[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-09-24 to 2014-09-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The department of health of the government of the United Kingdom

Test material

Test material information

Constituent 1

Reference substance name:

Amides, C18, branched and linear

Molecular formula:

Not applicable as UVCB

IUPAC Name:

Amides, C18, branched and linear

Test material form:

solid: pellets

Details on test material:

- Name of test material (as cited in study report): Amides, C18, branched and linear
- Physical state: extremely pale yellow granular solid
- Lot/batch No.: 202637
- Expiration date of the lot/batch: 20 April 2016
- Storage condition of test material: at RT, protect from light
- Analytical purity: 100%

Test animals

Species:

human

Strain:

other: human-derived epidermal keratinocytes (EpiSkin)

Details on test animals or test system and environmental conditions:

TEST METHOD
EpiSkinSM tissues are provided as kits (SkinEthic)
The test was carried out with the reconstituted threedimensional human skin model EPISKINSM (SkinEthic). The skin model consists of normal (noncancerous), adult humanderived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagencoated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13 day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

ADAPTATION TO CELL CULTURE CONDITIONS
Upon receipt, tissues were transferred into 12 well plates containing 2 mL pre-warmed maintenance medium per well and pre-incubated in a humidified incubator for at least 24 h (37 °C, 5% CO2) before use.

INCUBATION CONDITIONS (INCUBATOR)
Temperature (°C): 37
CO2 gas concentration (%): 5

Test system

Type of coverage:

other: in vitro system

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

unchanged (no vehicle)

Controls:

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

Amount / concentration applied:

TEST MATERIAL:
Amount(s) applied (volume or weight with unit): 10 mg

NEGATIVE CONTROL SUBSTANCE:
Amount(s) applied (volume or weight with unit): 10 μL DPBS

POSITIVE CONTROL SUBSTANCE:
Positive control substance: 10 μL SDS, 5% (w/v)

Duration of treatment / exposure:

15 min

Observation 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.
- 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 pre-warmed MTT solution for 3 h at 37 °C and 5% CO2. After aspiration of the MTT solution, tissues were dried. Extraction of the formazan product was carried out in 500 μL isopropanol. The optical density was measured at 562 nm wave length in a microplate reader (Anthos 2001 microplate reader).

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: 42 h . Reversibility: other: not applicable. (migrated information)

Results 2

Irritation / corrosion parameter:

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

Value:

11.1

Remarks on result:

other:

Remarks:

Basis: other: mean value of positive controls (SDS). Time point: 42 h. Reversibility: other: not applicable. (migrated information)

Results 3

Irritation / corrosion parameter:

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

Value:

97.8

Remarks on result:

other:

Remarks:

Basis: other: mean value of the test item. Time point: 42 h . Reversibility: other: not applicable. (migrated information)

In vivo

Irritant / corrosive response data:

In comparison to the untreated control for the test item a cell viability of 97.8% was measured after 42 h. Based on the results of the study the test item is non-irritant.

Any other information on results incl. tables

Table 1: Mean OD562 values and percentage viabilities for the negative control item, positive control item and test item

	Negative control			Positive control			Test item
Tissue sample	1	2	3	1	2	3	1	2	3
OD562	0.787	0.825	0.781	0.095	0.071	0.099	0.735	0.822	0.785
OD562(mean values of replicates)	0.798			0.088			0.781
SD	0.02			0.02			0.04
Viability (%)	100			11.1			97.8

SD: Standard deviation

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