3-((C12-18)-acylamino)-N-(2-((2-hydroxyethyl)amino)-2-oxoethyl)-N,N-dimethyl-1-propanaminium chloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

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 (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

427-370-1

EC Name:

-

Cas Number:

164288-56-6

Molecular formula:

C21H44N3O3Cl

IUPAC Name:

{[(2-hydroxyethyl)carbamoyl]methyl}dimethyl(3-octadecanamidopropyl)azanium chloride

impurity 1

Reference substance name:

Sodium chloride

EC Number:

231-598-3

EC Name:

Sodium chloride

Cas Number:

7647-14-5

Molecular formula:

ClNa

IUPAC Name:

sodium chloride

impurity 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Water

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Animals and animal husbandry
Female CBA/J (CBA/J@Rj) strain mice were supplied by Elevage Janvier (F-53941 Le Genest Saint Isle). On receipt the animals were randomly allocated to cages. The animals were nulliparous and nonpregnant.
After an acclimatisation period of at least five days under stabling and nutritional conditions identical to those of the test, the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card.
At the start of the study the animals were in the weight range of 19 to 23 g, and were eight weeks old.
The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
The animals were weighed at the beginning and at the end of the study.
The temperature and relative humidity were controlled to remain within target ranges of 20 to 23°C and 32 to 59%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (07.00 to 19.00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Food and drink
The drinking water (tap water from public distribution system) and food were supplied freely.
Microbiological verification and chemical analysis were conducted every six months by the Institut Européen de l’Environnement de Bordeaux (I.E.E.B.).

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Two separate experiments were conduced
-In the first experiment undiluted and 50% or 25% (v/v) in vehicle (DMF)
-Second experiment 2.5%, 5% or 10% (v/v) in vehicle (DMF)

No. of animals per dose:

4

Details on study design:

The test item was tested in two independent experiments.
In the first experiment, groups of four mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% (v/v) in vehicle – dimethylformamide –. The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.

Second experiment
Groups of four mice were treated with the test item at concentrations of 2.5%, 5% or 10% (v/v) in vehicle – dimethylformamide –. The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days
(Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.

Positive control substance(s):

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

Results and discussion

Positive control results:

under these experimental conditions, the substance α-Hexylcinnamaldehyde:
must be classified R 43 “may cause sensitization by skin contact” in accordance with the criteria for classification, packaging and labelling of dangerous substances and preparations of the E.E.C.
Directives 67/548, 2001/59 and 99/45. This substance must be characterised by the symbol “Xi” and the warning label “Irritant”.
In accordance with the Globally Harmonized System (COM(2007)355 final), the test item must be classified in category 1 “Skin sensitisation”. The signal word “Warning” and hazard statement H317 “May cause an allergic skin reaction” are required.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 4.16

Test group / Remarks:

For the treated group 2 at 25% of the test item

Results 2

Key result

Parameter:

SI

Value:

ca. 4.75

Test group / Remarks:

For the treated group 3 at 50% of the test item

Results 3

Key result

Parameter:

SI

Value:

ca. 5.2

Test group / Remarks:

For the treated group 4 at 100% of the test item

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

In view of these results, under these experimental conditions, the test item LCE08002 was considered to be a sensitiser under the conditions of the test.
It must be classified R43 “May Cause Sensitisation by Skin Contact”, in accordance with the criteria for classification, packaging and labelling of dangerous substances and preparations of the E.E.C. Directives 67/548, 2001/59 and 99/45. This item must be characterised by the symbol “Xi” and
the warning label “Irritant”.
In accordance with the Globally Harmonized System (COM(2007)355 final), the test item must be classified in category 1 “Skin sensitisation”. The signal word “Warning” and hazard statement H317 “May cause an allergic skin reaction” are required.

Executive summary:

The test was performed to access the skin sensitisation potential of the test item LCE08002 in the CBA/J strain mouse following topical applications to the dorsal surface of the ear.

The test item was tested in two independent experiments, in the first one at the concentration of 25%, 50% and 100% and in the second one at the concentration of 2.5%, 5% and 10%. The six groups, each of four animals, were treated for three consecutive days (D1, D2, D3) with 50 μL (25 μL per ear) of the test item. For each experiment a further group of four animals was treated with the vehicle dimethylformamide (DMF).

At D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting. The obtained values were used to calculate stimulation indices (SI).

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1 and 6 and by weight a skin biopsy of the ear at D6.

The experimental protocol was established according the OECD Guideline n°429 dated April 24th, 2002 and the test method B.42 of the commission directive 2004/73/EC.

No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test.

A slight dryness was registered at D3, in the treated group at 100%, between D4 and D6, in the treated group at 25% and only at D6 in the treated group at 10%. A dryness was noted between D4 and D6, in the treated group at 50% and 100%.

An important increase in ear thickness (+43.4%, +56.9%, +52.8%) and in ear weight (+37.5%, +53.6%, +78%) was recorded at the concentrations of 25%, 50% and 100%. No significant increase in ear thickness and in ear weight was recorded at the other concentrations.

Therefore, the test item must be considered “non-irritant” at the concentrations of 2.5%, 5% and 10% and “irritant” at the concentrations of 25%, 50% and 100%.

However, it was noted an increase of 1.8 times the weight of the lymph nodes in the treated group at 10%.

The Stimulation Index (SI) calculated by pooled approach was respectively 1.37, 1.61, 2.29, 6.46, 7.79 and 9.35 for the treated group at 2.5%, 5%, 10%, 25%, 50% and 100%.

The EC1.4 value determined by linear interpolation of points on the dose-response curve during the 2nd experiment was 2.81%.

In view of these results, under these experimental conditions, the test item LCE08002 was considered to be a sensitiser under the conditions of the test.

It must be classified R43 “May Cause Sensitisation by Skin Contact”, in accordance with the criteria for classification, packaging and labelling of dangerous substances and preparations of the E.E.C. Directives 67/548, 2001/59 and 99/45.

In accordance with the Globally Harmonized System (COM(2007)355 final), the test item must be classified in category 1 “Skin sensitisation”. The signal word “Warning” and hazard statement H317 “May cause an allergic skin reaction” are required.