Benzyltriethylammonium chloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-02-07 to 2007-03-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

data is from experimental reports

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

The testing facility indicated that the protocol was followed without deviation.

Principles of method if other than guideline:

Mouse Lymph node Assay (LLNA) was performed to assess the dermal sensitization potential of the test chemical

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:

Benzyltriethylammonium chloride

EC Number:

200-270-1

EC Name:

Benzyltriethylammonium chloride

Cas Number:

56-37-1

Molecular formula:

C13H22N.Cl

IUPAC Name:

N-benzyl-N,N-diethylethanaminium chloride

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material :Benzyltriethylammonium chloride
- Molecular formula :C13H22N.Cl
- Molecular weight :227.777 g/mol
- Smiles notation :[N+](CC)(CC)(Cc1ccccc1)CC.[ClH-]
- InChl :1S/C13H22N.ClH/c1-4-14(5-2,6-3)12-13-10-8-7-9-11-13;/h7-11H,4-6,12H2,1-3H3;1H/q+1;/p-1
- Substance type:Organic
- Physical state:Solid (Powder)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0000008753
- Expiration date of the lot/batch: 1 March 2008
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, light protected
- Solubility and stability of the test substance in the solvent/vehicle: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test item was placed into a volumetric flask glass beaker on a tared balance and the vehicle (ethanol: deionised water (7 + 3)) was quantitatively added. The test item concentrations were prepared serially. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer. The preparations were made freshly before each dosing occasion.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

OlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 7-12 weeks
- Weight at study initiation: 19.2 +/- 1.1 g
- Housing: individually in Makrolon Type I cages, with wire mesh top
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: specific length of time not recorded

ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 22 +/- 3 °C
- Humidity (%): 30–70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2007-02-28 (GLP main study) To: 2007-03-07

Study design: in vivo (LLNA)

Vehicle:

other: ethanol: deionised water (7 + 3)

Concentration:

1, 25, and 50 % (w/v)

No. of animals per dose:

4 (nulliparous and non-pregnant), 16 females in total

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Solubility in water: 630 g/L
- Irritation: In a non-GLP conform pre-test in two mice, test substance concentrations of 6.25, 12.5, 25, and 50 % were tested on one ear each. No irritation effects were observed at these concentrations after a single application.
- Lymph node proliferation response: no data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA:
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).
- Criteria used to consider a positive response:
The test substance was regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
1) exposure to at least one concentration of the test substance resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index;
2) 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:
- The test substance was placed into a volumetric flask glass beaker on a tared balance and the vehicle (ethanol: deionised water (7 + 3)) was quantitatively added. The test substance concentrations were prepared serially. Homogeneity of the test substance in the vehicle was maintained during treatment with the magnetic stirrer. The preparations were made freshly before each dosing occasion.

- 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 substance concentrations of 1, 25, and 50 % (w/v) in ethanol: deionised water (7 + 3). The application volume, 25 µL, was spread over the entire dorsal surface (diameter of 8 mm) 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 uL of 80.4 uCi/mL 3HTdR (corresponds to 20.1 uCi 3HTdR per mouse) by intravenous injection via a tail vein.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables. A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation: EC3 = (a-c) [(3-d)/(b-d)] + c; where EC3 is the estimated concentration of the test substance required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the coordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:

- The validation- / positive control study was performed with alpha- Hexylcinnamaldehyde in acetone: olive oil (4+1) using CBA/CaOlaHsd mice in December 2006.
- The respective DPM measurements for concentrations at 5, 10, and 25% (w/v), and the vehicle control were 8152.94, 24974.40, 49155.30, and 4049.44. The SI results were 2.04, 6.31, and 12.45 for concentrations at 5, 10, and 25%, respectively.
- The EC3 value was calculated to be 6.1%.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.62

Test group / Remarks:

1% (w/v) group

Remarks on result:

other: no indication of dermal sensitization

Results 2

Parameter:

SI

Value:

4.01

Test group / Remarks:

25% (w/v) group

Remarks on result:

other: no indication of dermal sensitization

Results 3

Parameter:

SI

Value:

5.99

Test group / Remarks:

50% (w/v) group

Remarks on result:

other: no indication of dermal sensitization

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
DPM per llymph node:
control group: 608.9 (8 lymph nodes)
1% (w/v) group: 376.1 (8 lymph nodes)
25% (w/v) group: 2439.7 (8 lymph nodes)
50% (w/v) group: 3646.4 (8 lymph nodes)

DETAILS ON STIMULATION INDEX CALCULATION see results table

EC3 CALCULATION
EC3 = (a-c) [(3-d)/(b-d)] + c = 17.8 % (w/v)
a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.
a=1
b=0.62
c=25
d=4.01

CLINICAL OBSERVATIONS:
No deaths occurred during the study period. The animals did not show any clinical signs of toxiicty after the first and second application. After the third application the mid (25%) and high dose (50%) induced redness of the ear skin of all animals in the group.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

No deaths occurred during the study period. The animals did not show any clinical signs of toxicity after the first and second application. After the third application, the mid (25%) and high dose (50%) induced redness of the ear skin of all animals in the group. The SI values for 1, 25, and 50 % (w/v) were 0.62, 4.01, 5.99 respectively. N-benzyl-N,N-diethylethanaminium chloride was found to be a skin sensitizer and an EC3 value of 17.8 % (w/v) was derived. Based on the available data and the criteria of the CLP Regulation, the test chemical was classified as skin sensitizer category 1B.

Executive summary:

Mouse Lymph node Assay (LLNA) was performed to assess the dermal sensitization potential of the test chemical. The GLP study was conducted as per OECD 429 Guidelines without any deviations.

4 (nulliparous and non-pregnant)/dose, 16 CBA/Ca female mice were used for the study. In a non-GLP conform pre-test in two mice, test substance concentrations of 6.25, 12.5, 25, and 50 % were tested on one ear each. No irritation effects were observed at these concentrations after a single application. 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 substance concentrations of 1, 25, and 50 % (w/v) in ethanol: deionised water (7 + 3). The application volume, 25 µL, was spread over the entire dorsal surface (diameter of 8 mm) 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).Hexyl cinnamic aldehyde dissolved in acetone:olive oil (4+1) was used as positive control chemical. Five days after the first topical application, all mice were administered with 250 uL of 80.4 uCi/mL 3HTdR (corresponds to 20.1 uCi 3HTdR per mouse) by intravenous injection via a tail vein.

The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 um mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were re-suspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then re-suspended in 5 % trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 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). The test substance was regarded as a sensitiser in the LLNA if the following criteria are fulfilled:

1) exposure to at least one concentration of the test substance resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index;

2) 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.

No deaths occurred during the study period. The animals did not show any clinical signs of toxicity after the first and second application. After the third application, the mid (25%) and high dose (50%) induced redness of the ear skin of all animals in the group. The SI values for 1, 25, and 50 % (w/v) were 0.62, 4.01, 5.99 respectively. the test chemical was found to be a skin sensitizer and an EC3 value of 17.8 % (w/v) was derived. Based on the available data and the criteria of the CLP Regulation, the test chemical was classified as skin sensitizer category 1B.