p-menth-1-en-8-ol

Endpoint summary

Description of key information

Skin irritation. Weight of evidence. Read-across approach: Method according to OECD guideline 404, GLP study. Based on the read-across approach from the analogue alpha terpineol (results taken from 3 independent tests), laevo alpha terpineol can be considered irritating to the skin.

Skin irritation. Supporting study. Read-across approach: open mouse ear assay. Based on the read-across approach from the analogue alpha terpineol, laevo alpha terpineol is considered to be a skin irritant.

Eye irritation (in vitro): Key study: Test method according to the OECD Guideline 438 with GLP. No prediction can be made for the test item in the ICE test.

Eye irritation (in vivo): Key study: Test method according to the OECD 405 Guideline with GLP. The test substance has to be classified in category 1 (serious eye damage) as irreversible effects were found on the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted in compliance with OECD guideline 404 with minor deviations: no details on test animals and environmental conditions; no details on patch material; observations not followed until the reversibility of the effects (only 7 days)

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

no details on test animals and environmental conditions; no details on patch material; observations not followed until the reversibility of the effects (only 7 days)

GLP compliance:

yes

Species:

rabbit

Strain:

other: albino

Details on test animals or test system and environmental conditions:

None

Type of coverage:

semiocclusive

Preparation of test site:

other: intact skin

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume with unit): 0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

7 days

Number of animals:

Three

Details on study design:

TEST SITE
- tested in a multi-patch study

OBSERVATION TIME POINTS: 1h, 1 d, 2 d, 3 d and 7 d.

SCORING SYSTEM:
- Method of calculation: Draize scoring system

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.33

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.33

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritant / corrosive response data:

- Two animals showed marked desquamation at the 7 day time interval. The other animal showed thickening and marked desquamation of the skin surface at the 7 day time interval.
- See table 1 for individual animal scores

Other effects:

No data

Table 1: Skin irritation data for individual rabbits

	1 hr	1 day	2 days	3 days	7 days	Mean (24, 48, 72 hrs)
Animal No. 1
Erythema	0	1	2	2	1	1.67
Oedema	1	2	2	2	1	2
Animal No. 2
Erythema	2	2	2	2	2	2
Oedema	2	3	2	2	1	2.33
Animal No. 3
Erythema	2	2	2	3	3	2.33
Oedema	2	3	3	3	3	3

Interpretation of results:

other: Irritant cat. 2 (CLP Regulation EC no. 1272/2008)

Conclusions:

Under the test conditions, alpha terpineol is considered irritating to the skin.

Executive summary:

In a primary dermal irritation study performed according to OECD guideline 404 and in compliance with GLP, three albino rabbits were dermally exposed to 0.5 mL of undiluted alpha terpineol, under a semi-occlusive patch for 4 hours to intact skin of the flank. Animals were then observed for 7 days. Irritation was scored by the method of Draize at 1, 24, 48 and 72 hours and 7 days after exposure. Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals were 1.67, 2 and 2.33 for erythema score and 2, 2.33 and 3 for oedema score. Under the test conditions the substance is considered to be irritating to the skin because the average oedema score exceeded 2.3 for two animals (CLP) and the effects were not fully reversible within the observation period of 7 days.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted in compliance with OECD guideline 404 with minor deviations: no details on test animals and environmental conditions; no details on patch material; observations not followed until the reversibility of the effects (only 7 days)

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

no details on test animals and environmental conditions; no details on patch material; observations not followed until the reversibility of the effects (only 7 days)

GLP compliance:

yes

Species:

rabbit

Strain:

other: albino

Details on test animals or test system and environmental conditions:

None

Type of coverage:

semiocclusive

Preparation of test site:

other: intact skin

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume with unit): 0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

7 days

Number of animals:

Four

Details on study design:

TEST SITE
- tested in a multi-patch study

OBSERVATION TIME POINTS: 1h, 1 d, 2 d, 3 d and 7 d.

SCORING SYSTEM:
- Method of calculation: Draize scoring system

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

- Three animals showed marked desquamation and one animal showed slight desquamation at the 7 day time interval.
- See table 1 for individual animal scores

Other effects:

No data

Table 1: Skin irritation data for individual rabbits

	1 hr	1 day	2 days	3 days	7 days	Mean (24, 48, 72 hrs)
Animal No. 1
Erythema	2	2	2	2	2	2
Oedema	2	3	3	3	1	3
Animal No. 2
Erythema	2	2	3	3	2	2.67
Oedema	3	3	3	3	3	3
Animal No. 3
Erythema	2	2	2	2	2	2
Oedema	2	3	2	3	3	2.67
Animal No. 4
Erythema	1	2	2	2	1	2
Oedema	2	2	2	1	0	1.67

Interpretation of results:

other: Irritant cat. 2 (CLP Regulation EC no. 1272/2008)

Conclusions:

Under the test conditions, alpha terpineol is considered irritating to the skin.

