Storax (balsam)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Apr 2017 - 01 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Storax (balsam)

EC Number:

232-458-4

EC Name:

Storax (balsam)

Cas Number:

8046-19-3

Molecular formula:

Not applicable, UVCB - NCS

IUPAC Name:

Storax (balsam)

Test material form:

other: liquid

Details on test material:

CAS : 8046-19-3 EINECS : 232-458-4
- Name of test material (as cited in study report): STYRAX RESINOID OIL
- Test Facility test item number: 208399/A
- Appearance: Orange viscous liquid
- Substance type: pure substance
- Physical state: liquid
- Analytical purity: 100%

Specific details on test material used for the study:

Identification: Styrax resinoid oil
Appearance: Orange viscous liquid
Batch: K17 031-1
Test item storage: At room temperature
Stable under storage conditions until: 02 February 2019

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: multiple donors

Source strain:

other: not applicable

Details on animal used as source of test system:

- Source: SkinEthic Laboratories, Lyon, France.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model TM
- Tissue batch number(s): 17-Ekin-017, 16-Ekin-047, 17-Ekin-003
- Test System: This model is a three-dimensional human epidermis model, which consists of adult humanderived 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.
- Test System Set Up: On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for approximately 22 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.
- Killed tissues: Living epidermis was transferred to 12 well plates and incubated with 2 ml Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored at ≤ -15°C. Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 ml maintenance medium. Further use of killed tissues was similar to living tissues.
- MTT medium: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).
- Environmental conditions: All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 74 - 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 - 37.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 36.3 - 37.4°C

APPLICATION AND REMOVAL OF TEST MATERIAL AND CONTROLS
The test was performed on a total of 3 tissues per test item together with negative and positive controls. Twenty five μl of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. Since the test item reacted with the MTT medium, in addition three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
Test for Color Interference by the Test Item: Styrax resinoid oil was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, at least 10 mg of the test item was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the incubation period a color check was performed.

Test for Reduction of MTT by the Test Item: After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-solution (0.3 mg/ml in PBS). The tissues were incubated 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 pre-labeled microtubes and extracted with 500 μl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 69 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
17-EKIN-017:
- Passage: 2
- Histology scoring (HES stained vertical paraffin sections): 21.6 ± 1.1 (CV = 5.2%), satisfactory
- IC50 determination (SDS concentration, MTT test): 1.8 mg/L
- Statistical analysis: Histology, probability 0.95 that 100% of the batch > 20. IC50, probability 0.95 that IC50≥ 1.7 mg/mL (threshold value)
- Biological safety: absence of HIV1 and 2 antibodies, Hepatitis C antibodies, Hepatitis B antigen HBs, bacteria, fungus and mycoplasma.

16-EKIN-047:
- Passage: 2
- Histology scoring (HES stained vertical paraffin sections): 22.6 ± 0.2 (CV = 0.9%), satisfactory
- IC50 determination (SDS concentration, MTT test): 1.9 mg/L
- Statistical analysis: Histology, probability 0.95 that 100% of the batch > 20. IC50, probability 0.95 that IC50≥ 1.9 mg/mL (threshold value)
- Biological safety: absence of HIV1 and 2 antibodies, Hepatitis C antibodies, Hepatitis B antigen HBs, bacteria, fungus and mycoplasma.

17-EKIN-003:
- Passage: 2
- Histology scoring (HES stained vertical paraffin sections): 22.8 ± 0.3 (CV = 1.1%), satisfactory
- IC50 determination (SDS concentration, MTT test): 1.9 mg/L
- Statistical analysis: Histology, probability 0.95 that 100% of the batch > 20. IC50, probability 0.95 that IC50≥ 1.6 mg/mL (threshold value)
- Biological safety: absence of HIV1 and 2 antibodies, Hepatitis C antibodies, Hepatitis B antigen HBs, bacteria, fungus and mycoplasma.

NUMBER OF REPLICATE TISSUES:
The test was performed on a total of 3 tissues per test item together with negative and positive controls.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Styrax resinoid oil was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the color interference, at least 10 mg of Styrax resinoid oil was added to 90 μl Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μl Milli-Q water was tested concurrently. At the end of the shaking period a color check was performed.

PREDICTION MODEL / DECISION CRITERIA
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.

A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 μL
- Concentration: undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL, PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL, SDS (re-spread after 7 minutes contact time.)
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

after 15 min exposure

Run / experiment:

mean

Value:

80

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Standard deviation 9.8%

Other effects / acceptance of results:

RESULT:
The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Styrax resinoid oil compared to the negative control tissues was 80%. Since the mean relative tissue viability for Styrax resinoid oil was above 50% Styrax resinoid oil is considered to be non-irritant.

OTHER EFFECTS:
Styrax resinoid oil was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because a color change was observed by adding MTT-medium it was concluded that Styrax resinoid oil did interact with the MTT endpoint. In addition to the normal procedure, three killed tissues treated with test item and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by Styrax resinoid oil was 7.8% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 20%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 11%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, Styrax resinoid oil is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

To evaluate Styrax resinoid oil for its ability to induce skin irritation, a human three dimensional epidermal model (EPISKIN Small model (EPISKINSMTM)) was used according to OECDTG 439. Styrax resinoid oil was applied undiluted (25μl), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Styrax resinoid oil did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by Styrax resinoid oil was 7.8% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.

 

The positive control had a mean cell viability of 20% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range.The standard deviation value of the percentage viability of three tissues treated identically was less than 11%, indicating that the test system functioned properly. Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Styrax resinoid oil compared to the negative control tissues was 80%.

 

In conclusion, Styrax resinoid oil is non-irritant in the in vitro skin irritation test under the experimental conditions described and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.and according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).