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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
Not applicable
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The basic principle underlying the LLNA method used is to induce a primary proliferation of lymphocytes in the lymph node draining the site of test material application. An ear abrasion technique is employed which enhances penetration of the metal ion by removing the horny layer of the epidermis. It enables the detection of ear-swelling responses of weak contact allergens by increasing their LNC proliferation. This proliferation is proportional to the dose applied (and to the potency of the allergen) and provides a simple means of obtaining an objective and quantitative measurement of sensitisation. The LLNA assesses this proliferation, wherein the proliferation in test groups is compared to that in vehicle treated controls. The ratio of the proliferation in treated groups to that in vehicle controls (Stimulation Index), is determined, and must be at least three. The methods described here are based on the use of radioactive labelling to measure the proliferation of the lymph node cells.
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6-8 wk

Route:
other: not applicable
Vehicle:
other: not applicable
Concentration / amount:
Not applicable
Route:
other: not applicable
Vehicle:
other: not applicable
Concentration / amount:
Not applicable
No. of animals per dose:
Not applicable
Details on study design:
Not applicable
Challenge controls:
Not applicable
Vehicle:
other: 20 % ethanol
Concentration:
10 % test material in 20 % ethanol solution
No. of animals per dose:
Three
Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: Stimulation index ≥ 3


TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of test material was applied to the dorsum of both ears for three consecutive days. Prior to the test material treatment, the ears of each mouse were gently abraded using a 19 g needle. Four days following the initial application, draining lymph nodes were excised.

Preparation of cell suspension: A single cell suspension of LNC was prepared by mechanical disaggregation through sterile 200 mesh steel gauge. The cells were washed twice with an excess phosphate-buffered saline (PBS) and resuspended in RPMI-1640 culture medium supplemented with 10 % fetal calf serum (FCS), 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), 100 µg/mL penicillin and 100 U/mL streptomycin. The cell concentration was adjusted to give 5 x 10E6 cells/mL.
Lymphocyte suspensions were seeded into 96 well microtiter plates at a concentration of 1 x 10E6 cells/well (5 wells per animal), and cultured with 0.5 µCi [3H] methyl thymidine ([3H]TdR) for 18 h at 37 °C in a humidified atmosphere of 5 % CO2 in air.
The incorporation of [3H]TdR was measured using a liquid scintillation counter and expressed as mean counts per min (cpm) ± standard deviation per node of three animals for each test group.

Other: Compound solubility - Completely dissolved in 20 % ethanol solution
Positive control substance(s):
not specified
Statistics:
Not reported
Positive control results:
Not reported
Parameter:
SI
Remarks on result:
other: 10 % zinc sulfate = 1.41
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean value cpm ± SD ( x 10-3) = 2.14 ± 0.77

Validity of ear abrasion technique: It does not affect the LNC response in the vehicle treated group.

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the conditions of the test, the test material was determined to be non-sensitising to mice.
Executive summary:

A study was conducted to evaluate the skin sensitisation potential of the test material in mouse using a modified Local Lymph Node Assay. No guideline or GLP compliance was documented in the study report.

Groups of BALB/c mice (n=3) were treated with 10% concentration of test material or vehicle (20% ethanol solution) by applying 25 µL to the dorsum of both abraded ears for three consecutive days. Four days following the initial application, draining lymph nodes were excised. A single cell suspension of LNC was prepared and the incorporation of [3H]TdR was measured using a liquid scintillation counter.

[3H]TdR incorporation (expressed as mean counts per min (cpm) ± standard deviation per node x 10-3) was 2.14 ± 0.77 and the ratio of the proliferation in treated group to that in vehicle control (stimulation index) was 1.41.

Hence, under the conditions of the test, the test material was determined to be non-sensitising to mice.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Zinc sulphate (ZnSO4•7 H2O) was tested in a mouse local lymph node assay (Ikarashi et al., 1992), according to the testing methods developed by Kimber et al.,(1989 and 1990). After gentle dermal abrasion, 25ml of a 5% zinc sulphate solution in 20% ethanol was applied for three consecutive days at the dorsal side of both ears of 3 Balb/c mice. On the fourth day the animals were sacrificed and the ear-draining lymph nodes were collected. Lymph node lymphocyte proliferation was determined by tritiated thymidin incorporation. The results were compared to those of vehicle-treated controls. Zinc sulphate did not induce proliferative activity, whereas for potassium bichromate, nickel sulphate and cobalt chloride (known dermal sensitizers) positive results were obtained.