Executive summary:

In a primary dermal irritation study performed according to OECD guideline 404 and in compliance with GLP, four albino rabbits were dermally exposed to 0.5 mL of undiluted alpha terpineol, under a semi-occlusive patch for 4 hours to intact skin of the flank. Animals were then observed for 7 days. Irritation was scored by the method of Draize at 1, 24, 48 and 72 hours and 7 days after exposure. Mean individual scores at 24, 48 and 72 h after exposure for the 4 animals were 2, 2.67, 2 and 2 for erythema score and 3, 3, 2.67 and 1.67 for oedema score. Under the test conditions the substance is considered to be irritating to the skin because the average oedema score exceeded 2.3 for two animals (CLP) and the effects were not fully reversible within the observation period of 7 days.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted in compliance with OECD guideline 404 with minor deviations: no details on test animals and environmental conditions; no details on patch material; observations not followed until the reversibility of the effects (only 7 days)

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

no details on test animals and environmental conditions; no details on patch material; observations not followed until the reversibility of the effects (only 7 days)

GLP compliance:

yes

Species:

rabbit

Strain:

other: albino

Details on test animals or test system and environmental conditions:

None

Type of coverage:

semiocclusive

Preparation of test site:

other: intact skin

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume with unit): 0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

7 days

Number of animals:

Four

Details on study design:

TEST SITE
- tested in a multi-patch study

OBSERVATION TIME POINTS: 1h, 1 d, 2 d, 3 d and 7 d.

SCORING SYSTEM:
- Method of calculation: Draize scoring system

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritant / corrosive response data:

- Three animals showed slight desquamation and one animal showed marked desquamation at the 7 day time interval.
- See table 1 for individual animal scores

Other effects:

No data

Table 1: Skin irritation data for individual rabbits

	1 hr	1 day	2 days	3 days	7 days	Mean (24, 48, 72 hrs)
Animal No. 1
Erythema	2	2	2	2	1	2
Oedema	2	3	3	2	0	2.67
Animal No. 2
Erythema	2	2	2	2	2	2
Oedema	2	2	2	2	2	2
Animal No. 3
Erythema	1	2	2	1	0	1.67
Oedema	0	1	1	0	0	0.67
Animal No. 4
Erythema	2	2	2	2	1	2
Oedema	1	3	3	3	1	3

Interpretation of results:

other: Irritant cat. 2 (CLP Regulation EC no. 1272/2008)

Conclusions:

Under the test conditions, alpha terpineol is considered irritating to the skin.

Executive summary:

In a primary dermal irritation study performed according to OECD guideline 404 and in compliance with GLP, four albino rabbits were dermally exposed to 0.5 mL of undiluted alpha terpineol, under a semi-occlusive patch for 4 hours to intact skin of the flank. Animals were then observed for 7 days. Irritation was scored by the method of Draize at 1, 24, 48 and 72 hours and 7 days after exposure. Mean individual scores at 24, 48 and 72 h after exposure for the 4 animals were 2, 2, 1.67 and 2 for erythema score and 2.67, 2, 0.67 and 3 for oedema score. Under the test conditions the substance is considered to be irritating to the skin because the average oedema score exceeded 2.3 for two animals (CLP) and the effects were not fully reversible within the observation period of 7 days.

Reference 4

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: open mouse ear assay. The pilot and main irritancy assay on mice ears were adopted from Evans and Schmidt (1979).

GLP compliance:

not specified

Species:

mouse

Strain:

other: albino

Details on test animals or test system and environmental conditions:

None

Type of coverage:

open

Preparation of test site:

other: intact skin (inner surface of animal’s ear)

Vehicle:

other: (acetone)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 µg
- Concentration (if solution): 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 µg/5 µL.

Observation period:

The ears were examined for redness after 30 minutes and then 15 minute interval until two observations indicated that further redness would not occur.

Number of animals:

12 per dose

Details on study design:

TEST SITE
- Area of exposure: inner surface of animal's ear

OBSERVATION TIME POINTS : The ears were examined for redness after 30 minutes and then 15 minute interval until two observations indicated that further redness would not occur.

SCORING SYSTEM:
- Method of calculation:
The time of maximum erythema was noted. The number of ears eliciting the degree of redness corresponding to at least ++ intensity on Hecker's scale at peak irritancy (Hecker 1971) also mentioned by Evans and Schmidt (1979) were noted and expressed in μg / 5 μI per ear.
The chronic effects of the test compound on the animals skin were also recorded after one and two days to ascertain the chronic inflammatory dose. Animals with at least ++ intensity after 24 and 48 hours were recorded and denoted by IU (Irritant units) (Hecker 1971).
The total number of red ears per dilution was tabulated. ID50 (Irritant dose in 50% individuals ) along with the upper and lower confidence limits were calculated by probit analysis.

Irritation parameter:

other: ID50 (μg / 5 μL)

Basis:

mean

Time point:

24/48 h

Score:

0.853

Reversibility:

fully reversible within: 48 h

Irritant / corrosive response data:

Alpha terpineol was found irritant with ID50 (Irritant dose in 50% individuals) = 0.853 µg/5 µL. Its reaction lasted for 24 h, indicating IU = 0.625 µg/5 µL after 24 h. No effects were recorded after 48 h up to the highest dose tested (IU>5 µg/5 µL).

