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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted April 2002
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted April 2004
Deviations:
no
Qualifier:
according to
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)
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
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)
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%.
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%.
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).

Table 1: Radioactivity measurements, individual values

Dose (%)

Animal

DPM/animal

Control1

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.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitising potential of 2-methyl-N-(4-sulfamoylphenyl) prop-2-enamide (SPM-N) was evaluated in a local lymph node assay (LLNA) performed according to OECD Guideline 429 and in compliance with GLP (Stitzinger, 2008e). Based on a range-finding test, test substance concentrations of 10, 25 and 50% in dimethyl formamide were selected for treatment in the main study. The 50% concentration was the maximum concentration that was technically possible to use. The test suspensions or the vehicle dimethyl formamide were applied to the dorsal surface of both ears of CBA mice (25 µL/ear) for three consecutive days (Days 1-3). 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 nodes were pooled for each animal and the cell proliferation of pooled lymph nodes was measured by incorporation of ³H-methyl thymidine and expressed as the amount of radioactive disintegration per minute (DPM).

Slight edema (score 1 of 4) 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, and no macroscopic abnormalities were noted in the surrounding area. The positive control group (hexyl cinnamic aldehyde) was valid.

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. The SI values calculated for the 10, 25 and 50% groups were 0.5, 0.7 and 0.3, respectively. Since the SI values were lower than 3, the test substance is considered to be not skin sensitising.

 


Migrated from Short description of key information:
Skin (OECD 406): not sensitising (Local Lymph Node Assay)

Justification for selection of skin sensitisation endpoint:
There is only one study available.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:
Justification for selection of respiratory sensitisation endpoint:
Study not required according to Annex VII-X of Regulation (EC) No 1907/2006.

Justification for classification or non-classification

The available data on the skin sensitisation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.