Fir, Abies balsamea, ext.

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Nov 2017 - 17 Nov 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:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fir, Abies balsamea, ext.

EC Number:

285-364-0

EC Name:

Fir, Abies balsamea, ext.

Cas Number:

85085-34-3

IUPAC Name:

Fir Balsam Oil

Test material form:

liquid

Details on test material:

Name of test material as cited in study report: Fir Needle Oil Canadian (Fir, Abies Balsamea, ext)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Botanical source and Lot# 67919
- Expiration date of the lot/batch: 13 septembre 2018
- Purity test date: UVCB, considered 100% pure

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25 °C, ≤ 70 RH%), protected from light, avoided contact with iron.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None
- Preliminary purification step (if any): None
- Final dilution of a dissolved solid, stock liquid or gel: No dilution performed. Test item applied as-is
- Final preparation of a solid: not applicable/test item is a liquid

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN Small Model(TM), 0.38cm2, Batch# 17-EKIN-042,Manufactured by SkinEthic, France

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult Donors

Source strain:

other: Strain no:09-KERA-007 + 09-KERA-010

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Recommended by the relevant OECD guideline for corrosivity

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used:
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).
- 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 dodecylsulphate (SDS)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (20.8 - 24.5 deg C) for 4 hours
- Temperature of MTT test (if applicable): 37 deg C for 3 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Not specified
- Observable damage in the tissue due to washing: None

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Wavelength: 570 nm


NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
- Two replicates test item, two negative controls and two positive controls were run. additionally, as the test item had an MTT interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used.


PREDICTION MODEL / DECISION CRITERIA
- The prediction model below corresponds to the methods agreed by EU regulatory agencies in line with OECD No. 431 (OECD, 2016).
- The cut-off value of 35% and classification method was validated in an international validation study of this kit (Fentem, 1998).

For 2 disks:
If both disks have mean viability of ≥35% = Non Corrosive
If both disks have mean viability of <35% = Corrosive (at the corresponding incubation period)

For more than 2 disks:
If the mean value is ≥35% and the variability is less than 50% = Non Corrosive
If the mean value is <35% and the variability is less than 50% = Corrosive

Otherwise:
If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35%. Outlier values may be excluded where there are scientific reasons, such as where application or rinsing is difficult and that the Study Director considers that a result is not representative.

Classification Packing group: Criteria for In Vitro interpretation:
UN GHS / EU-CLP Sub Category 1A If corrosive after 3 min exposure
Sub Category 1B If not corrosive after 3 min exposure and corrosive after 1 hour exposure
Sub Category 1C If not corrosive after 1 hour exposure and corrosive after 4 hours exposure
Non corrosive If not corrosive after 4 hours exposure

Control samples:

yes, concurrent negative control

yes, concurrent positive control

other: As the test item had an MTT interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): Physiological saline (0.9% (w/v) NaCl solution)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): undiluted Glacial Acetic Acid by VWR Lot#15A160011


The same treatment steps were followed in case of the killed tissues as in case of the living tissues.

Duration of treatment / exposure:

4 hours (±10 min) at room temperature (20.8-24.5°C)

Duration of post-treatment incubation (if applicable):

After the incubation times, all test item treated tissues or also the positive control tissues or also the additional control tissues (as mentioned in 3.6.2.) were removed and rinsed thoroughly with PBS solution to remove all the remaining test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly.
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units. The lid was replaced and the plate incubated at 37°C in an incubator with 5% CO2 for 3 hours (±15 minutes), protected from light, in a >95% humidified atmosphere.

Number of replicates:

2

Test animals

Details on test animals or test system and environmental conditions:

Not applicable

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

4 hours exposure

Value:

109.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean OD 0.8 = 100% RV

Positive controls validity:

valid

Remarks:

Mean OD 0.006 = 0.8% RV

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: NSMTT% = -0.9
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not Specified

ACCEPTANCE OF RESULTS:
- The mean OD value of the two negative control tissues should be ≥ 0.6 and ≤ 1.5 and negative control OD values should not be below historically established boundaries.
- The acceptable mean viability % range for positive control is ≤ 20%.
- The difference of viability between the two tissue replicates should not exceed 30%.
- The mean OD value of the blank samples (acidified isopropanol) should be <0.1.

-Range of historical values if different from the ones specified in the test guideline: SEE TABLE

Any other information on results incl. tables

The Non-Specific Colour % (NSC_living%) was determined in the In Vitro Skin Irritation Test. This value was below 5% and additional data calculation was not necessary, therefore check-method for colouring potential of test-items the use of additional controls for the non specific OD evaluation was not necessary in this study

Negative values of additional controls (e.g.: NSMTT%) were excluded from the calculation, but this did not change the result or integrity of the study.

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive to the skin

Remarks:

Based on CLP criteria (Annex I 1272/2008/EC)

Conclusions:

In conclusion, in this in vitro EpiSkin™(SM) model test with Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) (Batch number: 67919), the results indicate that the test item is non-corrosive to the skin in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

An in vitro skin corrosivity test of Fir Needle Oil Canadian (Fir, Abies Balsamea, ext)test item was performed in a reconstructed human epidermis model. EPISKIN^TM(SM)is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (detailed in 3.6. section). The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

Disks of EPISKIN^TM(SM) (two units) were treated with Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units.For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

Following exposure with Fir Needle Oil Canadian (Fir, Abies Balsamea, ext), the mean cell viability was 109.1% compared to the negative control (after adjustment for non-specific MTT reduction). This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™(SM) model test with Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) (Batch number: 67919), the results indicate that the test item is non-corrosive to the skin. The test item was not corrosive after the exposure time of 4 hours, therefore additional experiment with additional exposure times were not necessary to the classification.