Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 February - 20 March 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with OECD guideline 429 without any deviation

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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)

Version / remarks:

version of April 24th, 2004

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate

EC Number:

939-579-8

Molecular formula:

Empirical Formula : C11H21N3O3Na C2nH4n+1 (n=4 – 9), C11H21N3O3NaC18H35, C11H21N3O3NaC18H33 Molecular formula of the two main constituents : C23H46N3O3Na

IUPAC Name:

Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate

Details on test material:

- Name of test material (as cited in study report): CHIMEXANE HB
- Physical state: Thick colourless to slightly yellowish gel
- Analytical purity: 55.9 %
- Lot/batch No.: 0126344
- Date of receipt: 21 February 2006
- Expiration date of the lot/batch: March 2007
- Storage condition of test material: At room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 10 weeks
- Weight at study initiation: 20.7 ± 1.1 g
- Housing: Individually in disposable crystal polystyrene cages
- Diet (e.g. ad libitum): SsniffR/M-H pelleted diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water (e.g. ad libitum): Tap water (filtered using a 0.22 µ filter), ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 30-70 %
- Air changes: Approximately 12 cycles/h of filtered, non-recycled air
- Photoperiod: 12 h dark / 12 h light

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Remarks:

Batch No 31125-303 from Aldrich, Saint-Quentin-Fallavier, France

Concentration:

Main experiment: 0.1, 0.25, 0.5, 1.0 and 2.5 % (w/v)

No. of animals per dose:

Preliminary tests: One female/dose
Main experiment: Four females/dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Due to the unsatisfactory solubility of the test item in the first recommended vehicle (acetone/olive oil (4/1, v/v)) and dimethylformamide, methyl ethyl ketone was chosen from the other proposed vehicles.
- Irritation and lymph node proliferation response: A preliminary test was performed as follows: the test item was prepared at the concentrations of 10, 25, 50 and 100 % (w/v) in the first assay and at 1, 2.5, 5 and 10 % (w/v) in the complementary assay. For 3 consecutive days, the animals received applications of 25 μL of the dosage form preparations to the external surface of both ears (one concentration per ear). A gentle massage was performed in order to facilitate the application of the undiluted test item only. Measurement of the ear thickness (using a micrometer) was performed each day before treatment and 24 h after the application (first assay), or 72 h after the last application (complementary assay).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The test item was considered as a skin sensitizer when the SI for a dose group is ≥ 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of test material at concentrations of 0 (vehicle control), 0.1, 0.25, 0.5, 1.0, 2.5 % (w/v) in methyl ethyl ketone were applied to the dorsal surface of each ear on Days 1, 2 and 3. On Day 6, 250 μL of 0.9 % NaCl containing 20 μCi of [3H] methyl-thymidine (specific activity: 25 Ci/mmol) was injected into the tail vein of each experimental mouse. Five hours later, all mice were killed by cervical dislocation and the draining auricular lymph node of each ear was excised. A single cell suspension of auricular lymph node cells (ALNC) was prepared by mechanical disaggregation in Petri dishes with the plunger of a syringe. Cell suspensions were washed with 15 mL of 0.9 % NaCl and pellets obtained were re-suspended in 0.9 % NaCl for numeration of lymphocytes (cellularity) and determination of their viability by exclusion of trypan blue. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5 % (w/v) trichloroacetic acid (TCA) in purified water at +4 °C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5 % TCA. Three mL of Ultima GoldxR scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using β-scintillation counting. The results were expressed as disintegrations per min (dpm) per group and per node and Stimulation Indices (SI) were calculated.

