((4S,4aR,7S,7aR)-4,7-dimethylhexahydrocyclopenta[c]pyran-1(3H)-one

Endpoint summary

Description of key information

A primary skin irritation test was conducted with rabbits to determine the potential for Dihydronepetalactone to produce irritation after a single topical application. Under the conditions of this study, the test substance is classified as slightly irritating to the skin.

Initially, one rabbit was tested to determine the irritation/corrosion potential of the test substance. Three dose sites, each approximately 6-cm2, were delineated on this animal. Five-tenths of a milliliter of the neat test substance was applied to the skin of each dose site. The pads and entire trunk of the animal were then wrapped with semi-occlusive 3-inch MicroporeTM tape to avoid dislocation of the patches. The patches were removed at the appropriate intervals (3 minutes, 1 hour, and 4 hours). All dose sites were evaluated for skin irritation according to the Draize method of scoring  and for corrosion 30-60 minutes after the appropriate patch removal. Subsequent evaluations were performed approximately 24, 48, 72 hours and at 7 days after removal of the 4-hour patch. Since there was no corrosion observed at any of the dose sites for this animal, two additional rabbits were selected for testing and had the test substance applied for 4 hours only. All dose sites were evaluated for skin irritation according to the Draize scoring system at 30-60 minutes, and 24, 48, 72 hours and at 7 days after patch removal.

No visible necrosis (corrosion) of the skin tissue was observed at the 3-minute, 1-hour and 4-hour dose sites during the study.

3-Minute Exposure Site: Within 30-60 minutes of patch removal, very slight erythema was noted at the 3-minute exposure site, which resolved by 24 hours.

1-Hour Exposure Site: Within 30-60 minutes of patch removal, very slight erythema was noted at the 1-hour exposure site. The overall incidence and severity of irritation decreased with time. Dermal irritation cleared from the treated site by 48 hours.

4-Hour Exposure Site: Over the first 72 hours after patch removal, all three treated sites exhibited very slight to well-defined erythema and/or very slight edema. The overall incidence and severity of irritation decreased gradually with time. All animals were free of dermal irritation by Day 7 (study termination).

The Primary Dermal Irritation Index (PDII) calculated for this test substance was 1.7. The test substance is Mildly irritating according to GHS (8th revised edition).

 

A primary eye irritation test was conducted with rabbits to determine the potential for Dihydronepetalactone to produce irritation from a single instillation via the ocular route. One-tenth of a milliliter of the test substance was instilled into the right eye of three healthy rabbits. The left eye remained untreated and served as a control. Ocular irritation was evaluated by the Draize method of scoring.

Within 24 hours after test substance instillation, two treated eyes exhibited corneal opacity and ‘positive’ conjunctivitis. There was no iritis observed in any treated eye during this study. The overall incidence and severity of irritation decreased gradually with time. Positive irritation cleared from the two treated eyes by Day 4. All animals were free of ocular irritation by Day 7 (study termination).

The Maximum Mean Total Score of Dihydronepetalactone is 14.7. Under the conditions of this study, Dihydronepetalactone is classified as mildly irritating to the eye.

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:

May 19 to June 3, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- lot number of test material:
CH-DA-01-18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Stored refrigerated

- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage:
Test substance was expected to be stable for the duration of testing.

- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis:
The test substance was applied as received

- Solubility and stability of the test material in the solvent/vehicle and the exposure medium:
The test substance was applied as received

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding):
Mixed well prior to use

- Preliminary purification step (if any):
None

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Robinson Services, Inc.
- Age at study initiation: 14 weeks
- Weight at study initiation: 2584-2870 grams
- Housing:
The animals were singly housed in suspended stainless steel caging, which conforms to the size recommendations in the most recent Guidefor the Care and Use ofLaboratory Animals (Natl. Res. Council, 2011). Enrichment (e.g., toy) was placed in each cage. Litter paper was placed beneath the cage and was changed at least three times per week.

