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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in a GLP facility to OECD guidelines
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Test system: Mice, CBA/CaOlaHsd
Rationale: Recognised as the recommended test system
Source: Harlan Netherlands B.V. Postbus 6174 NL - 5960 AD Horst / The Netherlands
Number of animals for the pre-test: 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant) Number of test
Groups: 3
Number of control (vehicle) groups: 1
Age: 7 - 8 weeks (beginning of acclimatisation)
Identification: Single caging. The animals were distributed into the
test groups at random and identified by cage number.
Acclimatisation: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.

Housing: single
Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
Bedding: granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen)
Feed: pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
Water: tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf)
Environment: temperature 22 + 3°C
relative humidity 30-78%
artificial light 6.00 a.m. - 6.00 p.m.

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 25 and 50%
No. of animals per dose:
4
Details on study design:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 10, 25, and 50 % (w/v) in acetone:olive oil (4+1). The application volume, 25 ul, was spread over the entire dorsal surface ( 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle
alone (control animals).

3H-methyl thymidine (3HTdR) was purchased from GE Healthcare (GE Healthcare product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 ul of 80.7 uCi/ml 3HTdR (corresponds to 20.2 uCi 3HTdR per mouse) by intravenous injection via a tail vein.

Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Ketamin/Xylazin/Midazolam (4+2+1).
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 um mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and
incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The -scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.

A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3
value was calculated according to the equation

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

where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in
draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the
co-ordinates of the two pair of data lying immediately above and below the S.I. value of
3 on the local lymph node assay dose response plot.
Positive control results:
The positive control gave positive results
Parameter:
SI
Value:
2.05
Test group / Remarks:
10%
Remarks on result:
other: 2.05 @ 10%, 6.58 @ 25% and 9.76 @ 50%
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 7837 @ 10%, 25142 @ 25% and 37308 @ 50%
Parameter:
SI
Value:
6.58
Test group / Remarks:
25%
Parameter:
SI
Value:
9.76
Test group / Remarks:
50%

Calculation and Results of Individual Data
Vehicle: acetone:olive oil (4+1)
Test item concentration
% (w/v)
Group Measurement
DPM
Calculation Result
DPM-BGa) number of lymph nodes DPM per lymph node S.I.
--- BG I 24 --- --- --- ---
--- BG II 24 --- --- --- ---
--- 1 3846 3822 8 477.8  
10 2 7861 7837 8 979.6 2.05
25 3 25166 25142 8 3142.8 6.58
50 4 37332 37308 8 4663.5 9.76
BG = Background (1 ml 5% trichloroacetic acid) in duplicate
1   = Control Group
2-4 = Test Group
S.I. = Stimulation Index
a)      = The mean value was taken from the figures BG I and BG II
b)      = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
Migrated information
Executive summary:

In the study the test item Abitol-E dissolved in acetone:olive oil (4+1) was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 10, 25, and 50 %. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. In this study Stimulation Indices (S.I.) of 2.05, 6.58, and 9.76 were determined with the test item at concentrations of 10, 25 and 50 % in acetone:olive oil (4+1), respectively. The test item Abitol-E was found to be a skin sensitiser and an EC3 value of 13.1 % (w/v) was derived.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In the study the test item Abitol-E dissolved in acetone:olive oil (4+1) was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 10, 25, and 50 %. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. In this study Stimulation Indices (S.I.) of 2.05, 6.58, and 9.76 were determined with the test item at concentrations of 10, 25 and 50 % in acetone:olive oil (4+1), respectively. The test item Abitol-E was found to be a skin sensitiser and an EC3 value of 13.1 % (w/v) was derived.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test article meets the definition as a skin sensitizer