Reaction mass of N-(2-hydroxypropyl)decan-1-amide and N-(2-hydroxypropyl)octanamide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 26, 2017 to April 28, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The EPISKINTM(SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this 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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of N-(2-hydroxypropyl)decan-1-amide and N-(2-hydroxypropyl)octanamide

EC Number:

915-384-3

Molecular formula:

CnH2n+2NO2, where n=11 or 13

IUPAC Name:

Reaction mass of N-(2-hydroxypropyl)decan-1-amide and N-(2-hydroxypropyl)octanamide

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

EPISKINTM (SM) is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability. Cell viability determination is based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and conversion into a blue formazan salt that is quantitatively measured after extraction from tissues (Faller C. et al., 2002, Mosmann T., 1983). The reduction of cell viability in treated tissues is compared to negative controls and expressed as a %. The % reduction in viability is used to predict the irritation potential of the test substance.

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKINTM (SM) (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-017, Expiry Date: 01 May 2017).
EPISKINTM (SM) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. 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 (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

For killed epidermis, living epidermis units (Manufacturer: SkinEthic, France, Batch No.:17-EKIN-008, Expiry Date: 27 February 2017) were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37 °C in an incubator with 5 % CO2, in a >95 % humidified atmosphere for 48 hours (±1 hour). At the end of the incubation the water was discarded and the dead epidermis units were frozen (the frozen units can be used up to 6 months). Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Maintenance Medium). Further use of killed tissues was similar to living tissues.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

20 µL of pre- warmed test substance (~37 °C) were applied evenly to the epidermal surface.If necessary, the test substance was spread gently on the skin surface with a pipette tip (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours incubation at 37 °C, 5 % CO2. Subsequent tissues incubation for 3 hours with MTT solution (at 37 °C, 5 % CO2 protected from light)

Number of replicates:

3 replicates for the treatment with the test substance and three units per negative and positive controls.
Additional controls to examine following effects:
- colour contribution (NSCliving) from the test substance
- the possible MTT interaction potential of the test substance
- non-specific colour in killed tissues (NSCkilled)

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

6.2

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

As colour change (purple) was observed after three hours of incubation of the test substance in MTT working solution, thus the test substance might interact with MTT. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Additional controls on killed epidermis were included in the study. Based on observed mean OD (-0.012), the calculated NSMTT% is 1.5 %, the OD values were negative so these values excluded from the NSMTT calculation.

As the test substance was coloured, two additional test substance-treated tissues were used for the non specific OD evaluation. The mean optical density (measured at 570 nm) of tissues was 0.023, Non Specific Colour % was calculated as 3.0 %. This value was below 5 %, therefore additional data calculation was not necessary.

As the test substance was showed being an MTT-interacting substance and the test substance had an intrinsic colour, two additional test substance-treated killed tissues were used for the non specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.020, Non Specific Colour % (NSCkilled%) was calculated as 2.6 %. Because the NSCliving% was not used, therefore correction with NSCkilled% was not necessary.

Validity criteria:
- The mean OD value of the three negative control tissues was in the recommended range (0.776). Standard deviation of the viability results for negative control samples was 4.7.
- The positive control treated tissues showed 3.4 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.5.
- The standard deviation of viability values of the three test substance-treated tissue samples in the MTT assay was 1.3.
- The mean OD value of the blank samples (acidified isopropanol) was 0.047.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the study conditions, following exposure with the test substance, the mean cell viability was 6.2 % compared to the negative control. This is below the threshold of 50 %, therefore the test substance was considered as being irritant to skin.

Executive summary:

A study was conducted to determine the skin irritation potential of C8 -10 MIPA using Reconstructed Human Epidermis (RHE) Test Method according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. Three disks of EPISKINTM (SM were treated with the pre-warmed test substance and incubated for 15 minutes at room temperature. Exposure of the test substance was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5 % CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test substance. The possible MTT interaction potential of the test substance was examined using two additional test substance treated and two negative control treated killed epidermis units. Furthermore, to avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled), two additional disks were used. For each treated tissue, the viability was expressed as a % relative to the negative control. The experiment met the validity criteria, therefore the study was considered to be valid. Under the study conditions, following exposure with the test substance, the mean cell viability was 6.2% compared to the negative control. This is below the threshold of 50%, therefore the test substance was considered as being irritant to skin (Orovecz, 2017).