Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extraction

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extraction

EC Number:

948-256-0

IUPAC Name:

Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extraction

Specific details on test material used for the study:

exudate by hexane extraction
Batch number: 06/1234
EC number: 948-256-0
Appearance: Amber crystal
Purity: 100% UVCB
Expiry date: 29 February 2020
Storage conditions: Refrigerated (2-8°C), protected from light and humidity (stored in a tightly closed container)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: Human

Justification for test system used:

The EPISKINTM (SM) model has been validated for irritation testing in an international validation study 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.

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied in its original form; no formulation was required (although it was grounded to fine powder).

Details on test system:

SKIN DISC PREPARATION
- Procedure used:
- Quality control for skin discs: EPISKINTM (SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma.
The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKINTM(SM) test kits used in the present study) and are documented

PERFORMANCE OF THE STUDY
Pre-incubation: (day -1)
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight (at least 18 hours) at 37°C in an incubator with 5 % CO2 in a >95% humidified atmosphere.

Application and rinsing: (day 0)
1/ Test Item
First an appropriate amount (10 µL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 10 mg of the powdered test item was applied evenly to the epidermal surface. The amount was sufficient to cover the epidermal surface.
2/ Negative and positive controls
20 µL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette.


The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (23.4-25.8°C).
After the 15 minutes incubation time, the EPISKINTM (SM) units were removed and rinsed thoroughly one or two times with 25 mL PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2, in a >95 % humidified atmosphere.

MTT test: (day 2)
After the 42 hours incubation, all EPISKINTM (SM) units (except of the two living colour control units, which were incubated with Assay Medium) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKINTM (SM) units were incubated for 3 hours at 37°C in an incubator with 5% CO2 protected from light, in a >95 % humidified atmosphere.

Formazan extraction: (day 2)
After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 µL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

Cell viability measurement: (day 2)
Following the formazan extraction, 2×200 µL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 µL/well) was used as blank.
The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 13 August 2018, calibration is valid until August 2020) at the required wavelength on each day before use.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

Test item: 10µL
Negative and positive controls: 20 µL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours incubation

Number of replicates:

In this assay, three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

ca. 105.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

ca. 103

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

ca. 122.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

As no colour change (yellow colour) was observed after three hours of incubation of the test item in MTT working solution, the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.

As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues was 0.015, Non-Specific Colour % (NSCliving%) was calculated as 2.0% (see Table 1). This value was below 5%, therefore additional data calculation was not necessary.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Executive summary:

Following exposure to Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extraction, the mean cell viability was 110.4% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKINTM (SM) model test with Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extraction (Batch number: 06/1234), the results indicate that the test item is non-irritant to skin.