2-hydroxy-3-(oleoyloxy)propyl 5-oxo-L-prolinate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 - 27 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted in 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU method B.40 BIS. (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted in 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxy-3-(oleoyloxy)propyl 5-oxo-L-prolinate

EC Number:

283-393-3

EC Name:

2-hydroxy-3-(oleoyloxy)propyl 5-oxo-L-prolinate

Cas Number:

84608-82-2

Molecular formula:

C26H45NO6

IUPAC Name:

2-hydroxy-3-(oleoyloxy)propyl 5-oxo-L-prolinate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: Keratinocyte strain: 00267

Justification for test system used:

The EpiDerm™ tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized 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.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™, EPI-200-SCT
- Tissue batch number(s): 25832
- Delivery date: 25 July 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The inserts were thoroughly rinsed with DPBS, blotted with sterile cellulose tissue and set into the respective holding plate, using the wells containing assay medium.
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Anthos Reader 2010 Flexi, Anthos Microsysteme GmbH
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.466 ± 0.03 (acceptance criteria: OD (540 - 570 nm) = 1.0 - 3.0)
- Barrier function: 6.06 h (acceptance criteria: ET 50 = 4.77 - 8.72 h)
- Morphology: viable basal cell layer, intermediate spinous and granular layers and a functional stratum corneum
- Contamination: no
- Reproducibility: yes

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
And if criteria was met for corrosiveness and if the viability after 3 minutes exposure was less than 25%, then the substance is classified as Skin Corrosive Subcategory 1A, while a viability after 3 minutes exposure greater or equal to 25% would lead to a classification as Skin Corrosive Subcategory 1B or 1C
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 μL (a nylon mesh was added in order to ensure sufficient contact with the tissue surface (tissue size is not reported)
- Concentration: undiluted

NEGATIVE CONTROL
- Amount(s) applied: 50 μL
- Concentration: undiluted

POSITIVE CONTROL
- Amount(s) applied: 50 μL
- Concentration: 8M

Duration of treatment / exposure:

3 min / 60 min

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value of 2 replicates, test substance, 3 min exposure

Value:

96.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value of 2 replicates, test substance, 60 min exposure

Value:

91.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value of 2 replicates, positive control, 3 min exposure

Value:

27.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value of 2 replicates, positive control, 60 min exposure

Value:

8.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: tested, but no reduction was observed
- Colour interference with MTT: tested, but no interference was observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: not in the report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (OD = 1.7 (3 min and 1 h) ≥ 0.8 and ≤ 2.8 and within historical control range (3 min: 1.197 - 3.077 and 60 min: 1.377 - 2.571))
- Acceptance criteria met for positive control: yes (viabilty at 3 min = 27.9% < 50% and at 1 h = 8.1% < 15% and within historical control range (3 min: 9.6 - 57.3% and 60 min: 4.1 - 24.2%))
- Acceptance criteria met for variability between replicate measurements: yes, regarding to OECD TG 431 criteria (In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%

Any other information on results incl. tables

Table 1: MTT assay after 3 min exposure

	Negative control		Positive control		Test item
Tissue sample	1	2	1	2	1	2
OD570	1.681	1.749	0.503	0.522	1.674	1.661
	1.719	1.729	0.498	0.518	1.676	1.663
	1.729	1.728	0.493	0.520	1.671	1.677
OD570(mean-blank)	1.671	1.697	0.459	0.481	1.635	1.628
OD570(mean values)	1.684		0.470		1.632
rel. viability (%)	100.0		27.9		96.9
rel. SD	1.1		3.3		0.3

Table 2: MTT assays after 60 min exposure

	Negative control		Positive control		Test item
Tissue sample	1	2	1	2	1	2
OD570	1.710	1.721	0.170	0.167	1.532	1.584
	1.715	1.675	0.171	0.180	1.542	1.600
	1.709	1.702	0.172	0.181	1.537	1.614
OD570(mean-blank)	1.673	1.661	0.132	0.137	1.498	1.561
OD570(mean values)	1.667		0.135		1.530
rel. viability (%)	100.0		8.1		91.8
rel. SD	0.5		2.6		2.9

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive (Skin Irrit. 2 or not classified according to Regulation (EC) No 1272/2008)

Conclusions:

Under the conditions of the test, the test substance was shown to have no corrosive potential towards reconstructed human epidermis tissue in the EpiDerm™ model. The result does not allow for the non-classification or classification as irritant of the test substance and therefore further evaluation and/or data generation is required.