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Skin sensitisation

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skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for publication.
Reason / purpose for cross-reference:
reference to same study

Data source

Reference Type:
Nickel sensitisation in mice: A critical appraisal
Johansen P, Wackerle-Men Y, Senti G, Kundig TM
Bibliographic source:
Journal of Dermatological Science. 58:186-192

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
Mouse ear swelling test (MEST) was also conducted.
GLP compliance:
not specified
Type of study:
other: Both the 1) Mouse ear swelling test (MEST), and the 2) mouse lymphocyte proliferation test

Test material

Constituent 1
Chemical structure
Reference substance name:
Nickel chloride
EC Number:
EC Name:
Nickel chloride
Cas Number:
Molecular formula:
nickel(2+) dichloride
Details on test material:
- Name of test material (as cited in study report): nickel chloride hexahydrate
- Molecular formula (if other than submission substance): NiCl2(H2O)6
- Supplier: Fluka (Buchs, Switzerland)

In vivo test system

Test animals

other: C57BL/6, nude (B6.Cg-Foxn1nu/J), and T-cell receptor deficient (B6.129P2-Tcrbtm1Mom Tcrdtm1Mom/J) mice
Details on test animals and environmental conditions:
- Source: Harlan (Horst, The Netherlands - C57BL/6 strain) and Charles River Laboratories (Sulzfeld, Germany - nude and T-cell deficient strains).
- Age at study initiation: 6-10 (C57BL/6 strain) or 16-20 (nude and T-cell deficient strains) weeks.
- Housing: typically kept in conventional cages with certified AISI 304 stainless steel wire lids (Series 116) and bottle caps (Model ACCP2521) from Techniplast(R) as purchased from Indulab(R) (Gams, Switzerland); both lids and bottle caps were made from nickel-containing stainless steel. In selected experiments, in order to control for development of nickel tolerance, we bred C57BL/6 mice using PVC animal cages and water bottles which were not equipped with nickel-containing parts. The F1 offsprings were weaned after 3 weeks, kept in the same kind of nickel-free environment, and used for sensitisation experiments at the age of 8-9 weeks.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
intradermal and epicutaneous
other: sodium chloride
Concentration / amount:
10 mM
Challengeopen allclose all
intradermal and epicutaneous
other: sodium chloride
Concentration / amount:
10 mM
No. of animals per dose:
Details on study design:
- No. of exposures: one injection
- Test groups: NiCl2
- Control group: NaCl
- Site: inguinal flank
- Frequency of applications: one injection
- Duration: one injection
- Concentrations: 10 mM NiCl2
- Other: In selected experiments, the sensitising nickel solution was mixed with ovalbumin (20 μg per injection), LPS (1 μg), SDS (2 μg), or CpG ODN (20 nmole). Alternatively, the mice were sensitised by (i) epicutaneous application of 10 mM Ni(III) to the shaven abdominal skin, (ii) oral administration as Ni(II) or Ni(III) on days 0, 2 and 4 using syringe and needle for gavage feeding, or (iii) by topical administration of a 5% suspension of nickel chloride in vaseline (petrolatum) to the shaven abdominal skin on days 0, 2 and 4. All animal experiments were reviewed and approved by an animal ethical review committee and performed according to regulations described by Swiss Veterinary authorities.

B. CHALLENGE EXPOSURE (Mouse ear swelling test)
- No. of exposures: 1
- Test groups: NiCl2
- Control group: NaCl
- Site: pinna of left ear (NiCl2), pinna of right near (NaCl control).
- Concentrations: 10 mM (20 ul)
- Evaluation (hr after challenge): 48 hours
- Other: In the case of epicutaneous sensitisation, the mice were also challenged with Ni(II) by epicutaneous application to the ear, while topical and oral sensitisations were followed by intradermal Ni(II) challenge. Two days after the challenge, delayed-type hypersensitivity reactions were quantified by measuring the increase in ear thickness compared with pre-challenge values. Measurements were performed using a precision digital thickness gage from Miltutoya as provided by Brutsch-Ruegger Corp. (Urdorf, Switzerland). Swelling and erythema were also documented by photography and by
hematoxylin and eosin (H&E) staining of skin sections of ears that had been collected 2 days after the challenge and snap frozen in liquid nitrogen.
Challenge controls:
negative control: NaCl
Positive control substance(s):
not specified

Study design: in vivo (LLNA)

other: NaCl
2 mM NiCl2
No. of animals per dose:
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION: In addition to determining ear-swelling after recall with Ni(II), in vitro proliferation of auricular lymph node cells was measured. The mice were sensitised Ni(III) as described above. After 10 days, the sensitisation was repeated, and 6 days thereafter, the mice were challenged with 2 mM Ni(II) into one ear and with saline into the other ear. The auricular lymph node isolated 48 h later, the lymph node cells were counted and then analysed by flow cytometry for determination of CD4 T-cell numbers as well as by activation of these cells in terms of CD69 and CD44 expression. Moreover, 3 x 10^5 freshly prepared lymph node cells were incubated for 24 h in the presence or absence of 1-100 μM Ni(II) in DMDM medium supplemented with penicillin, streptomycin, 10% FCS, 50 μM 2-mercaptoethanol, and 10 U/ml mIL-2. Then, cell proliferation was determined by incubating the cells for another 16 h with 1 μCi [3H]-thymidine per well. The cells were harvested onto nitrocellulose filters, and the incorporated radioactivity was measured by beta-scintillation. The results are expressed as stimulation index (S1) and calculated as the ratio of Ni-stimulated to non-stimulated cells.

