2-methyl-N-(4-sulfamoylphenyl) prop-2-enamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 - 22 Oct 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted April 2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

adopted April 2004

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

adopted March 2003

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

438-940-2

EC Name:

-

Cas Number:

56992-87-1

Molecular formula:

C10 H12 N2 O3 S

IUPAC Name:

2-methyl-N-(4-sulfamoylphenyl) prop-2-enamide

Test material form:

other: powder aggregated into lumps

Details on test material:

- Name of test material (as cited in study report): SPM-N
- Physical state: white lumps with powder
- Lot/batch No.: T-9314
- Expiration date of the lot/batch: 29 Aug 2008
- Storage condition of test material: at room temperature in the dark
- Other: pH 7.6-7.5 (1% in water, indicative range)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L'Arbresle Cedex, France
- Age at study initiation: approximately 11 weeks
- Weight at study initiation: 23-25 g (range)
- Housing: during the study period, animals were housed individually in Macrolon cages (MI type, height 12.5 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd.), Surrey, United Kingdom) was supplied as cage enrichment.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1-23.5
- Humidity (%): 38-64
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 3 Oct 2007 To: 22 Oct 2007

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

10, 25 and 50%

No. of animals per dose:

5 females

Details on study design:

RANGE FINDING TESTS: 2 animals were tested with a 25 and 50% solution of the test substance in order to select the highest concentration to be used in the main study. The study protocol was the same as for the main study.
- Compound solubility: the highest concentration of 50% was the maximum concentration that could technically be applied
- Irritation: no skin irritation reactions were observed on the left or right ear.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: if the results indicate a SI ≥ 3, the test substance is considered to be a skin sensitiser

TREATMENT PREPARATION AND ADMINISTRATION:
A formulation of the test substance in dimethyl formamide was prepared within 4 h before application, homogeneity was obtained to visually acceptable levels. The formulation was mixed thouroughly with a vortex mixer prior to application. 25 µl was applied to the dorsal surface of both ears for 3 consecutive days. On Day 6, each animal was injected via the tail vein with 0.25 mL sterile phosphate buffered saline containing 20 µCi of 3H-methyl thymidine. After approximately 5 h, the mice were sacrificed and the draining lymph nodes of the ears were excised. The relative size was estimated by visual examination and any abnormalities of the nodes and surrounding areas were recorded. The nodes were pooled for each animal in PBS. A single cell suspension of lymph node cells was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 µm). Lymph node cells were washed twice with an excess of PBS and the DNA precipitated with 5% trichloroacetic acid (TCA) at 4 ºC overnight. The precipitate was recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL Ultima Gold cocktail as the scintillation fluid. The counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes, whichever came first. The scintillation counter automatically subtracted the background and converted Count Per Minute (CPM) to Disintegrations Per Minute (DPM).

Positive control substance(s):

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

Results and discussion

Positive control results:

A reliability check was performed in September 2007, to check the sensitivity of the test system and the reliability of the experimental techniques of the testing laboratory. Groups of 4-5 female CBA mice were exposed to 5, 10 and 25% alpha-hexylcinnamicaldehyde in acetone:olive oil (4:1), or the vehicle only. The SI values calculated for the substance concentrations 5, 10 and 25% were 1.0, 2.0 and 5.7, respectively. The negative control had an SI value of 1.0. An EC3 value of 14.1% was calculated using linear interpolation. The calculated EC3 value was in the acceptable range of 2-20%. The results of the previous 6-monthly reliability checks were EC3 values of 10.3, 9.5, 13.1 and 15.6%.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The SI values calculated for the 10, 25 and 50% groups were 0.5, 0.7 and 0.3, respectively (see Table 2). The EC3 value is therefore higher than 50%.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The mean DPM/animal values for the 10, 25 and 50% groups were 216, 320 and 438, respectively. The mean DPM/animal value for the control group was 438 (see Table 1 for individual values and Table 2 for mean values with standard deviation).

Any other information on results incl. tables

Table 1: Radioactivity measurements, individual values

Dose (%)	Animal	DPM/animal
Control^1	1	350
Control	2	251
Control	3	178
Control	4	943
Control	5	469
10	1	117
10	2	151
10	3	472
10	4	269
10	5	70
25	1	401
25	2	163
25	3	465
25	4	247
25	5	322
50	1	33
50	2	229
50	3	219
50	4	66
50	5	184

¹Control: dimethyl formamide

Table 2: Disintegrations per minute and stimulation index

Dose group (%)	DPM (mean ± SD)	SI ( mean ± SD)
Control	216 ± 72	1.0 ± 0.4
10	320 ± 54	0.5 ± 0.2
25	146 ± 41	0.7 ± 0.3
50	438 ± 135	0.3 ± 0.1

Slight edema (score 1) was noted on the ear of 1/5 mice treated with the 50% concentration. This is not considered to have had a significant effect on the activity of the lymph nodes. All the nodes of the animals in the control and treatment groups were normal in size. No macroscopic abnormalities were noted in the surrounding area. The body weights and body weight gains were comparable between the control and the treatment groups and no clinical signs were observed.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified