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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to 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)

Test material

Constituent 1
Test material form:

In vivo test system

Test animals

other: CBA/CaOlaHsd
Details on test animals and environmental conditions:
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 7 - 12 weeks (beginning of acclimatisation)
- Weight at study initiation: 20.1 ± 1.2 g (mean ± SD)
- Housing: Single; Makrolon Type I, with wire mesh top
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

- Temperature (°C): 22 ± 3ºC
- Humidity (%): 30 – 70 %
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

dimethyl sulphoxide
2.5, 5, and 10 % (w/v)
No. of animals per dose:
4 females (nulliparous and non-pregnant) per group
Details on study design:
In a pre-test in two mice, test item concentrations of 1.25, 2.5, 5, and 10 % were tested on one ear each. No irritation effects were observed at these
concentrations after a single application.

- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, 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.
- Second, 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.
The decision to select a STIMULATION INDEX (S.I.) of 3 as an arbitrary indication of sensitizing activity was made on the basis of investigations performed with a wide range of chemicals.

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 2.5, 5, and 10 % (w/v) in dimethylsulfoxide. The application volume, 25 μl, was spread over the entire dorsal surface (ø 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).

Five days after the first topical application, all mice were administered with 250 μl of 80.4 μCi/ml 3HTdR (corresponds to 20.1 μCi 3HTdR per mouse) by intravenous injection via a tail vein.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
The mean values and standard deviations were calculated in the body weight tables.

Results and discussion

Positive control results:
Group 1: concentration 5%: Stimulation index: 2.04
Group 2: concentration 10%: Stimulation index: 6.31
Group 3: concentration 25%: Stimulation index: 12.45
EC3: 6.1% (w/v)

In vivo (LLNA)

Key result
Remarks on result:
other: see Remark
In this study Stimulation Indices of 2.55, 1.63, and 2.22 were determined with the test item at concentrations of 2.5, 5, and 10 % in dimethylsulfoxide. The EC3 value (concentration of test item required to produce a S.I. of 3) could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

Any other information on results incl. tables

No deaths occurred during the study period. No symptoms of local toxicity at the ears of the animals and no systemic findings were

observed during the study period. The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
CLP Implementation.
The test item was not a skin sensitiser in this assay.
Executive summary:

In the study the test item dissolved in dimethylsulfoxide was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5, and 10 %. 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 2.55, 1.63, and 2.22 were determined with the test item at concentrations of 2.5, 5, and 10 % in dimethylsulfoxide, respectively. The test item was not a skin sensitiser in this assay.