Cedrus atlantica, ext.

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Cedrus atlantica, ext.

EC Number:

295-985-9

EC Name:

Cedrus atlantica, ext.

Cas Number:

92201-55-3

Molecular formula:

Not applicable due to UVCB nature of the substance

IUPAC Name:

Essential oil of Cedarwood Atlas obtained from the wood of Cedrus atlantica by steam distillation

Test material form:

liquid

Details on test material:

Name of test material as cited in study report: CEDAR ATLANTICA OIL

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

Specific details on test material used for the study:

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

Test animals / tissue source

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

Test system

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.

Results and discussion

In vitro

Results

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

In vivo

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).