Juniper, Juniperus oxycedrus, ext.

Endpoint summary

Description of key information

- Skin irritation/corrosion: irritating, based on existing data (Bouhlal, 1988) and additivity principles
- Eye irritation: irritating, based on existing data (OECD 437, GLP, K, Rel.1) and additivity principles

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion, other

Remarks:

Classification based on calculation rules for mixtures of the CLP Regulation

Type of information:

calculation (if not (Q)SAR)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

accepted calculation method

Reason / purpose for cross-reference:

reference to other study

Remarks:

supporting data in humans

Qualifier:

no guideline required

Principles of method if other than guideline:

Classification based on calculation rules for mixtures of the CLP Regulation

Irritation parameter:

other: classification

Remarks on result:

other: skin irritant category 2

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Executive summary:

The substance is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation.

The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2015) was used to determine the skin irritation/corrosion hazard of the registered substance.

Cade oil has more than 10% of its constituents classified as Skin irritant Category 2 and should be classified as a skin irritant without further testingaccording to the Regulation (EC) No 1272/2008

Constituent	Classification according to the Regulation (EC) No. 1272/2008 (CLP)		Classification according to the Directive 67/548/EEC		Source
	Skin irritation	Eye irritation	Skin irritation	Eye irritation
Germacrene delta	SCI2, H316	-	Xi ; R38	Xi ; R36	Self classification
Maltol	SCI2, H315	EDI2, H319	Xi ; R38	-	Self classification
Gaiacol ortho	SCI2, H315	EDI2, H319	Xi ; R38	Xi ; R36	Self classification
2 -methoxy para cresol	SCI2, H315	EDI2, H319	Xi ; R38	-	Self classification
E-isoeugenol	SCI2, H315	EDI2, H319	Xi ; R38	Xi ; R36	Self classification
Vanillin	-	EDI2, H319	-	Xi ; R36	REACH dossier
Dihydroeugenol	-	EDI2, H319	-	Xi ; R36	Self classification

Source: ECHA disseminated dossiers or self classification

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 December 2014 to 08 January 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Remarks:

Supporting data

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

10 January 2013

Species:

other: Bovine eye

Details on test animals or tissues and environmental conditions:

Origin: Bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint-Pierre-sur-Dives, France.
Age: bovine cattle were up to 12 months old.
Reason for choice: Bovine corneas are recommended by regulatory authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
Transport from supplier to CiToxLAB France: Eyes were transported to CiToxLAB France at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 μg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.

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 (± 8 μL) was applied on each cornea
- Concentration (if solution): undiluted

Duration of treatment / exposure:

The test item was evaluated by using a treatment time of 10 minutes ± 30 seconds.

Number of animals or in vitro replicates:

Total: 9 corneas - 3 corneas/group for test item, negative and positive controls

Details on study design:

Dose formulation application:
As the test item is a non-surfactant liquid, it was tested undiluted. As the test item could be sampled using a micropipette, a volume of 750 μL (± 8 μL) was applied on each cornea using the closed-chamber method as follows: the test and control items were introduced into the anterior chamber of the corneal holder through the dosing holes, to cover the epithelial side of the cornea. Then the dosing holes were sealed.

Treatment of corneas:
Corneas obtained from freshly slaughtered cattle (from abattoir) were mounted in corneal holders with the endothelial side against the O-ring of the posterior chamber. For pre-incubation, both chambers of the corneal holder were filled with MEM culture media supplemented with 1% fetal bovine serum plus penicillin/streptomycin (cMEM) and incubated for 1 h and 5 minutes (± 5 minutes) at 32 ± 1 °C. Then medium was removed & then refilled with fresh cMEM and corneas were examined macroscopically for any defects. Then, the opacity of the cornea was measured to obtain OPT0. The medium was removed from anterior chamber and the test item was applied onto the epithelium of the cornea. After application of the dose formulation, the holders were incubated, vertically (cornea positioned horizontally with the treated side uppermost) in a water bath at 32 ± 1 °C, for 10 minutes ± 30 seconds. At the end of the exposure period, the test item was removed from the anterior chamber and the corneas were rinsed up to five times with pre-warmed cMEM containing phenol red. Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red. Following the 10-minute treatment, the holders were incubated horizontally (corneas placed vertically) for 2 h ± 10 minutes in a water bath at 32 ± 1 °C. After completion of the 2 h incubation period, the medium of both anterior and posterior chambers was renewed with pre-warmed cMEM (32 ± 1 °C), then the second opacity measurement (OPT2) was performed.

