Bulnesia sarmienti, ext., sapond.

Endpoint summary

Description of key information

Skin irritation: irritating (OECD 439 & 431)
Eye irritation: not irritating (OECD 437)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18-01-2016 to 25-01-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

In accordance with GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Guaiacwood oil was heated until approximately 55°C to obtain a homogeneous liquid sample.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult donors

Source strain:

other: Strains no: 09-KERA-009, 08-KERA-008, 08-KERA-001, 10-KERA-005

Details on animal used as source of test system:

Not relevant

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-003)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C (actual range 36.2 - 36.7°C), with the exception of the test item incubation for 15 minutes at room temperature
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 36.2 - 36.7°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were washed with phosphate buffered saline to remove residual test item
- Observable damage in the tissue due to washing: The test item clotted during rinsing and was difficult to remove, however since in the end the tissue was clean and no test item was left this did not affect the results.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml
- Incubation time: 3 hours at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three viability tissues after 15 minutes exposure and 42 hours post incubation is less than or equal to 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin 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 negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL of the test item was applied (undiluted) to each of triplicate tissues

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL SDS
- Concentration (if solution): 5% in PBS

Duration of treatment / exposure:

15 ± 0.5 min at room temperature

Duration of post-treatment incubation (if applicable):

42 hours at 37°C

Number of replicates:

Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean (percentage of control)

Value:

11

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean tissue viability (percentage of control): 17%

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
Guaiacwood oil was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that Guaiacwood oil did not interact with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The mean relative tissue viability of the positive control should be ≤50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 17%.
- Acceptance criteria met for variability between replicate measurements: The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18. The standard deviation value of the percentage viability of three tissues treated identically was less than 10%, indicating that the test system functioned properly.

Interpretation of results:

study cannot be used for classification

Remarks:

No discrimination between irritation and corrosivity possible (additional test needed).

Conclusions:

Under the conditions of this test, the relative cell viability values decreased to 11% which is below the threshold for irritancy of ≤50%. Therefore, it can be concluded that the test item Guaiacwood oil is at least an irritant to skin and further research is needed to assign a classification in accordance with 1272/2008/EC (CLP).

Executive summary:

This in vitro study was performed according to OECD 439 to assess the irritation potential of Guaiacwood oil by means of the Human Skin Model Test (OECD TG 439). Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes. Tissue viability was determined for the test item and the positive control (as a percentage of the negative control viability) and mean tissue viability was calculated accordingly.

After treatment with Guaiacwood oil, the mean tissue viability was found to be 11%. This value is below the threshold for irritancy of ≤50%. Based on the results obtained, it can be concluded that Guaiacwood oil is at least an irritant to skin and further research is needed to be able to assign a classification in accordance with 1272/2008/EC (CLP).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06-06-2016 to 10-06-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

in accordance with GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"

Version / remarks:

Official Journal of the European Union No. L142, 31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Human epidermis tissue from donors

Details on animal used as source of test system:

Not relevant

Justification for test system used:

Recommended test sytem in international guidelines (OECD and EC)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue batch number(s): 23953
- Date of certificate of analysis: 8 June 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C
- Temperature of post-treatment incubation: 37 °C (MTT medium) and room temperature (isopropanol)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing: Once with PBS
- Observable damage in the tissue due to washing: No data
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 5 mg/ml diluted with MTT diluent (1:5)
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
No data

NUMBER OF REPLICATE TISSUES:
Test is done in duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No MTT interference was observed.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%. In addition, a test item considered non-corrosive (viability >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 the relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%. In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

ACCEPTANCE CRITERIA
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
- The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
- The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
- In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be <=30%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

An excess amount of the viscous test item (with spatulas) was added on top of the skin tissues to cover the skins completely. The negative controls were treated with 50 μl Milli-Q water, and the positive controls with 50 μl 8.0 KOH.

Duration of treatment / exposure:

3 minutes or 1 hour

Duration of post-treatment incubation (if applicable):

3 hours (MTT medium) and overnight (isopropanol)

Number of replicates:

The tests were performed in duplicate.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute application

Value:

112

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour application

Value:

103

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: Not observed in this test
- Colour interference with MTT: Not observed in this test

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes, except for positive control after 3-minute application but this was considered acceptable by the study director due to the viability reported (20%). In addition, both individual viabilities were clearly positive (<50%) after 3 minutes- Range of historical values if different from the ones specified in the test guideline: Not relevant

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this test, it can be concluded that the test item Guaiacwood oil is non-corrosive to skin in accordance with the criteria outlined in the guideline of this test.

Executive summary:

Guaiacwood oil was tested in vitro for skin corrosion in accordance with OECD Guideline 431. The testing material was topically applied on a human three dimensional epidermal model for 3 minutes and 1 hour. Positive and negative controls were included in the test. Skin corrosion is measured by remaining cell viability after exposure to the test item. The relative mean tissue viability after 3-minutes exposure compared to the negative control was 112% and after 1-hour exposure 103%. This means the mean relative tissue viability for Guaiacwood oil was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment. Based on these results, it can be concluded that the test item Guaiacwood oil is non-corrosive to skin in accordance with the criteria outlined in the guideline of this test.

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 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

in accordance with GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

other: Bovine

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Source: Local abattoir
- Age at study initiation: 12-60 months
From freshly slaughtered animals, eyes were transported to the test facility at day of slaughter and used within 24 hours.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL
- Concentration: 100%

Duration of treatment / exposure:

10 minutes (incubation at 32+/-1 °C)

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Test substance: 3 corneas
Negative control: 3 corneas
Positive control: 3 corneas

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (MEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes.

