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Administrative data

Description of key information

Based on the in vitro 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 December 05, 2018 to April xx, 2019
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)
Deviations:
yes
Remarks:
see "Principles of method if other than guideline"
Principles of method if other than guideline:
Optical Density (OD) values obtained with blanks were higher than 0.1 (0.191) causing a deviation from Acceptance Criteria 4. However, this SOP and guideline deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained.
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Remarks:
MatTek EpiDermTM tissue model EPI-200
Justification for test system used:
Initially the predictive capacity of the modified EpiDerm™ Skin Irritation Test (SIT) test method, using MatTek EpiDermTM tissue model EPI-200, underwent full prospective validation from 2003-2007. The test method components of this method were used to define the essential test methods components of the original and updated ECVAM Performance Standards (PS). A modification of the original EpiDerm™ SIT was validated using the original ECVAM PS in 2008. In 2008, ESAC concluded that the Modified EpiDerm™ SIT has sufficient accuracy and reliability for prediction of R38 skin irritating and no-label (non-skin irritating) test substances.
Vehicle:
unchanged (no vehicle)
Details on test system:
Test system:
The reconstructed human epidermal model EpiDermTM (EPI-200-MatTek Corporation) consists of normal human-derived epidermal keratinocytes which have been cultured to form a multi-layered highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

Characterisation of the test system:
MatTek’s EpiDermTM model has been extensively characterised for multiple parameters including morphology, tissue viability, skin barrier function and sterility. QC results for the specific lot of models received (Lot# 25819) were checked in-house for MatTek acceptance ranges with the following outcome:

- Morphology - PASS
- Tissue viability - PASS
- Skin barrier function (ET50 value for 1 % Triton X-100) where ET50 is the time taken for 1 % Triton X-100 to reduce the viability of the skin model to 50 % relative to the negative control) - PASS
- Sterility testing showed no contamination during long term antibiotic and antimycotic free culture - PASS
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 mg (nominal) of the neat test substance
Duration of treatment / exposure:
60 minutes (25 minutes at room temperature and 35 minutes at 37°C, 5 % CO2, >=95% relative humidity).
Duration of post-treatment incubation (if applicable):
42 h post-treatment incubation.
Number of replicates:
3 replicates for test substance, negative and positive control.
Irritation / corrosion parameter:
% tissue viability
Value:
103.13
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- Prior to the study, the required compatibility checks (as per SOP L0029) confirmed that the test substance did not interfere with MTT and no water colouration was observed.
- The test substance did not reduce the viability below 50% and therefore should be considered as non-irritant to the skin.

All acceptance criteria were met with the exception of 1 criterion:

- The mean OD570 of the negative control (treated with DPBS) tissues is ≥0.8 and ≤2.8.
Result: 1.777
- The mean of the positive control relative percentage viability must be ≤20 % of the mean of the negative controls.
Result: 3.8 %
- The standard deviation of OD values for triplicate skin models in each experimental condition must be <18 %.
Results:
NC: 5 %
PC: 0.72 %
Test substance: 16.17 %
- The mean OD of the 6 wells containing extraction solvent alone (blanks) should be ≤0.1.
Result: 0.191

Optical Density (OD) values obtained with blanks were higher than 0.1 (0.191) causing a deviation from Acceptance Criteria 4. However, the spectrophotometer was fully validated and had passed all required tests. The OD values for blanks observed in this study are consistent with historical data using this spectrophotometer in the laboratory and meet our current internal acceptance criteria of blank OD values <0.194 (mean of historical data, based on blanks obtained during the last 66 studies), therefore this is not considered to be an issue in the interpretation of this study data.
This SOP and guideline deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained.
Interpretation of results:
other: CLP criteria not met
Remarks:
non-irritant to skin
Conclusions:
Under the study conditions, the test substance was determined to be non-irritant to the skin.
Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the test substance, ' Reaction products of hexadecyl dihydrogen phosphate, dihexadecyl hydrogen phosphate, hexadecan-1-ol, stearic acid, esters of C18 (branched and linear) fatty acids with C18 (branched and linear) alcohols, and potassium hydroxide' (UVCB), in Reconstructed Human Epidermis (RHE) cells, according to OECD Guideline 439, in compliance with GLP. Three tissues of the human skin model EpiDermTM were treated with the test substance, positive or negative control for 60 minutes (25 minutes at room temperature and 35 minutes at 37°C, 5 % CO2, 95% RH) and 42 h post incubation period. Test was performed with 3 replicates for each type of treatment. Tissues were first pre-wetted with 25 μL DPBS (Sterile Dulbecco’s Phosphate Buffered Saline), subsequently 25 mg (nominal) of the neat test substance was applied. 30 μL of DPBS was used as negative control and 5% of sodium dodecyl sulphate as positive control. Viability of the tissues was assessed in MTT test and compared to the negative control. The percentage of viability obtained with the test substance was 103.13%, which is well above the irritant limit of 50%. The study met all the validity criteria. Under the study conditions, the test substance was determined to be non-irritant to the skin (XCellR8, 2017).

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:
weight of evidence
Study period:
From December 05, 2017 to April xx, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This in vitro risk assessment assay predicts the acute eye irritation potential of a chemical by measurement of its cytotoxic effect on the EpiOcular™ corneal epithelial model.
Qualifier:
according 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:
no
GLP compliance:
yes (incl. certificate)
Species:
other: EpiOcularTM tissue model (OCL-200-MatTek Corporation)
Strain:
other: Keratinocyte 4F1188
Details on test animals or tissues and environmental conditions:
Test system
The EpiOcularTM tissue model (OCL-200-MatTek Corporation) is composed of stratified human keratinocytes in a three-dimensional structure, reflecting the morphology and function of the human corneal epithelium found in vivo.