- Diet (e.g. ad libitum):
PMI 5326 High Fiber Rabbit Diet. A designated amount of the diet (approximately 150 grams/day) and alfalfa hay cubes (Grainland Select Alfalfa Cubes) were available to each rabbit.

- Water: Filtered tap water was supplied ad libitum

- Acclimation period: 14 or 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 49 - 70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

Five-tenths of a millilitre

Duration of treatment / exposure:

3 minutes, 1 hour, and 4 hours

Observation period:

All dose sites were evaluated for corrosion 30-60 minutes after patch removal. Subsequent evaluations were performed approximately 24, 48, 72 hours and at 7 days after removal of the four-hour patch.

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 6 Cm2
- coverage: 1-inch x 1-inch, 4-ply gauze pad.
- Type of wrap if used: semi-occlusive 3-inch Micropore tape

REMOVAL OF TEST SUBSTANCE
- Washing (if done): 3% soap solution followed by tap water and a clean paper towel
- Time after start of exposure: 3 minutes, 1 hour, and 4 hours

OBSERVATION TIME POINTS
30-60 minutes; 24, 48, 72 hours and at 7 days

SCORING SYSTEM:
- Method of calculation: Draize scoring system

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Remarks:

Average values for three rabbits.

Time point:

other: 30-60 minutes; 24, 48 and 72 hours

Score:

1.7

Reversibility:

fully reversible

Remarks:

All animals were free of dermal irritation by Day 7 (study termination).

Remarks on result:

probability of mild irritation

Irritant / corrosive response data:

No visible necrosis (corrosion) of the skin tissue was observed at the 3-minute, 1-hour and 4-hour
dose sites during the study. In the 3-minute exposure site, within 30-60 minutes of patch removal, very slight erythema was noted at the 3-minute exposure site, which resolved by 24 hours.


In 1 hour exposure site, within 30-60 minutes of patch removal, very slight erythema was noted at the 1-hour exposure site. The overall incidence and severity of irritation decreased with time. Dermal irritation cleared from the treated site by 48 hours.


In 4 hours exposure sites, over the first 72 hours after patch removal, all three treated sites exhibited very slight to well-defined erythema and/or very slight edema. The overall incidence and severity of irritation decreased gradually with time. All animals were free of dermal irritation by Day 7 (study termination).


The Primary Dermal Irritation Index for Dihydronepetalactone was 1.7.

INDIVIDUAL SKIN IRRITATION SCORES

3-MINUTE EXPOSURE

Animal No	Sex	Time after removal of patch
		Minute	Hours			Days
		30 - 60	24	48	72	7
3501	F	1/0	0/0	0/0	0/0	0/0

 

1-HOUR EXPOSURE

Animal No	Sex	Time after removal of patch
		Minute	Hours			Days
		30 - 60	24	48	72	7
3501	F	1/0	1/0	0/0	0/0	0/0

 

4-HOUR EXPOSURE

Animal No	Sex	Time after removal of patch
		Minute	Hours			Days
		30 - 60	24	48	72	7
3501	F	1/0	2/0	2/0	2/0	0/0
3502	F	1/0	2/0	1/0	1/0	0/0
3503	F	1/1	2/1	1/1	1/1	0/0
Total		3/1	6/1	4/1	4/1	0/0
Mean		1.0/0.3	2.0/0.3	1.3/0.3	1.3/0.3	0/0

Interpretation of results:

Category 3 (mild irritant) based on GHS criteria

Conclusions:

Mildly irritating according to GHS (8th revised edition)

Executive summary:

A primary skin irritation test was conducted with rabbits to determine the potential for Dihydronepetalactone to produce irritation after a single topical application. Under the conditions of this study, the test substance is classified as slightly irritating to the skin.

