Decan-5-olide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Decan-5-olide

EC Number:

211-889-1

EC Name:

Decan-5-olide

Cas Number:

705-86-2

Molecular formula:

C10H18O2

IUPAC Name:

6-pentyloxan-2-one

Test material form:

liquid

Details on test material:

Name : Decan-5-olide
Chemical Name (IUPAC) : Decan-5-olide
Molecular Formula : C10-H18-O2
Molecular Weight : 170.25
Origin : Organic
Physical state : Colourless liquid
Purity as per Certificate of Analysis : 97.2 %
Batch No. : P586
InChI: InChI=1S/C10H18O2/c1-2-3-4-6-9-7-5-8-10(11)12-9/h9H,2-8H2,1H3
Smiles : CCCCCC1CCCC(=O)O1

Specific details on test material used for the study:

Name : Decan-5-olide
Chemical Name (IUPAC) : Decan-5-olide
Molecular Formula : C10-H18-O2
Molecular Weight : 170.25
Origin : Organic
Physical state : Colourless liquid
Purity as per Certificate of Analysis : 97.2 %
Batch No. : P586
InChI: InChI=1S/C10H18O2/c1-2-3-4-6-9-7-5-8-10(11)12-9/h9H,2-8H2,1H3
Smiles : CCCCCC1CCCC(=O)O1

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo Kreuzelweg 53, 5961 NM Horst, Nederland
- Females (if applicable) nulliparous and non-pregnant: yes, Animals used were nulliparous and non-pregnant
- Microbiological status of animals, when known: Not specified
- Age at study initiation: 9 to 11 weeks
- Weight at study initiation: 17.0 to 20.5 gm
- Housing: Animals were housed individually (to avoid licking of test item by cage mates) in solid floor standard polysulfone cages (Size: Approximately L 360 x B 205 x H 140 mm), with stainless steel top grill having facilities for providing pelleted food and drinking water in polycarbonate bottle with stainless steel sipper tubes. Steam sterilized corn cob was used as bedding and changed along with the cage once a week
- Diet (e.g. ad libitum): Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was provided ad libitum
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier (manufactured by Eureka Forbes Limited, Mumbai - 400 001, India)
- Acclimation period: The animals were acclimatized for 6 days before start of the treatment
- Indication of any skin lesions: After physical examination for good health and the suitability for the study animals were used. No indication of any skin lesions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 24°C
- Humidity (%): 65 to 67 %
- Air changes (per hr): 13.5 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness
- IN-LIFE DATES: From: 18 January 2022 To: 24 February 2022

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

In the irritation screen, the vehicle MEK, concentrations of 10, 25, 50 and 75% v/v in MEK and 100% (undiluted) test item.

In main study (dermal sensitization), the vehicle MEK, concentrations of 25, 50% v/v in MEK and 100% (undiluted) test item.

No. of animals per dose:

In Iriritation screen - One female mice / dose
In main study - Four female mice / dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The miscibility / solubility of the test item was tested using AOO, DMF, MEK, PG, MEK and 1% L92 at the following concentrations: 90, 75, 60, 50, 40, 25 and 10 % w/v. The test item is a Colourless liquid. Based on the maximum miscible concentration, MEK was selected as the vehicle. The solubility results were captured in table and attached in any other information on materials and method incl. tables.
- Irritation: In the irritation screen, the vehicle MEK, concentrations of 10, 25, 50 and 75% v/v in MEK and 100% (undiluted) test item were evaluated for irritation potential (1 mouse / group) at each dose level for 3 consecutive days, as measured by erythema of the ears. There were no clinical signs, no erythema at the site of application, no significant increase in the ear thickness and ear punch weights and no effect on body weight. Results from this screening study were used to determine the dosing concentrations of test item for the main LLNA study
- Systemic toxicity: No effects observed.
- Ear thickness measurements: Ear thickness was measured using Digimatic micrometer (Mitutoyo, Japan) prior to dosing on Days 1 and 3 and on day 6. Additionally, on Day 6, ear thickness was determined by ear punch weight determination, after animals were euthanized.
- Erythema scores: Erythema scores, ear thickness and body weight data following the test item applications were compared to the response of the animals treated with vehicle alone.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
Animals were randomly distributed to different groups by body weight stratification method. The mice procured for the study were weighed and grouped into body weight ranges. These body weight stratified animals were distributed to all the study groups. Mice with extreme body weights were removed from the study
- Name of test method: Skin Sensitization: Local Lymph Node Assay
- Criteria used to consider a positive response: If the test item produces a Stimulation Index (SI) < 3, it is considered “Negative” for sensitization, and therefore an EC3 is not determined. Any test material that produces a SI >3 in the LLNA is normally considered “positive” for dermal sensitization potential (Kimber et al., 1994). While a SI >3 was originally developed empirically, a robust statistical evaluation indicated that it is an acceptable practical value for hazard identification (Basketter et al., 1999). While a test material that produces a SI of >3 in the LLNA should be considered “positive” for contact sensitization (Kimber et al., 1994).

