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Diss Factsheets

Administrative data

Description of key information

- skin: not sensitizing (OECD 429)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
according to guideline
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
according to guideline
other: U. S. EPA Health Effects Test Guidelines OPPTS 870.2600
GLP compliance:
yes (incl. QA statement)
Experimental Toxicology and Ecology, BASF AG
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, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: 6 – 12 weeks
- Weight at study initiation: 19.5 g – 22.6 g
- Housing: single housing in Makrolon cage, type II
- Diet (e.g. ad libitum): Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water (e.g. ad libitum): tap water; ad libitum
- Acclimation period: 15 days

- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12
propylene glycol
3, 10, 50%
No. of animals per dose:
Details on study design:
The selection of concentrations took into account available information on the chemical/physical properties, the composition and on acute toxicity and primary irritation/corrosion potential of the test substance. Additionally, the results of a pretest with a 50% test substance preparation were considered, which showed slightly increased ear weights and lymph node weights as indication of ear irritation. The 50% preparation was the maximum technically applicable concentration.

- Name of test method: Murine Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: The increase SI of cell count by a factor of >1.5 and/or of 3H-thymidine incorporation by a factor of ≥ 3 as compared to the concurrent vehicle control group is generally considered as indicating a sensitizing potential of a test substance

- Body weight determination: individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- Signs and symptoms: no detailed clinical examination of the individual animals was performed but any obvious signs of systemic toxicity and/or local inflammation at the application sites were noted in the raw data.
- Form of application: epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
- Application volume: 25 μL per ear
- Site of application: dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- Observations: twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
- ³H-thymidine injection: on study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected intravenously with 20 μCi of 3H-thymidine in 250 μl of sterile saline into a tail vein
- Terminal procedures: the animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
other: disintegrations per minute (DPM)
Remarks on result:
other: ³H-thymidine incorporation [DPM/Lymph Node Pair]: - control: 254.3 +/- 58.4 - 3%: 250.6 +/- 84.2 - 10%: 301.5 +/- 83.6 - 50%: 525.7 +/- 141.8 - positive control: 1225.6 +/- 453.6
Test group / Remarks:
animals 1-5, 0%
Test group / Remarks:
aniamls 6-10, 3%
Test group / Remarks:
animals 11-15, 5%
Test group / Remarks:
animals 16-20, 50%
Test group / Remarks:
animals, 21-25, positive control


Test group Treatment Cell Counts [Counts/Lymph Node Pair]
Mean  SI
1 vehicle 6222600 ± 528453 1.00
2 3% 8026800 ± 3292733 1.29
3 10% 7702800 ± 767852 1.24
4 50% 9681600 ± 2860637 1.56
5 positive control 14400000 ± 2083175 2.31
Test group Treatment 3H-thymidine incorporation [DPM/Lymph Node Pair]
Mean  SI
1 vehicle 254.3 ± 58.4 1.00
2 3% 250.6 ± 84.2 0.99
3 10% 301.5 ± 83.6 1.19
4 50% 525.7 ± 141.8 2.07
5 positive control 1225.6 ± 435.5 4.82
Test group Treatment 3H-thymidine incorporation [DPM/Lymph Node Pair]
Mean  SI
1 vehicle 4.5 ± 0.3 1.00
2 3% 5.2 ± 1.3 1.15
3 10% 5.1 ± 0.5 1.12
4 50% 5.7 ± 1.0 1.26
5 positive control 7.9 ± 0.9 1.73

SI: stimulation index

Interpretation of results:
GHS criteria not met
Migrated information
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The sensitization potential of the test substance was evaluated in a Local Lymph Node Assay (LLNA) performed under GLP according to OECD guideline 429, where the dorsal part of the ears of five female CBA mice per group were treated with three daily applications of 25 µL of a concentrations of 3, 10 and 50%, respectively (BASF, 2008). As a result, no signs of toxicity were noted. On study day five, the mice were injected intravenously with 20 μCi of 3H-thymidine in 250 μL of sterile saline into a tail vein and sacrificed five hours later to remove the auricular lymph nodes. Analysis of the mean stimulation indices SI (fold of change as compared to the vehicle control) revealed a small increase of the cell counts above the cut off stimulation index of 1.5 for the 50% test substance preparation, while no response was found for the 3% and 10% preparation. However, as the SI for cell count was at the border of biological relevance and the increase of 3Hthymidine incorporation was clearly not biologically relevant, the test substance did not show a skin sensitizing effect.

A further study according OECD TG 429 was conducted (Rockwood Pigments UK LTD, 2013). In total five groups of female mice of CBA/J strain (age 8 to 10 weeks) comprising 5 females per group were selected for the experiment. Three groups were topically applied with the test item in Propylene glycol (PG) at the dose concentrations of 10% (G2), 25% (G3) and 50% (G4) (w/v) in PG for three consecutive days (on days 0, 1 and 2) on the dorsum of both ears (25 µL per ear). Group 1(G1)served as vehicle control and treated with PG. Group (G5) served as a concurrent positive control and was treated with alpha-hexylcinnamaldehyde (HCA) at the dose concentration of 25% (v/v) in PG in same manner to confirm the sensitivity and reliability of the test method. On day 5 of treatment, the proliferation of lymph node cells in the lymph node draining the application site was measured by incorporation of 3H-methyl thymidine. The obtained values were used to calculate the stimulation index (SI). Group mean body weight of mice from positive control and test item treated groups was found to be comparable with the vehicle control group. The stimulation index (SI Value) calculated for test item treatment groups were found to be 1.29, 1.32 and 1.48 for the dose concentrations of 10%, 25% and 50% (w/v) in PG, respectively. The stimulation index (SI Value) calculated for positive control group treated with 25% v/v HCA was found to be 4.72%. The SI obtained for the test item at all the concentrations did not show greater than three-fold increase over the control value therefore it was concluded that under these experimental conditions, the test item did not induce contact sensitization to CBA/J strain mice in the Local Lymph Node Assay.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

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

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this information, the substance is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.