Registration Dossier

Administrative data

Description of key information

Skin sensitizer: not sensitising

Respiratory sensitiser: no study available

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
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.
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)
Positive control results:
The SI for the positive control substance hexyl cinnamic aldehyde (HCA), was 8.5 which demonstrates the validity of this study.
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

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.

Interpretation of results:
GHS criteria not met
Conclusions:
Rikabinol HB is not regarded as a potential skin sensitizer.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The study was performed (Huntingdon Life Sciences, 2012, Study MOG0009) to assess the skin sensitization potential of HBPA using the local lymph node assay (LLNA). The study was performed according to EC Method B.42 and OECD test guideline 429, and in compliance with GLP.

 

Three treated groups, each comprising four female mice receiving HBPA at concentrations of 10, 25 or 50% w/v. Similarly constituted groups received the vehicle dimethylformamide or positive control substance. The mice were treated by daily application of 25 µL of the appropriate concentration to the dorsal surface of both ears for three consecutive days.

 

The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) by β-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio), termed as Stimulation Index (SI).

 

The Stimulation Index obtained for 10, 25 and 50% w/v were 1.8, 2.7 and 1.9 respectively which indicates that HBPA did not show the potential to induce skin sensitization.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Respiratory sensitiser: no study available

Justification for classification or non-classification

As noted above, a Skin Sensitization study concluded that HBPA showed no sensitizing activity; on this basis HBPA is not classified for skin sensitization under either the Classification, Labelling and Packaging Regulation.

 

No information regarding the potential for HBPA to cause respiratory sensitization.