N,N-Dimethyl-9-dodecenamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 May - 02 August 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

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:

N,N-dimethyldodec-9-enamide

EC Number:

851-152-7

Cas Number:

1374570-57-6

Molecular formula:

C14H27NO

IUPAC Name:

N,N-dimethyldodec-9-enamide

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaCrl

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 9 to 10 weeks old on Day 1
- Weight at study initiation: Animals in the main studies were in a body weight range of 17 to 22 g on Day 1 of dosing. Individual body weights were within ±20% of the mean body weight for mice on the study.
- Housing: The animals were housed in groups of up to six during acclimatisation and housed in pairs from Day 1 in cages that conformed to the 'Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes’ (Home Office, London, 2014).
Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding (Datesand Ltd., Manchester, UK). The bedding had been analysed for specific contaminants and the results retained on file at Labcorp. No contaminants were present in bedding at levels which might have interfered with achieving the objective of the study.
- Diet (e.g. ad libitum): 5LF2 EU Rodent Diet 14%, was freely available to the animals at all times. Each batch of diet had been analysed for specific constituents and contaminants by the manufacturer. No contaminants were present in diet at levels which might have interfered with achieving the objective of the study.
- Water (e.g. ad libitum): Mains water was provided, ad libitum, via cage-mounted water bottles. The water had been periodically analysed for specific contaminants. No contaminants were present in water at levels which might have interfered with achieving the objective of the study.
- Acclimation period: 8 to 15 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 40 to 70
- Air changes (per hr): minimum of 15
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was controlled automatically to give a cycle of 12 hours light and 12 hours dark.
- IN-LIFE DATES: From: To: 31 May - 02 August 2022

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

100 %v/v; 50 % v/v; 25 %v/v; 10 %v/v; 5 %v/v; 2.5 %v/v

No. of animals per dose:

Preliminary screening test: 1 animal
Main study: 4 animals per dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The vehicle for the test article was 80% v/v acetone in olive oil, supplied by Labcorp. The vehicle was chosen because it was the first of the vehicles listed in the protocol that produced an overtly stable solution, emulsion or dispersion incorporating 50% v/v of the test article.
- Irritation: None observed
- Systemic toxicity: No death. Greasy fur to back of ears and neck observed on days 1, 3, 4, 5 & 6; Greasy fur to back, back of ears and neck observed on day 2
- Ear thickness measurements: See Table 9.5 below
- Erythema scores: 0 on days 1, 2, 3, 4, 5 & 6

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
Mice were arbitrarily selected from the delivery boxes and allocated to the appropriate number of cages. The condition of the animals was assessed daily throughout the acclimatisation period of 8 to 15 days. A second inspection was performed prior to study commencement to ensure the animals were suitable for the study. Overtly healthy animals were arbitrarily allocated to the study groups on the day of commencement of treatment.
- Criteria used to consider a positive response: The test article is regarded as a sensitiser when the maximum value of the SI is 3.0 or above. The test article is classified as a non-sensitiser when the maximum value of the SI is
less than 3.0. (This result is unchanged by observations of irritation at sites of application of the test formulation).

TREATMENT PREPARATION AND ADMINISTRATION:
Test Article Formulation
Formulations were freshly prepared as required using 80% v/v acetone in olive oil on Days 1, 2 and 3. The formulations were stored at room temperature, in sealed, air-tight containers prior to dosing and were used within two hours of preparation.
The formulations were mixed by multiple inversion of the containers prior to administration to ensure homogeneity.
Concentrations of test article were expressed volumetrically and in terms of test article received (without regard to purity or active content).

Test Article Administration
Each mouse was manually restrained with both auditory pinnae left free. The outer aspect of both pinnae of each mouse was treated by direct application of the appropriate vehicle control or test formulation (0.025 mL/pinna) dispensed from an automatic micro pipette.

Treatment Regimen
The groups of four female mice were subjected to application of the vehicle control or one of the test formulations to the outer aspect of the auditory pinnae, once daily on Days 1, 2 and 3. On Day 6 the mice were placed in a thermacage in order to dilate the peripheral blood vasculature and thus facilitate intravenous dosing. Each mouse was transferred to a cylindrical restrainer. A plastic syringe and fine gauge hypodermic needle were used to administer 0.25 mL phosphate buffered saline incorporating 20 μCi of 3HTdR into a tail vein of each mouse by slow bolus injection. After this treatment, the mice were returned to their cages. Approximately five hours after intravenous injection of the 3HTdR, all mice were killed by exanguination under a deep plane of inhalation anasthesia. Killing was organised to minimise the interval between death and the recovery of the auricular lymph nodes to no more than fifteen minutes.

Clinical Signs
Treated mice were observed twice daily on Days 1 to 5 and once on Day 6 for clinical signs of reaction to treatment or for irritation or other changes at the sites of application of the test article.
Routine Health Checks
All animals were examined at the beginning and end of the working day throughout the acclimatisation and study periods to ensure they were in good health.
Body Weights
Mice were weighed on Day 1 (the first day of dosing) and on Day 6 prior to intravenous administration of 3HTdR.

Positive control substance(s):

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

Results and discussion

Positive control results:

See attached historical positive control data. These data confirm adequate performance of the assay.