The skin sensitising potential of zinc sulphate (ZnSO4•7 H2O) was also investigated in guinea pigs. A well-performed maximisation test, conducted according to Directive 96/54/EC B.6 and OECD guideline 406, was carried out in female Dunkin Hartley guinea pigs. Based on the results of a preliminary study, in the main study 10 experimental animals were intradermally injected with a 0.1% concentration and epidermally exposed to a 50% concentration. Five control animals were similarly treated, but with vehicle (water) alone. Approximately 24 hours before the epidermal induction exposure all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were challenged with a 50% test substance concentration and the vehicle. A second challenge followed one week after the first. In response to the 50% test substance concentration, in some experimental animals and controls skin reactions of grade 1 were observed 48 hours after the first (5/10 and 2/5, respectively) and the second challenge (4/10 and 2/5, respectively). As the skin reactions were comparable among the experimental and control animals, and as there was poor consistency of the skin reactions among individual experimental animals after the first and second challenge, the observed skin reactions can be considered to be non-specific signs of irritation. Hence, it can be concluded that zinc sulphate did not induce hypersensitivity in experimental animals (Van Huygevoort, 1999).


Zinc bis(dihydrogen phosphate) was tested in a mouse local lymph node assay (Plodikova, 2010). Five Balb/c mice per group were exposed on the dorsum of both ears once a day by test and control substances during 3 consecutive days. Concentrations were 30%, 3%, 0.3% (w/v) in DAE 433 (mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol). Lymph node lymphocyte proliferation was determined by tritiated thymidin incorporation. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index, was determined. The test substance caused a dose dependent increase in radioisotope incorporation into the DNA of dividing lymphocytes, with a significant ratio of 3.13 at 30%. The test substance also showed a tendency to increased ear weight at the 30% concentration, which could also be a sign of irritation of the skin. The animals exposed to the test substance at all concentrations showed no further pathological skin reactions and no other negative clinical symptoms of intoxication throughout the experiment. The positive result could be due a contact allergen in mice but potential irritation effect does not rule out the possibility that it could be false positive result.

The skin sensitising potential of zinc bis(dihydrogen phosphate) was also investigated in guinea pigs to rule out whether it has an allergenic effect or not (Slais, 2010). A well-performed maximisation test, conducted according to OECD guideline 406, was carried out in albino guinea pigs. A pilot study for establishing of the dose for the main study was conducted first. Three guinea pigs were exposed to three doses (10, 70 and 500 mg/animal) of the Test item for 4 hours. The animals were observed for skin reaction 24, 48 and 72 hours after the exposure. Since no skin reaction was observed, a dose of 500 mg/animal was chosen for the main study. The tested animals in the main study were treated by three inductions – topic application with one week intervals. The negative control animals were treated only with Sterilux in the same manner. The positive control animals were treated with the reference item (2-mercaptobenzothiazole) in the dose of 0.5 mL/animal. Two weeks after the last induction, a topical challenge was performed in the treated and negative control animals. The positive control animals were treated with the reference item. At the intervals of 24 and 48 hours after the challenge exposure, the skin reaction of all the animals was evaluated. Daily clinical observations and weekly body weight values were recorded. No sensitizing effect of the Test item Zinc bis(dihydrogen phosphate) was observed in any of the animals after the challenge exposure. The clinical observations of the animals were without any pathological findings related to the treatment. The body weight values of all the animals increased during the study without abnormalities and corresponded to the age of the animals. Non-continuous or patchy erythema was observed in two positive control animals after the challenge exposure; no reactions were observed in the negative control animals. Based on these results the test system was considered to be reliable. Under the test conditions used the Test item Zinc bis(dihydrogen phosphate) was assessed to be not sensitizing.


Migrated from Short description of key information:
All available data suggests this compound does not have skin sensitisation potential.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Considering the absence of evidence of respiratory sensitization responses, this endpoint is not expected to be of concern for zinc and zinc compounds.


Migrated from Short description of key information:
While there is no particular study addressing respiratory sensitisation in experimental animals, there is no information suggesting zinc compounds to cause such effects animals.Taking into account the complete absence of skin sensitization potential of zinc compounds, respiratory sensitisation is not expected to be of concern for the zinc and zinc compounds considered in this chemical safety report.

Justification for classification or non-classification

The data on soluble zinc sulphate indicates no sensitisation potential. The data on soluble zinc bis(dihydrogen phosphate) indicates a positive sensitisation potential in a study done with the Mouse local lymph node assay but no sensitisation potential was indicated in another study done with the Guinea pig maximization test. Since this latter test is known to be more trustful in studies with metals, no sensitization is expected and therefore classification for skin sensitization is not required according to EC criteria.

Sensitisation is not expected from soluble zinc chloride, diammonium tetrachlorozincate and triammonium pentachlorozincate based on the data for zinc sulphate and zinc (bishydrogenphosphate) since the soluble zinc compounds share similar solubility characteristics.