Reaction Mass of 1,4-dimethyl-7-(prop-1-en-2-yl)-1,2,3,4,5,6,7,8-octahydroazulene and 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,3,3a,4,5,6,7-octahydroazulene and 4,8a,9,9-tetramethyldecahydro-1,6-methanonaphthalen-1-ol

Endpoint summary

Description of key information

• Not irritating to skin (equivalent to OECD 404)
• Not irritating to eyes (OECD 437 and equivalent to OECD 405)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-05-9 to 1988-05-17

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

3 animals (the minimum permitted) were treated

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Commerical supplier
- Weight at study initiation: Animal 1: 2.14 kg; Animal 2: 2.37 kg; Animal 3: 2.27 kg
- Housing: Individually caged throughout testing
- Diet: Free access to pelleted food
- Water: Free access

ENVIRONMENTAL CONDITIONS
- After patch application, each animal was immobilised in canvas body sleeve and placed in a quiet place for the 4 hour contact period.

Type of coverage:

semiocclusive

Preparation of test site:

other: clipping

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

Observations were made at 30-60 minutes, and then at 24, 48 and 72 hours after patch removal.

Number of animals:

Three

Details on study design:

DESIGN

- No standard substance was applied for corroborative purposes
- The untreated skin of each animal served as its own control
- The degree of irritation was recorded in accordance with criteria given in Table 1 (below)

ANIMALS
- Testing was conducted when the hair was in the telogen (non-growth) phase of the hair cycle.
- Three to four days prior to testing, the dorsal area of the trunk of healthy adult animals was clipped to identify those which were in telogen and which had no skin blemishes.

PATCHES
- Each patch consisted of a 25 mm x 25 mm gauze pad, backed by a 20 mm x 30 mm strip of thin polythene film stuck to a 25 mm x 75 mm strip of zinc oxide sticking plaster.
- Animals were removed from the canvas body sleeves after 4 hours and the corners of each treatment site were marked.
- Patches were then removed and excess substance wiped from the skin with a damp tissue.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.67

Reversibility:

not fully reversible within: 72 hours

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Reversibility:

not fully reversible within: 72 hours

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.33

Reversibility:

not fully reversible within: 72 hours

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Reversibility:

not fully reversible within: 72 hours

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.67

Reversibility:

not fully reversible within: 72 hours

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Reversibility:

not fully reversible within: 72 hours

Irritant / corrosive response data:

One hour after patch removal, two animals showed slight erythema and one showed distinct erythema. Two also showed slight oedema. These effects intensified to show marked erythema, together with slight or marked oedema, in all three animals at 24 to 72 hours after patch removal.

The individual scores are given in Table 2.

Other effects:

No data

Table 2: Results data

Animal number	Erythema					Oedema
	1 h	24 h	48 h	72 h	Mean*	1 h	24 h	48 h	72 h	Mean*
1	1	2	2	1	1.67	1	1	1	1	1
2	1	2	2	2	2	0	1	2	2	1.67
3	2	2	2	3	2.33	1	2	2	2	2

* Mean of 24 h, 48 h and 72 h scores

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test material was not irritating to rabbit skin.

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:

2011-09-23

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection 2011-07-19, 2011-07-20 & 2011-07-21; Date of signature 2011-08-31

Species:

other: Excised Bovine Cornea

Strain:

other: Not Applicable

Details on test animals or tissues and environmental conditions:

Not applicable

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
-Amounts(s) applied (volume or weight with unit): 0.75 mL of the test material was applied to triplicate corneas.
-Concentration (if solution): the test material was used as supplied.

VEHICLE
- No vehicle used

Duration of treatment / exposure:

The undiluted test material was applied for 10 minutes followed by an incubation period of 120 minutes.

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

Not applicable

Details on study design:

TEST SITE
- Area of exposure: 0.75 mL of the test material was applied to triplicate corneas.
- Percentage coverage: the test material was topically applied to the cornea. The holders were gently tilted back and forth to ensure a uniform application of the material over the entire cornea.
-Type of wrap used: none.

REMOVAL OF TEST SUBSTANCE
-Washing (if done): at the end of the exposure period the test material was removed from the anterior chamber and each cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM.
-Time after start of exposure: 10 minutes post exposure

SCORING SYSTEM
- Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
- Opacity Measurement: 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 from each 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.
- 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.
- In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value).

DATA INTERPRETATION
- A test material that induces an in vitro irritancy score > than or equal to 55.1 is defined as an ocular corrosive or severe irritant.
- Additionally, the opacity and permeability values were evaluated independently to determine whether the test material induced a response through only one of the two endpoints.
- Visual Observation: the condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.

Irritation parameter:

in vitro irritation score

Run / experiment:

120 minutes post rinsing

Value:

0.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The neat test material induced an in vitro irritancy score of 0.8 and was therefore considered not to be an ocular corrosive or severe irritant.
The corneas treated with the test item were clear post treatment and post incubation.

RESULTS

CORNEAL OPACITY AND PERMEABILITY MEASUREMENT

- Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Table 1.

CORNEAL EPITHELIUM CONDITION

- The condition of the cornea immediately after rinsing and at the final opacity measurement is given in Table 2.