QUALITY CHECK OF THE ISOLATED CORNEAS
At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
The medium from both chambers of each holder was replaced with fresh complete MEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.

NUMBER OF REPLICATES
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

NEGATIVE CONTROL USED
Identification: 0.9% w/v sodium chloride solution
Batch: 301038501
Purity: 0.9%
Expiry Date: 01 September 2015
Storage Conditions: Room temperature

POSITIVE CONTROL USED
Identification: Ethanol
Batch: STBD7546V
Purity: >99.8%
Expiry Date: 11 August 2015
Storage Conditions: Room temperature

APPLICATION DOSE AND EXPOSURE TIME
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the anterior
chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM without phenol red. The anterior chamber was refilled with fresh complete MEM without phenol red.
- POST-EXPOSURE INCUBATION: The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
Light transmission measured quantitatively with the aid of an opacitometer.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. After exposure and removal of the test substance, a post-treatment opacity reading was taken. After the post-exposure incubation, a final opacity reading was taken.
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas.
The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability:
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.
- Others (e.g, pertinent visual observations, histopathology):
Each cornea was observed visually before treatment, after the exposure and removal of the test substance, and after the post-exposure incubation.
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) was calculated as follows:
Mean opacity value + (15*mean OD492 value)
- The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. This value was corrected by subtracting the average change in opacity for the negative control. The average is used to calculate the IVIS.
- The corrected OD492 was calculated by subtracting the OD492 of the negative control from the OD492 value of each treated cornea. The average is used to calculate the IVIS.

DECISION CRITERIA:
IVIS≤3 = "No category. Not requiring classification to UN GHS or EU CLP",
IVIS >3; ≤55 = "No prediction of eye irritation can be made",
IVIS >55 = "Category 1. UN GHS or EU CLP Causes serious eye damage".

Irritation parameter:

in vitro irritation score

Value:

0.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

IVIS: 2.2

Positive controls validity:

valid

Remarks:

IVIS: 41.2

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:
The corneas treated with the test item were clear post treatment and clear post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2013 for bovine corneas treated with the respective negative control. When testing liquids the negative control range for opacity should be ≤4.7 and for permeability ≤0.080.
The negative control gave a mean opacity of 1.0 and mean permeability of 0.078. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control:
The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during 2013 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 27.8 to 51.0.
The positive control In Vitro Irritancy Score was 41.2. The positive control acceptance criterion was therefore satisfied.

Individual and Mean Corneal Opacity and Permeability Measurements 

Treatment	Cornea number	Opacity					Permeability (OD)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post-Incubation	Post-Incubation – Pre-Treatment	Corrected Value		Corrected Value
Negative Control	9	1	1	2	1		0.094
	18	1	1	3	2		0.089
	22	1	0	0	0		0.051
					1.0^a		0.078^c		2.2
Positive Control	1	0	25	29	29	28.0	0.820	0.742
	3	0	23	28	28	27.0	0.944	0.866
	16	2	28	30	28	27.0	1.244	1.166
						27.3^b		0.925^b	41.2
Test Item	17	2	4	4	2	1.0	0.147	0.069
	19	2	2	1	-1	0.0	0.052	0.000
	20	0	1	1	1	0.0	0.040	0.000
						0.3^b		0.023^b	0.7

OD = Optical density

^a= Mean of the post-incubation – pre-treatment values

^b= Mean corrected value

^c= Mean permeability

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the IVIS score for Guaiacwood oil was determined to be 0.7, which is below the limit for classification (IVIS ≤3). Therefore, the test item does not need to be classified in accordance with the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Executive summary:

An in vitro Bovine Corneal Opacity and Permeability (BCOP) Assay was performed with Guaiacwood oil according to OECD guideline 437 and under GLP conditions. Bovine cornea were acquired from a local abattoir, prepared and treated with the test substance, positive control or negative control. In Vitro Irritancy Scores (IVIS) were calculated based on the measured opacity and permeability of the cornea after exposure.

Under the conditions of this study, the IVIS score for Guaiacwood oil was determined to be 0.7, which is below the limit for classification (IVIS ≤3). Therefore, the test item does not need to be classified in accordance with the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion

In an in vitro study (OECD 439, GLP) with the human skin model EpiSkin™, the skin irritation potential of Guaiacwood oil was assessed. The results showed 11% cell viability, after treatment with the test item. This value is below the threshold for irritancy of ≤ 50% and hence the substance is irritating to the skin. An in vitro skin corrosion test was performed.

In an in vitro study (OECD 431, GLP) with the human skin model EpiDerm™, the skin corrosion potential of Guaiacwood oil was assessed. The results showed 112% and 103% cell viability after treatment for 3 minutes and 1 hour respectively. These values are above the thresholds for corrosivity of 50% and 15% and hence the substance can be considered non-corrosive.

Taking the results of the two in vitro studies together, Guaiacwood oil should be regarded as a skin irritant.

Eye irritation

In an in vitro Bovine Corneal Opacity and Permeability (BCOP) Assay (OECD 437, GLP), the eye irritation potential of Guaiacwood oil was examined. In Vitro Irritancy Scores (IVIS) were calculated based on the measured opacity and permeability of the cornea after exposure. The results show an IVIS of 0.7, which is below the threshold of 3, indicating that the substance is not irritating to the eye.

Justification for classification or non-classification

Based on the available information from the in vitro skin irritation and corrosion test, Guaiacwood oil can be considered irritating to the skin and should therefore be classified as Skin Irrit. 2 (H315) in accordance with Annex I of 1272/2008/EC (CLP).

Based on the available information from the in vitro eye irritation test, Guaiacwood oil can be considered not irritating to the eye and therefore does not need to be classified in accordance with Annex I of 1272/2008/EC (CLP).