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Toxicological information

Skin sensitisation

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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other: GLP-compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.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:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Potassium tetrafluoroborate
- Physical state: White powder
- Analytical purity: 99.1%
- Storage condition of test material: At room temperature in the dark
- Lot/batch No.: BWF10626
- Expiration date of the lot/batch: 31 July 2013

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
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: 20.7 - 25.0 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
- Humidity (%): 45-65
- 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:
10%, 25% and 50%
No. of animals per dose:
4
Details on study design:
ANIMAL ASSIGNMENT:
Upon arrival, the animals were taken to a quarantine room and checked for overt signs of ill health and anomalies. During the quarantine period serological investigation of the microbiological status was conducted in three randomly selected animals. The results of serological examination indicated an acceptable microbiological status. Thereupon, the quarantine measures were withdrawn and the room was cleared for use as experimental room and acclimatised to the laboratory conditions until the experimental start date. The animals of the study were allocated to the various groups by computer randomisation and proportionately to body weight. All animals assigned to the study were housed in one room during the study period and no other test system was housed in the same room during the study. During the study, each animal was identified by a unique tail marking. Each cage was provided with a card showing the colour code, the animal identification number, the cage number, the group number and the Provantis study number.

DOSE FORMULATIONS:
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: 50%, 25% and 10%. 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).
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 2, 3 and 4 were compared to vehicle-treated animals. Probability values of p<0.05 was considered significant. The reliability of the test model was tested in a separate study under similar conditions and using similar procedures. In this study the sensitivity of the test system and the reliability of the experimental procedures as used at TNO Triskelion were checked using Hexyl Cinnamic Aldehyde (a 25% concentration in vehicle, v/v) as reference compound. The validity of the model was tested by comparing the positive control and the vehicle-treated group.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
other: disintegrations per minute (DPM)
Value:
919
Test group / Remarks:
Negative control
Parameter:
other: disintegrations per minute (DPM)
Value:
1 235
Test group / Remarks:
50 (w/v)%
Parameter:
other: disintegrations per minute (DPM)
Value:
1 346
Test group / Remarks:
25 (w/v)%
Parameter:
other: disintegrations per minute (DPM)
Value:
1 356
Test group / Remarks:
10 (w/v)%
Parameter:
SI
Value:
1
Test group / Remarks:
Negative control
Parameter:
SI
Value:
1.34
Test group / Remarks:
50% (w/v)%
Parameter:
SI
Value:
1.46
Test group / Remarks:
25 (w/v)%
Parameter:
SI
Value:
1.47
Test group / Remarks:
10 (w/v)%

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.

 

3H-thymidine incorporation in the auricular lymph nodes

Group mean 3H-thymidine incorporations of 1356, 1346 and 1235 DPM were found in the auricular lymph nodes of animals treated with respectively 10%, 25% and 50% Potassium tetrafluoroborate 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 Potassium tetrafluoroborate treated animals compared to controls, were 1.47, 1.46 and 1.34 for the tested doses of 10%, 25% and 50% Potassium tetrafluoroborate, respectively.

Since the SI was < 3, the limiting value required for classification as a skin sensitiser, in response to all concentrations of Potassium tetrafluoroborate tested and no dose response relationship was apparent it was concluded that Potassium tetrafluoroborate did not have a skin sensitising potential when applied up to concentrations of 50%.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, no evidence was obtained that KBF4 has a skin sensitising potential when applied up to concentrations of 50%.
Executive summary:

In a GLP-compliant OECD Guideline 429 study, skin sensitisation properties of KBF4 were studied in mice. Three test groups of 4 female mice each were treated with 10, 25 and 50% KBF4 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 1356, 1346 and 1235 DPM were found in the auricular lymph nodes of animals treated with respectively 10%, 25% and 50% KBF4 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 KBF4 treated animals compared to controls, were 1.47, 1.46 and 1.34 for the tested doses of 10%, 25% and 50% KBF4, respectively. Since the SI was < 3, the limiting value required for classification as a skin sensitiser, in response to all concentrations of KBF4 tested and no dose response relationship was apparent it was concluded that KBF4 did not have a skin sensitising potential when applied up to concentrations of 50%.