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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Justification for not submitting the in vitro study.
The new legal requirements (21 June 2016) require an in vitro skin sensitisation study. However the previous data requirements were met with an in vivo study during dossier compilation for the >1000 T/y tonnage band. Therefore there is no need to repeat the study using the alternative test method.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Common name: BBSA
- Purity: 99.94% as certified by the sponsor
- Appearance: clear liquid
- Lot nr: 200911030035
- Production date: 3rd november 2009
- Shelf life: 2 years
- Date of sample receipt: 3rd december 2009
- Storage: Required quantity of the test substance was received from the test substance controller (TSC, IIBAT) and was stored in the utility room
- Safety precautions: test substance was handled with necessary protective clothing and all recommended safety measures were followed

In vivo test system

Test animals

other: Mus musculus /cba/CaCrl
Details on test animals and environmental conditions:
- Rationale: recognized by the international guidelines as the recommended test system
- Source: procured from charles river laboratory, UK and bred at IIBAT animal house facility
- Hygienic level during the study: good conventional
- No of animals for pre-test: 6 females
- No of animals for main experiment: 25 females
- No of animals per group: 5 f (nulliparous and non pregnant)
- Age at study initiation: 9-10 weeks old
- No of test substance treatment groups: three
- No of negative control group: one
- No of positive control group: one
- No of groups: five
- Health: only animals in acceptable health condition certified by the ANimal House in charge were used for the study
- Weight at study initiation: the weight variation in animals involved in the study was not exceed +/-20% of the mean weight
- ID: animals were housed in individual cages. Each cage was identified by cage card. Cage cards marked with unique animal number, group number, sex and treatment details were attached to cages
- Randomization: randomized as per IIBAT SOP (SOP/TOX/001). Randomization procedure involved assigning serial numbers to animals, generating random numbers from scientific calculator, ranking random numbers and assigning the animals to the various groups.
- Acclimation period: 5 days prior to the pretest and main experiment
- Diet: standard gamma irradiated pellet feed. Both drinking water and feed were provided ad libitum. The feed and water were routinely analyzed and were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

- Test room: tox lab 1A: Room 06
- Temperature (°C): 20.9-22.2 °C
- Humidity (%): relative humidity 50-57%
- Housing: individual cages, cage type standard polypropylene mouse cages with stainless steel top grill supplied by M/s. Vishnu Traders, UP, India
- Bedding: sieved and autoclaved paddy husk
- Sanitation: bedding material, cages, grills and water bottles were changed on alternate days
- Animal welfare: test facility (No 31/1999/CPCSEA dated 11.03.99) for breeding and exeriments of animals was registred by the committee for the purpose of control and supervision of experiments on animals, ministry of forest and environment, govt. of India
- Photoperiod (hrs dark / hrs light):12:12 controlled by automatic timer

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
test substance formulations were made fresly on each dosing occasion
3 different concentrations: 25, 50 and 100% v/v
No. of animals per dose:
Details on study design:
- each test group of mice was treated by topical application to the dorsal surface of each ear lobe with teh test substance at three different concentrations25 and 50% in acetone:olive oil (4:1), 100% test substance was applied as such
- the application volume, 25 ùl, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days
- a further group of mice were treated with an equivalent volume of the relevant vehicle alone (control animals) and relevant positive control (25% HCA)
- 5 days after the first topical application, all the group of mice were administred 250 ùl of phosphate-buffered saline (PBS) containing 20 ùCi of tritiated methyl thymidine by intravenous injection via lateral tail vein.

- approx 5h after treatment with HTdR all mice were euthanized using CO2. The drining auricular lymph nodes were rapidly excised and pooled for each group (10 nodes per group). Single cell suspension was prepared by mashing the lymph nodes between the frosted ends of microscopic slides.
- after washing twice with HBSS the lymph node cells were re-suspended in 3 ml of 5% trichloroacetic acid and incubated at approx +4°C for 18 hours
- the precipitates was re-suspended in 5% trichloroacetic acid (1ml) and transferred to glass scintillation vials with 10 ml of scientillation cocktail and thoroughly mixed. The level of HTdR incorporation was then measured on a liquid scintillation counter
- Similarly, background HTdR levels were also measured in 2 1ml-aliquots of 5% trichloroacetic acid. The liquid scintillation counter expresses HTdR incorporation as the number of radioactive disintegrations per minute.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
HCA: 12159 measurement dpm, 12090 Dpm-BG, 10 lymph nodes, 1209.0 Dpm per lymph node,
Result: 8.87 SI

In vivo (LLNA)

Resultsopen allclose all
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM value of positive control was 8.88 times more than the vehicle control.
Test group / Remarks:
Test group / Remarks:
Test group / Remarks:

Any other information on results incl. tables

A dose response relationship was observed. EC3 value was not calculated since none of the test substance concentrations tested did not produced stimulation index greater than 3.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
N-n-butylbenzenesulphonamide (proviplast 024) when applied to the dorsum of ear lobes, no erythema, edema was observed at the site of application in any of the doses applied. No mortality, clinical signs of toxicity were observed in any of the animals treated with different concentrations. Change in body weight was normal.
In conclusion, a dose level of 100%, 50% and 25% produced stimulation indices of 1.37, 1.09 and 0.67 resepectively. Stimulation indices of less than 3.0 were observed at all the three concentrations tested. Therefore, the EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI=3) for the test substance under the condition of this study was not calculable and hence test item N-n-butylbenzenesulphonamide (Proviplast 024) may be considered as a non sensitizer under present experimental condition.
Executive summary:

N-n-butylbenzenesulphonamide was tested in a Local Lymph Node Assay in CBA/CaCrl mice applied to the dorsum of ear lobes at 0, 25, 50 and 100% concentrations. No erythema, edema was observed at the site of application in any of the doses applied. No mortality, clinical signs of toxicity were observed in any of the animals treated with different concentrations. Change in body weight was normal. The stimulation index measured was 0.67, 1.09 and 1.37 for the, 25, 50 and 100% concentration, respectively. These values were all under the threshold of 3 (compared to the positive control value of 8.87), therefore the test substance was not sensitizing under the experimental conditions.