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Diss Factsheets

Administrative data

Description of key information

skin sensitisation (OECD 429, GLP): not sensitising

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:
07 - 23 Sep 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 23 July 2010
GLP compliance:
yes (incl. QA statement)
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of study:
mouse local lymph node assay (LLNA)
Details on test animals and environmental conditions:
- Source: Envigo RMS B.V., Inc, Horst, The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 - 12 weeks
- Weight at study initiation: 20.6 ± 0.3 g (Group 1), 21.2 ± 0.5 g (Group 2), 21.0 ± 1.3 g (Group 3), 21.3 ± 1.4 g (Group 4)
- Housing: 4 females of the same group per cage in Makrolon Type II (pre-test) or Makrolon Type III (main test) with wire mesh top and granulated soft wood bedding.
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

- Temperature (°C): 22 ± 2
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12
10, 25 and 50%
No. of animals per dose:
Details on study design:
PRE-SCREEN TESTS: To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50% once daily each on three consecutive days.
- Compound solubility: the highest test item concentration was a 50% solution in dimethylformamide based on a solubility experiment.
- Irritation: signs of local irritation were documented.
- Systemic toxicity: any clinical signs of systemic toxicity during the study were recorded. Clinical signs were recorded at least once daily.
- Ear weight: the ear weight was determined after sacrifice using biopsy punches from each ear.
- Ear thickness measurements: ear thickness were measured prior to the first application of the test substance, on Day 3 and prior to necropsy.
- Erythema scores: no visible erythema (0); very slight erythema (1); well defined erythema (2); moderate to severe erythema (3); severe erythema to formation of eschar which prevents grading of erythema (4)


- Name of test method: 3H-methyl thymidine incorporation determined by β-scintillation and γ-counting
- Criteria used to consider a positive response: The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/lymph node values are determined, mean scintillation-background DPM were subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
1) that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
2) that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
- Other: the animals were observed for signs of toxicity once daily, including pre- and post-dose observations on Days 1, 2 and 3. The body weight was recorded on Day 1 prior to dosing and prior to treatment with 3HTdR on day 6.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of the test material was applied to the entire dorsal surface of each ear of each mouse on Day 1, 2 and 3 in concentrations of 10, 25 and 50% in DMF. On Day 6 an injection of 250 µL phosphate buffered saline (PBS) containing 79.1 µCi of 3H-methyl thymidine (3H-TdR) was made into the tail vein of each mouse. Approximately five hours later, the draining auricular lymph node of each ear was excised into PBS and pooled per experimental group. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze and rinsed with PBS. To precipitate out the radioactive material, the pellet was resuspended in 5% trichloroacetic acid. The precipitates were incubated for at least 18 h at approximately 4 °C, centrifuged, resuspended in 1 mL TCA and transferred to 10 mL scintillation fluid before β-scintillation counting.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
The mean values and standard deviations of the body weight measurements were calculated. All calculations conducted on the DPM values were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.
Positive control results:
A study (performed in April 2016; Envigo study number 1764300) was performed to assess the sensitivity of the CBA/CaOlaHsd mice at the testing facility to a known sensitizer. The positive control substance hexyl cinnamic aldehyde (25% (v/v) in acetone:olive oil (4+1)) was considered to be a sensitizer under the conditions of the test (SI 7.84).
Key result
Test group / Remarks:
Key result
Test group / Remarks:
Key result
Test group / Remarks:
Cellular proliferation data / Observations:
The SI of the 10, 25 and 50% treatment group was 1.12, 0.92 and 0.82%, respectively.

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

No deaths occurred during the study period. No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period.

The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Table 1: Results of the main test

Test substance concentration [%] Measurement DPM DPM-BG * Number of lymph nodes  DPM per lymph node ** SI
BG I 23        
BG II 14        
Vehicle control 6053 6034.5 8 754.3 1.00
10 6797 6778.5 8 847.3 1.12
25 5583 5564.5 8 695.6 0.92
50 4961 4942.5 8 617.8 0.82

* The mean value was taken from the figures BG I and BG II

BG: Background (Background 3HTdR levels were measured in wo 1 mL aliquots of 5% trichloroacetic acid)

** 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:
other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitising properties of the test substance were tested in a local nymph node assay study performed according to OECD TG 429 and in compliance with GLP (reference 7.4.1-1). Groups of four female mice were exposed daily, for three consecutive days, to 10, 25 and 50% of the test substance or to the vehicle alone, on the dorsum of both ears. 50% of the test substance was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. On Day 6 of the study, mice were injected into the tail vein with 79.1 µCi [3H]-thymidine. Approximately 5 hours later, the draining auricular lymph node of each year was excised and pooled per experimental group. Radioactivity was measured as a function of isotope incorporation in draining auricular lymph nodes. The test concentrations of 10, 25 and 50% resulted in SI values of 1.12, 0.92 and 0.82. The positive control substance hexyl cinnamic aldehyde (25% (v/v) in acetone:olive oil (4+1)) was considered to be a sensitizer under the conditions of the test (SI 7.84).Therefore, the test substance is considered to be a non-sensitizer in the conducted LLNA test.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation do not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.