled 96-well plate. 200 uL of the isopropanol alone was added to three wells designates as “blanks”. The optical density was measured at 540 nm.

Tissue Histology:
One tissue of each treatment group was retained for possible tissue histopathology.
Irritation parameter:
other: Relative mean tissue viability %
Value:
ca. 104.9
Vehicle controls validity:
valid
Remarks:
100 % viability
Negative controls validity:
valid
Remarks:
100 % viability
Positive controls validity:
valid
Remarks:
32.2 % viability
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Assessment of the direct test substance reduction of MTT:
The MTT solution containing the test substance remained yellow, which indicated that test substance did not directly reduce MTT.

Assessment of eye irritation potential
The individual and mean OD540 values and mean viabilities for each treatment group,
Test substance: 104.9 %
Positive control: 32.2 %
Negative control: 100 %
It was considered unnecessarily to proceed with tissue histopathology.

The quality criterion required for the acceptance of results in the test was satisfied:
- the assy meets the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is <60% relative to the negative control treated tissues.
Interpretation of results:
other: not classified based on EU CLP criteria
Conclusions:
Under the study conditions, the test substance was determined to be non-irritating to the eye.
Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate' (100%), using the SkinEthic Reconstructed Human Corneal model, according to a method similar to OECD Guideline 492, in compliance with GLP. For the main test, triplicate SkinEthic tissues were treated with 30 mg of the test substance for 10 minutes. Triplicate tissues treated with 30 µL of Dulbecco’s Phosphate Buffered Saline (DPBS) with CA2+ and Mg2+ served as the negative control and triplicate tissues treated with 30 µL of 2% w/v Dodecyl Sulphate served as the positive control. At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissue. After extraction the absorbency of triplicate aliquots of the extraction MTT solution for each SkinEthic tissue was measured. The optical density was measured at 540 nm. Data were presented in the form of percentage viability (MTT conversion relative to negative controls). The relative mean tissue viability for the negative control and positive control were found to be 100 and 32.2%, respectively. Therefore, the study was considered to have met the validity criteria. The relative mean viability for the test substance treated tissues after 10 min exposure period was 104.9%, which was well below the threshold for non-classification. Under the study conditions, the test substance was determined to be non-irritating to the eye (Harlan, 2012).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin:

An in vitro study was conducted to determine the skin irritation potential of the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate', using Episkin method, according to EU Method B.46, in compliance with GLP. Skin irritation potential was examined using the EpiSkin reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 h. To identify possible interference of the test substance with MTT endpoint, prior to the main test the test substance was checked for the ability to directly reduce MTT. On Day 1 of the main test, triplicate tissues were treated with the undiluted test substance for an exposure period of 15 minutes. First 5 µL of the distilled water was topically applied to ensure contact between test substance and tissue, and then 10 mg of the test substance was applied on epidermal 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. At the end of the exposure period each tissue was rinsed before incubating for 42 h at 37 °C, 5 % CO2 in air. Formazan extraction was performed at Day 3. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. Absorbance optical density measurements were conducted at Day 6. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm. The relative mean viability of the test substance treated tissues was 103% (which is well above the threshold for classification) after the 15-minute exposure period and 42 h post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied. Under the study conditions, the test substance was determined to be non-irritating to the skin (Harlan, 2012).

Eye:

Study 1: An in vitro study was conducted to determine the corneal damage potential by the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate' (100%), using the Bovine Corneal Opacity and Permeability (BCOP) method according to OECD Guideline 437, in compliance with GLP. One valid experiment was performed with three replicates for negative control, positive control and the test substance. The undiluted test substance was applied in a way that as much as possible of the corneas surface was covered. Subsequently corneas were incubated for 240 minutes at 32 ± 1°C in BCOP corneal holder. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior and posterior chambers were refilled with fresh complete MEM. A post treatment opacity reading was taken and each cornea was visually observed. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium was replaced with 1 mL of sodium fluorescein solution (5 mg/mL) and corneas were incubated again at 32 ± 1°C for 90 minutes. After incubation 360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured. The two endpoints opacity and permeability were combined in an empirically derived formula to generate an in vitro irritancy score (IVIS). The obtained in vitro irritancy scores for the test substance, negative control and positive control were 1 (which is well below the threshold for no classification for eye), 3.3, and 99.6, respectively. The study was considered to have met all the validity criteria. Under the study conditions, the test substance was determined to be non-corrosive or non-irritating to the eye (Harlan, 2012).

Study 2: An in vitro study was conducted to determine the eye irritation potential of the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate' (100%), using the SkinEthic Reconstructed Human Corneal model, according to a method similar to OECD Guideline 492, in compliance with GLP. For the main test, triplicate SkinEthic tissues were treated with 30 mg of the test substance for 10 minutes. Triplicate tissues treated with 30 µL of Dulbecco’s Phosphate Buffered Saline (DPBS) with CA2+ and Mg2+ served as the negative control and triplicate tissues treated with 30 µL of 2% w/v Dodecyl Sulphate served as the positive control. At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissue. After extraction the absorbency of triplicate aliquots of the extraction MTT solution for each SkinEthic tissue was measured. The optical density was measured at 540 nm. Data were presented in the form of percentage viability (MTT conversion relative to negative controls).The relative mean tissue viability for the negative control and positive control were found to be 100 and 32.2%, respectively. Therefore, the study was considered to have met the validity criteria. The relative mean viability for the test substance treated tissues after 10 min exposure period was 104.9%, which was well below the threshold for non-classification. Under the study conditions, the test substance was determined to be non-irritating to the eye (Harlan, 2012).

Overall, based on the in vitro and ex vivo study results, the test substance is considered to be non-irritating to both skin and eyes.

Justification for classification or non-classification

Based on the in vitro and ex vivo study results, the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate', does not warrant a classification for skin and eye according to the EU CLP criteria (Regulation 1272/2008/EC).