TREATMENT PREPARATION AND ADMINISTRATION:

Dose Preparation: Concentrations tested for the irritancy screen were selected based upon solubility in an appropriate LLNA vehicle. The toxicity data regarding the irritation potential was also taken into consideration. The required quantity of the test item was mixed with MEK to get concentration of 25 and 50% v/v The dose formulations were prepared daily just prior to dosing.

Irritation Screen: Concentrations tested for the irritancy screen were selected based upon miscibility in an appropriate LLNA vehicle. The toxicity data regarding the irritation potential was also taken into consideration. Since a concentration of 90% was the maximum miscible concentration in MEK, the highest possible dose concentration of 100% undiluted test item was selected as highest dose concentration for testing.
In the irritation screen, the vehicle MEK, concentrations of 10, 25, 50 and 75% v/v in MEK and 100% (undiluted) test item were evaluated for irritation potential (1 mouse / group), as measured by erythema of the ears. Both ears of six female mice (one mouse/concentration) were topically treated once daily for three consecutive days with one of the concentrations of the test item.
The test item was applied using an adjustable micropipette with disposable tips. All mice received 25 µL of one concentration of the test item, spread over the dorsal surface of each ear in a manner to prevent test item loss (50 µL total / mouse). Similarly, the vehicle was applied to the ears of one animal. Both ears were observed for skin reaction prior to application of the test item (on day 1, 2 and 3), and on day 6. All mice were weighed on Days 1 and 6. Ear thickness was measured using Digimatic micrometer (Mitutoyo, Japan) prior to dosing on Days 1 and 3 and on day 6. Additionally, on Day 6, ear thickness was determined by ear punch weight determination as detailed in paragraph 9.6, after animals were euthanized. Erythema scores, ear thickness and body weight data following the test item applications were compared to the response of the animals treated with vehicle alone. I
Based on the results of this screening study, the dose concentrations of 25, 50% of test item in MEK and 100% (undiluted) test item were selected for the main study, because none of the tested concentrations elicited any irritation reaction, no increased ear thickness and ear punch weights.
Note: Ear thickness and ear punch weight were determined for irritation screen only.

Main Study, Dermal Sensitization: The test item (25 µL/ear) was applied on the dorsum of both ears as described above. Four female mice/group received the vehicle (MEK, the positive control substance (25% v/v α-hexylcinnamaldehyde), and 25, 50% of test item in MEK and 100% (undiluted) test item, once daily for three consecutive days. Both ears were observed for skin reaction prior to application of the test item (on day 1, 2 and 3), and on day 6. All mice were weighed on days 1 and 6.

Preparation of 3H-Methyl Thymidine (3H -TdR): A solution of 3H-TdR (80 µCi/mL) [specific activity 0.9637 mci/mmol; Perkin Elmer, USA] in sterile phosphate buffer saline (PBS) was prepared freshly. The prepared working solution of 3H-TdR was analyzed for radioactivity.

Injection of 3H-Methyl Thymidine (3H -TdR): On day 6, a volume of 250 µL (20 µCi) of 3H-TdR in PBS was administered to each mouse via the lateral tail vein using a 1.0 mL disposable syringe fitted with 26 G x ½ inch needle

Collection of auricular lymph nodes: Approximately five hours post injection of 3H-TdR, animals were euthanized using isoflurane anaesthesia. The auricular lymph nodes (bilateral) were excised and placed in PBS, processed and the radioactivity was counted.

