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Description of key information

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:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Accorduing to OECD Guideline and GLP.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Interfauna UK Limited, Blackthrone, Bicester Oxon, UK
- Age at study initiation: young adults
- Housing: 4 males per cage
- Diet (e.g. ad libitum): RM1, Special Diets Services Limitied, Witham essex, UK
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- air changes: min. 15 per hour
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 h/ 12 h
Vehicle:
other: 3:1 EtOH:DEP (test substance) and acetone (positive control).
Concentration:
0.3%; 1%; 3%; 10%; 30%.
No. of animals per dose:
4
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The results are expressed as a counts per minute (cpm) value per lymph node for each group. The activity of each test group is then divided by the activity of the vehicle control group to give a test:control ratio for each concentration. The criterion for a positive response is that one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group. Consequently, a test substance which does not fulfil the above criterion is designated as unlikely to be a sensitiser.

EC3 calculations
The estimated concentration of the test substance required to produce a 3-fold increase in the draining lymph node cell proliferative activity (EC3) was calculated. The EC3 value was derived by interpolating between two points on the Sensitisation Index (SI) axis, one immediately above and the other immediately below the SI value of 3 (vehicle treated control values [SI= l] not being used for the latter). Where the data points lying immediately above and below the SI value ofthree have the co-ordinates (a,b) and (c,d) respectively, the EC3 value was calculated using the fo llowing equation:

EC3 = c + [(3-d)/(b-d)] x (a-c)
Positive control results:
A concentrations used in the positive control experiments yielded an at least 3-fold increase in isotope incorporation and thus were valid.
Key result
Parameter:
EC3
Value:
> 3 - < 10

Skin sensitization potential of dl-alpha-tocopherol:

Concentration of test substance (% w/v) Number of lymph nodes assayed Counts per minute (cpm) cpm per lymph node (x10 exp-2) Test control ratio
0 (vehicle only) 8 2058 2.57 N/A
0.3 8 1260 1.58 0.61
1 8 1729 2.16 0.84
3 8 2257 2.82 1.1
10 8 8553 10.69 4.16
30 8 13843 17.3 6.73
N/A - not applicable

Skin sensitization potential of the positive control substance (hexylcinnamaldehyde):

Concentration of test substance (% w/v) Number of lymph nodes assayed Counts per minute (cpm) cpm per lymph node (x10 exp-2) Test control ratio
0 (vehicle only) 8 876 1.1 N/A
1 8 2755 3.44 3.13
3 8 4809 6.01 5.46
10 8 8160 10.2 9.27
N/A - not applicable
Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the results of this study, D,L-alpha-tocopherol needs to be classified as Skin Sens 1B, according 1272/2008/EEC.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

D,L-alpha-tocopherol was tested in a guideline conforming Local Lymph Node Assay (OECD Guideline No. 429) under GLP-conditions (CTL, 2001). Groups of four male mice were used per group. The test substance was applied as 0.3%, 1%, 3%, 10% or 30% w/v preparations in 3:1 EtOH:DEP. The test substance was shown to have the capacity to cause skin sensitisation when applied as 10% or 30% w/v preparations. In a positive control study, hexyl cinnamaldehyde was shown to have the capacity to cause skin sensitisation when applied as 1%, 3% or 10%w/v preparations in acetone, confirming the validity of the protocol used for this study. In conclusion, D,L-alpha-tocopherol was found to be a skin sensitiser under the conditions of the test, with an EC3 concentration of 7.4%.

In order to assess the skin sensitization potential of D,L-alpha-tocopherol, a Maximization Test in Guinea pigs (OECD Guideline No. 406, GLP; Csato and Karunaratne, 1996) was carried out in 30 (20 test and 10 control) female albino Guinea pigs. The intradermal induction of sensitization was carried out with 0.2% solution of the test article in light liquid paraffin or in emulsion with Freund’s Complete Adjuvant. The epicutaneous induction of sensitization was conducted with a 25% concentration of the test article in ethanol under occlusion. Two weeks after the epidermal induction, the challenge was completed by epicutaneous application of the test article in the highest non irritating concentration, i.e. 12.5% (as determined in the rangefinding phase of the study) in ethanol under occlusive dressing for 24 hours. Skin reactions, i.e. erythema and eschar as oedema formation were evaluated at 24 and 48 hours after removal of the dressing. Seven test animals (37%) exhibited slight to moderate erythema following challenge with 12.5% test article, at the 24 and/or 48 hours examination. None of the test animals responded positively to challenge with the vehicle at any examination. None of the control animals reacted positively to challenge with either 12.5% test article or the vehicle, ethanol at any examination. Given the results of this study, it is concluded that the test article, dl-alpha-tocopherol, exhibited a moderate sensitizing potential in the guinea pig under the conditions of this study. According to Annex VI of 67/548/EEC, the substance is to be considered sensitizing.

In another study the skin sensitisation potential of D,L-alpha-tocopherol was investigated in the Open Epicutaneous Test (OET), which was carried out in the albino Guinea pig (OECD guideline 406, non-GLP; Csato, 1997).

During the induction phase of sensitisation the test article was applied epicutaneously onto the skin of the test animals 5 days a week for 4 consecutive weeks; 6 animals per treatment group were treated on the right flank with the test article at the concentrations of 50%, 30%, 10% and 3% in ethanol, respectively. Treatment sites were left open between the subsequent applications. The control group was treated similarly with the vehicle, ethanol only. To assess the sensitization responses, 4 weeks after beginning of the induction treatment a challenge application has been carried out. The animals (test and control) have been exposed on the left flank, by open epicutaneous way, to progressive dilutions of D,L-alpha-tocopherol in ethanol. Two weeks later a second challenge was done similarly with further dilutions of the test article. The cutaneous reactions, such as erythema and oedema formation were evaluated daily at each individual treatment site during the induction period. Challenge reactions were assessed at 24 and 48 hours after application. The test article induced slight to strong irritant skin reactions in the experimental animals after repeated application during the induction treatment.

In the majority of the test animals sensitisation responses were detected at the challenge application sites exposed to the test article in the concentration of 3 % or higher. However, when applying D,L-alpha-tocopherol at the concentration of 1 % or lower, no skin sensitisation responses as such were observed during the experiment.

Considering the above experimental data, it can be concluded that topically applied D,L-alpha-tocopherol revealed a skin sensitizing potential at higher concentrations (> 3%) in Guinea pigs and in the mouse LLNA. However, cutaneous exposure to D,L-alpha-tocopherol at lower (non-irritating) concentrations (< = 1 % in Guines pigs and < = 3% in mice) did not result in sensitisation responses, and accordingly, is unlikely to give rise to skin sensitisation in man.


Migrated from Short description of key information:
D,L-alpha-tocopherol is considered a weak to moderate skin sensitiser in animal models as tested in a Guinea pig maximisation test, according to OECD guideline 406 and a Local Lymph Node Assay according to OECD guideline 429.

Justification for selection of skin sensitisation endpoint:
GLP compliant and guideline conforming study

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, the test substance needs to be classified as a sensitiser Cat 1B according to the EU Regulation on Classification, Labelling and Packaging of Substances and Mixtures (CLP) (EC) No. 1272/2008.