The EpiOcular TM eye irritation test (EIT) using the MatTek EpiOcularTM tissue model OCL-200, was validated by the European Union Reference Laboratory for Alternative to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular TM EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose, as an alternative to Draize Rabbit Eye Test with excellent correlation of in vivo to in vitro test results.

QC results for the specific lot of models received (Lot# 27002) were checked in-house for MatTek acceptance ranges with the following outcome:
- Morphology - PASS
- Tissue viability - PASS
- Skin barrier function (ET50 value for 0.3 % Triton X-100) where ET50 is the time taken for 0.3 % Triton X-100 to reduce the viability of the skin model to 50 % relative to the negative control) - PASS
- Sterility testing showed no contamination during long term antibiotic and antimycotic free culture - PASS



Vehicle:
other: PBS (Sterile Dulbecco’s Phosphate Buffered Saline)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
20 µL of PBS (Sterile Dulbecco’s Phosphate Buffered Saline) plus 50 mg of test substance.
Duration of treatment / exposure:
6 h ± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion
Duration of post- treatment incubation (in vitro):
18 h ± 15 minutes post-treatment incubation
Number of animals or in vitro replicates:
Three tissues per condition (n=3)


Irritation parameter:
other: Percentage of viability (relative to negative control)
Value:
84.2
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Prior to the study, the required compatibility checks (as per SOP L0069) confirmed that the test substance did not interfere with MTT or solvent.
Interpretation of results:
other: CLP criteria not met
Remarks:
non-irritant to eye
Conclusions:
Under the study conditions, the test substance was determined to be non-irritant to the eye.
Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the test substance, 'Reaction products of hexadecyl dihydrogen phosphate, dihexadecyl hydrogen phosphate, hexadecan-1-ol, stearic acid, esters of C18 (branched and linear) fatty acids with C18 (branched and linear) alcohols, and potassium hydroxide', using Reconstructed human cornea-like Epithelium (RhCE), according to OECD Guideline 492, in compliance with GLP. EpiOcularTM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% RH. On Day 1, after pre-wetting tissues with 20 µL PBS (Sterile Dulbecco’s Phosphate Buffered Saline) for 30 ± 2 min, the tissues in triplicate were exposed to single topical application of 50 mg neat test substance or 50 µL of reference substances (negative control: sterile water; positive control: methyl acetate) for 6 h ± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion, and 18 h ± 15 minutes post-treatment incubation, prior to the MTT endpoint. On Day 2, MTT tests and measurement were performed 570 nm without reference filter. The viability of the tissues were assessed and compared to a negative control. The percentage viability obtained with the test substance was determined to be 84.2%,which is well above the threshold (i.e., >60%) indicating no irritation potential. Under the study conditions, the test substance was determined to be non-irritant to the eyes (XCellR8, 2019).

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, ' Reaction products of hexadecyl dihydrogen phosphate, dihexadecyl hydrogen phosphate, hexadecan-1-ol, stearic acid, esters of C18 (branched and linear) fatty acids with C18 (branched and linear) alcohols, and potassium hydroxide' (UVCB), in Reconstructed Human Epidermis (RHE) cells, according to OECD Guideline 439, in compliance with GLP. Three tissues of the human skin model EpiDermTM were treated with the test substance, positive or negative control for 60 minutes (25 minutes at room temperature and 35 minutes at 37°C, 5 % CO2, 95% RH) and 42 h post incubation period. Test was performed with 3 replicates for each type of treatment. Tissues were first pre-wetted with 25 μL DPBS (Sterile Dulbecco’s Phosphate Buffered Saline), subsequently 25 mg (nominal) of the neat test substance was applied. 30 μL of DPBS was used as negative control and 5% of sodium dodecyl sulphate as positive control. Viability of the tissues was assessed in MTT test and compared to the negative control. The percentage of viability obtained with the test substance was 103.13%,which is well above the irritant limit of 50%. The study met all the validity criteria. Under the study conditions, the test substance was determined to be non-irritant to the skin (XCellR8, 2017).

Eye:

An in vitro study was conducted to determine the eye irritation potential of the test substance, 'Reaction products of hexadecyl dihydrogen phosphate, dihexadecyl hydrogen phosphate, hexadecan-1-ol, stearic acid, esters of C18 (branched and linear) fatty acids with C18 (branched and linear) alcohols, and potassium hydroxide', using Reconstructed human cornea-like Epithelium (RhCE), according to OECD Guideline 492, in compliance with GLP. EpiOcularTM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% RH. On Day 1, after pre-wetting tissues with 20 µL PBS (Sterile Dulbecco’s Phosphate Buffered Saline) for 30 ± 2 min, the tissues in triplicate were exposed to single topical application of 50 mg neat test substance or 50 µL of reference substances (negative control: sterile water; positive control: methyl acetate) for 6 h ± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion, and 18 h ± 15 minutes post-treatment incubation, prior to the MTT endpoint. On Day 2, MTT tests and measurement were performed 570 nm without reference filter. The viability of the tissues were assessed and compared to a negative control. The percentage viability obtained with the test substance was determined to be 84.2%,which is well above the threshold (i.e., >60%) indicating no irritation potential. Under the study conditions, the test substance was determined to be non-irritant to the eyes (XCellR8, 2019).

Justification for classification or non-classification

Based on the in vitro study results, the test substance does not warrant a classification for skin and eye according to the EU CLP criteria (Regulation 1272/2008/EC).