Initially, one rabbit was tested to determine the irritation/corrosion potential of the test substance. Three dose sites, each approximately 6-cm2, were delineated on this animal. Five-tenths of a milliliter of the neat test substance was applied to the skin of each dose site. The pads and entire trunk of the animal were then wrapped with semi-occlusive 3-inch MicroporeTM tape to avoid dislocation of the patches. The patches were removed at the appropriate intervals (3 minutes, 1 hour, and 4 hours). All dose sites were evaluated for skin irritation according to the Draize method of scoring (Draize et al., 1944; see Table 6) and for corrosion 30-60 minutes after the appropriate patch removal. Subsequent evaluations were performed approximately 24, 48, 72 hours and at 7 days after removal of the 4-hour patch. Since there was no corrosion observed at any of the dose sites for this animal, two additional rabbits were selected for testing and had the test substance applied for 4 hours only. All dose sites were evaluated for skin irritation according to the Draize scoring system at 30-60 minutes, and 24, 48, 72 hours and at 7 days after patch removal.

No visible necrosis (corrosion) of the skin tissue was observed at the 3-minute, 1-hour and 4-hour dose sites during the study.

3-Minute Exposure Site: Within 30-60 minutes of patch removal, very slight erythema was noted at the 3-minute exposure site, which resolved by 24 hours.

1-Hour Exposure Site: Within 30-60 minutes of patch removal, very slight erythema was noted at the 1-hour exposure site. The overall incidence and severity of irritation decreased with time. Dermal irritation cleared from the treated site by 48 hours.

4-Hour Exposure Site: Over the first 72 hours after patch removal, all three treated sites exhibited very slight to well-defined erythema and/or very slight edema. The overall incidence and severity of irritation decreased gradually with time. All animals were free of dermal irritation by Day 7 (study termination).

The Primary Dermal Irritation Index (PDII) calculated for this test substance was 1.7. The test substance is Mildly irritating according to GHS (8th revised edition).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 1 - June 8, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- lot number of test material:
CH-DA-01-18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Stored refrigerated

- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage:
Test substance was expected to be stable for the duration of testing.

- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis:
The test substance was applied as received

- Solubility and stability of the test material in the solvent/vehicle and the exposure medium:
The test substance was applied as received

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding):
Mixed well prior to use

- Preliminary purification step (if any):
None

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Robinson Services, Inc.
- Age at study initiation: 15 - 16 weeks
- Weight at study initiation: 2965 - 3478 grams
- Housing:
The animals were singly housed in suspended stainless steel caging, which conforms to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). Enrichment (e.g., toy) was placed in each cage. Litter paper was placed beneath the cage and was changed at least three times per week.

- Diet (e.g. ad libitum):
PMI 5326 High Fiber Rabbit Diet. A designated amount of the diet (approximately 150 grams/day) and alfalfa hay cubes (Grainland Select Alfalfa Cubes) were available to each rabbit.

- Water: Filtered tap water was supplied ad libitum

- Acclimation period: 19 or 33 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 49 - 68
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12

Vehicle:

unchanged (no vehicle)

Controls:

other: The other eye of each rabbit remained untreated with the test substance and served as a control.

Amount / concentration applied:

One-tenth of a milliliter of the test substance.

Duration of treatment / exposure:

The upper and lower lids were gently held together after instillation for about one second before releasing to minimize loss of the test substance.

Observation period (in vivo):

1, 24, 48, and 72 hours and at 4 and 7 days post-instillation.

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
Ocular irritation was evaluated using Draize method of scoring at 1, 24, 48, and 72 hours and at 4 and 7 days post-instillation. The fluorescein dye evaluation procedure (which involves rinsing with physiological saline) was used in the treated eye at 24 hours and as needed at subsequent scoring intervals


SCORING SYSTEM: Draize method

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

maximum mean total score (MMTS)

Basis:

mean

Remarks:

The time interval with the highest mean score (Maximum Mean Total Score - MMTS) for all rabbits was used to classify the test substance by the system of Kay and Calandra

Time point:

24 h

Score:

14.7

Reversibility:

fully reversible within: 96 hours

Remarks on result:

probability of mild irritation

Irritant / corrosive response data:

Within 24 hours after test substance instillation, two treated eyes exhibited corneal opacity and
‘positive’ conjunctivitis. There was no iritis observed in any treated eye during this study. The
overall incidence and severity of irritation decreased gradually with time. Positive irritation
cleared from the two treated eyes by Day 4. All animals were free of ocular irritation by Day 7
(study termination).
The Maximum Mean Total Score of Dihydronepetalactone is 14.7.

