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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 May to 23 June 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in accordance with an appropriate guideline and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier (France)
- Age at study initiation: 8 weeks
- Weight at study initiation: 20.1 to 23.4 g
- Housing: 2 or 4 in polycarbonate cages (Techniplast 1145T) and then individually in disposable crystal polystyrene cages
- Diet (ad libitum): SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiaten, Germany)
- Water (ad libitum): facility tap water
- Acclimation period: 13 or 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 30 to 70
- Air changes (per hr): approximately 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 8 June 2011
To: 20 June 2011

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 25, 50 or 100% preliminary test
5, 10 25, 50 or 100% main test
No. of animals per dose:
2 females preliminary test
4 females main test
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: solution obtained at 50% concentration of test substance.
- Irritation: no local reaction noted
- Ear thickness response: no increase in ear thickness noted

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: SI >= 3 together with dose-response relationship

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was administered as a solution in the vehicle. The test item was mixed by vortex with the required quantity of vehicle. The test item concentrations were expressed in % (v/v). The positive control was prepared at the concentration of 25% (v/v) a mixture of Acetone/Olive Oil (4/1; v/v) (AOO). Dosage forms preparations were prepared extemporaneously on the day of each administration. The dosage forms were be stored at room temperature and delivered to the study room in stoppered plastic tubes.

On days 1, 2 and 3, a dose-volume of 25 μL of the appropriate dosage form preparation was applied to the dorsal surface of one or both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking, to ensure an optimized application of the test materials and to facilitate ear thickness measurement, the animals were placed under light isoflurane anaesthesia during the administration. No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The EC3 value (theoretical concentration resulting in a SI value of 3) was determined by linear interpolation of points on the dose-response curve, immediately above and below the 3-fold threshold. The equation used for calculation of EC3 was:

EC3 = c + [(3 - d)/(b - d)] x (a - c)

Where a = the lowest concentration giving Stimulation Index > 3; b = the actual Stimulation Index caused by a; c = the highest concentration failing to produce a Stimulation Index of 3; d = the actual Stimulation Index caused by c.

Group means and Standard deviations calculated where appropriate.

Results and discussion

Positive control results:
The positive control Stimulation Index was 12.66, therefore the experiment was considered valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
A dose-related increase in the Stimulation Index was recorded at TBM concentrations of ≥ 10%, and the threshold positive value of 3 was exceeded at the concentration of 25%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity. The EC3 value was calculated to be 20%.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Group mean dpm increased with increasing concentration in treated groups - see table.

Any other information on results incl. tables

Mortality

There were no unscheduled deaths during the study.

Clinical Signs

There were no remarkable clinical signs noted during the study.

Body weight

There was no effect on body weight.

Ear thickness measurement and local reactions

No local reactions were noted in any animals and no notable increase in ear thickness was noted at any tested concentration.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Tertiobutylmercaptan was found to be sensitising in a mouse local lymph node assay, resulting in a Stimulation Index of greater than 3 at concentrations of 25% and above (3.62, 4.26 and 30.43 at substance concentrations of 25, 50% and 100% respectively). The EC3 value was calculated to be 20%.