Methyl trimethyl-3-[(1-oxododecyl)amino]propylammonium sulphate

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

Remarks:

Study carried out according to OECD 429 (2010) and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl trimethyl-3-[(1-oxododecyl)amino]propylammonium sulphate

EC Number:

234-204-8

EC Name:

Methyl trimethyl-3-[(1-oxododecyl)amino]propylammonium sulphate

Cas Number:

10595-49-0

Molecular formula:

C19H42N2O5S

IUPAC Name:

3-(dodecanoylamino)-N,N,N-trimethylpropan-1-aminium methyl sulfate

Details on test material:

- Name of test material (as cited in study report): RL 860/007
- Physical state: liquid
- Analytical purity: 40% substance in water
- Batch No: 20.11.2007
- Stability in solvent: >1 month in water and propylene glycol at room temperature
- Storage condition of test material: room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands
- Age at study initiation: 8-10 w
- Weight at study initiation:
- Housing: individually in Markolon Type I with wire mesh top
- Diet: ad libitum, pelleted standard diet
- Water: ad libitum, tap water
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

5%, 10% and 25%

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: a solubility experiment was carried out prior to the main study
- Irritation: pre-test performed in 2 female animals with the concentrations of 10, 25, 50 and 100%, each applied on the ear for 3 consecutive days. At 25 and 50% slight redness was seen, as well as at 100% were it was more pronounced. Thus, 25% was chosen as the highest concentration for the main study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Lymph node proliferation response: No of radioactive disintegrations/ min/ lymph node (DPM)
- Stimulation index: 3HTdR incorporated into lymph node cells treated animals/control
- Criteria used to consider a positive response: 1. Exposure to at least one concentration resulted in at least a 3-fold increase of 3HTdR incorporation; 2. Data with a dose-response

TREATMENT PREPARATION AND ADMINISTRATION:
Each test group was treated by topical application to the dorsal surface of each ear lobe with the relevant concentrations in dimethylformamide, with an application volume of 25 ul, once daily for 3 consecutive days. A vehicle control group was also used. The preparations were made freshly before each dosing. Five days after the first epidermal application, all mice were intravenously injected with 250 ul of 81.1 uCi/ml 3HTdR. Five hours after this treatment, the animals were sacrifised, and the draining lymph node were used for the evaluation of cells and determination of the Stimulation Index. The level of 3HTdR incorporation was measured with a beta-scintillation counter.

Positive control substance(s):

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

Statistics:

Mean and standard deviations were calculated for the body weights.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Vehicle control: - 5%: 3.64 10%: 7.45 25%: 11.08

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Vehicle control: 3338 5%: 11993 10%: 24517 25%: 36420

Results 3

Key result

Parameter:

SI

Value:

ca. 3.64

Test group / Remarks:

2: 5%

Results 4

Key result

Parameter:

SI

Value:

ca. 7.45

Test group / Remarks:

3: 10%

Results 5

Key result

Parameter:

SI

Value:

ca. 11.08

Test group / Remarks:

4: 25%

Any other information on results incl. tables

Table 1: Calculation and results of individual data.

Concentration % (w/v)	Group	Measurement DPM	Calculation			Result
			DPM-BG	No of lymph nodes	DPM/lymph node	S.I.
-	BG I	59	-	-	-	-
-	BG II	52	-	-	-	-
-	1	3338	3283	8	410.3	1
5	2	11993	11938	8	1492.2	3.64
10	3	24517	24462	8	3057.7	7.45
25	4	36420	36365	8	4545.6	11.08

BG: Background (1 ml 5% trichloroacetic acid) in duplicate

1: vehicle control group

2-4: test groups

S.I.: Stimulation Index

No deaths occured during the conduction of the experiment. The animals did not show any treatment related clinical signs, except for redness detected in the application site, after the third dosing of the highest concentration. The body weights were within the normal range (details in the attached document).

The results for the positive control confirm the validity of this test (details in the attached document).

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The test material RL 860/07 was a skin sensitizer in this LLNA test. Since RL 860/07 contains 40% CAS 10595-49-0, it can be concluded that CAS 10595-49-0 is a skin sensitizer.

Executive summary:

In the present Local Lymph Node Assay (LLNA), the test material RL 860/07 (40% CAS 10595 -49 -0 in water) dissolved in dimethylformamide was assessed for it skin sensitizing potential. The concentrations 5, 10, and 25% (w/v) were applied on the ear of female CBA/CaOlaHsd mice, once per day, for three consecutive days (4 animals/group). Vehicle controls were tested only with dimethylformamide. The concentrations chosen were based on a range-finding study performed prior to the main test. Hexyl cinnamic aldehyde was used as a positive control. Five days after the first treatment, the animals were injected intravenously with radiolabelled thymidine and were sacrificed after five hours.The proliferative capacity of the lympocytes in draining lymph nodes was examined. The results revealed Stimulation Indices higher than 3, increasing on a dose-dependent manner. On the basis of this result, the test material RL 860/07, and hence, CAS 10595 -49 -0, shall be classified as skin sensitizers.