Cedrus atlantica, ext.

Endpoint summary

Description of key information

Skin irritation: not irritating (OECD 439)
Eye irritation: not irritating (BCOP, OECD437)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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)

GLP compliance:

yes

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: Tissues were washed with phosphate buffered saline to remove residual test item.
- Observable damage in the tissue due to washing: Not reported

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

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 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 hours at 37°C

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean (percentage of control)

Value:

63

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean tissue viability (percentage of control): 17%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
Cedar atlantica 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 Cedar atlantica 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:

GHS criteria not met

Conclusions:

After treatment with the test item Cedarwood Atlas oil the relative cell viability values (compared to the negative control tissues) decreased to 63%. This value is above the threshold for irritancy of 50%. Therefore it can be concluded that Cedarwood Atlas oil is not irritant to skin and does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).

Executive summary:

This in vitro study was performed to assess the irritation potential of Cedarwood Atlas 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. 25 µL of the test item was applied to each tissue, spread to match the tissue size. The test item and the positive and negative controls were washed off the skin tissues after 15 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide] solution for 3 hours following approximately 71 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm. 25 µL of either the negative control (PBS) or the positive control (SDS) were applied to each tissue.

After treatment with the test item Cedarwood Atlas oil the relative cell viability values (compared to the negative control tissues) decreased to 63%. This value is above the threshold for irritancy of 50% as reported in the OECD guideline. Therefore it can be concluded that Cedarwood Atlas oil is not irritant to skin and does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

27 March 2015

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): Cedarwood Atlas oil

Species:

cattle

Strain:

other: not relevant

Details on test animals or tissues and environmental conditions:

TEST SYSTEM:
-Source: local abattoir
-Age at study initiation: 12-60 months

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)

Observation period (in vivo):

Not relevant

Number of animals or in vitro replicates:

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

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Time after start of exposure: 10 minutes

SCORING SYSTEM:
The In Vitro Irritancy Score was calculated as follows: mean opacity value + (15*OD492 value)
- Prediction model: 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".
- 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 substracting the OD492 of the negative control from the OD492 value of each treated cornea. The average is used to calculate the IVIS.

TOOL USED TO ASSESS SCORE:
- Opacity: Light transmission measured quantitatively with the aid of an opacitometer
- Permeability: After incubation of the cornea with sodium fluorescein for 90 minutes (anterior chamber), the concentration of this substance in the medium of the posterior chamber was determined using the Anthos 2001 microplate reader.

OPACITY MEASUREMENT
Calculated: initial opacity minus final opacity reading. The value is thereafter corrected with the negative control values
The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

PERMEABILITY MEASUREMENT
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

Irritation parameter:

in vitro irritation score

Value:

2.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

IVIS: 1.6

Positive controls validity:

valid

Remarks:

IVIS: 49.3

Other effects:

The corneas treated with the test item or 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

The positive control IVIS was within the range of 27.8 to 51.0.

The negative control gave opacity of ≤4.7 permeability of ≤0.080. Both positive and negative control acceptance criteria were therefore satisfied.

Table 1. Individual and Mean Corneal Opacity and Permeability Measurements.

Treatment	Cornea No	Opacity					Permeability (OD)		In vitro irritancy score
		Pre-treatment	Post-treatment	Post-incubation	Post-incub.-pre-treatm.	Corrected value		Corrected value
Negative control	5	4	4	4	0		0.018
	12	5	4	7	2		0.026
	16	4	4	6	2		0.018
					1.3*		0.021s		1.6
Positive control	1	3	39	39	36	34.7	1.123	1.102
	14	2	34	33	31	29.7	1.260	1.239
	19	5	39	37	32	30.7	1.215	1.194
						31.7**		1.178**	49.3
Test item	3	2	4	5	3	1.7	0.045	0.024
	18	5	7	9	4	2.7	0.031	0.010
	20	6	8	10	4	2.7	0.035	0.014
						2.4**		0.016**	2.6

*: Mean of the post-incubation minus pre-treatment values

s: Mean permeability

**: Mean corrected value

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the IVIS for Cedarwood Atlas oil was determined to be 2.6, which is below the limit for classification (IVIS ≤3). Therefore according to this study the test item does not require classifying according to the criteria laid down in Annex I of 1272/2008/EC (CLP).

Executive summary:

An in vitro Bovine Corneal Opacity and Permeability (BCOP) Assay was performed with Cedarwood Atlas 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 for Cedarwood Atlas oil was determined to be 2.6 which is below the threshold of 3 that indicates that the substance is not irritating. Therefore Cedarwood Atlas oil does not require classification for eye irritation or serious eye damage, according to the criteria laid down 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

In an in vitro study (OECD 439, GLP) with the human skin model EpiSkin™, the skin irritation potential of Cedarwood Atlas oil was assessed. After treatment with the test item Cedarwood Atlas oil the relative cell viability values (compared to the negative control tissues) decreased to 63%. This value is above the threshold for irritancy of 50% as reported in the OECD guideline. Therefore it can be concluded that Cedarwood Atlas oil is not irritant to skin

Eye irritation

In an in vitro Bovine Corneal Opacity and Permeability (BCOP) Assay (OECD 437, GLP), the eye irritation potential of Cedarwood Atlas oil was examined. In Vitro Irritancy Scores (IVIS) were calculated based on the measured opacity and permeability of the cornea after exposure. The results showed an IVIS of 2.6, 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 test, Cedarwood Atlas oil is not irritating to the skin, and according to the regulatory framework and applicability of the Test Guideline 439, it should not be classified for skin irritation.

Based on the available information, from the ex vivo BCOP eye irritation test, Cedarwood Atlas oil, is not irritating to the eye, and according to the Test Guideline 437 criteria, it should not be classified for eye irritation.