- 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.

IN VITRO IRRITANCY SCORE

- The results are summarised as follows:

Treatment	In Vitro Irritancy Score
Test Material	0.8
Negative Control	1.3
Positive Control	36.1

CRITERIA FOR AN ACCEPTABLE TEST

- The positive control In Vitro irritancy Score was within the range of 30.9 to 67.7. The positive control acceptance criterion was therefore satisfied.

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity					Permeability (OD)		In vitroIrritancy Score
		Pre-Treatment	Post-Treatment	Post-Incubation	Post-Incubation-Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	1	2	3	3	1		0.085
	2	4	4	5	1		0.028
	3	2	2	2	0		0.020
					0.7*		0.044+		1.3
Positive Control	4	4	32	30	26	25.3	1.173	1.129
	5	4	32	29	25	24.3	0.753	0.709
	6	2	24	21	19	18.3	0.891	0.847
						22.7·		0.895·	36.1
Test Item	7	4	5	5	1	0.3	0.048	0.004
	8	3	4	4	1	0.3	0.046	0.002
	9	3	4	5	2	1.3	0.058	0.014
						0.7·		0.006·	0.8

OD= Optical density                 * = Mean of the post treatment-pre‑treatment values      += Mean permeability              ·= Mean corrected value

Table 2: Corneal epithelium condition

Treatment	Cornea Number	Observation
		Post Treatment	Post Incubation
Negative Control	1	clear	clear
	2	clear	clear
	3	clear	clear
Positive Control	4	cloudy	cloudy
	5	cloudy	cloudy
	6	cloudy	cloudy
Test Item	7	clear	clear
	8	clear	clear
	9	clear	clear

Interpretation of results:

GHS criteria not met

Conclusions:

The neat test item was considered not to be an ocular corrosive or severe irritant and as the IVIS was below 3, the test item should not be classified for eye irritancy.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

A reliable, Klimisch score 2, in vivo skin irritation study was performed according to EC Method B.4 (Unilever Research Laboratory, 1988). 0.5 mL of the test material was applied to the skin of three New Zealand white rabbits using a semi-occlusive dressing. Animals were observed for signs of erythema and oedema and the responses were graded at 30-60 minutes, and then at 24, 48 and 72 hours after patch removal. One hour after patch removal, two animals showed slight erythema and one showed distinct erythema. Two also showed slight oedema. These effects intensified to show marked erythema, together with slight or marked oedema, in all three animals at 24 to 72 hours after patch removal. Based on the classification criteria according to Regulation (EC) No. 1272/2008, Patchouli oil is not considered a skin irritant based on the irritancy scores for individual animals.

This result is supported by reliable, Klimisch score 2 data from Leberco Laboratories, 1971 in which 0.5 mL of Patchouli oil was applied to the skin of three healthy albino rabbits. The treated skin was then evaluated at the end of the 24 h contact period and then again after 72 hours according to the method of Draize. All scores were zero which indicates that the test material was not a skin irritant.

Additional reliable Klimisch score 2 supporting information is available from two human repeat insult patch tests (Hill Top Research, Inc, 1971 and Harrison Research Laboratories, Inc, 1983) which demonstrate that the test material is not a skin irritant. In the Harrison Laboratories study (1983), a total of 51 subjects completed the study in which 0.2 mL of a 2% solution of Patchouli was applied to the skin in a series of nine induction patches for a period of three weeks. During both the induction and challenge phases, the test areas were observed and the reaction scored and recorded. The original patch sites exhibited no reactions during rest or at the challenge and no reactions were observed during the induction or challenge phases of the study. In the study performed at Hill Top Research (1971) 22 subjects were treated with 0.5 mL of a 9.25% solution of Patchouli oil, light. All 22 subjects showed little or no primary irritation. It is therefore considered that Patchouli oil is not an irritant to human skin.

 

Eye irritation:

A reliable in vitro Bovine Corneal Opacity and Permeability Assay (BCOP), Klimisch score 1, was performed in accordance with GLP and OECD Guideline 437 to assess the ocular irritancy potential of Patchouli Oil to the isolated bovine cornea (Harlan Laboratories Ltd., Report 41102451R, 2012). The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The in vitro irritancy score for Patchouli Oil was 0.8 and hence the test material was considered not to be an ocular corrosive or severe irritant and not to require any classification for eye irritation.

The in vitro result is supported by a reliable, Klimisch score 2 in vivo study from Leberco Laboratories, 1971 in which 0.1 mL of Patchouli oil was applied to the eyes of three healthy albino rabbits. Both the treated and control eyes were examined every 24 hours for four days and then again on the seventh day. The scores were recorded according to the Draize scale for scoring ocular lesions. A mild to moderate conjunctival reaction was observed which had disappeared by the fourth day of observation. Based on the individual animal scores and the reversibility of the observed effects, the test material is not considered to be an eye irritant.

Justification for classification or non-classification

Patchouli was not irritating in the in vivo skin, in vitro eye and in vivo eye irritation studies and therefore requires no classification according to CLP Regulation (EC) No 1272/2008.