Amyris balsamifera, ext.

Endpoint summary

Description of key information

Skin irritation (OECD TG 439): irritating

Skin corrosion (OECD TG 431): not corrosive

Eye irritation (OECD TG 437): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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

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)

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.

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

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

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

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).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05-09-2016 to 12-09-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test item :207778/A
- Source and lot/batch No.of test material: L4275185
- Expiration date of the lot/batch: 31 October 2017 (retest date)

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

Test system:

human skin model

Remarks:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-036

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France.

Source strain:

other: Not applicable

Details on animal used as source of test system:

Not applicable

Justification for test system used:

EPISKIN 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:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:EPISKIN Small ModelTM
- Tissue batch number(s):16-EKIN-036
- Production date/ Shipping date / Delivery date: No data
- Date of initiation of testing: 5 September 2016

PRE-TEST PROCEDURE:
- Pre-incubation: On day of receipt the tissues were transferred to 12-well plates and preincubated with
prewarmed maintenance Medium for approximately 22 hours at 37°C.
- Test item colour interference: To assess colour interference, 10 μL of test item 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 colour check was performed.
- Test item MTT reduction: To assess the ability of the test item to reduce MTT, 25 μL 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 colour check was performed.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure: room temperature
- Temperature used during treatment / post-treatment incubation : 36.1 - 37.1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml in PBS
- Incubation time: 3 hours at 37 °C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: No data
- Linear OD range of spectrophotometer: No data

DECISION CRITERIA
- A test substance 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 substance and 42 hours of post incubat ion is ≤ 50% of the mean viability of the negative controls.
- A test substance 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 substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25µl

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25µl (aq)Phosphate buffered saline (PBS, Merck KGaA, Darmstadt, Germany).

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25µl Sodium dodecyl sulphate (SDS, Sigma, Steinheim, Germany) [CAS Number 151-21-3] in PBS
- Concentration (if solution): 5% (aq)

Duration of treatment / exposure:

15 ± 0.5 minutes (the positive control was re-spread after 7 minutes contact time).

Duration of post-treatment incubation (if applicable):

After rinsing with PBS tissues were moved to a new well with 2 ml pre-warmed maintenance medium and incubated for 42 hours at 37°C.
After exposure and incubation, tissues were dried and transferred to 2 ml MTT-solution (0.3 mg/ml in PBS). The tissues were incubated for 3 h at 37°C. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 71 hours.

Number of replicates:

3

Details on test animals or test system and environmental conditions:

Not applicable

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Relative mean tissue viability compared to the negative control tissues (100%)

Value:

8.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

5.3

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: No colour changes observed
- Colour interference with MTT: Not colour changes observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: No data

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean cell viability of 5.3% after 15 ± 0.5 minutes exposure.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was 11% or less, indicating that the test system functioned properly.

Interpretation of results:

other: Skin Irritant

Remarks:

Category 1 or Category 2

Conclusions:

Under the conditons of this test, the relative mean tissue viability for the test item determined to be 8.1%. Based on this result, the substance is considered to be an irritant. Under the conditions of this test, the test item should be classified as category 1 or category 2 skin corrosion/irritation in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).

Executive summary:

The possible skin irritation potential of Amyris oil was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD

TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 μL undiluted test substance. After 42 hours 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 (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 5.3% after 15

minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 11%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 8.1%. Based on this result, the substance is considered to be irritant and needs to be classified for skin irritation as category 1 or category 2 skin corrosion/irritation in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16-09-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test item :207778/A
- Source and lot/batch No.of test material: L4275185
- Expiration date of the lot/batch: 31 October 2017 (retest date)

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

Species:

other: Bovine

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source:Vitelco, 's Hertogenbosch, The Netherlands
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)).
- Time interval prior to initiating testing: Bovine eyes were used as soon as possible after slaughter
- indication of any existing defects or lesions in ocular tissue samples: no

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):750 µl
- Concentration (if solution): unchanged

Duration of treatment / exposure:

10 ± 1 minutes at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

After exposure, corneas were incubated for 120 ± 10 minutes at 32 ± 1 °C

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
-The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32°C. The corneas were incubated for the minimum of 1 hour at 32°C.

QUALITY CHECK OF THE ISOLATED CORNEAS:
- After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Physiological saline

POSITIVE CONTROL USED: Ethanol

APPLICATION DOSE AND EXPOSURE TIME:
-750 µl of either the positive control, the negative control or the test item, Corneas were incubated in a horizontal position for 10 minutes at 32°C
TREATMENT METHOD: [closed chamber / open chamber]
POST-INCUBATION PERIOD: -

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2
- Post-exposure incubation: Corneas were incubated for 120 minutes at 32°C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity value is calculated (measured with the device OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microplate reader (OD490)
- Others: After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
-The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN
GHS Category 1) and test items not requiring classification for eye irritation or serious eye
damage (UN GHS No Category)
In vitro score range UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1
-Acceptability of the assay: The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean: The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.5

Positive controls validity:

valid

Remarks:

26.3

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.6

Positive controls validity:

valid

Remarks:

69.5

Remarks on result:

no indication of irritation

Interpretation of results:

other: not classified

Remarks:

based on GHS and CLP

Conclusions:

The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.2 after 10 minutes of treatment. The test item induced an IVIS ≤ 3, therefore no classification is required for eye irritation or serious eye damage under the conditions of this test, in accordance with UN GHS, and the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

In this Bovine Corneal Opacity and Permeability test (BCOP test), the eye hazard potential of Amyris oil was tested. 750 µl of the testing material was tested through topical application directly on top of the corneas for 10 minutes. The negative and positive controls for opacity and permeability were within the laboratory historical range indicating that the test conditions were adequate and that the test system functioned properly. The test item did not induce ocular irritation through both endpoints, resulting in a mean vitro irritancy score of 1.2 after 10 minutes of treatment. The test item induced an IVIS ≤ 3, therefore no classification is required for eye irritation or serious eye damage under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation

The possible skin irritation potential of Amyris oil was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD

TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 μL undiluted test substance. After 42 hours 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 (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 5.3% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 11%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 8.1%. Based on this result, the substance is considered to be irritant and needs to be classified for skin irritation in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).

Skin corrosion

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), orPotassium 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).

Eye irritation

In this Bovine Corneal Opacity and Permeability test (BCOP test), the eye hazard potential of Amyris oil was tested. 750 µl of the testing material was tested through topical application directly on top of the corneas for 10 minutes. The negative and positive controls for opacity and permeability were within the laboratory historical range indicating that the test conditions were adequate and that the test system functioned properly. The test item did not induce ocular irritation through both endpoints, resulting in a mean vitro irritancy score of 1.2 after 10 minutes of treatment. The test item induced an IVIS ≤ 3, therefore no classification is required for eye irritation or serious eye damage under the conditions of this test.

Justification for classification or non-classification

Based on the available data, Amyris oil should be classified as skin irritant (Skin Irrit. 2 / H315) but not as eye irritant in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).