Registration Dossier

Administrative data

Endpoint:
skin sensitisation, other
Remarks:
in vivo (LLNA) study
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2012-03-28 to 2012-04-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Zircon, cadmium yellow is a zircon-type oxide/pigment with the main structural element ZrSiO4 (Zr in an dodecahedral O environment) which forms a strong and inert matrix. Zr and Si are the main components with percentages of ~46% w/w and 15% w/w respectively. The amount of Cd as component is about 2% w/w.

The read-across substance Silicic acid, zirconium salt, cadmium pigment encapsulated is a zircon-type pigment as well with an identical crystallographic structure and a very similar composition. Zr content is ~46% w/w and the Si content is ca. 14% w/w. Cd is present in about 4.7% w/w. Minor amounts of Se and S (1.53 and 1.04% w/w, respectively) are present as well.

Besides the obvious structural analogy the solubility of both pigments in aqueous and physilogical media are as follows (determination of Zr and Cd):

Solubility of Cd from the pigment Zircon, cadmium yellow in physiological media was in a range of 0.180 µg/L (PBS) and 0.398 µg/L (ALF) after 2 hours. After 24 hours a dissolution range from 0.140 µg/L (GMB) - 0.722 µg/L (GST) was measured.

Solubility of Zr from the pigment Zircon, cadmium yellow in physiological media was in a range of below LOD and 0.812 µg/L (ALF) after 2 hours. After 24 hours a dissolution range from below LOD (ASW) - 1.44 µg/L (ALF) was measured.

The read-across substance Silicic acid, zirconium salt, cadmium pigment encapsulated afforded after 2 hours a solubility for the element Cd in range of 0.07 µg/L (GMB) 2.58 µg/L (GST) and after 24 hours a solubility of 0.07 µg/L (GMB) to 6.13 µg/L (GST).

Solubility of Zr from the pigment Silicic acid, zirconium salt, cadmium pigment encapsulated in physiological media was in a range of below LOD and 0.52 µg/L (ALF) after 2 hours. After 24 hours a dissolution range from below LOD - 1.25 µg/L (ALF) was measured.

T/D testing of the pigment Zircon, cadmium yellow afforded the following solubility at 1mg loading after 28 days:
Cadmium: below LOD (<0.0048) and 0.2077 ± 0.0993μg/L at pH 8 and pH 6, respectively
Zirconium:
T/D testing of the read-across substance Silicic acid, zirconium salt, cadmium pigment encapsulated afforded the following solubility at 1mg loading after 28 days:
Cadmium: 0.34 ± 0.09 μg/L and 0.86 ± 0.39 μg/L at pH 8 and pH 6, respectively
Zirconium:
In sum, the two zircon type pigments are structurally identical with very similar composition and both show a very low solubility in different artificial and aqueous media. In fact, the read-across substance Silicic acid, zirconium salt, cadmium pigment encapsulated shows slightly higher dissolution and bioaccessibility without showing any signs of systemic or local toxicity in various studies (acute inhalation, skin/eye irritation, sensitisation). No toxicity on algae, fish and invertebrates could be observed in respective studies.
Hence, read-across to Silicic acid, zirconium salt, cadmium pigment encapsulated is fully justified.

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)
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. certificate)
Remarks:
signed 2009-11-12
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): Silicic acid, zirconium salt, cadmium pigment encapsulated
- Physical state: solid, bright red powder, odourless
- Melting point: >1000°C
- Water solubility: Not soluble
- Relative density: 4.70
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, kept dry in closed container

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 at study initiation: 69 days
- Weight at study initiation: 27 - 33 g
- Housing: animals were kept singly in MAKROLON cages (type III) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 15 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. Animals were housed singly in order to prevent their licking off the test item from the ears of the other animals.
- Diet (ad libitum): Commercial diet ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- 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 times per hour.
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
other: acetone/olive oil (3 + 1, v/v)
Concentration:
50%, 25% and 10 % (w/w) of silicic acid, zirconium salt, cadmium pigment encapsulated
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% of silicic acid, zirconium salt, cadmium pigment encapsulated in acetone/olive oil (3+1, v/v) were examined.
Silicic acid, zirconium salt, cadmium pigment encapsulated was a red powder. Hence, a 50% suspension was the highest feasible concentration of silicic acid, zirconium salt, cadmium pigment encapsulated in acetone/olive oil (3+1, v/v).
In addition, possible clinical signs after administration were noted.
Results:
No pronounced 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 of each animal was 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 noted.
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 PBS6/0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.

OBSERVATIONS
The following observations were made during the course of the study:
- Clinical signs: animals were observed once daily for any clinical signs of local systemic irritation at the application site or of systemic toxicity. Observations were recorded for each individual animal. Cageside observations included skin/fur, eyes, mucous mem-branes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
In addition, animals were checked regularly throughout the working day from 7:30 a.m. to 4:30 p.m. On Saturdays and Sundays animals were checked regularly from 8:00 a.m. to 12:00 noon with a final check performed at approximately 4:00 p.m., if applicable.
- 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. Outliers would have been 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 and lymph node weight (statistically significant at p ≤ 0.01). The values for the stimulation index of lymph node cell count and lymph node weight were 1.519 and 1.826, respectively. Therefore, the study could be regarded as valid.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.018
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:
1.053
Test group / Remarks:
10 % (w/w) test item
Remarks on result:
other: 10 % (w/w) test item; ear weight
Key result
Parameter:
SI
Value:
0.798
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:
0.982
Test group / Remarks:
25 % (w/w) test item
Remarks on result:
other: 25 % (w/w) test item; ear weight
Key result
Parameter:
SI
Value:
1.1
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:
0.971
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 silicic acid, zirconium salt, cadmium pigment encapsulated at concentrations of 10%, 25% or 50% did not reveal statistical significantly increased values for 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.

STIMULATION INDEX RESULTS OF LYMPH NODE WEIGHT AND EAR THICKNESS
10 % w/w: SI: 1.130 (lymph node weight); SI: 0.996 (ear thickness)
25 % w/w: SI: 1.043 (lymph node weight); SI: 1.024 (ear thickness)
50 % w/w: SI: 1.239 (lymph node weight); SI: 1.052 (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.