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Description of key information

The skin sensitisation study, conducted according to OECD TG 429 (Local Lymph Node Assay) and in compliance with GLP, tested the test substance at concentrations of 25 % and above and concluded the test substance to be sensitising (CiToxLAB, 2011).

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:
26 May to 23 June 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
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
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 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.
Positive control results:
The positive control Stimulation Index was 12.66, therefore, the experiment was considered valid.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Group mean dpm increased with increasing concentration in treated groups - see table.
Parameter:
SI
Value:
1.73
Test group / Remarks:
5 % 2-methylpropane-2-thiol
Parameter:
SI
Value:
1.77
Test group / Remarks:
10 % 2-methylpropane-2-thiol
Parameter:
SI
Value:
3.62
Test group / Remarks:
25 % 2-methylpropane-2-thiol
Parameter:
SI
Value:
4.26
Test group / Remarks:
50 % 2-methylpropane-2-thiol
Parameter:
SI
Value:
30.43
Test group / Remarks:
100 % 2-methylpropane-2-thiol
Parameter:
SI
Value:
12.66
Test group / Remarks:
25 % HCA (positive control)
Parameter:
EC3
Value:
20
Test group / Remarks:
An EC3 value of 20% was calculated from the results and presented in the study report

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.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
2-Methylpropane-2-thiol 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%.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In a mouse local lymph node assay conducted according to OECD Test Guideline 429 (CiToxLAB, 2011) the stimulation index (SI) was greater than 3 at concentrations of 25% and above (3.62, 4.26 and 30.43 at 25%, 50% and 100% concentrations respectively). Therefore, 2-methylpropane-2-thiol was considered to be sensitising. The EC3 value (estimated test substance concentration that will give an SI of 3) was calculated to be 20%.

2-methylpropane-2-thiol was also found to be a dermal sensitizer in a supporting study in guinea pigs by a modified Buehler method (Douds, 1995).

Following a pilot study to determine the appropriate concentrations of test material to use in the induction and challenge phases, Hartley-derived albino guinea pigs (10/sex) were dermally treated with undiluted 2-methylpropane-2-thiol (0.4 ml, 4 cm2 area) once /week for 6 hours/exposure for three consecutive weeks. Following a 2 week rest period, the test animals and a naïve control group (5/sex) were dermally challenged with 75 % test substance W/V in mineral oil. The animals were scored (0-3 scale) for skin sensitising reactions at 24 and 48 hours following the challenge phase. Hexylcinnaldehyde historical positive control data were cited as demonstrating susceptibility of the test system to a sensitising agent. 

The primary challenge resulted in 13/20 animals (65%) exhibiting scores of ≥2 at 24 hours and 11/20 animals (55%) exhibiting similar scores at 48 hours post-challenge. The mean dermal score for the entire test group was 1.9 at 24 hours and 1.7 at 48 hours. All control group animals exhibited a grade 1 response at 24 hours with all but one reporting the same response at 48 hours. Based on the severity and incidence of the sensitisation response observed in the test group, 2-methylpropane-2-thiol is considered to be a dermal sensitizer in guinea pigs with moderate potency according to ECHA guidance (REACH Chapter R.8 (Characterisation of dose [concentration] –response for human health); Appendix R.8-10.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data 2-methylpropane-2-thiol requires classification for skin sensitisation as Skin Sensitiser Category 1B: (H317: May cause allergic skin reaction), according to Regulation (EC) No 1272/2008, as amended.