Cobalt chromite green spinel

Skin sensitisation

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:

2012-05-14 to 2012-07-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Read-across category approach:
Cobalt chromite green spinel is a spinel-type oxide/pigment with the general structural element AB2O4 which forms a strong and inert matrix. Co and Cr are the main components with percentages of ~12.7% w/w and 44.3% w/w respectively. The amount of Zn as component is about 14.9% w/w.

The read-across substance Cobalt chromite blue green spinel is a spinel-type pigment as well with an identical crystallographic structure and a very similar composition. Co content is ~14.8% w/w and the Cr content is ca. 30% w/w. Al is present in about 9.4% w/w. Contents of Zn is about 15.1% w/w.

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

Solubility of Co from the pigment Cobalt chromite green spinel in physiological media was in a range of 17.2 µg/L (GMB) and 99.1 µg/L (ALF) after 2 hours. After 24 hours a dissolution range from 12.8 µg/L (GMB) - 108 µg/L (ALF) was measured.

Solubility of Cr from the pigment Cobalt chromite green spinel in physiological media was in a range of 0.264 µg/L (GMB) and 5.95 µg/L (GST) after 2 hours. After 24 hours a dissolution range from 0.452 µg/L (GMB) - 6.80 µg/L (GST) was measured.

The read-across substance Cobalt chromite blue green spinel afforded after 2 hours a solubility for the element Co in range of 0.34 µg/L (PBS) and 6.45 µg/L (ALF) and after 24 hours a solubility of 0.37 µg/L (GMB) to 9.89 µg/L (GST).

Solubility of Cr from the pigment Cobalt chromite blue green spinel in physiological media was in a range of below LOD and 1.11 µg/L (ALF) after 2 hours. After 24 hours a dissolution range from 0.18 µg/L (PBS) - 0.99 µg/L (ALF) was measured.

T/D testing of the pigment Cobalt chromite green spinel afforded the following solubility at 1mg loading after 28 days:
Cobalt: 2.038 ± 0.008 and 0.790 ± 0.039 μg/L at pH 8 and pH 6, respectively
Chromium: 1.489 ± 0.008 μg/L and Zinc: 1.840 ± 0.378 μg/L and
T/D testing of the read-across substance Cobalt chromite blue green spinel afforded the following solubility at 1mg loading after 28 days:
Cobalt: below LOD at pH 8 and pH 6, respectively
Chromium: below LOD at pH 8 and pH 6, respectively

In sum, the two spinel type pigments are structurally identical with similar composition. Both show a very low solubility in different artificial and aqueous media. In fact, the read-across substance Cobalt chromite blue green spinel shows slightly lower dissolution and bioaccessibility without showing any signs of systemic or local toxicity in various studies (acute inhalation, skin/eye irritation, sensitisation).

Based on the information summarised above, read-across to Cobalt chromite blue green spinel is fully justified.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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 2009-11-12

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt chromite blue green spinel

EC Number:

269-072-0

EC Name:

Cobalt chromite blue green spinel

Cas Number:

68187-11-1

Molecular formula:

ZnxCo(1-x)Cr(2-y-z)AlyO(4[3/2]z)

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Cobalt chromite blue green spinel (Pigment Blue 36)
- Chemical description: cobalt chromite blue green spinel
- Physical state: solid, blue green powder, odourless
- Melting point: >1000°C
- Water solubility: practically insoluble
- Relative density: 4.96

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: 62 days
- Weight at study initiation: 27 - 35 g
- Housing: before application the animals were housed in groups in MAKROLON cages (type III) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 15 cm. After application the animals were housed singly in order to prevent their licking off the test item from the ears of the other animals. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages.
- 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:

10 %, 25% and 50% (w/w) of cobalt chromite blue green spinel

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 cobalt chromite blue green spinel (Pigment Blue 36) in acetone/olive oil (3+1, v/v) were examined. Possible clinical signs would have been recorded.
Cobalt chromite blue green spinel (Pigment Blue 36) was a blue green powder. Hence, a 50% suspension was the highest feasible concentration of cobalt chro-mite blue green spinel (Pigment Blue 36) in acetone/olive oil (3+1, v/v).
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. No clinical signs were recorded.

MAIN STUDY
The experimental schedule of the assay was as follows:
Day 1:
The weight of each animal and possible clinical signs 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.
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
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 membranes, 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 2.232 and 1.745, respectively. Therefore, the study can be regarded as valid.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.145

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:

1.038

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:

1.118

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:

1.019

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:

1.152

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:

0.968

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 chromite blue green spinel (Pigment Blue 36) 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: 1.128 (lymph node weight); SI: 1.090 (ear thickness)
25 % w/w: SI: 1.191 (lymph node weight); SI: 1.090 (ear thickness)
50 % w/w: SI: 1.149 (lymph node weight); SI: 1.081 (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.