Table 1: irritant responses of the compounds Isolated from Lavandula stoechas oil on Albino Mice

Dose levels (μg / 5 μL)		Alpha terpineol
5		11/12
2.5		9/12
1.25		7/12
0.625		5/12
0.3125		3/12
0.15625		1/12
ID 50	μg / 5 μL	0.853
	S.D	0.128
	t	3.25
	U.C.L.	1.369
	L.C.L.	0.552
IU (μg / 5μL) after	24 h	0.625
	48 h	>5

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the test conditions, alpha terpineol was found to be a skin irritant.

Executive summary:

The irritant potentials of Lavandula stoechas were investigated on albino mice. Six of its components were evaluated for irritancy by open mouse ear assay. Alpha terpineol was dissolved in acetone (10 mg/10 mL (w /v) solution). Further dilutions were prepared. Doses of 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 µg/5 µL were applied to inner surface of animal’s ear. The ears were examined for redness after 30 minutes and then 15 minute interval until two observations indicated that further redness would not occur. The time of maximum erythema was noted. The number of ears eliciting the degree of redness according to Hecker's scale were noted and expressed in μg / 5 μI per ear. The chronic effects after 24 and 48 hours were recorded and denoted by IU (Irritant units). ID50 (Irritant dose in 50% individuals ) were calculated by probit analysis. ID50 of alpha terpineol was found to be 0.853 µg/5 µL and its reaction lasted for 24 h, indicating IU = 0.625 µg/5 µL after 24 h. It is concluded that alpha terpineol is a skin irritant.

Reference 5

Endpoint:

skin irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
The study does not need to be conducted since an in vivo study on irritation is available.

Reference 6

Endpoint:

skin irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance alpha terpineol which shares the same functional groups with the substance L-alpha terpineol also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 1.67 was obtained in erythema score.

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 2 was obtained in edema score.

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 2 was obtained in erythema score.

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.33

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 2.33 was obtained in edema score.

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.33

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 2.33 was obtained in erythema score.

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 3 was obtained in edema score.

Irritant / corrosive response data:

- Two animals showed marked desquamation at the 7 day time interval. The other animal showed thickening and marked desquamation of the skin surface at the 7 day time interval.
- See table 1 for individual animal scores

Table 1: Skin irritation data for individual rabbits

	1 hr	1 day	2 days	3 days	7 days	Mean (24, 48, 72 hrs)
Animal No. 1
Erythema	0	1	2	2	1	1.67
Oedema	1	2	2	2	1	2
Animal No. 2
Erythema	2	2	2	2	2	2
Oedema	2	3	2	2	1	2.33
Animal No. 3
Erythema	2	2	2	3	3	2.33
Oedema	2	3	3	3	3	3

Interpretation of results:

other: Irritant cat. 2 (CLP Regulation EC no. 1272/2008)

Conclusions:

Based on the read-across approach from the analogue alpha terpineol, laevo alpha terpineol can be considered irritating to the skin.

Executive summary:

In a primary dermal irritation study performed according to OECD guideline 404 and in compliance with GLP, three albino rabbits were dermally exposed to 0.5 mL of undiluted alpha terpineol, under a semi-occlusive patch for 4 hours to intact skin of the flank. Animals were then observed for 7 days. Irritation was scored by the method of Draize at 1, 24, 48 and 72 hours and 7 days after exposure. Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals were 1.67, 2 and 2.33 for erythema score and 2, 2.33 and 3 for oedema score. Under the test conditions the substance is considered to be irritating to the skin because the average oedema score exceeded 2.3 for two animals (CLP) and the effects were not fully reversible within the observation period of 7 days. Based on these results, the read-across approach was applied and laevo alpha terpineol can be considered irritating to the skin.

Reference 7

Endpoint:

skin irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance alpha terpineol which shares the same functional groups with the substance L-alpha terpineol also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 2 was obtained in erythema score.

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 3 was obtained in edema score.

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 2.67 was obtained in erythema score.

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 3 was obtained in edema score.

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 2 was obtained in erythema score.

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 2.67 was obtained in edema score.

Irritation parameter:

erythema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 2 was obtained in erythema score.

Irritation parameter:

edema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 1.67 was obtained in edema score.

Irritant / corrosive response data:

- Three animals showed marked desquamation and one animal showed slight desquamation at the 7 day time interval.
- See table 1 for individual animal scores

Table 1: Skin irritation data for individual rabbits

	1 hr	1 day	2 days	3 days	7 days	Mean (24, 48, 72 hrs)
Animal No. 1
Erythema	2	2	2	2	2	2
Oedema	2	3	3	3	1	3
Animal No. 2
Erythema	2	2	3	3	2	2.67
Oedema	3	3	3	3	3	3
Animal No. 3
Erythema	2	2	2	2	2	2
Oedema	2	3	2	3	3	2.67
Animal No. 4
Erythema	1	2	2	2	1	2
Oedema	2	2	2	1	0	1.67

Interpretation of results:

other: Irritant cat. 2 (CLP Regulation EC no. 1272/2008)

Conclusions:

Based on the read-across approach from the analogue alpha terpineol, laevo alpha terpineol can be considered irritating to the skin.