INDIVIDUAL SCORES FOR OCULAR IRRITATION

	Rabbit No.: 3401 (Female)
	Hours				Days
	1	24	48	72	4	7
I. Cornea
A. Opacity	0	2	2	1	0	0
B.Area	4	1	1	1	4	4
(AxB)x5	0	10	10	5	0	0
II. Iris
A.Values	0	0	0	0	0	0
Ax5	0	0	0	0	0	0
III. Conjunctivae
A.Redness	1	2	2	2	1	0
B. Chemosis	2	1	1	0	0	0
C. Discharge	3	1	0	0	0	0
(A+B+C)x2	12	8	6	4	2	0
Total	12	18	16	9	2	0

 

	Rabbit No.: 3402 (Female)
	Hours				Days
	1	24	48	72	4	7
I. Cornea
A. Opacity	0	2	2	1	0	0
B.Area	4	1	1	1	4	4
(AxB)x5	0	10	10	5	0	0
II. Iris
A.Values	0	0	0	0	0	0
Ax5	0	0	0	0	0	0
III. Conjunctivae
A.Redness	1	2	2	2	1	0
B. Chemosis	2	2	1	0	0	0
C. Discharge	3	2	0	0	0	0
(A+B+C)x2	12	12	6	4	2	0
Total	12	22	16	9	2	0

 

	Rabbit No.: 3403 (Female)
	Hours				Days
	1	24	48	72	4	7
I. Cornea
A. Opacity	0	0	0	0	0	0
B.Area	4	4	4	4	4	4
(AxB)x5	0	0	0	0	0	0
II. Iris
A.Values	0	0	0	0	0	0
Ax5	0	0	0	0	0	0
III. Conjunctivae
A.Redness	1	1	0	0	0	0
B. Chemosis	1	1	0	0	0	0
C. Discharge	2	0	0	0	0	0
(A+B+C)x2	8	4	0	0	0	0
Total	8	4	0	0	0	0

Interpretation of results:

Category 2B (mildly irritating to eyes) based on GHS criteria

Conclusions:

Under the conditions of this study, Dihydronepetalactone is classified as mildly irritating to the
eye.

Executive summary:

A primary eye irritation test was conducted with rabbits to determine the potential for Dihydronepetalactone to produce irritation from a single instillation via the ocular route. One-tenth of a milliliter of the test substance was instilled into the right eye of three healthy rabbits. The left eye remained untreated and served as a control. Ocular irritation was evaluated by the Draize method of scoring.Within 24 hours after test substance instillation, two treated eyes exhibited corneal opacity and ‘positive’ conjunctivitis. There was no iritis observed in any treated eye during this study. The overall incidence and severity of irritation decreased gradually with time. Positive irritation cleared from the two treated eyes by Day 4. All animals were free of ocular irritation by Day 7 (study termination).

The Maximum Mean Total Score of Dihydronepetalactone is 14.7. Under the conditions of this study, Dihydronepetalactone is classified as mildly irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Justification for classification or non-classification

Adverse effects are observed for the substance Dihydronepetalactone in both the skin irritation and eye irritation studies.  The skin irritation effects were classified as Category 3 under GHS and it does not meet the criteria for classification according to Regulation (EC) No 1272/2008.  The eye irritation effects were classified as category 2 according to Regulation (EC) No 1272/2008 and category 2B under GHS.