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Toxicological information

Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From July 12 to 24, 2017
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Version / remarks:
Adopted on 22 July 2010
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Constituent 1
Reference substance name:
Vat Orange 002 - Similar Substance 01
Vat Orange 002 - Similar Substance 01
Test material form:

In vivo test system

Test animals

Details on test animals and environmental conditions:
Species and strain: Mice, CBA/JN
Sex: Females (nulliparous and non-pregnant)
Age: 7 weeks old
Supplier: Charles River Italia S.p.A., Calco (Lecco), Italy
Breeder: Charles River, Jackson, USA.
Date of arrival: 29 June 2017
Weight range at arrival: 19.6 to 21.4 grams
Acclimatisation period: At least 5 days
Veterinary health check: During acclimatisation period

Animals per cage: Up to 5 during acclimatisation; 1/cage during the study
Housing: Polysulfone solid bottomed cages measuring 35.5 × 23.5 × 19
cm with nesting material
Cage control: Daily inspected and changed as necessary (at least twice/week)
Water: Drinking water supplied to each cage via a water bottle
Water supply: ad libitum
Diet: 4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy)
Diet supply: ad libitum throughout the study
Room lighting: Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours
Air changes: Approximately 15 to 20 air changes per hour
Temperature range: 22 °C±2 °C
Relative humidity range: 55%±15%

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
Test item: 10, 5 and 2.5% (w/w).
Positive control: approximately 29% (w/w)
No. of animals per dose:
4 animals per dose
Details on study design:
- Compound solubility: A solubility trial was performed in order to establish if acetone/olive oil 4:1 v/v could be used as a vehicle. Initially, it appeared that an administrable homogeneous creamy suspension had been obtained at the concentration of 25%w/w in acetone/olive oil 4:1 v/v. However, in the animal unit the highest concentration that could be used for dosing was 10%w/w in acetone/olive oil 4:1 v/v, since the formulated test item became more dry and not administrable. The concentration at 10% was a more liquid suspension and therefore administrable.
- Irritation:The treated sites of all animals were examined daily (once before first dosing, before dosing on Days 2 and 3 and daily thereafter).
- Systemic toxicity: The animals were observed for clinical signs on: Day 1: before and 1 hour after dosing Day 2 to 6: daily (approximately 1 hour after daily dosing, when applicable
- Ear thickness measurements: The ear thickness was measured by a suitable micrometer on Day 1 (before dosing), on Day 3 (before dosing) and on Day 6.
- Erythema scores: Irritation to the skin was assigned a numerical value according to the table below:
Erythema and eschar formation Value
No erythema 0
Very slight erythema 1
Well defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4


- Name of test method: LOCAL LYMPH NODE ASSAY (LLNA: BrdU-ELISA method)
- Criteria used to consider a positive response: The test item is considered to induce sensitisation when the SI for any single treatment dose group is ≥ 1.6. It is not required that an increased response is observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6 ≤ SI ≤ 1.9).

TREATMENT PREPARATION AND ADMINISTRATION: The animals were treated for three consecutive days (Days 1, 2, 3) with the vehicle or test item formulations. A dose volume of 25 μL/ear/day of the appropriate concentration was applied to the dorsal surface of each ear (50 μL/animal/day), using a micropipette.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Differences between each treated group and the concurrent negative control group (individual BrdU labelling indices) were assessed by Dunnett’s test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test.

Results and discussion

Positive control results:
In the group treated with the positive control item, a Stimulation Index of 3.09 was calculated. As it was greater than 2, the study was regarded as valid.

In vivo (LLNA)

Resultsopen allclose all
Test group / Remarks:
Group tested at the lowest dose
Test group / Remarks:
Group tested at mid dose
Test group / Remarks:
Group tested at the highest dose
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: No significant increase in cell proliferation of draining lymph nodes was observed in any treatment group.

DETAILS ON STIMULATION INDEX CALCULATION: The calculated Stimulation Indices (SI) were 1.01, 1.11 and 1.47, respectively at low, mid- and high dose levels.

CLINICAL OBSERVATIONS: Neither mortality nor clinical signs were recorded in animals treated at all dose levels investigated [10, 5 and 2.5% (w/w)].

BODY WEIGHTS: Changes in body weight observed during the study were within the expected range for this strain and age of animals.

Applicant's summary and conclusion

Interpretation of results:
other: CLP criteria not met
Not skin sensitising
Executive summary:


The potential of the test item to cause skin sensitisation reactions following topical application to the skin of CBA/JN mice, was assessed using the LLNA:BrdU-ELISA method, according to the OECD Guideline for testing of chemicals No. 442b.

In the preliminary test five concentrations [10 (maximum feasible concentration), 5, 2.5, 1 and 0.5 % w/w in acetone: olive oil 4:1 (v/v)] were tested in the preliminary phase, in order to identify a non toxic and minimally irritant concentration and avoid false positive results.

No signs of toxicity (significant clinical signs or body weight losses) were observed at the tested concentrations.

According to the results of the irritation screening, the concentration of 10% w/w was judged to be not irritant.

In the main assay, the test item was topically administered at the concentrations of 10, 5 and 2.5 % (w/w), in acetone:olive oil 4:1 (v/v).


No mortality nor clinical signswere recorded in any animal.

Changes in bodyweight observed during the study were within the expected range for this strain and age of animals.

No increase in cell proliferation of draining lymph nodes was observed in any treatment group.


The calculated Stimulation Indices (SI) were 1.01, 1.11 and 1.47 at the low, midand high dose levels [2.5, 5 and 10 %,], respectively. Neither correlation with the doses nor statistical significance was observed.


The above results indicate that the test item does not elicit any sensitisation response in mice following dermal exposure.