Trans-esterification product of castor oil and 2-ethyl-2-(hydroxymethyl)propane-1,3-diol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April-December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, 28 July 2015.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

“In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”. 06-Jul-2012.

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

07-Nov-2014

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,2-bis(hydroxymethyl)butyl (R)-12-hydroxyoleate

EC Number:

266-551-6

EC Name:

2,2-bis(hydroxymethyl)butyl (R)-12-hydroxyoleate

Cas Number:

67025-99-4

Molecular formula:

C24H46O5

IUPAC Name:

2,2-bis(hydroxymethyl)butyl 12-hydroxyoctadec-9-enoate

Constituent 2

Reference substance name:

Castor oil fatty acid di- and tri-esters with trimethylolpropane

Molecular formula:

UVCB - Variable molecular formula and weight

IUPAC Name:

Castor oil fatty acid di- and tri-esters with trimethylolpropane

Constituent 3

Reference substance name:

castor oil fatty acid (non-ricinoleate) mono-ester with trimethylolpropane

Molecular formula:

UVCB - Variable molecularformula and weight

IUPAC Name:

castor oil fatty acid (non-ricinoleate) mono-ester with trimethylolpropane

Constituent 4

Reference substance name:

Castor oil, ester with glycerol

EC Number:

270-616-4

EC Name:

Castor oil, ester with glycerol

Cas Number:

68459-67-6

Molecular formula:

UVCB - Varied molecular weight and formula

IUPAC Name:

castor oil fatty acid esters with glycerol

Constituent 5

Reference substance name:

Castor oil

EC Number:

232-293-8

EC Name:

Castor oil

Cas Number:

8001-79-4

Molecular formula:

UVCB

IUPAC Name:

castor oil

Constituent 6

Reference substance name:

Glycerol

EC Number:

200-289-5

EC Name:

Glycerol

Cas Number:

56-81-5

Molecular formula:

C3H8O3

IUPAC Name:

propane-1,2,3-triol

Constituent 7

Reference substance name:

Fatty acids, castor-oil

EC Number:

263-060-9

EC Name:

Fatty acids, castor-oil

Cas Number:

61789-44-4

Molecular formula:

UVCB - Varied formula and molecular weight. Mostly C18, some C16 and possibly other carbon chain components

IUPAC Name:

fatty acids, castor oil

Constituent 8

Reference substance name:

Propylidynetrimethanol

EC Number:

201-074-9

EC Name:

Propylidynetrimethanol

Cas Number:

77-99-6

Molecular formula:

C6H14O3

IUPAC Name:

2-ethyl-2-(hydroxymethyl)propane-1,3-diol

Test material form:

liquid

Details on test material:

UVCB. Batch No. 0000177755. Expiration date: 26 Aug 2020. Clear yellow-amber liquid with mild odour at 20 degrees C. Stored at room temperature. Stable for the duration of testing.
Composition: 100%, Trans-esterification product of castor oil and 2-ethyl-2(hydroxylmethyl)propane-1,3-diol (TMP), CAS No. 68551-65-5.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Vertellus Industries, Batch 0000177755

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: assumed to be stable for duration of study

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek EpiDerm™ SIT Kit

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model
- Tissue batch number(s): Lot No.: Not specified
- Date of initiation of testing: Not specified

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.4-37.5°C, 5% CO2
- Temperature of post-treatment incubation (if applicable): Ambient

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 15 DPBS washes, followed by 3 submersions in 150 mL DPBS
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 5 mg/mL MTT diluted in DMEM to a final concentration of 1 mg/mL
- Incubation time: 3 hr
- Spectrophotometer: Standard plate spectrophotometer
- Wavelength: 570 nM
- Filter: yes
- Filter bandwidth: band pass of maximum ± 30 nm
- Linear OD range of spectrophotometer: 1.0-2.5
- Functional Evaluation of Test Chemicals for Potential Interference with the Assay: In order to determine if the physical and/or chemical characteristics of the test substance could potentially interfere with the MTT endpoint, such as if the material is colored and/or able to reduce MTT directly; evaluations were performed according to MatTek EpiDerm™ SIT Kit instructions. MTT reduction is a concern if the test substance cannot be fully rinsed from the tissue prior to MTT extraction.
- Test for Potential Staining and/or Color Change: Approximately 30 µL of liquid test substance was added to 0.3 ml of deionized water in a clear glass vial. After 62 minutes of incubation at 37°C in an atmosphere containing 5% CO2, the mixture was shaken and evaluated for presence and intensity of staining/coloration. Color change was not significant.
- Test for MTT reducers: Approximately 30 µL of liquid test substance was added to 1.0 ml of MTT medium in a clear glass vial. After 62 minutes of incubation at 37°C in an atmosphere containing 5% CO2, the mixture was shaken and evaluated for presence and intensity of staining/coloration. A vial of MTT medium served as a control. No significant color change was noted and thus it was not deemed a potential MTT reducer.
- Mesh compatibility: Approximately 30 µL of liquid test substance was added to a nylon mesh on a slide and incubated for 62 minutes at ambient temperature. Afterward the slide was checked under the microscope and evaluated for evidence of reaction with the mesh. No direct reaction with the mesh was observed. However, the material was difficult to rinse, therefore mesh was not used with the test substance in this assay.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3 per condition (vehicle control, positive control, test item)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the tissue viability after exposure and post-incubation is less than or equal to 50%. Further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification of the test substance. The test substance may be considered as non-irritant to skin in accordance with UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL of the undiluted test substance, negative control or positive control substances was added to the surface of three single viable tissues per substance. A piece of sterile mesh was placed on each tissue to assure good distribution and contact with the surface except for those treated with test substance as the mesh was known to adhere to the surface during rinsing .

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

45 h (26 + 19)

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative Control

Value:

100

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive Control

Value:

2

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item

Value:

97

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

All values for test validity were met; viability of cells in the test assay was 97%, indicative of high survival and low risk of irritation.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was evaluated for skin irritation potential by assessing its effect on a reconstituted three-dimensional human epidermis model (Epiderm™) using MTT reduction as an indication of tissue viability. The test substance was observed to have high cell viability (97%) and thus be non-irritating.