Positive control substance(s):

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

Statistics:

No data

Results and discussion

Positive control results:

- Mean DPM = 3776.29
- Stimulation index = 23.94

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.78

Test group / Remarks:

0.1% (w/v)

Results 2

Parameter:

SI

Value:

2.04

Test group / Remarks:

0.25% (w/v)

Results 3

Parameter:

SI

Value:

4.42

Test group / Remarks:

4.42% (w/v)

Results 4

Parameter:

SI

Value:

10.95

Test group / Remarks:

1.0% (w/v)

Results 5

Parameter:

SI

Value:

22.25

Test group / Remarks:

2.5% (w/v)

Any other information on results incl. tables

Preliminary test:

Test item was found to be irritant in the first preliminary test at the concentrations of 10, 25, 50 and 100 % (w/v). A complementary preliminary test was therefore performed at the following lower concentrations: 1, 2.5, 5 and 10 % (w/v). In the complementary preliminary test, the test item was irritant at the concentrations of 5 and 10 % (w/v). The highest tested concentration retained for the main test was therefore 2.5 % (w/v).

 

Main test:

- Clinical examinations: No mortality and no clinical signs were observed during the study. The body weight change of treated animals was similar to that of control animals.

- Local irritation: An erythema was noted on Day 6 in 1/4 animals at the concentration of 0.1 % (w/v) and, in association with dryness of the skin, in all animals at the concentration of 2.5 % (w/v). No increase in ear thickness was observed in the animals of the treated groups.

 

Table 7.4.1: Results of skin sensitization:

Treatment and concentrations	No. of nodes per group	dpm per group	dpm per node	Stimulation index (SI)	Increase in ear thickness (% between Day 1 and Day 6)	Irritation level	EC3 value
Vehicle	8	157.71	19.71	-	-0.98	-	-
0.1 %	8	122.55	15.32	0.78	-1.98	I	0.35%
0.25 %	8	322.11	40.26	2.04	2.04
0.5 %	8	697.54	87.19	4.42	4.42
1.0 %	8	1726.50	215.81	10.95	0.99
2.5%	8	3508.91	438.61	22.25	1.92
HCA 25 %	8	3776.29	472.04	23.94	-	-	-

                            

dpm = disintegrations per minute

I = non-irritant (increase in ear thickness < 10 %)

HCA = α-hexylcinnamaldehyde

EC3 value = theoretical concentration resulting in a SI value of

stimulation index = dpm of treated group / dpm of control group

Applicant's summary and conclusion

Interpretation of results:

sensitising

Conclusions:

Under the test conditions, CHIMEXANE HB is classified as ‘R43 May cause sensitisation by skin contact’, according to the criteria of Annex VI to the Directive 67/548/EEC and ‘Category 1’ according to the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In a GLP-compliant local lymph node assay performed according to OECD guideline 429, CBA/J mice (4 females/dose) received 25 µL of CHIMEXANE HB at concentrations of 0 (vehicle control), 0.1, 0.25, 0.5, 1.0 and 2.5 % (w/v) in methyl ethyl ketone or of the positive control (alpha-hexylcinnamaldehyde (25 % v/v) in methyl ethyl ketone) to the dorsal surface of each ear for three consecutive days. On Day 6, all animals were injected with [3H] methyl-thymidine and after five hours the draining (auricular) lymph nodes were excised and measured for radioactivity expressed as a number of disintegrations per minute (DPM).

 

Mean DPM for 0, 0.1, 0.25, 0.5, 1.0 and 2.5 % (w/v) CHIMEXANE HB were 157.71, 122.55, 322.11, 697.54, 1726.5 and 3508.91, respectively. Stimulation index for 0.1, 0.25, 0.5, 1.0 and 2.5 % (w/v) CHIMEXANE HB were 0.78, 2.04, 4.42, 10.95 and 22.25, respectively. The estimated concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was 0.35 % (w/v).

An erythema was noted on Day 6 in 1/4 animals at the concentration of 0.1 % and, in association with dryness of the skin, in all animals at the concentration of 2.5 %. No increase in ear thickness was observed in the animals of the treated groups. As a moderate increase in cellularity and a stimulation index > 3 (SI = 23.94) were noted in the postive control group, the study was considered valid.

 

Under the test conditions, CHIMEXANE HB is classified as ‘R43 May cause sensitisation by skin contact’, according to the criteria of Annex VI to the Directive 67/548/EEC and ‘Category 1’ according to the CLP Regulation (EC) N° (1272-2008).