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Skin sensitisation

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skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24-29 Oct 2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
TNO Triskelion, Utrechtseweg 48 3704 HE Zeist, the Netherlands
Type of study:
mouse local lymph node assay (LLNA)

Test material

Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): p-Toluenesulfonamide
- Chemical name 4-methylbenzenesulfonamide
- Molecular formula: C7H9NO2S
- Molecular weight: 171.2 g/mol
- Colour / Appearance: white untransparent crystalline solid
- Storage conditions: ambient temperature, dark

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Charles River Laboratories
- Age at study initiation: approximately 9-11 weeks
- Weight at study initiation: 21.1 - 25.5 g
- Housing: During acclimatisation the animals were group housed in macrolon type III cages (maximally 5 mice/cage). After allocation the animals were housed in macrolon type III cages (4 mice/group/cage) for the main study. For the dose range finding test, one animal was housed in a macrolon type II cage. The cages were provided with wood shavings (Lignocel) as bedding material and shreds of paper (Enviro-dri) and gnaw wood as environmental enrichment
- Diet: Standard diet ad libitum (Rat and Mouse no.3 breeding diet RM3) was purchased from SDS Special Diets Services, Whitham, England.
- Water: domestic mains tap-water, ad libitum
- Acclimation period: at least 5 days

- Temperature (°C): 22 ± 2, one deviation from the optimal temperature was observed (period of <30 minutes of 24.7°C).
- Humidity (%): 45-65, the actual upper limit during the study period was 99.9%, which was considered not to have affected study integrity.
- Air changes (per hr): ca 10
- Photoperiod (hrs dark / hrs light): 12/12 (light on from 7:00 AM - 7:00 PM)

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
0, 10, 25, 50% w/v
No. of animals per dose:
Details on study design:
The dose formulations of the test substance in vehicle were freshly prepared on each day of dosing, i.e. day 0, 1 and 2. The formulation was prepared on a weight (test substance)/volume (vehicle) basis. The formulation was mixed (vortex) until visible homogeneity was obtained. Based on known hazard data of p-Toluenesulfonamide it was to be expected that dermal application of a 50% (and lower) concentration should not cause systemic toxicity and/or excessive skin irritation. Therefore, concentrations of 50%, 25% and 10% were selected for this study. No adjustment was made for the purity of the test substance. The dose formulations were used for dosing of the animals within 2 hours after preparation. Homogeneity, concentration and stability of the test substance in vehicle were not determined in this study.

On day 0, 1 and 2 the different concentrations of test substance and control substances were administered to the dorsum of both ears (25 μL on each ear by means of a pipette). On day 5 all animals received an intravenous injection of 250 μL of phosphate-buffered saline (PBS) containing 20 μCi of 3H-thymidine in the tail vein. Five hours after the 3H-thymidine injection the animals were sacrificed by intra peritoneal (i.p.) injection with an overdose of sodium pentobarbital and the draining auricular lymph nodes were excised, pooled per animal in PBS and further processed to determine the 3H-thymidine incorporation. Per animal, the paired auricular lymph nodes (ALN) were excised and were placed together in a vial containing 5 mL phosphate buffered solution (PBS). A single cell suspension was prepared by gently rubbing the nodes between the rough ends of microscope slides. The cell suspension was washed twice. The cell pellet obtained after the second wash was resuspended in 3 mL of 5% trichloroacetic acid (TCA) and left overnight (~18 hours) at 2-10ºC. After centrifugation and removal of the supernatant, 1 mL of 1.5 M KOH in 20% EtOH was added to the precipitate and left for 24 hours at room temperature. Thereafter, the solution was transferred into a clean scintillation vial and 20 mL liquid scintillation cocktail (Hionic Fluor, Perkin Elmer) was added. The vial was thoroughly shaken and the number of disintegrations per minute (DPM) was determined by counting for five minutes in a liquid scintillation counter (Perkin Elmer).

The general condition and behaviour of the animals were checked at least once daily during the study. All findings including any abnormalities were recorded in the morning, when applicable before dosing and in case of changes in clinical signs also in the afternoon. Body weights were determined at allocation, at the start (day 0) and at the end (day 5) of the study. The animals were sacrificed on day 5 for excision of the auricular lymph nodes. The animals were macroscopically examined including the auricular lymph nodes and the ears.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistical evaluation of the data (body weights and 3H-thymidine incorporation) were performed by one-way ANOVA, followed by Dunnett’s multiple comparison tests in case of a significant result. In case of difference of variance between the different groups, log-transformation of the data before statistical evaluation was considered. Body weights and 3H-thymidine incorporation of the test groups or the positive control group were compared with the vehicle control animals. Probability values of p<0.05 were considered significant.

Results and discussion

Positive control results:
The stimulation index of Hexyl Cinnamic Aldehyde (25% in vehicle v/v) was 4.42.

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: see Remark
Concentration (% w/v) Stimulation index 0 : 1.0 10 : 2.97 25 : 1.48 50: 1.23
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Concentration (% w/v) Disintegrations per minute (group means) 0 : 1672 10 : 4974 25 : 2473 50 : 2063

Any other information on results incl. tables

No signs of local irritation or clinical signs of systemic toxicity were observed in any of the animals during the study period. No aberrant body weights or body weight gains were observed and no statistical significant difference was found between the vehicle and the different test groups and between the vehicle and the positive control group.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information Criteria used for interpretation of results: EU
Under the experimental conditions of this study, p-Toluenesulfonamide did not have a skin sensitising potential when dermally applied up to concentrations of 50%.
Executive summary:

In a GLP-compliant OECD Guideline 429 study, skin sensitisation properties of p-Toluenesulfonamide were studied in mice. Three test groups of 4 female mice each were treated with 10, 25, and 50% w/v by means of open application of 25 μL to the dorsum of each ear (total 50µL) for three consecutive days. Three days after the last treatment, all animals received an intravenous injection of 3H-thymidine. Five hours later, the 3H-thymidine incorporation in the draining auricular lymph nodes was determined. The results were compared with those of a negative control group which was treated with the vehicle (acetone/olive oil, 4:1 v/v). Group mean 3H-thymidine incorporations of 4974, 2473 and 2063 DPM were found in the auricular lymph nodes of animals treated with respectively 10, 25, and 50% p-Toluenesulfonamide. The group mean value in the vehicle control animals was 1672 DPM. The calculated stimulation indices (SI), representative of the change in 3H-thymidine incorporation in p-Toluenesulfonamide treated animals compared to controls, were 2.97, 1.48 and 1.23 for the tested doses of 10, 25, and 50% p-Toluenesulfonamide, respectively. Since the SI was < 3, the limiting value required for classification as a skin sensitiser, in response to all concentrations of p-Toluenesulfonamide tested and no dose response relationship was apparent it was concluded that p-Toluenesulfonamide did not have a skin sensitising potential when dermally applied up to concentrations of 50%.