Propanamide, N-[4-(2,4-dihydroxyphenyl)-2-thiazolyl]-2-methyl-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Aug 2011 - 13 Sept 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Federal Office of Public Health, Bern, Switzerland

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

N-[4-(2,4-dihydroxyphenyl)-1,3-thiazol-2-yl]-2-methylpropanamide

EC Number:

807-621-3

Cas Number:

1428450-95-6

Molecular formula:

C13H14N2O3S

IUPAC Name:

N-[4-(2,4-dihydroxyphenyl)-1,3-thiazol-2-yl]-2-methylpropanamide

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Kent, United Kingdom
- Age at study initiation: 9 - 11 weeks
- Weight at study initiation: 15.8 - 22.2 g
- Housing: The animals were housed in groups in Makrolon Type-II cages with standard softwood bedding.
- Diet: Pelleted standard Harlan Teklad 2914C rodent maintenance diet, ad libitum
- Water: Community tap water from Itingen/Switzerland, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

1, 2.5, 5, 10 and 25 % (w/v)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed with concentrations of 75, 50, 25 and 10 % test substance and the solvents acetone/olive oil (4:1 v/v), dimethylformamide, methyl ethyl ketone, propylene glycol, dimethyl sulfoxide, ethanol 70 % and ethanol 30 %. The highest concentration which can be technically used is 25 % (w/w) in dimethylformamide.
- Irritation and lymph node proliferation response: To determine the highest non-irritant test concentration, a pre-test was performed. Two mice were treated with concentrations of 10 % and 25 % (w/v) each in dimethylformamide on three consecutive days. In the pre-test, clinical signs were recorded within approximately 1 hour and 24 ± 4 hours after each application as well as on the day corresponding to the day of lymph node preparation. Additionally, ear thickness measurements were performed pre-dose, approximately 48 hours after the first application as well as on the day corresponding to the day of lymph node preparation. No signs of irritation or systemic toxicity were observed at any reading time point and the ear thickness did not change from the pre-dose value.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-methyl thymidine incorporation determined by β-scintillation
- Criteria used to consider a positive response: The 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 S.I. and 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:
Each test group of mice was treated by topical application of 25 µL of the test substance to the 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 as vehicle control group.
Five days after the first topical application, all mice were administered 250 µL of phosphate-buffered saline (PBS) containing 81.82 µCi/mL 3HTdR by intravenous injection via the tail vein. Approximately five hours after treatment with 3HTdR, all mice were euthanized.
The draining lymph nodes were rapidly excised and pooled in PBS for each experimental group (8 lymph nodes per group). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through a gauze (200 µm mesh size) and transferred in centrifugation vials. After washing twice with PBS, the lymph node cells were resuspended in 5 % trichloroacetic acid and incubated at approximately 5 °C for at least 18 hours for precipitation of macromolecules. The supernatant was removed by centrifugation. The precipitates were then resuspended in 1 mL 5 % trichloroacetic acid and transferred to glass scintillation vials with scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was measured on a β-scintillation counter.

Positive control substance(s):

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

Statistics:

For the body weight mean values and standard deviations were calculated.

Results and discussion

Positive control results:

The positive control substance (hexyl cinnamic aldehyde) induced SI values of 1.8, 1.6 and 8.4 at 5, 10 and 25 %, thus meeting the reliability criteria for the local lymph node assay (SI ≥ 3).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0

Test group / Remarks:

Group 1 (0 % Test item)

Results 2

Parameter:

SI

Value:

1.5

Test group / Remarks:

Group 2 (1 % Test item)

Results 3

Parameter:

SI

Value:

2

Test group / Remarks:

Group 3 (2.5 % Test item)

Results 4

Parameter:

SI

Value:

2.9

Test group / Remarks:

Group 4 (5 % Test item)

Remarks on result:

other: used for calculation of EC3 value

Results 5

Parameter:

SI

Value:

3.5

Test group / Remarks:

Group 5 (10 % Test item)

Remarks on result:

other: used for calculation of EC3 value

Results 6

Parameter:

SI

Value:

3.8

Test group / Remarks:

Group 6 (25 % Test item)

Results 7

Key result

Parameter:

EC3

Value:

5.8

Test group / Remarks:

Calculated from SI values of group 4 and 5.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

CLP: Skin sens 1B, H317

Executive summary:

In order to study the possible contact allergenic potential of W630 (Sample ID: 25130), five groups of four female mice each were treated daily with the test item at concentrations of 1%, 2.5%, 5%, 10% and 25% (w/v) in DMF by topical application to the dorsum of each ear lobe for three consecutive days. A control group of four female mice was treated with the vehicle DMF only.

Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine, 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes were excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3HTdR measured in a β-scintillation counter.

All treated animals survived the scheduled study period. Neither clinical signs on the ears of the animals nor systemic findings were observed during the study period.

The results obtained [Stimulation Index (S.I.)] are reported in the following table. The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.