Enzymatic hydrolysis products of Ophiopogon japonicus, Liliaceae, root

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Enzymatic hydrolysis products of Ophiopogon japonicus, Liliaceae, root

EC Number:

812-241-6

Cas Number:

952500-62-8

IUPAC Name:

Enzymatic hydrolysis products of Ophiopogon japonicus, Liliaceae, root

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Very clear beige powder
- Storage conditions: Ambient temperature (20±5 °C), keep away from humidity
- Batch No.: 150904-0120
- Production date: 2015-09-04
- Expiry date: 2017-09-04

In vitro test system

Test system:

human skin model

Remarks:

human reconstructed epidermis (tissues)

Cell type:

other: human reconstructed epidermis (tissues)

Cell source:

other: SkinEthic Laboratories

Vehicle:

unchanged (no vehicle)

Details on test system:

SkinEthicTM RHE
Description: SKINETHIC RhE: RECONSTRUCTED HUMAN EPIDERMIS, 0.5cm² reconstructed epidermal human keratinocutes. Cells are grown on inert polycarbonate filter on chemically defined medium, airlifted for 17 days, till a highly differentiated and stratified epidermis model is obtained comprising the main basal, supra basa, spinous and granular layers and a functional stratum corneum. The RHE model presents a histological morphology comparable to the in vivo human tissue.

Test system

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

other: not applicable

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL of sterile water and 16 +/-2 mg of the test item was applied to the epidermis surface.

CONTROLS
- Negative control:16 +/- 0.5 μL of Dulbecco’s Phosphate-Buffered Saline (D-PBS)
- Positive control: 16 +/- 0.5 μL of 5 % (w/v) aqueous solution of Sodium Dodecyl Sulfate (SDS)

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 42 +/- 1 minutes at room temperature.
At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5 % CO2 in a humidified incubator for 42 h.

Number of animals:

Triplicate tissues for test item, negative and positive controls

Details on study design:

PRE-INCUBATION STEP
After arrival, 9 RHE will be transferred into new 6-well plates with 1 mL/well of roome temperature SkinEthic Growth Medium (SGM). The absence of air bubbles will be checked by observing underneath the well of the plate. The plate will be then incubated from 1 hour and 30 minutes to 24 hours at 37°C +/- 1°C, 5+/-1% CO2.

PREPARATION OF TEST SOLUTIONS:
32 mg/cm² of test substance will be used
16 +/- 2 mg of test substance, will be applied to each insert, in triplicate.
Positve control: 5% SDS in sterile water
Negative control: DPBS

TREATMENT OF TISSUES
Before starting the test, RHE inserts will be transferred into a new 24- well plate, supplemented with 300µL of fresh SkinEthic Maintenance Medium (SMM) in each well.
10µL of sterile water and 16+/-2mg of solid test substance will be applied in the center of the insert in triplicate.
For negative and positive controls, 16 +/- 0.5 µL will be applied in triplicate.
The exposure of the tissues to the test and control items was performed at room temperature for 42 minutes (± 1 minute).

RINSING OF TISSUES AND INCUBATION FOR 42 HOURS
At the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS to remove any residual test or control items. The rinsed tissues were incubated at 37 °C, 5 % CO2 in a humidified incubator for 42 h.

MTT VIABILITY ASSAY
Following the 42 hour post-exposure incubation period: MTT test (MTT Loading/Formazan Extraction) was performed and tissues were incubated for 3 h (± 5 minutes) at 37 °C, 5 % CO2 in a humidified incubator.

OPTICAL DENSITY MEASUREMENTS
At the end of the formazan extraction period: The optical density was measured at 570 nm using a plate reader.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

79.55

Positive controls validity:

valid

Remarks:

Viability: 1.36%

In vivo

Irritant / corrosive response data:

Following a 42 minutes exposure and 42 h of recovery period, the relative mean viability of the tissues treated with the test item was 79.55 % with a Variation coefficient of 3.49 as assessed by the MTT assay.

Other effects:

None

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, "OF14 AT" was concluded to be NON IRRITANT.

Executive summary:

The test substance "OF14 AT" was subjected to skin irritation assay, conducted according to OECD 439 (2015).

The test was carried out using reconstructed human epidermis (RHE), in triplicate. The exposure of the insert to the test susbtance was carried out for 42 minutes at room temperature.

After treatment the inserts were rinsed with D-PBS and post-incubated with growth medium for additional 42 hours at 37 +/- 1°C and 5 +/-1% CO2. Finally, inserts were incubated with MTT solution in order to evaluate cell viability which is a direct measure of the irritant potential of the test substance.

Under these conditions, the test substance "OF 14 AT" was concluded to be non-irritant.