Methyl 12-hydroxyoctadecanoate

Endpoint summary

Description of key information

Non-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:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study under GLP

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.

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.

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)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Vertellus, 0000140430

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

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

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.: 25883
- Date of initiation of testing: 20 February 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C, followed by room temperature laminar flow
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 15 1mL DPBS washes, followed by 3 submersions in 150 mL DPBS
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP:

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: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 4.3%
- Morphology and Reproducibility: The EpiDerm (TM) epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum analogous to patterns found in vivo.
- Contamination: None

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.
The mixture of 25 mg of the test item per 300 µl aqua dest. and/or per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

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): 25 µL of sterile DPBS were applied to the epidermal surface in order to improve the contact between the powder and the epidermis. Afterwards, 25 mg (39 mg/cm^2) of the test item were applied directly atop the EpiDerm TM tissue using an application spoon, avoiding compression of the test item. The test item was spread to match size of the tissue by using a bulb-headed Pasteur pipette.

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):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative Control

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive Control

Value:

3.7

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item

Value:

88.9

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

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 (OECD 439, EpiDerm(TM)) using MTT reduction as an indication of tissue viability. The test substance was observed to be non-irritating at doses up to 25 mg/plate.

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:

2018 February 07 - 2018 March 26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study under GLP

Qualifier:

according to guideline

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)

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Method Summary No. 164: EpiOcular™ Eye Irritation Test

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT)

Version / remarks:

For the prediction of acute ocular irritation of chemicals For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Vertellus, 0000140430
- Expiration date of the lot/batch: 09 November 2018

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

Details on test animals or tissues and environmental conditions:

The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinised surface, showing a cornea-like structure analogous to that found in vivo.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg

Duration of treatment / exposure:

6 hr

Duration of post- treatment incubation (in vitro):

18 hr

Number of animals or in vitro replicates:

2/dose

Details on study design:

- Details of the test procedure used : RhCE tissue was treated with test substance for 6 hrs. After an 18 hr post-treatment incubation, tissue samples were evaluated for viability using an MTT reduction assay.
- RhCE tissue construct used, including batch number : EpiOcular™ tissues on agarose (Lot No.:27021, 27027)
- Doses of test chemical and control substances used : Negative control: 50 µL. Positive control: 50 µL. Test item: 50 mg. The test item was ground into a fine powder using a mortar and pestle. Approximately 50 mg (83.3 mg/cm^2) of the test item were applied directly to the EpiOcular TM tissue using an application spoon, avoiding compression of the test item. The test item was spread to match size of the tissue.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) : Exposure: 6 hr, 37 °C. Post-exposure immersion: 25 min, room temperature. Post-exposure incubation: 18 hr, 37 °C.
- Justification for the use of a different negative control than ultrapure H2O (if applicable) : N/A.
- Justification for the use of a different positive control than neat methyl acetate (if applicable) : N/A
- Description of any modifications to the test procedure
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable) : The mixture of 50 mg test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value killed tissue controls were performed for quantitative correction of results.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable) : 2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : 570
- Description of the method used to quantify MTT formazan : Absorbance (OD570)
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable) : N/A
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities obtained after treatment compared to the negative control tissues concurrently treated with Aqua dest. (distilled water). The test item is considered to be irritant to the eye but it cannot be differentiated between UN GHS “Category 1” or “Category 2”, if the relative tissue viability is less or equal to 60%. The test item is considered to be non-irritant in accordance with UN GHS “No Category” if relative tissue viability is higher than 60%.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : Yes
- Complete supporting information for the specific RhCE tissue construct used : Yes
- Reference to historical data of the RhCE tissue construct: Yes
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals : Yes
- Positive and negative control means and acceptance ranges based on historical data : Yes
- Acceptable variability between tissue replicates for positive and negative controls: Yes
- Acceptable variability between tissue replicates for the test chemical: Yes

Irritation parameter:

other: Tissue Viability [%]

Run / experiment:

Negative Control

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: Tissue Viability [%]

Run / experiment:

Positive Control

Value:

22.3

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

positive indication of irritation

Irritation parameter:

other: Tissue Viability [%]

Remarks:

NSMTT corrected

Run / experiment:

Test item

Value:

100.1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: Observed values in range of historical values

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was evaluated for eye irritation potential according to OECD Guideline 492. In this study under the given conditions the test item showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance displayed mild irritation to skin or eye, which, in either case, did not qualify for classification as irritants according to the ECHA Guidance on the interpretation of the CLP criteria (ver 5.0, 2015). The criteria for classification according to Regulation EC No. 1272/2008 are not met and the substance is not classified.