Amyris balsamifera, 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:

20 Jul 2017 - 21 Jul 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

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:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Amyris balsamifera, ext.

EC Number:

291-076-6

EC Name:

Amyris balsamifera, ext.

Cas Number:

90320-49-3

Molecular formula:

Not applicable due to the UVCB nature of the substance.

IUPAC Name:

Essential oil of Amyris obtained from the wood of Amyris balsamifera (Rutaceae) trees by steam distillation.

Test material form:

liquid: viscous

Remarks:

Pale yellow slightly viscous liquid

Details on test material:

Name of test material as cited in study report: Amyris oil
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from light

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: obtained from sponsor, batch L4275185
- Expiration date of the lot/batch: 31 October 2017
- Purity test date:04 July 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The liquid test item was applied undiluted (50 µl) directly on top of the tissue.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Tissue obtained by MatTec corporation from accredited insitutions, with the consent of the donor or the donor's legal next of kin.

Source strain:

not specified

Justification for test system used:

A human three dimensional epidermal test, is recommended in international guidelines (e.g. OECD and EC) to minimise the need of in vivo testing

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: The commercially available EpiDerm tissues were obtained from MatTek Corporation, Ashland MA, U.S.A. The Skin models tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was [complete, e.g. 10 kΩ]
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Test item incubation of 3 minutes at room temperature, or 1 hour at 37.0 ± 1.0°C (actual range 36.8 - 37.5°C).
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 36.8 - 37.5°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: at least one
- Modifications to validated SOP: none

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: Exposure was performed in duplicate
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability equal to or above 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

- The test substance is considered to be non-corrosive to skin if
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

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): undiluted Milli-Q

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): 8N

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

None

Number of replicates:

Duplicate

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

111

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was < 8%, indicating that the test system functioned properly. Furthermore Historical control data was shown for November 2013 to November 2016.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range
- Acceptance criteria met for positive control: The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
- Acceptance criteria met for variability between replicate measurements: In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be  30%.
- Range of historical values if different from the ones specified in the test guideline: see table

Any other information on results incl. tables

The above historical control data range of the controls were obtained by collecting all data over the period of November 2013 to November 2016

	Neg	Neg	Pos	Pos	Pos	Pos
	3 min OD570	1 h OD570	3 min OD 570	1h OD570	3 min viability	1h viability
Range	1.324 -2.615	1.361 – 2.352	0.0172 – 0.56	0.046 – 0.339	6 – 25	3 – 13
Mean	1.84	1.85	0.19	0.14	11.03	7.45
SD	0.26	0.22	0.09	0.06	4.39	2.51
n	81	83	80	77	38	38

SD = Standard deviation

n = Number of observations

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive to the skin

Remarks:

Based on CLP

Conclusions:

Based on the results of this study Amyris Oil does not need to be classified for skin corrosion in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

The skin corrosion potential of Amyris Oil was tested according to OECDTG431. A human three dimensional epidermal model (EpiDerm) was exposed topically to 50µL undiluted Amyris Oil, Milli-Q (negative control), or Potassium hydroxide (positive control) for 3 minutes, or 1 hour. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 111% and 117%, respectively. Both the negative and the positive control were considered valid. The mean relative tissue viability for Amyris Oil was above 50% after the 3-minute treatment and above 15% after the 1-hour treatment. Based these results, Amyris Oil does not need to be classified for skin corrosion in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).