Fatty acids, dehydrated castor-oil

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 - 10 Sept 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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 22 July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, dehydrated castor-oil

EC Number:

263-061-4

EC Name:

Fatty acids, dehydrated castor-oil

Cas Number:

61789-45-5

Molecular formula:

not applicable, substance is UVCB

IUPAC Name:

Fatty acids, dehydrated castor-oil

Details on test material:

- Name of test material (as cited in study report): fatty acids, dehydrated castor-oil
- Analytical purity: 100%
- Substance type: UVCB
- Physical state: clear light yellow liquid
- Lot/batch No.: 20110372
- Expiration date of the lot/batch: 25 May 2014
- Stability under storage conditions: stable
- Storage condition of test material: at room temperature in the dark

Test animals

Species:

human

Strain:

other: EPISKIN SMALL MODEL TM, three-dimensional human epidermis model

Details on test animals or test system and environmental conditions:

TEST SKIN MODEL
- Source: SkinEthic Laboratories, Lyon, France
- Batch no.: 12-EKIN-032


TEST METHOD
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.


ADAPTATION TO CELL CULTURE CONDITIONS
Upon receipt, tissues were transferred into 12-well plates and preincubated with prewarmed Maintenance Medium (Skinethic Laboratories, Lyon, France) for approximately 25 h at 37 °C before use.

INCUBATION CONDITIONS
- Temperature (°C): 37 ± 1
- CO2 gas concentration (%): 5.0 ± 0.5
- Humidity: 80-100%

Test system

Type of coverage:

other: open (in vitro system)

Preparation of test site:

other: reconstructed human epidermis

Vehicle:

unchanged (no vehicle)

Controls:

other: concurrent control tissues treated with PBS served as negative controls, positive controls were exposed to 5% SDS

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: 5% (aq) sodium dodecyl sulphate SDS in PBS (the positive control substance was re-spread after 7 minutes contact time)

Duration of treatment / exposure:

15 min (at room temperature)

Observation period:

Not applicable. Post-treatment incubation period: 42 h

Number of animals:

Not applicable.The test was performed in triplicates for each treatment and control group.

Details on study design:

TEST SITE
- Area of exposure: 0.38 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: The test item was washed from the skin surface with phosphate buffered saline (PBS).
- Time after start of exposure: 15 min
- Post-treatment incubation period: 42 h

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed 42 h after the incubation period. Therefore, tissues were incubated in 2 mL MTT medium (0.3 mg/mL) for 3 h at 37 °C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µl isopropanol. Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the Multiskan spectrum.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: cell viability (%)

Value:

100

Remarks on result:

other:

Remarks:

Basis: other: mean value of negative controls (PBS). Time point: 15 min. Reversibility: other: not applicable. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

5

Remarks on result:

other:

Remarks:

Basis: other: mean value of positive controls (5% SDS). Time point: 15 min. Reversibility: other: not applicable. (migrated information)

Results 3

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

106

Remarks on result:

other:

Remarks:

Basis: other: mean value of the test item. Time point: 15 min. Reversibility: other: not applicable. (migrated information)

In vivo

Irritant / corrosive response data:

The test substance did not interact directly with MTT as determined before study start. The absolute mean OD570 of the negative control tissues were within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 12%, indicating that the test system functioned properly.The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 106%. Since the mean relative tissue viability of the test substance was above 50%, the test substance is considered to be non-irritant.

Any other information on results incl. tables

Table 1: MTT assay after 15 min exposure

	Negative control (PBS)			Positive control* (5% SDS)			Test item
Tissue sample	1	2	3	1	2	3	1	2	3
OD570	1.163 1.265	1.065 1.157	1.139 1.233	0.055 0.057	0.050 0.053	0.059 0.060	1.029 1.260	1.184 1.188	1.410 1.382
OD570 (mean)	1.214	1.111	1.186	0.056	0.051	0.059	1.145	1.186	1.396
OD570 (mean values of replicates)	1.170			0.056			1.242
SD	0.053			0.004			0.135
Mean tissue viability (%)	100			5			106

SD: standard deviation

OD: optical density

*The positive control was repeated in a second experiment with the same batch of skin and 5% (aq) SDS. In the first experiment the positive control had a mean cell viability after 15 minutes exposure of 46% and did not meet the acceptability criteria. In the second experiment, the positive control had a mean cell viability of 5% and met the acceptability criteria. It was concluded that the deviation in the mean cell viability of the positive control in the first in vitro skin irritation test was caused by a technical error.

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified