[(±)-(4-amino-2-hydroxy-4-oxobutyl)trimethylammonium] chloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 October 2012 to 14 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline and EU method. GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

[(±)-(4-amino-2-hydroxy-4-oxobutyl)trimethylammonium] chloride

EC Number:

226-073-0

EC Name:

[(±)-(4-amino-2-hydroxy-4-oxobutyl)trimethylammonium] chloride

Cas Number:

5261-99-4

Molecular formula:

C7H17N2O2.Cl

IUPAC Name:

4-amino-2-hydroxy-N,N,N-trimethyl-4-oxobutan-1-aminium chloride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Physical state: white powder
- Analytical purity: 99,4%
- Lot/batch No.: WI 2274
- Storage condition of test material: room temperature.

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J Rj

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: ELEVAGE JANVIER, Route des Chènes Secs B.P. 4105, 53940 LE GENEST-ST-ISLE, France
- Age at study initiation: 8 weeks old
- Weight at study initiation: 19.7-20.9 g
- Housing: Group caging / mice were provided with glass tunnel-tubes. Cage type: Type II. polypropylene / polycarbonate.
- Diet (e.g. ad libitum): ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice”, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15-20 air exchange/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 24 October 2012 To: 11 December 2012

Study design: in vivo (LLNA)

Vehicle:

other: 1% aqueous Pluronic® PE9200

Concentration:

10, 25 and 50 (w/v) %

No. of animals per dose:

Four animals

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The solubility of the test item was examined in a short Preliminary Compatibility Test. Due to the physical characteristics of the test item, 100 (w/v) % concentration was not achievable. No proper formulation was achieved using AOO (acetone:olive oil 4:1 (v:v) mixture), DMF (N,N-Dimethylformamide), Methyl ethyl ketone, Propylene glycol or DMSO (Dimethyl sulfoxide) at 50 (w/v) % concentration; however, the formulation at the same concentration after an approximately 5-minute incubation in an ultrasonic water bath using 1% aqueous Pluronic® PE9200 (1% Pluronic) as vehicle was suitable for the test. As 1% Pluronic is one of the vehicles recommended by the relevant OECD guideline, it was selected for vehicle of the study.
- Results: In the Preliminary Irritation / Toxicity Test, no mortality or signs of systemic toxicity were observed. No body weight loss was detected in the preliminary experiment. Minimal amount of test item precipitate was observed on the ears of the animals in the 50 (w/v) % dose group after treatment for one animal on Days 1-3 and for the other animal in this group on Day 3. Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. The revealing ear punch weights were within the historical control range. The appearance of the lymph nodes was normal in both groups.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
Topical application
During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Proliferation assay
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).

The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels.

Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C.

After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5 (w/v) % TCA solution was added to the tubes for precipitation of macromolecules.

After overnight (approximately 18 hours) incubation at 2-8 ºC, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4 ºC), and supernatants were removed. Pellets were resuspended in 1 mL of 5 (w/v) % TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).

The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 (w/v) % TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

Positive control substance(s):

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

Results and discussion

Positive control results:

No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index value of 15.5) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
Furthermore, the DPN values observed for the positive control substance in this experiment were within the historical control range. Each treated group included 4 animals.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The stimulation index values were 0.8, 1.4 and 1.7 at concentrations of 50, 25 and 10 (w/v) %, respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The disintegrations per minute (DPM) were 563.5, 1018.5 and 1214.5 at concentrations of 50, 25 and 10 (w/v) %, respectively.

Any other information on results incl. tables

CLINICAL OBSERVATION

No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application. Test item precipitate was observed on the ears of the animals after treatment in the 50 % (w/v) dose group on Days 1 and 3.

BODY WEIGHT MEASUREMENT

No treatment related effects were observed on animal body weights.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion, under the conditions of the present assay, the test substance, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Executive summary:

The aim of this study was to determine the skin sensitisation potential of the test substance following dermal exposure according to the OECD guideline 429 and EU method B.42. Based on the results of the Preliminary Compatibility Test, the test item was tested for formulation compatibility in 1% aqueous Pluronic® PE9200 (1% Pluronic). The highest achievable concentration was 50 (w/v) %. In the main assay, twenty female CBA/J Rj mice were allocated to five groups of four animals each:

- three groups received the test substance (formulated in 1% Pluronic) at 50, 25 and 10 (w/v) % concentrations,

- the negative control group received the vehicle (1% Pluronic),

- the positive control group received 25 (w/v) % HCA (dissolved in 1% Pluronic).

The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality or systemic clinical signs were observed during the study. No treatment related effects were observed on animal body weights in any treated groups. There were no indications of any irritancy at the site of application. The observed stimulation index values were 0.8, 1.4 and 1.7 at concentrations of 50, 25 and 10 (w/v) %, respectively. The result of the positive control substance confirmed the validity of the assay. In conclusion, under the conditions of the present assay, the test substance, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.