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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes
Details on test material:

- Name of test material (as cited in study report): Rikabinol HB
- Substance type: Monomer
- Physical state: White solid
- Analytical purity: 95.6%
- Impurities (identity and concentrations):
- Lot/batch No.: 7095
- Expiration date of the lot/batch: 31 December 2012
- Storage condition of test material: Room temperature in the dark
- Other: Date received 12 December 2011

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Ltd
- Age at study initiation: approximately eight to twelve weeks
- Weight at study initiation: 17.0 to 23.1 g
- Housing: housed two animals per cage, in solid bottomed polycarbonate cages with a stainless steel mesh lid.
- Diet: The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes
- Acclimation period: for at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23
- Humidity (%): 40 to 70
- Air changes (per hr): The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod (hrs dark / hrs light): Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
10, 25 and 50% w/v
No. of animals per dose:
4
Details on study design:
MAIN STUDY
Preparation of single cell suspensions
A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless steel gauze (200 mesh size). The pooled LNC were then washed by adding 10 mL PBS, pelleted at 190 x g for 10 minutes and resuspended. The cells were washed twice again and resuspended in 3 mL trichloroacetic acid (TCA: 5%) following the final wash.

Administration of 3H-methyl Thymidine
Five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 250 L of phosphate buffered saline containing 3H-methyl Thymidine (3HTdR: 80 Ci/mL) giving a nominal 20 Ci to each mouse. The injection into the tail vein was carried out using a plastic syringe and needle after the mouse had been heated in a warming chamber.


Determination of incorporated 3H-methyl Thymidine
After overnight incubation (minimum of 18 hours) with 5% TCA at 4°C, the precipitate was recovered by centrifugation and resuspended in 1 mL 5% TCA and transferred to 10 mL Ultima gold scintillation fluid on Day 7. The 3HTdR incorporation was measured by ß scintillation counting.The proliferative response of LNC was expressed as radioactive disintegrations per minute per lymph node (dpm/node).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The SI for the positive control substance hexyl cinnamic aldehyde (HCA), was 8.5 which demonstrates the validity of this study.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Group Concentration dpm 1 DMF 5933.85 2 10% w/v 10769.05 3 25% w/v 16301.55 4 50% w/v 11522.65 5 HCA 25% v/v 50403.75
Key result
Parameter:
SI
Value:
ca. 1.8
Test group / Remarks:
10 % w/v
Key result
Parameter:
SI
Value:
ca. 2.7
Test group / Remarks:
25 % w/v
Parameter:
SI
Value:
ca. 1.9
Test group / Remarks:
50 % w/v

Any other information on results incl. tables

Mortality and clinical results

There were no deaths and no signs of ill health or toxicity were observed during this study.

Wet/greasy fur on the head was noted following each dosing occasion, except for animals dosed with the test material at 50% w/v, this was related to unoccluded dermal administration of a liquid formulation/vehicle and not an effect of the test substance. White residue was observed shortly after dosing in all animals dosed with the test material at 25 and 50% w/v. This observation was still present in two mice (A15, A16) at study termination on Day 6.

Dermal reactions

No signs of dermal irritation were seen on the ear during the study.

Body weight

A loss in bodyweight was noted over the study period, however, a small loss in bodyweight is not uncommon in young laboratory mice and is not considered to be an effect of treatment.

Simulation Index and Estimated Concntration of Three

The SI (test/control ratios) obtained for 10, 25 and 50% w/v Rikabinol HB were 1.8, 2.7 and 1.9 respectively. As a SI of 3 or more was not recorded for any of the concentrations tested, Rikabinol HB is not considered to have the potential to cause skin sensitization.

As all concentrations tested resulted in an SI of less than 3 the EC3 concentration is greater than the highest concentration tested (50% w/v).

The SI for the positive control substance hexyl cinnamic aldehyde (HCA), was 8.5 which demonstrates the validity of this study.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Rikabinol HB is not regarded as a potential skin sensitizer.