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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
Study period:
from 2017-11-22 to 2017-12-5
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 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Version / remarks:
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Constituent 1
Chemical structure
Reference substance name:
Trisodium 4-amino-5-hydroxy-3-[[4-[[2-oxo-1-[(phenylamino)carbonyl]propyl]azo]phenyl]azo]-6-[(4-sulphonato-1-naphthyl)azo]naphthalene-2,7-disulphonate
EC Number:
EC Name:
Trisodium 4-amino-5-hydroxy-3-[[4-[[2-oxo-1-[(phenylamino)carbonyl]propyl]azo]phenyl]azo]-6-[(4-sulphonato-1-naphthyl)azo]naphthalene-2,7-disulphonate
Cas Number:
Molecular formula:
trisodium 4-amino-5-hydroxy-3-[[4-[[2-oxo-1-[(phenylamino)carbonyl]propyl]azo]phenyl]azo]-6-[(4-sulphonato-1-naphthyl)azo]naphthalene-2,7-disulphonate
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks old
- Weight at study initiation: 18 to 20 grams
- Veterinary health check: During acclimatisation period
- Animals per cage: 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)
- Diet: 4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy), ad libitum throughout the study
- Water: drinking water supplied to each cage via a water bottle, ad libitum
- Acclimation period: At least 5 days

- Temperature: 22 °C ± 2 °C
- Humidity: 55 % ± 15 %
- Air changes: Approximately 15 to 20 air changes per hour
- Photoperiod: Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours

Animal identification was permanent, following arrival, by ink marking on the tail. Animals were identified by odd numbers. Animals were randomised at arrival. Healthy animals without observable skin lesions were chosen. At the time of treatment (for preliminary test and main assay), the animals aged approximately 8-9 weeks.

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
Preliminary test: 25, 10, 5, 2.5, 1 and 0 % (w/w).
Main assay: 25, 10, 5 and 0 % (w/w).

Concentrations were calculated considering the test item as supplied. No correction factor was applied.
No. of animals per dose:
Preliminary test: one per dose
Main test: four per dose (negative and positive control included)
Details on study design:
A solubility trial was performed in order to establish if acetone/olive oil 4:1 v/v could be used as a vehicle.

During the study, the test item was mixed in acetone/olive oil 4:1 (v/v) as vehicle.

- Frequency of treatment: three consecutive days (Days 1, 2, 3) with the vehicle or test item formulations, depending on the animal.
- Dose volume : 25 µL/ear/day of the appropriate concentration was applied to the dorsal surface of each ear (50 µL/animal/day), using a micropipette.
- In vivo observations: mortality and morbidity, throughout the study, all animals were checked twice daily.
-Clinical signs: 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)
- Body weight: at allocation (Day 1) and on sacrifice (Day 6).
- Scoring of dermal reaction: the treated sites of all animals were examined daily (once before first dosing, before dosing on Days 2 and 3 and daily thereafter). Irritation to the skin was assigned a numerical value according to (Erythema and eschar formation):
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
- Ear thickness measurement: by a suitable micrometer on Day 1 (before dosing), on Day 3 (before dosing) and on Day 6.
- Termination: Euthanasia method, the animals were sacrificed on Day 6 by carbon dioxide narcosis. After sacrifice, regularly shaped biopsies were obtained from both ears and weighed together.
- Selection of dose levels: in the main assay, the test item was used at the higher concentrations that were systemically tolerated and judged not to cause excessive local skin irritation. In particular, concentrations are considered irritant if:
i) erythema grading (score) is ≥ 3 at any day of measurement and/or
ii) ear thickness is ≥ 25 % with respect to Day 1 and/or
iii) ear punch weight is ≥ 25 % with reference to the negative control group.