Ear punch weight: The animals were euthanized using isoflurane anesthesia and both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 6 mm). For each animal both punches were immediately weighed using an analytical balance.

Processing of auricular lymph nodes : A single cell suspension of the lymph node cells (LNC) from each mouse was prepared by gentle mechanical disaggregation using a tissue homogeniser (Stomacher 80 MicroBiomaster, Seward Ltd, United Kingdom) for 30 seconds at medium speed using PBS (approx. 10 mL).
After all the nodes had been processed, the tubes were centrifuged at 200 x g for 10 minutes at 4°C. The supernatant was poured into a container for radiolabel waste collection. 10 mL of PBS was added to each tube and inverted to resuspend the pellet. The tubes were centrifuged as described above and the supernatant was poured off. After the second wash, the cell pellet was suspended in 3.0 mL of 5% trichloroacetic acid (TCA) and stored overnight at 2-8°C for approximately 19 hours. Clumping of LNC was avoided by ensuring that the pellet was completely resuspended in a small volume of PBS before making up to the final volume. The suspended precipitates were centrifuged at 200 x g for 10 minutes at 4°C and the supernatant was poured off into container for radiolabel waste collection. The pellet from each mouse was reconstituted in 1 mL of 5% TCA and subsequently transferred to a scintillation vial containing 10 mL of a scintillation cocktail (Insta-Gel Plus, PerkinElmer, USA). Two additional 2 mL aliquots of Milli-Q-water were used to rinse the tubes and the rinses were added to the scintillation vial containing 1 mL of the pellet in TCA and cocktail. The samples were mixed using a snapping wrist action.
The radioactivity in each precipitate was measured for 5 minutes using a ß-scintillation counter (Tricarb 4910-TR, Packard Instruments, USA) as disintegrations per minute (dpm) per mouse.

Positive control substance(s):

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

Statistics:

Means and SD were generated for body weight data (absolute and gain) and LLNA response (dpm and SI values). The body weight and dpm data were analysed by one-way analysis of variance. When the differences were indicated by the ANOVA, a comparison of treated vs. control groups was done using a Dunnett’s t-test (p<0.05).
Statistically significant differences (p<0.05), indicated by the aforementioned tests, are designated by the superscripts throughout the report as stated below:
+/- : Significantly higher (+)/lower (-) than the vehicle control group

Results and discussion

Positive control results:

Positive controls are used to demonstrate appropriate performance of the assay by responding with adequate and reproducible sensitivity to a sensitising test substance for which the magnitude of the response is well characterised. α-hexylcinnamaldehyde [HCA] (CAS No. 101-86-0) was used as a positive control for contact sensitization. HCA was diluted to 25% v/v in MEK as suggested in test guideline.

The sensitivity of this LLNA assay was demonstrated via the response from the positive control (25% v/v HCA in MEK), which elicited a stimulation index (SI) of 4.16, in comparison with the vehicle-treated mice.

In vivo (LLNA)

Results

Key result

Parameter:

SI

Value:

2.795

Test group / Remarks:

G5 100% Concentration of test item(undiluted)

Remarks on result:

other: Since, the test item produced a Stimulation Index (SI) < 3, it is considered “Negative” for sensitization

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA : Individual and summary data of Disintegrations Per Minute (DPM) for 3H-Methyl Thymidine incorporation in Auricular Lymph Nodes and Stimulation Index (SI) was mentioned in the section" Any other infomation on results incl. tables"

DETAILS ON STIMULATION INDEX CALCULATION : The SI was calculated for each mouse using the equation mentioned in the section " Any other information on materials and methods incl. tables". The results of calculated SI index values were captured in tabular format and attached in section " Any other infomation on results incl. tables"

EC3 CALCULATION : Since, the test item, produced a Stimulation Index (SI) < 3, it is considered “Negative” for sensitization, and therefore an EC3 was not determined.

CLINICAL OBSERVATIONS: A cage-side examination was conducted twice daily, and the following parameters were evaluated: decreased / increased activity, repetitive behavior, vocalization, incoordination / limping, injury, neuromuscular function (convulsion, fasciculation, tremor, and twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal / urinary quantity. In addition, all animals were observed for morbidity and mortality (twice daily).
The ears were graded for erythema once test item exposure as per scoring system mentioned in the section " Any other information on materials and methods incl. tables".