Permeability determination
- Application of sodium fluorescein: Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium of the anterior chamber was removed and the chamber received 1 mL of a fluorescein solution (4 mg/mL). For each series of three corneas, a chronometer started from the fluorescein application time of the first cornea of the series. The holders were incubated vertically (cornea positioned horizontally with the fluorescein-treated side uppermost) in a water bath at 32 ± 1 °C for 90 ± 5 minutes. After incubation the medium in the posterior chamber of each holder was decanted and retained. Then optical density at 490 nm (OD490) was measured using the spectrophotometer.

CONTROLS:
Negative control: 0.9 % sodium chloride solution
Positive control: 10% sodium hydroxide solution

OTHERS:
- Macroscopic examination: After permeability determination, the corneas were removed from the holders and observed for opaque spots, other irregularities and any separation of the epithelium. Then, the corneas were discarded.

Irritation parameter:

in vitro irritation score

Value:

31

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean opacity of the negative control corneas was 0 < 1.8 and the mean OD490nm of the negative control corneas was 0.004 < 0.0269.
- Acceptance criteria met for positive control: the mean IVIS was 271 which falls within two standard deviations of the historical mean (172.3-292.7)

Irritant / corrosive response data:

- Macroscopic examinations: No notable opaque spots or irregularities were observed on negative control corneas. Fluorescein fixation was observed on the corneas treated with the test item. Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.
- In Vitro Irritancy Score (IVIS) for test item and positive control were 31 and 271, respectively.

Other effects:

None

Table 7.3.2/1: Eye irritation – results

GROUP		OPACITY				PERMEABILITY		SCORE
	Holder	OPT0	OPT2	OPT2-OPT0	cOPT	OD490 nm	cOD490 nm
Negative control	44	2	0	-2.0	-	0.009	-	-
	45	2	1	-1.0	-	0.001	-	-
	10	2	1	-1.0	-	0.003	-	-
	Mean	-	-	-1.3	-	0.004	-	-
	SD	-	-	0.6	-	0.004	-	-
Test item	5	0	25	25.0	26.3	0.521	0.517	34
	36	3	33	30.0	31.3	0.080	0.076	32
	40	3	22	19.0	20.3	0.462	0.458	27
	Mean	-	-	-	26.0	-	0.350	31
	SD	-	-	-	5.5	-	0.239	3.6
Positive control	37	1	160	159.0	160.3	7.904	7.900	279
	28	1	161	160.0	161.3	7.360	7.356	272
	17	3	160	157.0	158.3	7.008	7.004	263
	Mean	-	-	-	160.0	-	7.420	271
	SD	-	-	-	1.5	-	0.451	7.7

 

OD: optical density

cOD: corrected optical density

cOPT: corrected corneal opacity

SD: standard deviation

OPT0: corneal opacity before treatment

OPT2: corneal opacity after the 2 h recovery period

Interpretation of results:

other: could not be predicted

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of the test item, Cade Oil, could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS and EU CLP Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS and EU CLP No Category).

Executive summary:

In an in vitro eye irritation study performed according to the OECD Guideline 437 and in compliance with GLP, 750 μL (± 8 μL) of undiluted test item, Cade Oil was applied to isolated bovine corneas for 10 minutes followed by an incubation period of 2 h at 32 °C. Three corneas were used for each treated series (undiluted test item; negative control; positive control). Before the treatment, a first opacity measurement was performed using an opacitometer. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes at 32 °C. At the end of the incubation period, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

 

No notable opaque spots or irregularities were observed on negative control corneas. Fluorescein fixation was observed on the corneas treated with the test item. Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.In Vitro Irritancy Score (IVIS) for test item and positive control were 31 and 271, respectively.

 

As the test item-induced a mean IVIS > 3 and ≤ 55, the eye hazard potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Reference 2

Endpoint:

eye irritation, other

Remarks:

Classification based on calculation rules for mixtures of the CLP Regulation

Type of information:

calculation (if not (Q)SAR)

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

accepted calculation method

Reason / purpose for cross-reference:

reference to other study

Remarks:

Supporting data

Qualifier:

no guideline required

Principles of method if other than guideline:

Classification based on calculation rules for mixtures of the CLP Regulation

Irritation parameter:

other: classification

Remarks on result:

other: eye irritant category 2

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Executive summary:

The substance is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation.