- Scheme of treatment:
Day 1, 2, 3: Dermal application of the test item, the positive control or vehicle at a dose volume of 25 μL/ear (50 μL/animal).
Day 4: no application
Day 5: Intraperitoneal administration of BrdU
Day 6: Sacrifice, processing of lymph nodes and determination of cell proliferation.
- Frequency of treatment: for three consecutive days (Days 1, 2, 3) with the vehicle, test or control item formulations.
- Dose volume: 25 μL/ear/day of the appropriate concentration was applied to the dorsal surface of each ear (50 μL/animal/day), using a micropipette.
- 5-bromo-2-deoxyuridine (BrdU) treatment: intraperitoneally, on Day 5 (once only), with 0.5mL/animal of a solution of BrdU at a concentration of 10mg/mL in physiological saline (0.9% NaCl), using a plastic graded syringe.
-In vivo observations: Mortality and morbidity, throughout the study, all animals were checked twice daily.
-Clinical signs: before dosing commenced and daily up to sacrifice (approximately 1 hour after dosing on Days 2, 3 and 5).
-Body weight: at allocation (Day 1) and on sacrifice (Day 6).
-Terminal phase: Euthanasia method and lymph node collection, the animals were killed on Day 6, approximately 24 hours after BrdU injection, by carbon dioxide narcosis. No necropsy was performed on the sacrificed animals. Shortly after sacrifice of each animal, the auricular lymph nodes were rapidly excised, pooled on individual basis and individually collected in a solution of 2% BSA-PBS [2% bovine serum albumine (BSA) in phosphate buffered saline, PBS].

-Preparation of single cell suspension: A single cell suspension of lymph node cells (LNC) was prepared from each animal by gentle mechanical disaggregation and passage through a 70 µmnylon mesh. The suspensions thus obtained were centrifuged and each supernatant resuspended in 20mL of 2 % BSA-PBS.
-Measurement of BrdU content in lymphocytes DNA: BrdU was measured by ELISA using a commercial kit (Roche Applied Science,Mannheim, Germany, Catalogue No. 11 647 229 001, batch no. 19845500), according to manufacturer instructions. Briefly, 100 µL of the LNC suspension were added to the wells of a flat-bottom 96-well microplate in triplicate. Two replicate microplates were prepared. After fixation and denaturation of the LNC, 100 µL of anti-BrdU antibody labelled with peroxidase were added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and 100 µL of the substrate solution were then added and allowed to produce chromogen. The reaction was finally stopped by adding 25 µL of stop solution (1MH2SO4). Absorbance (OD) was detected at 450 nm (with reference wavelength: 690 nm).

-Calculation of the BrdU labelling index:
BrdU labelling index = (OD 450–OD blank450 )–(OD 690–OD blank690 )
The BrdU labelling index was calculated for each mouse and a group mean was subsequently calculated. Results for each treatment group were expressed as the mean Stimulation Index (SI). The SI was derived by dividing the mean BrdU labelling index/mouse within each test item group and the positive control groups by the mean labelling indices for the respective vehicle group.

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).

The assay is considered satisfactory if the Stimulation Index (SI) of the positive control group is higher than 2.0.
Positive control substance(s):
other: 25% (w/w) alpha-hexylcinnamaldehyde technical grade 85% in acetone:olive oil 4:1 v/v
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 3.38 was calculated. As it was greater than 2, the study was regarded as valid.

In vivo (LLNA)

Resultsopen allclose all
Test group / Remarks:
conc. 0 % w/w
Test group / Remarks:
conc. 5 % w/w
Test group / Remarks:
conc. 10 % w/w
Test group / Remarks:
conc. 25 % w/w
Cellular proliferation data / Observations:
No significant increase in cell proliferation of draining lymph nodes was observed in any treatment group.

Any other information on results incl. tables


At the concentration of 25 %w/w in acetone/olive oil 4:1 v/v, a good and administrable formulation was obtained.


No signs of toxicity (clinical signs or toxicologically relevant body weight losses) were observed at any of the tested concentrations. The evaluation of visible reactions showed no erythema at any of the concentrations investigated [25, 10, 5, 2.5 and 1% (w/w)].

The evaluation of ear thickness indicated that no increase was induced by treatment (values of Day 6 compared to Day 1). The evaluation of ear punch weight indicated that no increase was found at any dose level investigated. Based on the results described above, the highest concentration selected for the main assay was 25% (w/w).


Neither mortality nor relevant clinical signs were recorded in animals treated at all dose levels investigated [25, 10 and 5% (w/w)]. 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: not classified according to the CLP Regulation (EC 1272/2008)
The test item was considered not to be skin sensitizer.
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 (2010). Five concentrations [25 (maximum feasible concentration), 10, 5, 2.5 and 1 % 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. Based on the results observed, in the main assay the test item was topically administered at the concentrations of 25, 10 and 5% (w/w), in acetone: olive oil 4:1 (v/v).

No mortality nor relevant clinical signs were recorded in any animal. Changes in body weight 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 all dose groups, with a Stimulation Indices (SI) of 1.21, 0.90 and 0.72 at low, medium and high dose groups, respectively. No correlation with the doses nor statistical significance was observed. These results indicate that the test item does not elicit a sensitisation response.