Executive summary:

In a primary dermal irritation study performed according to OECD guideline 404 and in compliance with GLP, four albino rabbits were dermally exposed to 0.5 mL of undiluted alpha terpineol, under a semi-occlusive patch for 4 hours to intact skin of the flank. Animals were then observed for 7 days. Irritation was scored by the method of Draize at 1, 24, 48 and 72 hours and 7 days after exposure. Mean individual scores at 24, 48 and 72 h after exposure for the 4 animals were 2, 2.67, 2 and 2 for erythema score and 3, 3, 2.67 and 1.67 for oedema score. Under the test conditions the substance is considered to be irritating to the skin because the average oedema score exceeded 2.3 for two animals (CLP) and the effects were not fully reversible within the observation period of 7 days. Based on these results, the read-across approach was applied and laevo alpha terpineol can be considered irritating to the skin.

Reference 8

Endpoint:

skin irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance alpha terpineol which shares the same functional groups with the substance L-alpha terpineol also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 2 was obtained in erythema score.

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 2.67 was obtained in edema score.

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 2 was obtained in erythema score.

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 2 was obtained in edema score.

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 1.67 was obtained in erythema score.

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 0.67 was obtained in edema score.

Irritation parameter:

erythema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 2 was obtained in erythema score.

Irritation parameter:

edema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: read-across from an analogue for which 3 was obtained in edema score.

Irritant / corrosive response data:

- Three animals showed slight desquamation and one animal showed marked desquamation at the 7 day time interval.
- See table 1 for individual animal scores

Other effects:

No data

Table 1: Skin irritation data for individual rabbits

	1 hr	1 day	2 days	3 days	7 days	Mean (24, 48, 72 hrs)
Animal No. 1
Erythema	2	2	2	2	1	2
Oedema	2	3	3	2	0	2.67
Animal No. 2
Erythema	2	2	2	2	2	2
Oedema	2	2	2	2	2	2
Animal No. 3
Erythema	1	2	2	1	0	1.67
Oedema	0	1	1	0	0	0.67
Animal No. 4
Erythema	2	2	2	2	1	2
Oedema	1	3	3	3	1	3

Interpretation of results:

other: Irritant cat. 2 (CLP Regulation EC no. 1272/2008)

Conclusions:

Based on the read-across approach from the analogue alpha terpineol, laevo alpha terpineol can be considered irritating to the skin.

Executive summary:

In a primary dermal irritation study performed according to OECD guideline 404 and in compliance with GLP, four albino rabbits were dermally exposed to 0.5 mL of undiluted alpha terpineol, under a semi-occlusive patch for 4 hours to intact skin of the flank. Animals were then observed for 7 days. Irritation was scored by the method of Draize at 1, 24, 48 and 72 hours and 7 days after exposure. Mean individual scores at 24, 48 and 72 h after exposure for the 4 animals were 2, 2, 1.67 and 2 for erythema score and 2.67, 2, 0.67 and 3 for oedema score. Under the test conditions the substance is considered to be irritating to the skin because the average oedema score exceeded 2.3 for two animals (CLP) and the effects were not fully reversible within the observation period of 7 days. Based on these results, the read-across approach was applied and laevo alpha terpineol can be considered irritating to the skin.

Reference 9

Endpoint:

skin irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance alpha terpineol which shares the same functional groups with the substance L-alpha terpineol also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

other: ID50 (μg / 5 μL)

Basis:

mean

Time point:

24/48 h

Score:

0.853

Reversibility:

fully reversible within: 48 h

Remarks on result:

other: read-across from an analogue for which 0.853 μg/5 μL score was obtained for ID50 (irritant dose in 50% individuals).

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the read-across approach from the analogue alpha terpineol, laevo alpha terpineol can be considered a skin irritant.

Executive summary:

The irritant potentials of Lavandula stoechas were investigated on albino mice. Six of its components were evaluated for irritancy by open mouse ear assay. Alpha terpineol was dissolved in acetone (10 mg/10 mL (w /v) solution). Further dilutions were prepared. Doses of 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 µg/5 µL were applied to inner surface of animal’s ear. The ears were examined for redness after 30 minutes and then 15 minute interval until two observations indicated that further redness would not occur. The time of maximum erythema was noted. The number of ears eliciting the degree of redness according to Hecker's scale were noted and expressed in μg / 5 μI per ear. The chronic effects after 24 and 48 hours were recorded and denoted by IU (Irritant units). ID50 (Irritant dose in 50% individuals ) were calculated by probit analysis. ID50 of alpha terpineol was found to be 0.853 µg/5 µL and its reaction lasted for 24 h, indicating IU = 0.625 µg/5 µL after 24 h. It is concluded that alpha terpineol is a skin irritant. Based on these results, the read-across approach was applied and laevo alpha terpineol can be considered a skin irritant.

