Reaction product of castor oil with glycerol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 - 14 Dec 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Demonstration of proficiency not in report; no historical control data range reported.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

adopted in 2015

Deviations:

yes

Remarks:

During the wash phase, MTT solution was added to all tissues for 2 minutes in error. This was then rectified by washing with DPBS. The study acceptance criteria were assessed and all met. Therefore, the deviation had no impact on the study.

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted in 2019

Deviations:

yes

Remarks:

During the wash phase, MTT solution was added to all tissues for 2 minutes in error. This was then rectified by washing with DPBS. The study acceptance criteria were assessed and all met. Therefore, the deviation had no impact on the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

Medicines & Healthcare products Regulatory Agency, Department of Health of the Government of the United Kingdom

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of castor oil with glycerol

EC Number:

949-117-7

Molecular formula:

not applicable, substance is UVCB

IUPAC Name:

Reaction product of castor oil with glycerol

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm (EPI-200)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™(EPI-200) (MatTek Corporation, Ashland, USA)
- Tissue batch number: 28675
- Keratinocyte strain: 00267
- Delivery date: 11 Dec 2018
- Date of initiation of testing: 11 Dec 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature and 37°C
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: several steps (not further specified)
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Incubation time: not specified
- Spectrophotometer: BMG LabTech FluoStar Optima
- Wavelength: 570 nm
- Filter: no filter used
- Filter bandwidth: not applicable, no filter used
- Spectrophotometer calibration date: 05 Nov 2018

FUNCTIONAL MODEL CONDITIONS (specified by MatTek Corporation)
- Viability: 2.004 ± 0.101 (acceptance range: 1.0 - 3.0)
- Barrier function: 5.21 h (acceptance range: 4.77 - 8.72 h)
- Morphology: 14 layers are present; thickness: 114.6 µm (acceptance range: >70 µm - <130 µm)
- Contamination: no contamination reported

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance did not directly reduce the MTT solution, an additional functional check was not performed.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating or corrosive to skin if the skin model viability after exposure and post-treatment incubation is ≤ 50%.
- The test substance is considered to be non-irritant and non-corrosive if the skin model viability after exposure and post-treatment incubation is > 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL

Duration of treatment / exposure:

60 ± 1 min (25 min at room temperature and 35 min at 37 °C, 5% CO2, ≥ 95% relative humidity)

Duration of post-treatment incubation (if applicable):

42 ± 4 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues

Run / experiment:

60 min

Value:

8.888

Vehicle controls validity:

not applicable

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: no
- Colour interference with MTT: yes, the level of background interference detected was sufficiently low that no correction was necessary (0.634%, threshold of 5%)

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 1: Viability measurements of test and reference items and controls.

Condition	Tissue #	Raw data		Blank corrected data		Mean OD	% of Viability
		Aliquot 1	Aliquot 2	Aliquot 1	Aliquot 2
NC	Tissue 1	2.041	2.033	1.887	1.879	1.883	111.874
	Tissue 2	1.67	1.668	1.516	1.514	1.515	90.008
	Tissue 3	1.82	1.791	1.666	1.637	1.651	98.118
PC	Tissue 1	0.196	0.187	0.042	0.033	0.037	2.218
	Tissue 2	0.181	0.184	0.027	0.030	0.028	1.684
	Tissue 3	0.187	0.194	0.033	0.040	0.036	2.159
TA1	Tissue 1*	1.049	1.043	0.895	0.889	0.892	52.991
	Tissue 2	0.281	0.287	0.127	0.133	0.130	7.714
	Tissue 3	0.333	0.314	0.179	0.160	0.169	10.061

NC: negative control (DPBS)

PC: Positive control (SDS 5%)

TA1: Test Item 1

*: Note that TA1 tissue was excluded as an outlier value and is not included in the final calculations.

Table 2: Mean and SD of cell viability measurements and of viability percentages

Name	Code	Mean of OD	SD of OD	Mean of viability (%)	SD of viability (%)	CV %	Classification
DPBS	NC	1.683	0.186	100.000	11.054	11.054	Non-Irritant
SDS 5%	PC	0.034	0.005	2.020	0.293	14.508	Irritation potential
Test Item	TA1	0.150	0.028	8.888	1.660	18.672	Irritation potential

NC: negative control (DPBS)

PC: Positive control (SDS 5%)

TA1: Test Item 1

Applicant's summary and conclusion

Interpretation of results:

other: irritating potential

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered non-irritant and non-corrosive based on a negative result in the Reconstructed human epidermis test method (in vitro skin irritation). A positive in vitro irritation response is not conclusive with respect to classification of the test substance as irritant (Skin Irrit. Cat. 2) or corrosive (Skin Corr. Cat. 1) and therefore requires further evaluation and/or data generation.

Executive summary:

The available data on skin irritation and corrosion indicate a skin irritating potential (OECD 439) but not a skin corrosion potential (OECD 431). There is regulatory acceptance in the EU that under these circumstances a substance is considered to be a skin irritant. The registered substance, therefore, meets the classification critera of Regulation (EC) No. 1272/2008 (CLP) and is classified as Skin Irrit. 2 (H315).