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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-12-05 to 2017-01-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010-07-22
Deviations:
yes
Remarks:
modified OECD 429, method according to Ehlings et al. 2005
Principles of method if other than guideline:
The test was performed in accordance with the method according to Ehling et al (2005): An european inter-laboratory validation of alternative endpoints of the murine local lymph node assay: first round, Toxicology 212 (2005) 60-68 and Ehling et al (2005): An european inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round, Toxicology 212 (2005) 69-79.

Threshold values of the stimulation indices of lymph node cell count (i.e. sensitising properties) and ear weight (i.e. irritating properties) were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (cell count) or 1.1 (ear weight) are considered positive
(these values were fixed empirically during the inter-laboratory validation of this method). In addition, the lymph node weights were determined for concentration related properties.
GLP compliance:
yes (incl. QA statement)
Remarks:
2014-05-14
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Tripotassium hexacyanocobaltate
EC Number:
237-742-1
EC Name:
Tripotassium hexacyanocobaltate
Cas Number:
13963-58-1
Molecular formula:
C6CoN6.3K
IUPAC Name:
tripotassium hexacyanocobaltate
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material: Potassium hexacyanocobaltate (III)
- Physical state: solid, slightly yellowish powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, stored in a tightly closed original container in a dry and well-ventilated place.

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Females nulliparous, healthy and non-pregnant: yes
- Age (on test day 1): approx. 9 weeks
- Weight (on test day 1): 26 - 33 g
- Housing: kept singly in MAKROLON cages (type II) with a basal surface of approx. 360 cm² and a height of approx. 14 cm; bedding material: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany)
- Diet (ad libitum): commercial diet ssniff® R/M-H V1534
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 3 °C (maximum range)
- Relative humidity: 55 % ± 15 % (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
10%, 25% and 50% (w/w) of potassium hexacyanocobaltate (III)
No. of animals per dose:
6 animals
Details on study design:
RANGE FINDING TESTS:
A preliminary experiment was carried out in 3 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations. Three concentrations of 10%, 25% and 50% suspended in methyl ethyl ketone were examined.
Potassium hexacyanocobaltate (III) was a yellow powder. Hence, a 50% suspension was the highest feasible concentration of Potassium hexacyanocobaltate (III) in methyl ethyl ketone.
The preliminary experiment was conducted under conditions identical to the main LLNA study, except there was no assessment of lymph node proliferation and only 1 animal per concentration was used. In this preliminary experiment no negative control was tested due to animal welfare reasons.
Both ears of each mouse were observed for erythema and scored using the scoring table (table 1) as shown in the field "Any other information on materials and methods incl. tables" below:
The results obtained were compared to reactions known from administration of recommended vehicles for example: acetone:olive oil (4:1, v/v) or dimethyl sulphoxide or other media used as vehicles in the LLNA (historical background data). For example, the erythema score for the vehicle methyl ethyl ketone (test item) or the vehicle acetone:olive oil (4:1)(positive control) in NMRI mice is E:0.

Results:
No irritating properties were observed in this preliminary experiment, no differences in ear weight and ear thickness were noted.

MAIN STUDY
The experimental schedule of the assay was as follows:
Day 1:
The weight and any clinical signs of each animal were individually identified and recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer.
Open application of 25 μL of the appropriate dilution of the test item, the vehicle alone or the positive control (as appropriate) were administered to the dorsum of each ear.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
The animals were checked for any clinical signs of local systemic irritation at the application site or of systemic toxicity.
Day 4 (24 hours after the last application):
Ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer.
Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance.
Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in PBS/0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.

OBSERVATIONS
- Clinical signs: animals were observed once daily for any clinical signs of local systemic irritation at the application site or of systemic toxicity. In addition, animals were checked regularly throughout the working day and on the weekend.
- Body weight: the weight of each mouse was recorded at the time of allocation of animals to groups (test day 1) and at the time of necropsy (test day 4).

ANALYSIS OF RESULTS
The so-called stimulation (or LLN-) indices to determine the sensitising potential were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones.
Thus, in case of no stimulating effect the index for the lymph node cell count is always below 1.4 (cut-off value). An index above 1.4 is considered positive.
For lymph node weight significance at p ≤ 0.01 is considered positive (U-test according to MANN and WHITNEY). A possible concentration-response-relationship for the lymph node weight was examined by linear regression analysis employing PEARSON's correlation coefficient. U-test was performed for cell count, too. Outliers were determined according to the Nalimov test.
In addition, the acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control.
The stimulation indices were calculated by dividing the average ear weight and average ear thickness on test day 4 per group of the test item treated animals by the vehicle treated ones. The cut-off threshold value for ear weight was set at 1.1.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Please refer to "details on study design"

Results and discussion

Positive control results:
The positive control group caused the expected increases in lymph node cell count (SI: 1.614) and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
10 % (w/w) test item
Remarks on result:
other: 10 % (w/w) test item; lymph node cell count
Key result
Parameter:
SI
Value:
1.02
Test group / Remarks:
10 % (w/w) test item
Remarks on result:
other: 10 % (w/w) test item; ear weight
Key result
Parameter:
SI
Value:
1.21
Test group / Remarks:
25 % (w/w) test item
Remarks on result:
other: 25 % (w/w) test item; lymph node cell count
Key result
Parameter:
SI
Value:
1.094
Test group / Remarks:
25 % (w/w) test item
Remarks on result:
other: 25 % (w/w) test item; ear weight
Key result
Parameter:
SI
Value:
1.286
Test group / Remarks:
50 % (w/w) test item
Remarks on result:
other: 50 % (w/w) test item; lymph node cell count
Key result
Parameter:
SI
Value:
1.087
Test group / Remarks:
50 % (w/w) test item
Remarks on result:
other: 50 % (w/w) test item; ear weight
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
Threshold values of the stimulation indices of lymph node cell count and ear weight were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (cell count) or 1.1 (ear weight) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method).

RESULTS ON SKIN SENSITISATION
In the main study treatment with potassium hexacyanocobaltate (III) at concentrations of 10%, 25% or 50% did not reveal any statistical significantly increased values for the lymph node cell count. The stimulation indices of the lymph node cell count did not exceed the threshold level of 1.4. Hence, the test item is classified as not sensitising.
The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted for potassium hexacyanocobaltate (III) based on ear weight data.

STIMULATION INDEX RESULTS OF LYMPH NODE WEIGHT AND EAR THICKNESS
10 % w/w: SI: 1.093 (lymph node weight); SI: 1.008 (ear thickness)
25 % w/w: SI: 1.186 (lymph node weight); SI: 1.008 (ear thickness)
50 % w/w: SI: 1.163 (lymph node weight); SI: 1.000 (ear thickness)

CLINICAL OBSERVATIONS:
No signs of local or systemic intolerance were recorded.

BODY WEIGHTS
The animal body weight was not affected by the treatment.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The substance is not a skin sensitiser.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does not require classification as skin sensitiser.