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:

04 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption.
- Number of animals: not reported
- Characteristics of donor animals (e.g. age, sex, weight): 7 weeks old. 1.5 - 2.5 kg.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.
- Time interval prior to initiating testing: The heads have been collected on 04 June 2018 at 8: 25 am. The eyes were enucleated at lab on 04 June 2018 at 10:15 am.
- indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: no

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): 30 μL of test item

Duration of treatment / exposure:

10 second

Duration of post- treatment incubation (in vitro):

No post-treatment incubation is performed.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.0ºC and 32.2ºC.
After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure using sodium fluorescein. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope.
Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.
Once all eyes had been examined and approved, the eyes were incubated between 45 and 64 minutes to equilibrate them to the test system prior to dosing.

EQUILIBRATION AND BASELINE RECORDINGS:
Eyes were incubated between 45 and 64 minutes to equilibrate them to the test system prior to dosing
Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED
30 μL physiological saline - Dutscher Batch No. 3013132 (one eye)

SOLVENT CONTROL USED: not applicable.

POSITIVE CONTROL USED
5% (w/v) Benzalkonium chloride –Sigma–Batch No. BCBQ9761V - 30 μL (three eyes)

APPLICATION DOSE AND EXPOSURE TIME
30 μL of the test item was applied for 10 seconds.

OBSERVATION PERIOD: Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The test item was rinsed from the eye after 10 seconds of observation with 20 mL of physiological saline at ambient temperature.
- Indicate any deviation from test procedure in the Guideline: NO

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: It was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points (see table 4)
- Damage to epithelium based on fluorescein retention: Fluorescein retention value for all test eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item (Table No.5)
- Swelling: optical pachymeter on a slit-lamp microscope ((HaagStreit BP900 slit-lamp microscope with depth-measuring device no. I). The slit-width was set at 9 1/2 equalling 0.095 mm. The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item (see table 3).
- Macroscopic morphological damage to the surface: The aim of this evaluation was to determine whether any “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea were visible.These findings can vary in severity and may occur simultaneously.

SCORING SYSTEM:
- Mean corneal swelling: It was expressed as a percentage and was calculated from corneal thickness measurements according to the following formula:
(corneal thickness measurement at time t - corneal thickness at time=0 / corneal thickness at ime=0 )*100

- Mean maximum opacity score:
0 - No opacity,
0.5 -Very faint opacity
1- Scattered or diffuse areas; details of the iris clearly visible
2- Easily discernible translucent area; details of the ris are slightly obscured,
3-Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4-Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment :
0-No fluorescein retention,
0.5-Very minor single cell staining,
1-Single cell staining scattered throughout the treated area of the cornea,
2-Focal or confluent dense single cell staining,
3-Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA: Decision criteria was used as indicated in the TG.

Irritation parameter:

cornea opacity score

Run / experiment:

Maximal mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE Class II

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean at 30 min post-treatment

Value:

2.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class III

Irritation parameter:

percent corneal swelling

Run / experiment:

Maximal mean

Value:

6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class II

Irritation parameter:

morphological effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No effects

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: NO, No morphological effects were noted, whatever the examination time.

DEMONSTRATION OF TECHNICAL PROFICIENCY:Yes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:YES, the combination of the three endpoints for the negative control, physiological saline, was 3xI classified as “No Category”
- Acceptance criteria met for positive control: YES, the combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV,classified as “Corrosive/Severe Irritant ”

Table 9: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT

Test item

Endpoint measured	Eye No.	0	30	75	120	180	240
Corneal opacity	4	0	1	1	1	1	1
	5	0	1	1	1	1	1
	6	0	1	1	1	1	1
Mean		0.0	1.0	1.0	1.0	1.0	1.0
ICE class					II
Fluorescein retention	4	0.5	2
	5	0.5	2
	6	0.5	3
Mean		0.5	2.3
ICE class			III
Corneal thickness	4	0.58	0.59	0.60	0.60	0.60	0.60
	5	0.59	0.62	0.63	0.64	0.64	0.64
	6	0.62	0.64	0.65	0.65	0.65	0.65
Corneal swelling (%)	4	-	2	3	3	3	3
	5	-	5	7	8	8	8
	6	-	3	5	5	5	5
Mean			3	5	6	6	6
ICE class					II
Combination of the 3 Endpoints	1 x III, 2 x II
CLASSIFICATION	No prediction can be made

Note: No morphological effects were noted, whatever the examination time.

Table 8: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT

Positive control

Endpoint measured	Eye No.	0	30	75	120	180	240
Corneal opacity	1	0	3	3	3	3	3
	2	0	3	3	3	3	3
	3	0	3	3	3	3	3
Mean		0.0	3.0	3.0	3.0	3.0	3.0
ICE class					IV
Fluorescein retention	1	0.5	3
	2	0.5	3
	3	0.5	3
Mean		0.5	3.0
ICE class			IV
Corneal thickness	1	0.60	0.69	0.75	0.78	0.84	0.89
	2	0.59	0.66	0.71	0.79	0.82	0.85
	3	0.61	0.71	0.75	0.82	0.86	0.87
Corneal swelling (%)	1	-	15	25	30	40	48
	2	-	12	20	34	39	44
	3	-	16	23	34	41	43
Mean		-	14	23	33	40	45
ICE class					IV
Combination of the 3 Endpoints	3 x IV
CLASSIFICATION	Category 1 : Corrosive/Severe irritant

Note: Blisters on the cornea noted from 30 minutes post-dose in eyes No. 1, No. 2 and No. 3.

