Potassium trifluorozincate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Triskelion, Utrechtseweg 48 3704 HE Zeist, the Netherlands

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Potassium trifluorozincate

EC Number:

237-537-7

EC Name:

Potassium trifluorozincate

Cas Number:

13827-02-6

Molecular formula:

F3Zn.K

IUPAC Name:

potassium trifluorozincuide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: Nocolok Zn Flux
- Chemical name: Potassium trifluorozincate
- Physical state: white powder
- Analytical purity: 99.%
- Lot/batch No.: BWF10118
- Expiration date of the lot/batch: 31 December 2011
- Storage condition of test material: ambient temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: 20.4 - 25.3 g
- Housing: During acclimatisation the animals were group housed in macrolon type III cages (5 mice/cage). After allocation, shortly before start of treatment, the animals were housed in macrolon type III cages (4 mice/cage). The cages were provided with sterilised wood shavings (Lignocel) as bedding material and shreds of paper (Enviro-dri) as environmental enrichment.
- Diet: Standard diet ad libitum (Rat and Mouse no.3 breeding diet RM3) was purchased from SDS Special Diets Services, Whitham, England.
- Water: domestic mains tap-water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2, one deviation from the optimal temperature was observed (period of <10 minutes of 26.3°C).
- Humidity (%): 45-65, the actual upper limit during the study period was 77.8%, which were considered not to have affected study integrity.
- Air changes (per hr): ca 10
- Photoperiod (hrs dark / hrs light): 12/12 (light on from 7:00 AM - 7:00 PM)

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0, 2.5, 5.5 and 10%

No. of animals per dose:

4

Details on study design:

DOSE FORMULATIONS:
Before the start of the study trial-formulations of Nocolok Zn Flux in vehicle at the concentrations of 50%, 25% and 10% were prepared to check the feasibility of dosing by means of an air-displacement pipette. The results showed that the highest possible concentration that could be pipetted accurately was a 10% concentration. The dose formulations of the test substance in vehicle were freshly prepared on each day of dosing, i.e. day 0, 1 and 2. The formulations were prepared on a weight (test substance)/volume (vehicle) basis. After mixing, the dose formulations were shaken until visible homogeneity was obtained. Dose formulations were prepared at the concentrations: 10%, 5% and 2.5%. No adjustment was made for the purity of the test substance. The dose formulations were used for dosing of the animals within 2 hours after preparation.

STUDY DESIGN:
The dose formulations were applied topically once daily for three consecutive days on the dorsum of both ears (25 μL on each ear by means of a pipette). Each animal received a total amount of 50 μL per dosing. On day 5 all animals received an intravenous injection of 250 μL of phosphate-buffered saline (PBS) containing 20 μCi of 3H-thymidine in the tail vein. Five hours after the 3H-thymidine injection the animals were sacrificed by i.p. injection with an overdose of sodium pentobarbital and the draining auricular lymph nodes were excised, pooled per animal in PBS and further processed to determine the 3H-thymidine incorporation.

OBSERVATIONS AND MEASUREMENTS:
The general condition and behaviour of the animals was checked daily during the study. All findings including any abnormalities were recorded. Body weights were determined at allocation (day -1), at the start (day 0) and at the end (day 5) of the study. The animals were sacrificed on day 5 for excision of the auricular lymph nodes. The animals were examined for signs regarding the appearance of the lymph nodes and the ears, but were not subjected to a full macroscopic examination. Per animal, the paired auricular lymph nodes (ALN) were excised and were placed together in a vial containing 5 mL phosphate buffered solution (PBS). A single cell suspension was prepared by gently rubbing the nodes between the rough ends of microscope slides. The cell suspension was washed twice. The cell pellet obtained after the second wash was resuspended in 3 mL of 5% trichloroacetic acid (TCA) and left overnight (~18 hours) at 2-10ºC. After centrifugation and removal of the supernatant, 1 mL of 1.5 M KOH in 20% EtOH was added to the precipitate and left for 24 hours at room temperature. Thereafter, the solution was transferred into a clean scintillation vial and 20 mL liquid scintillation cocktail (Hionic Fluor, Perkin Elmer) was added. The vial was thoroughly shaken and the number of disintegrations per minute (DPM) was determined by counting for five minutes in a liquid scintillation counter (Perkin Elmer).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Statistical evaluations of the data (body weights and 3H-thymidine incorporation) were performed by analysis with Dunnett’s multiple comparison tests. Body weights and 3H-thymidine incorporation of the test substance groups were compared to vehicle-treated animals. Probability values of p<0.05 were considered significant.

Results and discussion

Positive control results:

The stimulation index of Hexyl Cinnamic Aldehyde (25% in vehicle v/v) determined in study 8525/03 was 4.3.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Concentration (% w/v) Stimulation index 0 1.0 2.5 1.97 5 1.22 10 1.78

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Concentration (% w/v) Disintegrations per minute 0 919 2.5 1810 5 1118 10 1641

Any other information on results incl. tables

- Clinical signs: No signs of local irritation or other clinical signs were observed in any of the animals during the study period.

- Body weights: No aberrant body weights or body weight gains were observed and no statistical significant difference was found between the vehicle and the different test groups.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the experimental conditions of this study, no evidence was obtained that KZnF3 has a skin sensitising potential when applied up to the maximum, technically attainable concentration of 10%.

Executive summary:

In a GLP-compliant OECD Guideline 429 study, skin sensitisation properties of KZnF3 were studied in mice. Three test groups of 4 female mice each were treated with 2.5, 5 and 10% KZnF3 by means of open application of 25 μL to the dorsum of each ear for three consecutive days. Three days after the last treatment, all animals received an intravenous injection of 3H-thymidine. Five hours later, the 3H-thymidine incorporation in the draining auricular lymph nodes was determined. The results were compared with those of a negative control group which was treated with the vehicle (acetone/olive oil, 4:1 v/v). Group mean 3H-thymidine incorporations of 1810, 1118 and 1641 DPM were found in the auricular lymph nodes of animals treated with respectively 2.5%, 5% and 10% KZnF3 formulations. The group mean value in the vehicle control animals was 919 DPM. The calculated stimulation indices (SI), representative of the change in 3H-thymidine incorporation in Nocolok Zn Flux treated animals compared to controls, were 1.97, 1.22 and 1.78 for the tested doses of 2.5%, 5% and 10% KZnF3, respectively. Since the SI was < 3, the limiting value required for classification as a skin sensitiser, in response to all concentrations of KZnF3 tested and no dose response relationship was apparent it was concluded that KZnF3 did not have a skin sensitising potential when applied up to concentrations of 10%.