Positive control substance(s):
not specified
Not applicable.

Results and discussion

Positive control results:
Not applicable.

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: see Remark
The ear-swelling 48 h after the challenge did not differ between sensitised and non sensitised mice, and the adjuvants and the excipients did not affect the ear-swelling and erythema when compared to mice sensitised with Ni(III) alone. See Table 1 below for more details.
other: disintegrations per minute (DPM)
Remarks on result:
other: Not applicable.

Any other information on results incl. tables

Mouse Ear Swelling Test Results: The intradermal application with a nickel chloride solution produced cutaneous inflammation in mice, although these reactions were not significantly different from those obtained in mock-sensitised mice.

  • Mice sensitized with Ni(II) and Ni(III) produced similar ear-swelling reactions, indicating that the severity of the skin reaction was independent of the oxidation state of nickel.
  • The observed skin reactions seemed dependent on nickel, as similar skin reactions could be produced with a sulphate salt of nickel.
  • The skin reaction was accompanied by a prominent erythema, and the increase in ear thickness was associated with characteristic pathological features of edema, and inflammatory cell infiltration. The H&E staining of histological skin sections suggested that the skin reactions were not mediated by nickel-specific lymphocytes but were irritant in nature and characterized mainly by the infiltration with neutrophil granulocytes.
  • Further experiments using various sensitization intervals and doses of nickel, different oxidation stages, and different routes of administration (epicutaneous, topical and oral) all revealed that the skin reactions upon nickel challenge were independent of the sensitisation regimen and that the reactions were not adaptive.

Effect of Sensitization Regimen on MEST and LLNA results: The ear-swelling 48 h after the challenge did not differ between sensitised and not sensitised mice, and the adjuvants and the excipients did not affect the ear-swelling and erythema when compared to mice sensitised with Ni(lII) alone; neither were the CD4-positive T-cell counts affected by sensitization regimen (Table 1 below). In addition, in vitro proliferation of auricular lymphocytes did not suggest that sensitisation with nickel induced a specific CD4 T-cell responses.

Table 1. Erythema, ear-swelling, and lymphocyte activation in mice sensitised by intradermal injections of nickel chloride.


Readout and challenge regime

      Erythema - a     MEST - b     CD4+ cells in LNs (x10^5)- c     Stimulation index - d
 Sensitization  NiCl2  NaCl  NiCl2  NaCl  NiCl2  NaCl  NiCl2  NaCl
 NaCl  1.25  0.25  97  4  3.2  4.6  1.8  1.7
 Ni(III)*  1.25  0  91  9  3.1  2.5  1.5  1.5
 Ni(III) + OVA  1.25  0  66  7  2.6  2.4  1.2  1.3
 Ni(III) + LPS  0.75  0  85  6  2.9  3.4  1.5  2.0
 Ni(III) + SDS  1.50  0  70  3  1.6  3.6  1.1  1.3
 Ni(III) + CpG  1.25  0  81  4  3.4  4.5  2.2  1.8

a - Mice were individually scored with 0 (no erythema), 1 (notable erythema), or 2 strong erythema and an average was calculated.

b - The average MEST (swelling of ear compared 10 baseline) Is indicated micrometers.

c - Number of CD4-positive lymphocytes in auricular lymph nodes was calculated based on the number of lymph node cells (counted in hemocytometer) and the percentage of CD4 T-cell subsets (analysed by flow cytometry). No statistical differences were detected using the Kruskal-Wallis non-parametric H-test with Dunn's multiple comparison test (n=5).

d - Stimulation index of lymph cells re-stimulated in vitro with 100 uM Ni(II) in the presence of [3H]-thymidine for 16 h. No statistical differences were detected using the Kruskal-Wallis non-parametric H-test with Dunn's multiple comparison test (n=5).

*Ni(III) was made by mixing NiCl2 with a 20 -fold molar excess of H2O2.

Effects of titrating NiCl2 amounts in sensitized and non-sensitized mice: There was a dose-dependent swelling of the ear as NiCl2 levels were increased, but no difference was found between sensitized and non-sensitised mice (p> 0.05 by two-way ANOVA). The cellular infiltrate in the swollen ears was identical in sensitised and non-sensitised mice as measured by histology.

Effects of NiCl2 in lymphocyte deficient mice: The exclusion of any lymphocytic involvement in the skin reactions was further supported by similar experiments performed in lymphocyte deficient mice. The cutaneous inflammation observed in T-cell receptor knock-out mice and in athymic nude mice did not differ from that observed in wild type mice.

MEST results in mice bred in Ni-free environment: There was no difference in the ear-swelling reactions in mice bred under conventional conditions and in mice bred and kept under nickel-free conditions. Moreover, for mice kept under nickel-free conditions, the skin reactions following a challenge with Ni(II) chloride were similar in nickel-sensitised and in nonsensitised mice.

Applicant's summary and conclusion

The authors concluded that the present investigation demonstrated that C57BL/6 mice could not be immunologically sensitised to nickel, and that non-sensitised mice experienced the same local skin reactions upon intradermal administration of nickel as did mice sensitised to nickel. Therefore the observed skin reaction upon nickel exposure should be characterised as an irritant rather than an allergic contact dermatitis due to nickel. These results question the validity of the described murine nickel allergy model.
Executive summary:

Study rated by an independent reviewer.