Zinc hydroxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Zinc bis(dihydrogen phosphate)

EC Number:

237-067-2

EC Name:

Zinc bis(dihydrogen phosphate)

Cas Number:

13598-37-3

Molecular formula:

Zn(H2PO4)2

IUPAC Name:

zinc bis(dihydrogen phosphate)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Zinc bis(dihydrogen phosphate)
- Physical state: white crystalline solid powder
- Analytical purity: ZnO(%): 25-27 P2O5(%): 43-49.5
- Composition of test material, percentage of components: Zinc bis(dihydroogen phosphate)*2H2O : phosphate (as P2O5) 44.6% ±5% rel.; Zinc: 21.5%±0.5 % rel.
- Lot/batch No.: Z 11-81
- Stability / expiration: 31.08.2011
- Storage condition of test material: in dark at the ambient temperature in the original container

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Breeding farm VELAZ s.r.o., Kolec u Kladna, Czech Republic
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 18.7-21.7g
- Housing: maximum six in macrolon cages (35X20X15cm) with sterilized softwood shavings
- Diet: pelleted standard diet for experimental animals ad libitum (ST1 BERGMAN)
- Water: drinking tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5-23.4
- Humidity (%): 41-48
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

other: DAE 433 (mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol)

Concentration:

30%, 3%, 0.3% (w/v)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
in pilot experiment, the highest concentration 30% (maximum technically practicable concentration) was administered to 3 animals to assess and/or discard a possible systemic toxicity or high irritation of skin. The route of administration was the same as in the main study. During the pilot experiment no test item related effects were found in all three animals, respectively no clinical symptoms of systemic toxicity and no macroscopic changes (after necropsy) were detected.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: if the simulation index (SI) is ≥3 , and the response increases in dose-related manner (dose-response relationship)
- dosage volume: 25µl/ear /animal

TREATMENT PREPARATION AND ADMINISTRATION:
all suspensions were prepared by mixing an appropriate amount of zinc bis(dihydrogen phosphate) (1.5g, 0.15g or 0.015g) and the vehicle to obtain the application form (3X5mL) in concentration of 30%, 3% or 0.3% (w/v). The suspensions were prepared before the start of application by mixing on mangetic stirrer and then were still mixed during application. Suspensions were prepared on each day immediately before administration (10-20min)

Positive control substance(s):

other: dinitrochlorobenzene

Statistics:

Kruskal-Wallis test: for the comparision of the measured effect in all treatment groups with the vehicle control group, as global test
Mann-Whitney test: for all 2-group comparisions

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

9.29

Test group / Remarks:

positive control

Results 2

Parameter:

SI

Value:

1

Test group / Remarks:

negative control

Results 3

Parameter:

SI

Value:

3.13

Test group / Remarks:

30% test substance

Results 4

Parameter:

SI

Value:

1.09

Test group / Remarks:

3% test substance

Results 5

Parameter:

SI

Value:

0.69

Test group / Remarks:

0.3% test substance

Any other information on results incl. tables

group	radioisotope incorporation		ear weight
	mean DPM	SI	mean (mg)
negative control	799.43	1.00	23.66
positive control	7427.57	9.29+	38.88*
30%	2503.43	3.13+	26.18*
3%	869.99	1.09	23.52
0.3%	550.90	0.69	22.78

*= statistically significant p<0.05 (Mann-Whitney test)

+= values ≥3

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

Test substance had a positive result in LLNA test, but potential irriation effect does not rule out the possibility that it could be false positive result.

Conclusions:

Test substance had a positive result in LLNA test, but potential irriation effect does not rule out the possibility that it could be false positive result.

Executive summary:

The test substance, zinc bis(dihydrogen phosphate) was tested for the assessment of skin sensitisation potential with the murine local lymph node assay.

The local lymph node assay (LLNA) with radionuclides was used. The testing was conducted according to the EU Method B.42, skin sensitization: local lymph node assay. In this study the contact allergenic potential of zinc bis(dihydrogen phosphate) was evaluated after topical application to female BALB/c mice. Five mice per group were exposed on the dorsum of both ears once a day by test and control substances during 3 consecutive days. Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated on the base on using radioactive labelling. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation index, was determined. Statistical evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

Concentrations: positive control DNCB: 0.5% (w/v) and zinc bis(dihydrogen phosphate): 30%, 3%, 0.3% (w/v) in DAE 433.

The animals exposed to the test substance at all concentrations showed no pathological skin reactions and no other negative clinical symptoms of intoxication throughout the experiment. There was no significant difference in body weight increment of all groups in comparison to the vehicle control. The positive control substance DNCB elicted a reaction pattern with statistically significant increase in ear weight and stimulation index of cell proliferation 9.29, which was in congruence with his expected mode of action as a contact allergen.

The test substance showed a tendency to increased ear weight in the highest of concentrations tested. The result of skin irritation effect was considered as positive- it means the thest substance caused irritation of skin. Comparison of stimulation indexes between treated groups and control group revealed that the test substance caused significant increase in radioisotope incorporation into the DNA of dividing lymphocytes. This effect was dose dependent, with a significant ratio of 3.13 at 30%.

The test substance zinc bis(dihydrogen phosphate) had a positive result in LLNA test. Positive results in cell proliferation and no clinical symptoms of systemic toxicity revealed that the test substance zinc bis(dihydrogen phosphate) could be a contact allergen in mice but potential irriation effect does not rule out the possibility that it could be false positive result.