Cobalt zinc silicate blue phenacite

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation, other

Remarks:

in vivo (LLNA) study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-12 to 2015-08-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

signed 2014-05-14

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt zinc silicate blue phenacite

EC Number:

270-208-6

EC Name:

Cobalt zinc silicate blue phenacite

Cas Number:

68412-74-8

Molecular formula:

Zn(2-x)Co(x)SiO4 0,7≤x≤1,1

IUPAC Name:

cobalt zinc silicate blue phenacite

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: Cobalt zinc silicate blue phenacite (Pigment Blue 74)
- Physical state: intense blue powder, odourless

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: kept dry in closed container at room temperature

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 and non-pregnant: yes
- Age (on test day 1): 62 days
- Weight (on test day 1): 27 - 34 g
- Housing: kept singly in MAKROLON cages (type III) 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 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany); Food residue was removed.
- 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)
- Air changes: 12 - 18/hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

10%, 25% and 50% (w/w) of cobalt zinc silicate blue phenacite (Pigment Blue 74)

No. of animals per dose:

6 female mice

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 acetone/olive oil (4:1 v/v) of cobalt zinc silicate blue phenacite (Pigment Blue 74) were examined.
Cobalt zinc silicate blue phenacite (Pigment Blue 74) was a dark blue powder. Hence, a 50% suspension was the highest feasible concentration of cobalt zinc silicate blue phenacite (Pigment Blue 74) in acetone/olive oil (4:1 v/v).
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.
Both ears of each mouse were observed for erythema and scored using the scoring table (table 1) shown in the field "Any other information on materials and methods incl. tables" below:
Results:
No irritating properties were observed in this preliminary experiment at concentrations of 10%, 25% or 50%, 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 observations 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.
On all administration days possible clinical signs were recorded.
Day 4 (24 hours after the last application the animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis):
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 in order to determine a possible sensitising potential 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: 1.755) and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.417

Test group / Remarks:

10 % (w/w) test item

Remarks on result:

other: 10 % (w/w) test item; lymph node cell count

Results 2

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

10 % (w/w) test item

Remarks on result:

other: 10 % (w/w) test item; ear weight

Results 3

Key result

Parameter:

SI

Value:

1.618

Test group / Remarks:

25 % (w/w) test item

Remarks on result:

other: 25 % (w/w) test item; lymph node cell count

Results 4

Key result

Parameter:

SI

Value:

0.973

Test group / Remarks:

25 % (w/w) test item

Remarks on result:

other: 25 % (w/w) test item; ear weight

Results 5

Key result

Parameter:

SI

Value:

1.573

Test group / Remarks:

50 % (w/w) test item

Remarks on result:

other: 50 % (w/w) test item; lymph node cell count

Results 6

Key result

Parameter:

SI

Value:

1.064

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 cobalt zinc silicate blue phenacite (Pigment Blue 74) at concentrations of 10%, 25% or 50% revealed statistical significantly increased values for the lymph node cell count. The stimulation indices of the lymph node cell count exceeded the threshold level of 1.4. In addition, the lymph node weight was statistical significantly increased for the concentrations of 25% and 50%. The Pearson's correlation test indicated a positive dose-response relationship for the lymph node weight.
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.

STIMULATION INDEX RESULTS OF LYMPH NODE WEIGHT AND EAR THICKNESS
10 % w/w: SI: 1.074 (lymph node weight); SI: 1.025 (ear thickness)
25 % w/w: SI: 1.389 (lymph node weight); SI: 1.017 (ear thickness)
50 % w/w: SI: 1.407 (lymph node weight); SI: 1.076 (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:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item is considered to be a skin sensitiser.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test substance is classified as skin sensitiser (Category 1; H317 "May cause an allergic skin reaction").