BODY WEIGHTS: The initial and terminal body weights were recorded.

Any other information on results incl. tables

 Resultsand discussion:

Irritation Screening Study

 

Once daily topical applications of vehicle-MEK, 10, 25, 50 and 75% v/v in MEK and 100% (undiluted)test item were performed to one animal at each dose level for 3 consecutive days. There were no clinical signs, no erythema at the site of application, no significant increase in the ear thickness and ear punch weights and no effect on body weight. Results from this screening study were used to determine the dosing concentrations of test item for the main LLNA study.

Main Study:

The analyzed radioactivity of³H-TdR working solution was 77.38 µCi/mL against the nominal concentration of 80 µCi/mL.

There were no clinical signs, no erythema at the site of application and no significant effect on body weight gains.     

The sensitivity of this LLNA assay was demonstrated via the response from the positive control (25% v/v HCA inMEK), which elicited a stimulation index (SI) of 4.16, in comparison with the vehicle-treated mice. The Mean SI values for 25, 50%of test iteminMEK and 100% (undiluted) test item were 2.22, 2.52 and 2.80 respectively.

Irritation Screening Test:

A. Body Weights (g) and Clinical Signs

Mice Nos.	Dose Concentration	Day 1 (Pre-dose)	Day 6	Body wt. Gain (g)	Clinical Signs
Mh3461	Vehicle: MEK	17.3	18.5	1.2	NAD
Mh3462	10% v/v	17.2	18.3	1.1	NAD
Mh3463	25% v/v	17.0	18.0	1.0	NAD
Mh3464	50% v/v	17.5	18.3	0.8	NAD
Mh3465	75% v/v	18.3	19.1	0.8	NAD
Mh3466	100 % (undiluted)	18.5	19.2	0.7	NAD

NAD: No Abnormality Detected

B.Erythema Scores (for both ears)

Mice Nos.	Dose Concentration	Day 1 (Pre-dose)	Day 2	Day 3	Day 6
Mh3461	Vehicle: MEK	0	0	0	0
Mh3462	10% v/v	0	0	0	0
Mh3463	25% v/v	0	0	0	0
Mh3464	50% v/v	0	0	0	0
Mh3465	75% v/v	0	0	0	0
Mh3466	100 % (undiluted)	0	0	0	0

Erythema scores – 0: No erythema

C.Ear thickness measurement and ear punch weight

Mice Nos.	Dose Concentration	Ear thickness (µm)						Ear punch weight (g)
		Day 1 (pre-dose)		Day 3		Day 6		Day 6
		L.E	R.E	L.E	R.E	L.E	R.E	L.E	R.E
Mh3461	Vehicle: MEK	252	251	252	252	253	253	0.008	0.009
Mh3462	10% v/v	253	250	253	250	254	251	0.009	0.008
Mh3463	25% v/v	251	254	252	254	252	255	0.007	0.008
Mh3464	50% v/v	256	253	257	253	257	254	0.009	0.008
Mh3465	75% v/v	261	259	262	260	263	261	0.009	0.008
Mh3466	100 % (undiluted)	264	262	264	263	265	263	0.010	0.009

L.E: Left ear; R.E: Right ear

D. Percent change in ear thickness

Mice Nos.	Dose concentration	% Change in ear thickness compared to day 1
		Day 3		Day 6
		L.E	R.E	L.E	R.E
Mh3461	Vehicle: MEK	0.00	0.40	0.40	0.80
Mh3462	10% v/v	0.00	0.00	0.40	0.40
Mh3463	25% v/v	0.40	0.00	0.40	0.39
Mh3464	50% v/v	0.39	0.00	0.39	0.40
Mh3465	75% v/v	0.38	0.39	0.77	0.77
Mh3466	100 % (undiluted)	0.00	0.38	0.38	0.38

L.E: Left ear; R.E: Right ear   

Summary of Body Weight and Body Weight Changes:

Group and Dose Concentration	No. of Mice		Body weight (g)
			Day 1 (Initial)	Day 6	Weight change (Day 6 – Initial)	Clinical signs
G1 Vehicle:MEK	4
		Mean	18.975	19.900	0.925	NAD
		SD	1.355	1.311	0.050
G2 25% v/v HCA	4
		Mean	18.925	19.575	0.650	NAD
		SD	1.044	1.034	0.058
G3 25% v/v test item	4
		Mean	18.875	19.750	0.875	NAD
		SD	1.040	1.008	0.050
G4 50% v/v test item	4
		Mean	18.975	19.775	0.800	NAD
		SD	0.768	0.793	0.082
G5 100% (undiluted)	4
		Mean	18.900	19.650	0.750	NAD
		SD	0.702	0.714	0.058

Summary of Disintegrations Per Minute (DPM) for³H-Methyl Thymidine Incorporation in Auricular Lymph Nodes and Stimulation Index (SI):

Group and Dose Concentration	No. of Mice		DPM / Mouse	SI
G1 Vehicle:MEK	4
		Mean	590.750	1.000
		SD	196.364	0.331
G2 25% v/v HCA	4		+
		Mean	2458.750	4.163
		SD	421.404	0.714
G3 25% v/v test item	4		+
		Mean	1311.250	2.220
		SD	366.537	0.622
G4 50% v/v test item	4		+
		Mean	1489.000	2.520
		SD	233.522	0.396
G5 100% (undiluted)	4		+
		Mean	1652.750	2.795
		SD	206.777	0.350

  +: Significantly higher than the vehicle control group, HCA: α – Hexylcinnamaldehyde

Individual Body Weights and Clinical signs:

Group and Dose concentration		Mouse No.		Body weight (g)			Weight change (g) (Day 6 – Initial)		Clinical signs
			Initial (Pre-Dose) Day 1		Day 6
G1 Vehicle:MEK		Mh3191		17.2		18.2		1.00		NAD
		Mh3192		20.5		21.4		0.90		NAD
		Mh3193		19.1		20.0		0.90		NAD
		Mh3194		19.1		20.0		0.90		NAD
G2 25% v/v HCA		Mh3195		19.2		19.8		0.60		NAD
		Mh3196		20.0		20.7		0.70		NAD
		Mh3197		17.5		18.2		0.70		NAD
		Mh3198		19.0		19.6		0.60		NAD
G3 25% v/v test item		Mh3199		19.4		20.3		0.90		NAD
		Mh3200		19.9		20.7		0.80		NAD
		Mh3201		18.7		19.6		0.90		NAD
		Mh3202		17.5		18.4		0.90		NAD
G4 50% v/v test item		Mh3203		18.1		18.8		0.70		NAD
		Mh3204		18.6		19.5		0.90		NAD
		Mh3205		19.4		20.2		0.80		NAD
		Mh3206		19.8		20.6		0.80		NAD
G5 100% (undiluted)		Mh3207		18.2		18.9		0.70		NAD
		Mh3208		18.4		19.2		0.80		NAD
		Mh3209		19.4		20.1		0.70		NAD
		Mh3210		19.6		20.4		0.80		NAD

NAD: No Abnormality Detected, HCA: α – Hexylcinnamaldehyde

Individual Local Reaction Scores at the Site of Application:

Group and Dose Concentration	Mouse No.	Erythema Score in both ears
		Day 1 Pre-dose		Day 2		Day 3		Day 6
		R.E	L.E	R.E	L.E	R.E	L.E	R.E	L.E
G1 Vehicle:MEK	Mh3191	0	0	0	0	0	0	0	0
	Mh3192	0	0	0	0	0	0	0	0
	Mh3193	0	0	0	0	0	0	0	0
	Mh3194	0	0	0	0	0	0	0	0
G2 25% v/v HCA	Mh3195	0	0	1	1	1	1	1	1
	Mh3196	0	0	1	1	1	1	1	1
	Mh3197	0	0	1	1	1	1	1	1
	Mh3198	0	0	1	1	1	1	1	1
G3 25% v/v test item	Mh3199	0	0	0	0	0	0	0	0
	Mh3200	0	0	0	0	0	0	0	0
	Mh3201	0	0	0	0	0	0	0	0
	Mh3202	0	0	0	0	0	0	0	0
G4 50% v/v test item	Mh3203	0	0	0	0	0	0	0	0
	Mh3204	0	0	0	0	0	0	0	0
	Mh3205	0	0	0	0	0	0	0	0
	Mh3206	0	0	0	0	0	0	0	0
G5 100% (undiluted)	Mh3207	0	0	0	0	0	0	0	0
	Mh3208	0	0	0	0	0	0	0	0
	Mh3209	0	0	0	0	0	0	0	0
	Mh3210	0	0	0	0	0	0	0	0