Table 7: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT

Negative control

Endpoint measured	Eye No.	0	30	75	120	180	240
Corneal opacity	16	0.0	0.0	0.0	0.0	0.0	0.0
ICE class					I
Fluorescein retention	16	0	0.5	-	-	-	-
ICE class			I
Corneal thickness	16	0.60	0.60	0.60	0.60	0.60	0.60
Corneal swelling (%)	16	-	0	0	0	0	0
ICE class					I
Combination of the 3 Endpoints	3 x I
CLASSIFICATION	No Category

Note: No morphological effects were noted, whatever the examination time.

Interpretation of results:

other: no prediction can be made (CLP Regulation EC no. 1272/2008)

Conclusions:

Under experimental conditions, no prediction can be made for the test item in the ICE test.

Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 under GLP conditions. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to either 30 μL of the test item, 30 μL of 5% Benzalkonium chloride (positive control) or 30μL of physiological saline (negative control). Three eyeballs were used in test item and positive groups, and one for the negative control group. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. Under experimental conditions, no prediction can be made for the test item in the ICE test since the combinations of the 3 endpoints were 1 x III, 2 x II.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 September 2018 - 29 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to OECD 405 with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

A relative humidity lower than 30% was registered on several days during the study. As no effect was noted on the health of the animals, this deviation was considered as without impact on the conclusion of the study.

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: C.E.G.A.V (La Passerie, F-61350 Saint Martin d’Egrenne) or Granja San Bernardo (Tulebras, Navarra - Spain)
- Age at study initiation: 9, 10 or 11 weeks old
- Weight at study initiation: 1.74 to 2.31 kg.
- Housing: The animals were individually housed.
- Diet (e.g. ad libitum): ad libitum. ENVIGO – 2030C
- Water (e.g. ad libitum): ad libitum. Drinking water (tap-water from public distribution system). Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas – Eurofins (FRANCE).
- Acclimation period: ≥ 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20ºC ± 3ºC
- Humidity (%): 30 to 70%
- Air changes (per hr): Minimum 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): N/A

Duration of treatment / exposure:

one application of test item not removed throughout the test.

Observation period (in vivo):

Clinical observations for changes in the cornea, iris and conjunctivae were performed 1, 24, 48 and 72 hours following treatment. If no reaction was observed 72 hours after instillation, the study was terminated. In case of persistent reactions, additional observations were carried out from D7 to D21 in order to determine the reversible character of the lesions observed.

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No
- Time after start of exposure: N/A

SCORING SYSTEM:

CHEMOSIS (A):
0-No swelling
1-Slight swelling, including the nictitating membrane
2-Swelling with eversion of the eyelid
3-Swelling with eyelid half-closed
4-Swelling with eyelid more than half-closed

DISCHARGE (B):
0-No discharge
1-Slight discharge (normal slight secretions in the inner corner not to be taken into account)
2-Discharge with moistening of the eyelids and neighbouring hairs
3-Discharge with moistening of the eyelids and large areas around the eye

REDNESS (C):
0-Blood vessels normal
1-Vessels significantly more prominent than normal
Vessels individually distinguishable with difficulty
2-Generalised red coloration
3-Generalised deep red coloration

IRIS (D):
0-Normal
1-Iris significantly more wrinkled than normal, congestion, swelling of the iris which continues to react to light, even slowly
2-No reaction to light, haemorrhage, significant damage (any or all of these characteristics)

CORNEA: DEGREE OF OPACITY (E):
0- No modification visible either directly or after instillation of fluorescein (no loss of glint or polish)
1- Translucent areas (diffuse or disseminated), iris details clearly visible
2- Easily identifiable translucent area, iris details slightly obscured
3- Opalescent area, no iris details visible, pupil outline scarcely distinguishable
4- Total corneal opacity, completely obscuring the iris and pupil

CORNEA: EXTENT OF OPACITY (F):
1- Opaque area present but covering one quarter or less
2- Between one quarter and half
3- Between half and three quarters
4- Between three quarters and the entire surface


TOOL USED TO ASSESS SCORE: not specified

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

A7288

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 3 days

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

A7305

Time point:

24/48/72 h

Score:

1.7

Max. score:

2

Reversibility:

not fully reversible within: 21 days

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Remarks:

A7306

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

A7288

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

A7305

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

A7306

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

A7288

Time point:

24/48/72 h

Score:

1.3

Max. score:

2

Reversibility:

fully reversible within: 14 days

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

A7305

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Remarks:

A7306

Time point:

24/48/72 h

Score:

1.3

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

A7288

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 2 days

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

A7305

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

A7306

Time point:

24/48/72 h

Score:

0.7

Max. score:

1

Reversibility:

fully reversible within: 3 days

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

- at the conjunctivae level: a moderate redness noted 1 hour after the test item instillation in all animals and totally reversible between Days 7 and 14. This reaction was associated with a moderate chemosis noted 1 hour after the test item instillation in all animals and totally reversible between Days 2 and 7.
- at the corneal level: a slight to moderate opacity, noted 24 hours after the test item instillation in all animals and totally reversible between Days 3 and 14 in two animals and remaining on the last day of the test (Day 21) with slight intensity (grade 1).
- at the iris level: it was noted an injection 24 hours after the test item instillation in one animal and was totally reversible on Day 7.

