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

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 20 - June 17, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2010

Deviations:

yes

Remarks:

The protocol states that all decedents and moribund mice will undergo a gross pathology examination. Necropsy was not performed on two preliminary animals that were found dead on Day 3. This deviation had no impact on the outcome of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

N/A

Molecular formula:

C10H16O2

Test material form:

liquid

Remarks:

Colorless

Details on test material:

Test substance was expected to be stable for the duration of testing.

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:
Test substance was expected to be stable

- Solubility and stability of the test material in the solvent/vehicle and the exposure medium:
Solubility testing indicated that the test substance was soluble in 1% Pluronic® L92

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

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RIVIS Inc.
- Age at study initiation: 12 weeks
- Weight at study initiation: 18.9-22.8 grams
- Housing:
The animals were individually housed in plastic solid bottom cages during the dosing and resting phase of the study. After final weighing until sacrifice, animals were housed in their respective dose groups in plastic cages with bedding. Bedding in the plastic, solid bottom cages was changed at least once per week. Enrichment (e.g., nesting material) was placed in each cage. All caging conformed to the size recommendations in the most recent Guide for the Care and Use of
LaboratoryAnimals (NatI. Res. Council, 2011).

- Diet (e.g. ad libitum):
Envigo Tekiad Global 16% Protein Rodent Diet® #2016. The diet was available ad-libitum.

- Water: Filtered tap water was supplied ad libitum

- Acclimation period: 21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 23
- Humidity (%): 53 - 69
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12

Study design: in vivo (LLNA)

Vehicle:

other: 1% Pluronic® L92

Concentration:

10%, 25% and 50% (Main study)
25%, 50% and 100% (preliminary study)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
Test substance (prepared as w/w mixtures in 1% Pluronic® L92) concentrations of 25%, 50% and 100% were tested to determine the overt systemic toxicity or excessive local irritation. Each group consisted of two mice. The ears of each mouse were evaluated for erythema and edema pre-dose on Day 1, 2, 3, and 6. 25 uL of the appropriate concentration of the test substance or the vehicle alone was applied to the dorsum of both ears of each mouse for three consecutive days. No treatment was made on Days 4 and 5. On Day 6, the sites for each mouse were evaluated for local reactions (erythema & edema). Animals were observed daily for signs of toxicity.

MAIN STUDY

- Criteria used to consider a positive response:
The mean and standard deviation of the dpm values were calculated for each dose group. A stimulation index (SI) was derived for each experimental group by dividing the mean dpm of each experimental group by the mean dpm of the vehicle control group. Any test substance that produces an SI > 3 in the LLNA is normally considered “positive” for dermal sensitization potential. The EC3 value was not calculated since all dose levels induced a stimulation index of less than 3.0.

TREATMENT PREPARATION AND ADMINISTRATION:

Concentrations of 10%, 25% and 50% were selected for the main test based on results of the preliminary screening test. Dilutions of the test substance were prepared as w/w mixtures in 1% Pluronic® L92. The vehicle control, 1% Pluronic® L92, and a single concentration of a 25% w/w mixture of HCA (positive control) in 1% Pluronic® L92 were also prepared. All dosage preparations were freshly prepared on the day of application.

Beginning on Day 1, a quantity of 25 uL of the appropriate test substance concentration, the positive control substance, or the vehicle alone was applied to the dorsum of both ears of each mouse once per day for three consecutive days (Days 1, 2, and 3) using a micropipette. During application, the material was gently spread as evenly as possible over the dorsal surface of the ear using the tip of the pipette.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Statistical analysis was performed on the DPM minus background values. Significance was judged at p < 0.05. The treated groups and negative vehicle control group were compared using a One-Way Analysis of Variance (ANOVA), followed by comparison of the treated groups to control by Dunnett’s t-test for multiple comparisons. Where variances are considered significantly different by Bartlett’s test, groups were compared using a non-parametric method (Kruskal-Wallis non parametric analysis of variance followed by Dunn’s test) (INSTAT Biostatistics, Graph Pad Software, San Diego, CA). Outlier analysis was conducted using Grubbs (1969).

Results and discussion

Positive control results:

The positive control (HCA) at 25% produced a dermal sensitization response in mice (SI = 5.75). Therefore, the LLNA test system was valid for this study with Dihydronepetalactone.

Very slight erythema (score of 1) was evident at six positive control sites on Day 2, at all sites on Day 3 and at eight sites on Day 6. Slight edema (score of 1) was present at three sites on Day 3.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.04

Test group / Remarks:

10% concentration group

Results 2

Key result

Parameter:

SI

Value:

1.16

Test group / Remarks:

25% concentration group

Results 3

Key result

Parameter:

SI

Value:

1.77

Test group / Remarks:

50% concentration group

Any other information on results incl. tables

All animals appeared active and healthy throughout the study. Three mice from the test groups, two from the vehicle control group and three from the positive control group lost body weight during the study. All other mice gained body weight during the study.

No dermal irritation was observed for any of the test group sites in any dose groups.

STIMULATION INDEX

Group		Group mean DPM	SI	Sensitization response
Vehicle Control	1	1662.69	-	Not applicable
Positive Control	2	9554.65*	5.75	Positive -— valid study
10% Test Substance	3	3396.61	2.04	Not a Sensitizer
25% Test Substance	4	1937.03	1.16	Not a Sensitizer
50% Test Substance	5	2936.69	1.77	Not a Sensitizer

 * Statistically significant difference from vehicle control at p < 0.01 by Dunnett Multiple Comparisons test

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these findings and on the evaluation system used, Dihydronepetalactone is not
considered to be a contact dermal sensitizer at concentrations less than or equal to 50% in the
LLNA.
Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate
contact sensitizer.

Executive summary:

A dermal sensitization test was conducted with mice to determine the potential for Dihydronepetalactone to produce sensitization after repeated topical applications.

Three concentrations of the test substance (10%, 25% and 50%) in 1% Pluronic® L92 Surfactant w/w in distilled water (1% Pluronic® L92) were topically applied to fifteen healthy test mice (five mice/group) for three consecutive days. Three days after the last application, the mice were given an IV injection containing 20 iiCi of 3H-methyl thymidine. Approximately five hours later, all animals were euthanized via an overdose of inhaled Isoflurane and the draining (auricular) lymph nodes were harvested and prepared for analysis in a scintillation counter. The results are presented in disintegrations per minute per mouse (dpm/mouse). Each animal’s ears were also evaluated for erythema and edema prior to each application and again on Day 6, prior to the IV injection.

A vehicle control group (five animals) and a positive control group (five animals) were maintained under the same environmental conditions and treated in the same manner as the test animals. The vehicle control animals were treated with 1% Pluronic® L92 and the positive control group animals were treated with a 25% w/w mixture of RCA in 1% Pluronic® L92. In an effort to reduce the total number of animals used, this study was run concurrently with another study to utilize a common positive control and common vehicle control group.

Treatment of mice with 10%, 25% and 50% of Dihydronepetalactone resulted in stimulation index values of 2.04, 1.16, and 1.77, respectively. As a stimulation index (SI) of less than 3.0 was observed in all the treatment groups, the test substance was not considered positive for a dermal sensitization potential. The positive control (HCA) at 25% produced a dermal sensitization response in mice (SI 5.75). Therefore, the LLNA test system was valid for this study with Dihydronepetalactone. Based on the results of this study, the test substance is not considered to be a contact dermal sensitizer at concentrations less than or equal to 50% in the LLNA.