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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline compliant studies with well characterized test material.
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Experimental Toxicology and Ecology, BASF SE, Germany
Type of study:
mouse local lymph node assay (LLNA)
Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.3 g – 23.8 g
- Housing: single housing in Makrolon cage, type II
- Diet: Kliba-Labordiaet (Maus / Ratte Haltung "GLP"), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water: Tap water ad libitum
- Acclimation period: at least 5 days

- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12 / 12

propylene glycol
5, 10, and 25%
No. of animals per dose:
Details on study design:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test-substance concentration which can be technically used was a 25% test-substance preparation. The test-substance preparations at different concentrations were formulated in propylene glycol.
- Irritation: To determine the highest test-substance concentration that does not induce local signs of skin irritation and/or systemic toxicity, a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Two mice were treated with a 25% test-substance concentration on three consecutive days. In the pre-test clinical signs were recorded after each application as well as on day 5. Signs of local irritation were recorded on day 1, 2 and 5. Furthermore, the ears were punched after sacrifice at the apical area using a biopsy punch (Ø 0.8 cm) and were immediately pooled per animal and weighed using an analytical balance. Additionally the weight of the pooled lymph nodes from both sides was determined for each animal. At the tested concentration of 25% the animals showed some increases in ear weights and lymph node weights. Moreover compound residues on the application area and blue discolored ear skin were noted during the whole observation period. Signs of systemic toxicity were not observed in the pre-test.
Therefore, the following dose levels were selected for the main study: 5%, 10% and 25%.

- Name of test method: Murine Local Lymph Node Assay
- Criteria used to consider a positive response: increase in 3H-thymidine incorporation by a factor of >/= 3, cell-count stimulation index (SI >/= 1.5), lymphnode-weight stimulation index and ear weight stimulation index together with dose-response.

TREATMENT PREPARATION AND ADMINISTRATION: The test-substance preparation was produced on a weight per weight basis shortly before the application by stirring with a high speed homogenizer (Ultra-Turrax) and/or a magnetic stirrer. The homogeneity of the test-substance preparation during application was provided by stirring with a magnetic stirrer. Propylene glycol was used as the vehicle because good homogeneity of the preparations was achieved. 25 μL per ear were applied on 3 consecutive days (day 0 – day 2) to the same application site.
Positive control substance(s):
other: A positive control is not included in the study. A separate study with a positive control (Alpha-Hexylcinnamaldehyde, techn. 85%) is carried out in the laboratory twice a year.
Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group. Further statistical analyses were conducted: 3H-thymidine incorporation, cell count, lymph node weight and ear weight (WILCOXON - Test).
Remarks on result:
other: vehicle: 1.00 5%: 1.579 (statistically significant for the value p ≤ 0.05) 10%: 2.55 (statistically significant for the value p ≤ 0.01) 25%: 3.44 (statistically significant for the value p ≤ 0.01)
other: disintegrations per minute (DPM)
Remarks on result:
other: dpm / lymph node pair: vehicle: 238 5% 385.1 10%: 626.4 25%: 845.7

Cell Count Stimulation Index

   Mean [Counts / lymph node pair]  Stimulation Index
 vehicle  8,098,800  1.00
 5%  8,752,800  1.04
 10%  10,173,600  0.99
 25% 11,318,400  1.34#

Ln-Weight Stimulation Index

   Mean [mg]  Stimulation Index
 vehicle  5.2  1.00
 5%  5.4  1.08#
 10%  6.0  1.26##
 25%  6.5  1.40##

Ear-Weight Stimualtion Index

   Mean [mg]  Stimulation Index
 vehicle 28.5  1.00
 5% 31.9   1.08
 10% 31.8  1.17#
 25% 34.0  1.26##

# = statistically significant for the value p ≤ 0.05

## = statistically significant for the value p ≤ 0.01

No signs of systemic toxicity were noticed in all animals during general observation. Blue stained ear skin was observed at all test substance concentrations during the whole observation period.

Interpretation of results:
Migrated information GHS Cat 1B Criteria used for interpretation of results: EU
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The skin sensitizing potential was assessed using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of cells in the auricular lymph nodes to repeated application of the test substance to the dorsal skin of the ears. Groups of 5 female CBA/J mice each were treated with 5%, 10% and 25% w/w preparations of the test substance in propylene glycol or with the vehicle alone. The 25% preparation was the maximum technically applicable concentration.

Besides a slight reduction of the mean body weights of test groups 3 and 4, no signs of systemic toxicity were noticed. When applied as 25% preparation in propylene glycol, the test substance induced a statistically significant and biologically relevant (increase above the cut off Stimulation Index of 3) increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes. The 5% and 10% concentrations caused a statistically significant, but not biologically relevant increase of 3H-thymidine incorporation.

Migrated from Short description of key information:
A valid LLNA is available. The EC3 for the stimulation index was 18% so that GHS subcategory 1B is assigned.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result EC3 = 18% the substance is considered to be classified for skin sensitization (R43) under Directive 67/548/EEC, as amended for the 31st time in Directive2009/2/EG.


Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result of the EC3 = 18% the substance is considered to be classified for skin sensitization (GHS Cat 1B) under Regulation (EC) No. 1272/2008, as amended for the third time in Directive (EC 618/2012).