Other effects:

- Other observations: A relative humidity lower than 30% was registered on 01, 02, 27 and 29 October 2018. The minimum value measured was 26% (30% minimum as per OECD guideline 405). As no effect was noted on the health of the animals, this deviation is considered as without impact on the conclusion of the study.

Table 1. Assessment of acute eye irritation. Individual and mean scores of conjunctivae, iris and cornea

TEST ITEM: p-MENTH-1-EN-8-OL

Instillation: 0.1 mL of the test item

Instillation dates (D0): 24 September 2018 (A7288) & 08 October 2018 (A7305 & A7306)

Animal n° Weight (kg)	Time after treatment	CONJUNCTIVAE		IRIS		CORNEA
		CHEMOSIS (A)	REDNESS ( C)	LESION (D)	OPACITY ( E)
A7288	24 hours	1	2	0	2
	48 hours	0	1	0	1
	72 hours	0	1	0	0
Start: 2.31	TOTAL	1	4	0	3
End: 2.81	Mean	0.3	1.3	0.0	1.0
A7305	24 hours	1	2	1	2
	48 hours	1	2	1	2
	72 hours	1	2	1	1
Start: 1.89	TOTAL	3	6	3	5
End: 2.75	Mean	1.0	2.0	1.0	1.7
A7306	24 hours	1	2	0	1
	48 hours	1	1	0	1
	72 hours	0	1	0	1
Start: 1.74	TOTAL	2	4	0	3
End: 2.51	Mean	0.7	1.3	0.0	1.0
CLASSIFICATION in accordance with the CLP regulation		Taking into account the irreversibility of the reaction, the test item has to be classified in Category 1 "Irreversible effects on the eyes" with the hazard statement H318 "Causes serious eye damage"

Table 2. Total and individual scores of ocular irritation. Animal No.: A7288

Observation time	CONJUNCTIVAE				IRIS		CORNEA			Individual irritation index
	A	B	C	(A+B+C)x2	D	Dx5	E	F	ExFx5
				X=		Y=			Z=	X+Y+Z=
1 Hour (D0)	2	2	2	12	0	0	0	0	0	12
24 Hours (D1)	1	0	2	6	0	0	2	1	10	16
48 Hours (D2)	0	0	1	2	0	0	1	1	5	7
72 Hours (D3)	0	0	1	2	0	0	0	0	0	2
Day 7 (D7)	0	0	1	2	0	0	0	0	0	2
Day 14 (D14)	0	0	0	0	0	0	0	0	0	0

Table 3. Total and individual scores of ocular irritation. Animal No.: A7305

Observation time	CONJUNCTIVAE				IRIS		CORNEA			Individual irritation index
	A	B	C	(A+B+C)x2	D	Dx5	E	F	ExFx5
				X=		Y=			Z=	X+Y+Z=
1 Hour (D0)	2	3	2	14	0	0	0	0	0	14
24 Hours (D1)	1	0	2	6	1	5	2	4	40	51
48 Hours (D2)	1	0	2	6	1	5	2	4	40	51
72 Hours (D3)	1	0	2	6	1	5	1	4	20	31
Day 7 (D7)	0	0	0	0	0	0	2	1	10	10
Day 14 (D14)	0	0	0	0	0	0	1	1	5	5
Day 21 (D21)	0	0	0	0	0	0	1	1	5	5

Table 4. Total and individual scores of ocular irritation. Animal No.: A7306

Observation time	CONJUNCTIVAE				IRIS		CORNEA			Individual irritation index
	A	B	C	(A+B+C)x2	D	Dx5	E	F	ExFx5
				X=		Y=			Z=	X+Y+Z=
1 Hour (D0)	2	3	2	14	0	0	0	0	0	14
24 Hours (D1)	1	0	2	6	0	0	1	4	20	26
48 Hours (D2)	1	0	1	4	0	0	1	2	10	14
72 Hours (D3)	0	0	1	2	0	0	1	1	5	7
Day 7 (D7)	0	0	0	0	0	0	1	1	5	5
Day 14 (D14)	0	0	0	0	0	0	0	0	0	0

Interpretation of results:

other: classified as Cat. 1 (serious eye damage) (CLP Regulation EC no. 1272/2008)

Conclusions:

Based on the results of an acute eye irritation study performed on New Zealand White rabbits, the test substance has to be classified in category 1 (serious eye damage) as irreversible effects were found on the eye.

