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

Endpoint summary

Description of key information

Not irritating to skin or eye

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:

April-December 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 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

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

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative Control

Value:

100

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive Control

Value:

2

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1991 May-June

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The analogue approach is used for the hazard assessment of toxicological endpoints for the registration of the target substance trimethylolpropane ricinoleate (CAS 68551-65-5) based on generation of different breakdown/metabolic products, resulting not only in similar physical and biological systems (Scenario 2 of the Read-Across Assessment Framework (RAAF, ECHA, 2015), but also consequently in similar physico-chemical and toxicological properties. The source compounds for read-across are fatty acids, C16-18, esters with pentaerythritol (CAS 85116-93-4) and pentaerythritol ricinoleate (CAS 78-22-8). It is proposed that the different alcohols resulting from ester hydrolysis of the source compounds and the target substance will not result in significant variation in biological effects.
Neither target nor source compounds are classified for mammalian hazardous effects. The use of reliable experimental data, all evaluated as reliable according to Klimisch scores of 1 or 2, to estimate the toxicity of the registered substance is adequate for the purposes of fulfilling the data requirements of registration and classifying potential hazards. Similar grouping into categories has been accepted by other regulatory agencies (U.S. EPA, 2010; U.S. FDA for food notifications). Thus, this read-across approach is adequate for the purposes of risk assessment and classification and labeling.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

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:
Henkel, 51531022
- Expiration date of the lot/batch: 1992 July 25
- Purity test date: 1991 January 25

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable up to 18 months
- Solubility and stability of the test substance in the solvent/vehicle:
Stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
N/A
- Final dilution of a dissolved solid, stock liquid or gel:
Test substance was suspending in Tween 80 (1:1 ratio), heated to 70 °C, and diluted with distilled water to desired concentration

Species:

rabbit

Strain:

other: Kleinrussen

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Thomae, Biberach/D.
- Age at study initiation: 4 months
- Weight at study initiation: 2090 g
- Housing: single cages
- Diet (e.g. ad libitum): ad libitum Ssniff rabbit diet K4, Ssniff GmbH, Soest/D.
- Water (e.g. ad libitum): ad libitum tap water
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature, 20-25 °C
- Humidity (%): 45-70%
- Photoperiod (hrs dark / hrs light): 12 hrs

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 ml

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): warm water
- Time after start of exposure: 24 hours

SCORING SYSTEM: Cornea opacity (0-4), Iris (0-2), Conjunctivae redness (0-3), Conjunctivae chemosis (0-4), Exsudation (0-3)

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

other: exsudation

Basis:

mean

Time point:

24/48/72 h

Score:

0.1

Max. score:

1

Reversibility:

fully reversible

Interpretation of results:

GHS criteria not met

Conclusions:

The analogue test substance was evaluated for eye irritation under OECD guideline 405. The test substance is not considered an eye irritant; nor is the target (registered) substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A guideline in vitro skin corrosion study was performed under GLP using the Epiderm™ model, with the results of 97% cell viability, interpreted as clearly non-irritating. This new contemporary study, performed by GLP according to guideline on the test material itself, allows the conclusion of "non-irritating to skin" to be drawn. This is consistent with the general conclusion on the category of polyol esters of fatty acids (analogue substances) which are not known to be dermal irritants (Becker, et al., 2015 on Pentaerythrityl Tetraesters; U.S. EPA Screening Level Hazard Characterization on Polyol Esters Category, 2011), due in part to low bioavailability via the dermal route. Using an analogue test material of castor oil esters of pentaerythritol mixed with a small amount of additive known to be corrosive to skin, a guideline in vivo irritation study using occlusive wrappings in 3 rabbits demonstrated no corrosion but some irritation of the skin. The irritation effect was attributed to the corrosive additive and the presence of the additive complicates the interpretation of this study.

An eye irritation study demonstrated that an analogue substance was not irritating.

Justification for classification or non-classification

The in vitro skin irritation study of the registered substance resulted in a finding of no irritation. Similarly, no irritation was seen in studies of exposure to the eye. The criteria for classification according to Regulation EC No. 1272/2008 are not met.