1,3-Propanediaminium, N1-[3-[[2-[dimethyl[3-[(2-methyl-1-oxo-2-propen-1-yl)amino]propyl]ammonio]acetyl]amino]propyl]-2-hydroxy-N1,N1,N3,N3,N3-pentamethyl-, chloride (1:3)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 22 April to 14 May, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, with OECD guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The lymph nodes were resuspended in 1mL 5% trichloroacetic acid instead of 3mL 5% TCA on the day of preparation. Nevertheless, all macromolecules have been precipitated since values obtained for the vehicle controls are well within the historical control

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The lymph nodes were resuspended in 1mL 5% trichloroacetic acid instead of 3mL 5% TCA on the day of preparation. Nevertheless, all macromolecules have been precipitated since values obtained for the vehicle controls are well within the historical controls

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:

[3-(2-{dimethyl[3-(2-methylprop-2-enamido)propyl]azaniumyl}acetamido)propyl][(2R)-2-hydroxy-3-(trimethylazaniumyl)propyl]dimethylazanium [3-(2-{dimethyl[3-(2-methylprop-2-enamido)propyl]azaniumyl}acetamido)propyl][(2S)-2-hydroxy-3-(trimethylazaniumyl)propyl]dimethylazanium hexachloride

EC Number:

700-055-7

Cas Number:

630113-05-2

Molecular formula:

C22 H48 N5 O3.3Cl

IUPAC Name:

[3-(2-{dimethyl[3-(2-methylprop-2-enamido)propyl]azaniumyl}acetamido)propyl][(2R)-2-hydroxy-3-(trimethylazaniumyl)propyl]dimethylazanium [3-(2-{dimethyl[3-(2-methylprop-2-enamido)propyl]azaniumyl}acetamido)propyl][(2S)-2-hydroxy-3-(trimethylazaniumyl)propyl]dimethylazanium hexachloride

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: 8-12 weeks
- Weight at study initiation: 17.5-21.0 g
- Housing: single
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

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


IN-LIFE DATES: From: To:

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

10, 25, 50 and 100% (w/v) in propylene glycol

No. of animals per dose:

4 per dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:
- Irritation:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was 100% of the undiluted test item. The solutions were formulated in propylene glycol.To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 10, 25, 50, and 100 % on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index and data are compatible with a conventional dose response (allowing for either local toxicity or immunological suppression).


TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 10, 25, 50, and 100% (w/v) in propylene glycol. The application volume, 25 µl, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application, all mice were administered with 250 µl of 78.9 µCi/ml 3H-methyl thymidine (corresponds to 19.7 µCi 3HTdR per mouse) by intravenous injection via a tail vein.
Prior to the first application of the test item and prior to treatment with 3HTdR.the ear thickness was determined using a micrometer .
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital Sodium. The draining lymph nodes were rapidly excised and pooled per group. Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze. After washing two times with phosphate buffered saline, the lymph node cells were resuspended in 5 % trichloroacetic acid and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid and transferred to plastic scintillation vials with 10, 25, 50, and 100% ml of ‘Ultima Gold’ scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
After the lymph nodes have been excised, both ears of mice were punched at the apical area using a biopsy punch. For each animal both punches were immediately weighed per animal using an analytical balance.
The proliferative response of lymph node cells is expressed as DPM/node and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

Positive control substance(s):

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

Statistics:

Mean values and standard deviations were calculated.

Results and discussion

Positive control results:

Experiment performed in February 2008 gave the following results for alpha-Hexylcinnamaldehyde in acetone/olive oil 4:1:
Stimulation Index:
1.78 at 5%
1.84 at 10%
4.87 at 25%

EC3=15.7% (w/v)

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.93

Test group / Remarks:

10%

Results 2

Parameter:

SI

Value:

1.12

Test group / Remarks:

25%

Results 3

Parameter:

SI

Value:

0.72

Test group / Remarks:

50%

Results 4

Parameter:

SI

Value:

1.42

Test group / Remarks:

100%

Any other information on results incl. tables

Calculation and Results of Individual Data

Vehicle: propylene glycol

Test item concentration % (w/v)	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	29	---	---	---	---
---	BG II	30	---	---	---	---
---	1	4879	4850	8	606.2
10	2	4550	4521	8	565.1	0.93
25	3	5484	5455	8	681.8	1.12
50	4	3510	3481	8	435.1	0.72
100	5	6925	6896	8	861.9	1.42

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

1     =   Control Group

2-5 =   Test Group

S.I.  =   Stimulation Index

^a)     =   The mean value was taken from the figures BG I and BG II

^b)     =   Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

In this study the stimulation indices (S.I.) of 0.93, 1.12, 0.72 and 1.42 were determined with the test item at concentrations of 10, 25, 50 and 100% in propylene glycol. The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.The test item Triquat Monomer was found to be not a skin sensitiser under the described conditions.

Triquat Monomer is not classified as a skin sensitiser according to the criteria of Annex VI Directive 67/548/EEC and EU GHS

Executive summary:

In this study the test item Triquat Monomer dissolved in propylene glycol was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 10, 25, 50, and 100%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. Local signs of irritation of the ear skin were not detected. Ear thickness or ear weights were not affected.

In this study Stimulation Indices (S.I.) of 0.93, 1.12, 0.72 and 1.42 were determined with the test item at concentrations of 10, 25, 50, and 100% in propylene glycol, respectively. The test item was not a skin sensitiser in this assay under the described conditions.

Triquat Monomer is not classified as a skin sensitiser according to the criteria of Annex VI Directive 67/548/EEC and EU GHS.