Executive summary:

The eye irritation potential of the test substance was determined in accordance with the OECD guideline 405 with GLP. Three young adult albino New Zealand rabbits were given a single ocular application of 0.1 mL test substance in one eye of the rabbit while the contralateral eye remained untreated and served as the control. Animals were observed at 1, 24, 48, 72 h. Thereafter, in case of persistent reactions, additional observations were carried out from D7 to D21 in order to determine the reversible character of the lesions observed. The corneal opacity, iris and the conjunctivae (redness and chemosis) scores were recorded. The mean eye irritation scores at 24, 48 and 72 h post-application observations for each animal were 1.0, 1.7 and 1.0 for corneal opacity, 0.0, 1.0 and 0.0 for iris effects, 1.3, 2.0 and 1.3 for conjunctival redness and 0.3, 1.0 and 0.7 for conjunctival chemosis. These effects were fully reversible between days 3 and 14 for all animals except in the case of corneal opacity for one animal in which remaining effects on the last day of the test (Day 21) were still recorded. Based on these results, the test substance has to be classified in category 1 (serious eye damage) according to CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation. Weight of evidence. Read-across approach: In a primary dermal irritation study performed according to OECD guideline 404 and in compliance with GLP, three independent tests were conducted on 3 or 4 albino rabbits which were dermally exposed to 0.5 mL of undiluted alpha terpineol, under a semi-occlusive patch for 4 hours to intact skin of the flank. Animals were then observed for 7 days. Irritation was scored by the method of Draize at 1, 24, 48 and 72 hours and 7 days after exposure. The following results were obtained for each test:

Test 1: A Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals tested were 1.67, 2 and 2.33 for erythema score and 2, 2.33 and 3 for oedema score. The substance is considered to be irritating to the skin because the average oedema score exceeded 2.3 for two animals (CLP) and the effects were not fully reversible within the observation period of 7 days.

Test 2: Mean individual scores at 24, 48 and 72 h after exposure for the 4 animals tested were 2, 2.67, 2 and 2 for erythema score and 3, 3, 2.67 and 1.67 for oedema score. The substance is considered to be irritating to the skin because the average oedema score exceeded 2.3 for two animals (CLP) and the effects were not fully reversible within the observation period of 7 days.

Test 3: Mean individual scores at 24, 48 and 72 h after exposure for the 4 animals tested were 2, 2, 1.67 and 2 for erythema score and 2.67, 2, 0.67 and 3 for oedema score. The substance is considered to be irritating to the skin because the average oedema score exceeded 2.3 for two animals (CLP) and the effects were not fully reversible within the observation period of 7 days.

Based on these results, the read-across approach was applied and laevo alpha terpineol can be considered irritating to the skin.

Skin irritation. Supporting study: Read-across approach. The irritant potentials of Lavandula stoechas were investigated on albino mice. Six of its components were evaluated for irritancy by open mouse ear assay. Alpha terpineol was dissolved in acetone (10 mg/10 mL (w /v) solution). Further dilutions were prepared. Doses of 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 µg/5 µL were applied to inner surface of animal’s ear. The ears were examined for redness after 30 minutes and then 15 minute interval until two observations indicated that further redness would not occur. The time of maximum erythema was noted. The number of ears eliciting the degree of redness according to Hecker's scale were noted and expressed in μg / 5 μI per ear. The chronic effects after 24 and 48 hours were recorded and denoted by IU (Irritant units). ID50 (Irritant dose in 50% individuals ) were calculated by probit analysis. ID50 of alpha terpineol was found to be 0.853 µg/5 µL and its reaction lasted for 24 h, indicating IU = 0.625 µg/5 µL after 24 h. It is concluded that alpha terpineol is a skin irritant. Based on these results, the read-across approach was applied and laevo alpha terpineol can be considered a skin irritant.

Eye irritation (in vitro): Key study: An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 under GLP conditions. Eyes from chicken were exposed to either 30 μL of the test item, 30 μL of 5% Benzalkonium chloride (positive control) or 30μL of physiological saline (negative control). Three eyeballs were used in test item and positive groups, and one for the negative control group. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. Under experimental conditions, no prediction can be made for the test item in the ICE test since the combinations of the 3 endpoints were 1 x III, 2 x II.

Eye irritation (in vivo): Key study: The eye irritation potential of the test substance was determined in accordance with the OECD guideline 405 with GLP. Three young adult albino New Zealand rabbits were given a single ocular application of 0.1 mL test substance in one eye of the rabbit while the contralateral eye remained untreated and served as the control. Animals were observed at 1, 24, 48, 72 h. Thereafter, in case of persistent reactions, additional observations were carried out from D7 to D21 in order to determine the reversible character of the lesions observed. The corneal opacity, iris and the conjunctivae (redness and chemosis) scores were recorded. The mean eye irritation scores at 24, 48 and 72 h post-application observations for each animal were 1.0, 1.7 and 1.0 for corneal opacity, 0.0, 1.0 and 0.0 for iris effects, 1.3, 2.0 and 1.3 for conjunctival redness and 0.3, 1.0 and 0.7 for conjunctival chemosis. These effects were fully reversible between days 3 and 14 for all animals except in the case of corneal opacity for one animal in which remaining effects on the last day of the test (Day 21) were still recorded. Based on these results, the test substance has to be classified in category 1 (serious eye damage) according to CLP Regulation.

Justification for classification or non-classification

Based on the available information, the test item is classified as skin irritant (category 2) in accordance with CLP Regulation (EU) No. 1272/2008.

Based on the available information, the test item needs to be classified in category 1 (serious eye damage) in accordance with CLP Regulation (EU) No. 1272/2008.