L.E: Left ear ; R.E: Right ear;0 : No erythema; 1 :Very slight erythema (barely perceptible)HCA: α – Hexylcinnamaldehyde

Disintegrations Per Minute (DPM) for³H-Methyl Thymidine  Incorporation in Auricular Lymph Nodes and Stimulation Index (SI):

Group and Dose concentration		Mouse No.		*DPM/Mouse		SI
G1 Vehicle:MEK		Mh3191		732		1.24
		Mh3192		539		0.91
		Mh3193		335		0.57
		Mh3194		757		1.28
G2 25% v/v HCA		Mh3195		2066		3.50
		Mh3196		3024		5.12
		Mh3197		2521		4.27
		Mh3198		2224		3.76
G3 25% v/v test item		Mh3199		1551		2.63
		Mh3200		1283		2.17
		Mh3201		804		1.36
		Mh3202		1607		2.72
G4 50% v/v test item		Mh3203		1802		3.05
		Mh3204		1447		2.45
		Mh3205		1470		2.49
		Mh3206		1237		2.09
G5 100% (undiluted)		Mh3207		1898		3.21
		Mh3208		1484		2.51
		Mh3209		1750		2.96
		Mh3210		1479		2.50

HCA: α – Hexylcinnamaldehyde, *: DPM / Mouse = Measured DPM – Average Background DPM

Applicant's summary and conclusion

Interpretation of results:

other: Not-sensitising

Conclusions:

A topical application with 25, 50% of test item in MEK and 100% (undiluted) test item elicited a stimulation index (SI) of 2.22, 2.52 and 2.80 respectively. The test item DECAN-5-OLIDE (CAS: 705-86-2) did not demonstrate dermal sensitization potential in the mouse LLNA, as the lymph nodes draining the area of topical application did not elicit a proliferative response greater than the 3X threshold.

Executive summary:

The test item,DECAN-5-OLIDE (CAS: 705-86-2) was evaluated for the potential to cause contact sensitization by measuring the lymphocyte proliferative response from auricular lymph nodes following topical application to the female CBA/Ca mouse ear.The test item was formulated in Methyl Ethyl Ketone (MEK) following a solubility assessment.

Screening Study: One female CBA/Ca mice/dose received the vehicle MEK and10, 25, 50 and 75% v/v of test item inMEK and 100% (undiluted) test item respectively once daily for three consecutive days. There were no clinical signs, no erythema at the site of application, no significant increase in the ear thickness and ear punch weights and no effect on body weight. Based on the results dose concentrations of25, 50% v/vof test item inMEK and 100% (undiluted) test item was selected for the main study.

Main study: Four female CBA/Ca mice/group received the vehicle (MEK), 25% v/v α-hexylcinnamaldehyde (HCA: positive control inMEK), and 25, 50% of test item inMEK and 100% (undiluted) test item once daily for three consecutive days on days 1 to 3. On day 6, the uptake of 3H-methyl thymidine into the auricular lymph nodes draining the site of test item application was measured approximately five hours post administration. Performance of the study was confirmed via a positive response using 25% v/v α-hexylcinnamaldehyde, a contact sensitizer, which elicited proliferation with a Stimulation Index (SI) value of 4.16, in comparison to vehicle-treated miceThere were no clinical signs, no local skin reactions at the tested concentrations and treatment had no significant effect on body weight gain. The test item at dose concentrations of 25, 50% v/vof test iteminMEK and 100% (undiluted) test item  elicited proliferative response with SI of 2.22, 2.52 and 2.80 respectively in comparison with the vehicle-treated mice.

The test item “DECAN-5-OLIDE(CAS: 705-86-2)” did not demonstrate dermal sensitization potential in the mouse LLNA, as the lymph nodes draining the area of topical application did not elicit a proliferative response greater than the 3X threshold.