In vivo (LLNA)

Results

Results 1

Parameter:

EC3

Value:

24.6

Results 2

Parameter:

SI

Value:

5.59

Test group / Remarks:

100 %v/v

Results 3

Parameter:

SI

Value:

3.65

Test group / Remarks:

50 %v/v

Results 4

Parameter:

SI

Value:

3.04

Test group / Remarks:

25 %v/v

Results 5

Parameter:

SI

Value:

1.47

Test group / Remarks:

10 %v/v

Results 6

Parameter:

SI

Value:

0.81

Test group / Remarks:

5 %v/v

Results 7

Parameter:

SI

Value:

0.47

Test group / Remarks:

2.5 %v/v

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation counts obtained from the test groups relative to the corresponding mean scintillation count obtained from the vehicle control (see Table 9.1 & 9.2 below).
The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.

EC3 CALCULATION
The EC3 value was calculated using the following equation:
EC3 = c+[(3-d)/(b-d)](a-c)
where
a = lowest concentration giving SI ≥ 3
b = actual SI caused by a
c = highest concentration failing to produce SI of 3
d = actual SI caused by c

The EC3 was calculated to be 24.6%.

CLINICAL OBSERVATIONS:
There were no clinical signs indicative of a systemic effect of treatment among mice treated with the vehicle or with 2.5%, 5%, 10%, 25%, 50% or 100% v/v formulations of the test article.
In the first main study, greasy wet fur on the back of the neck and ears was noted in all animals on Day 1. Greasy fur on the back of the neck and ears was noted in all animal from Days 2 to 4, persisting in animals in Groups 2, 3 and 4 on Days 5 and 6.
Incidents of greasy fur on the dorsal thoracic area and on the front legs were also noted on Days 4 and 5 in animals in Groups 3 and 4.
In the second main study, greasy fur on the back of the neck and ears was noted in all animals on Day 2, with greasy fur on the back of the neck and ears noted on Days 3 and 4 in animals treated at a concentration of 10% v/v.

BODY WEIGHTS
There was no indication of a treatment related effect on body weight (see Tables 9.6 & 9.7 below).

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness).
All animals survived treatment with N,N-dimethyldodec-9-enamide.
The vehicle and test formulation application sites remained free of irritation.

Any other information on results incl. tables

Table 9.1 Group DPMs and Stimulation Index (SI), Experiment 1

Concentration (% v/v in 80% v/v acetone in olive oil)	Group Number	Group DPM	Stimulation Index (SI)^a
Vehicle	1	726	NA
25	2	2207	3.04
50	3	2648	3.65
100	4	4060	5.59

a = Stimulation Index of 3.0 or greater indicates a positive result

NA = Not applicable

Table 9.2 Group DPMs and Stimulation Index (SI), Experiment 2

Concentration (% v/v in 80% v/v acetone in olive oil)	Group Number	Group DPM	Stimulation Index (SI)^a
Vehicle	5	707	NA
2.5	6	335	0.47
5	7	576	0.81
10	8	1040	1.47

a = Stimulation Index of 3.0 or greater indicates a positive result

NA = Not applicable

Table 9.5: Preliminary Screening Test - Ear Thickness Measurements

Concentration (% v/v)	Animal Number	Ear	Thickness (mm) on Day:
			1	3	6
100	257	Left	0.21	0.21	0.21
		Right	0.20	0.21	0.22

  

Table 9.6 Body Weights, Main Experiment 1

 

Concentration (% v/v in 80% v/v acetone in olive oil)	Group Number	Animal Number	Body Weights (g)
			Day 1	Day 6
Vehicle	1	258	20	21
		259	19	20
		260	19	20
		261	22	22
25	2	262	19	20
		263	20	22
		264	22	24
		265	21	20
50	3	266	20	21
		267	20	20
		268	20	21
		269	20	21
100	4	270	20	19
		271	22	19
		272	20	20
		273	22	19

 

 Table 9.7 Body Weights, Main Experiment 2

 

Concentration (% v/v in 80% v/v acetone in olive oil)	Group Number	Animal Number	Body Weights (g)
			Day 1	Day 6
Vehicle	5	318	19	20
		319	19	21
		320	17	18
		321	18	20
2.5	6	322	19	19
		323	17	21
		324	20	22
		325	17	18
5	7	326	19	21
		327	19	20
		328	17	19
		329	19	21
10	8	330	17	19
		331	19	21
		332	17	19
		333	19	21

 

 

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Executive summary:

This study was conducted to assess the potential of the test article, N,N-dimethyldodec-9-enamide, to cause skin sensitisation in the mouse.

Following a preliminary screening test using the undiluted test article, the main study was conducted using the undiluted test article and 50 and 25% v/v formulations in 80% v/v acetone in olive oil. In order to calculate an EC3 value, a second experiment was conducted using 2.5%, 5% and 10% v/v formulations.

Groups of four female CBA/CaCrl mice were subjected to topical applications of vehicle control or of one of the test formulations to the outer aspect of the auditory pinnae once daily on Days 1, 2 and 3. On Day 6 a 20 μCi dose of tritiated 3H-methyl thymidine was injected intravenously into each mouse. Approximately five hours later the auricular lymph nodes were recovered from each animal. The pairs of nodes from each animal were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter.

Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation counts obtained from the test groups relative to the corresponding mean scintillation count obtained from controls. The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.

	Concentration of Test Article in Applied Formulation (% v/v)
	2.5	5	10	25	50	100
Stimulation Index	0.47	0.81	1.47	3.04	3.65	5.59

The EC3 was calculated to be 24.6%.

The Local Lymph Node Assay demonstrated that N,N-dimethyldodec-9-enamide has the potential to cause skin sensitisation.