The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2015) was used to determine the eye irritation/corrosion hazard of the registered substance (additivity principles):

- Cade oil has more than 10% of its constituents classified as Eye irritant Category 2 and should be classified as a eye irritant without further testing according to the Regulation (EC) No 1272/2008

Constituent	Classification according to the Regulation (EC) No. 1272/2008 (CLP)		Classification according to the Directive 67/548/EEC		Source
	Skin irritation	Eye irritation	Skin irritation	Eye irritation
Germacrene delta	SCI2, H316	-	Xi ; R38	Xi ; R36	Self classification
Maltol	SCI2, H315	EDI2, H319	Xi ; R38	-	Self classification
Gaiacol ortho	SCI2, H315	EDI2, H319	Xi ; R38	Xi ; R36	Self classification
2 -methoxy para cresol	SCI2, H315	EDI2, H319	Xi ; R38	-	Self classification
E-isoeugenol	SCI2, H315	EDI2, H319	Xi ; R38	Xi ; R36	Self classification
Vanillin	-	EDI2, H319	-	Xi ; R36	http://echa.europa.eu/registration-dossier/-/registered-dossier/2209/
Dihydroeugenol	-	EDI2, H319	-	Xi ; R36	Self classification

Source: ECHA disseminated dossiersor self classification

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion:

The substance is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation.

The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2015) was used to determine the skin irritation/corrosion hazard of the registered substance.

The decision of classification as skin irritant was based on the following arguments:

1/ Existing human data: some evidence of irritation was observed in a human patch test (Bouhlal, 1988). In this study 25 healthy volunteers were treated with undiluted Cade oil using epicutaneous test. Irritation was observed.

2/ Existing data on constituents (additivity principles):

- Cade oil has more than 10% of its constituents classified as Skin irritant Category 2 and should be classified as a skin irritant without further testing according to the Regulation (EC) No 1272/2008

Constituent	Classification according to the Regulation (EC) No. 1272/2008 (CLP)		Classification according to the Directive 67/548/EEC		Source
	Skin irritation	Eye irritation	Skin irritation	Eye irritation
Germacrene delta	SCI2, H316	-	Xi ; R38	Xi ; R36	Self classification
Maltol	SCI2, H315	EDI2, H319	Xi ; R38	-	Self classification
Gaiacol ortho	SCI2, H315	EDI2, H319	Xi ; R38	Xi ; R36	Self classification
2 -methoxy para cresol	SCI2, H315	EDI2, H319	Xi ; R38	-	Self classification
E-isoeugenol	SCI2, H315	EDI2, H319	Xi ; R38	Xi ; R36	Self classification
Vanillin	-	EDI2, H319	-	Xi ; R36	http://echa.europa.eu/registration-dossier/-/registered-dossier/2209/
Dihydroeugenol	-	EDI2, H319	-	Xi ; R36	Self classification

Source: ECHA disseminated dossiers or self classification

Eye irritation:

A key study was identified (Gerbeix, 2015, Rel.1). In this BCOP assay conducted on Cade oil according to the OECD Guideline 437 and in compliance with GLP, the in vitro mean irritancy score for the test item was 31.

Cade oil is not an ocular corrosive or severe irritant. However, based on this study, no definitive conclusion on eye irritation can be established since the IVIS was > 3 and ≤ 55 (test item is not predicted as causing serious eye damage and not classified for eye irritation or serious eye damage).

Also, in the preliminary test of an epiocular eye irritation test (Study 42559 TIO), the test item was found to have direct MTT reducing properties and colouring potential (> 50% of the viable negative control) therefore the substance was considered incompatible with the method.

Additivity principles were therefore applied to conclude on classification:

- Cade oil has more than 10% of its constituents classified as Eye irritant Category 2 and should be classified as eye irritant without further testing according to the Regulation (EC) No 1272/2008.

Justification for classification or non-classification

Harmonized classification:

Cade oil has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information and typical composition provided by the Lead Registrant, Cade oil is classified as skin irritant: Skin Irritant Category 2 (H315: Causes skin irritation) according to the criteria of the Regulation (EC) No. 1272/2008 (CLP).

Based on the available information and typical composition provided by the Lead Registrant, Cade oil is classified as eye irritant: Irritating to eyes Category 2 (H319: Causes serious eye irritation) according to the criteria of the Regulation (EC) No. 1272/2008 (CLP)

No information was available regarding respiratory irritation.