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Description of key information

Skin sensitisation: not sensitising (modified OECD 429; method according to Ehlings et al. 2005; GLP compliant) (read across to cobalt zinc aluminate blue spinel)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2010-06-11 to 2010-07-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002-04-24
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 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). 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)
Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 9 weeks
- Weight at study initiation: 26 -31 g
- Housing: The 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.
- 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 +/- 3 °C (maximum range)
- Relative humidity: 55% +/- 15 % (maximum range)
- Air changes: 12 -18 times per hour
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
other: acetone/olive oil (3 + 1, v/v)
Concentration:
10 % w/w, 25 % w/w, and 50 % w/w of Pigment Al-Co-Zn
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 Pigment Al-Co-Zn in acetone/olive oil (3+1 v/v), w/w, were examined.
Results:
In a preliminary experiment, concentrations of 10%, 25% and 50%, employing 1 animal per concentration, were examined. 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 test item was suspended in a mixture of acetone (Batch no. 32201, SIGMA ALDRICH Chemie GmbH, 82024 Taufkirchen, Germany) / olive oil (Batch no. 4602602, Henry Lamotte GmbH, 28197 Bremen, Germany) (3+1, v/v). The vehicle was selected on the basis of maximising the test concentrations and solubility whilst producing a solution/suspension suitable for application of the test item.

The test item suspension was administered to the dorsum of both animal's ears at an application volume of 25 µL/ear.

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.
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 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"
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 1.81 and 1.77, respectively. Therefore, the study could be regarded as valid.
Ear weight: SI: 1.018
Key result
Parameter:
SI
Remarks:
lymph node cell count
Value:
1.172
Test group / Remarks:
10 % w/w test item
Remarks on result:
other: SI: 1.136 (lymph node weight)
Key result
Parameter:
SI
Remarks:
ear weight
Value:
0.953
Test group / Remarks:
10 % w/w test item
Remarks on result:
other: SI: 1.056 (ear thickness)
Key result
Parameter:
SI
Remarks:
lymph node cell count
Value:
1.322
Test group / Remarks:
25 % w/w test item
Remarks on result:
other: SI: 1.273 (lymph node weight)
Key result
Parameter:
SI
Remarks:
ear weight
Value:
0.871
Test group / Remarks:
25 % w/w test item
Remarks on result:
other: SI: 1.103 (ear thickness)
Key result
Parameter:
SI
Remarks:
lymph node cell count
Value:
1.267
Test group / Remarks:
50 % w/w test item
Remarks on result:
other: SI: 1.386 (lymph node weight)
Key result
Parameter:
SI
Remarks:
ear weight
Value:
1.006
Test group / Remarks:
50 % w/w test item
Remarks on result:
other: SI: 1.125 (ear thickness)
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
Treatment with Pigment Al-Co-Zn 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. In addition, the lymph node weight was not increased significantly. Hence, the test item is classified as not sensitising.
The threshold level for the ear weight of 1.1 was not exceeded, i.e. no irritating properties were noted.
(Please refer to "Attached background material" for the raw data on stimulation indices)

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

BODY WEIGHTS:
The animal body weight was not affected by the treatment.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the present test conditions, Pigment Al-Co-Zn at concentrations of 10%, 25% or 50% (w/w) in acetone/olive oil (3+1, v/v) did not reveal any sensitising properties in the local lymph node assay. Therefore, the test item must not be classified and labelled according to regulation (EC) No.: 1272/2008.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Read-across argumentation: due to the structural similarity of cobalt aluminate blue spinel and cobalt zinc aluminate blue spinel(both pigments are spinels with Co2+in octahedral lattice position; both pigments consist of the same components: Co, Al, Zn, see Table 1 below), as well as their similar physico-chemical properties (solubility) and their bioaccessibility (tables 2 and 3), read-across is made use of to address this endpoint.

Table 1.

Pigment

CAS

EC

Structure type

Composition

Cobalt zinc aluminate blue spinel

68186878

269-049-5

Spinel

Co: 15.26%, Al: 28.42%, Zn: 20.49%

Cobalt aluminate blue spinel

1345-16-0

310-193-6

 

Spinel

Co: 31%, Al: 30.7%, Zn: 1.12%

 

Table 2. Cobalt aluminate blue spinel:Concentration of cobalt in artificial media, calculated nominal cobalt concentration and dissolved amount of cobalt.

media and sample

total Co ± SD in method blanks [µg/L]

total Co ±SD in sample vessels [µg/L]

Co ± SD in sample vessels with blank subtraction [µg/L]

calculated nominal Co concentration in [µg/L]#

dissolved amount Co in artificial media [%] normalizedfor measured background in method blank

ALF 2h

0.07 ± 0.01

6.78 ± 0.22

6.70 ± 0.22

31469

0.02 ± <0.01

ALF 24h

0.08 ± 0.01

12.9 ± 0.37

12.9 ± 0.37

31469

0.04 ± <0.01

ASW 2h

<LOD

<LOD

<LOD

31123

-

ASW 24h

<LOD

1.56 ± 0.08

1.56 ± 0.08

31123

0.01 ± <0.01

GMB 2h

0.02 ± <0.01

0.08 ± 0.01

0.06 ± 0.01

31396

<0.001

GMB 24h

0.03 ± 0.01

0.40 ± 0.02

0.37 ± 0.02

31396

0.001 ± <0.001

GST 2h

<LOD

4.96 ± 0.16

4.96 ± 0.16

31517

0.02 ± <0.01

GST 24h

<LOD

9.18 ± 0.08

9.18 ± 0.08

31517

0.03± <0.01

PBS 2h

<LOD

0.05 ± 0.02

0.05 ± 0.02

31399

<0.001

PBS 24h

<LOD

0.46 ± 0.14

0.46 ± 0.14

31399

0.002 ± <0.001

# (initial weight (e.g. 50mg)*30.99 § (percentage cobalt in test item)/100)*2 (multiplication to calculate cobalt amount in one litre --> 100 mg/L) = nominal cobalt concentration in [mg/L]/1000 = nominal cobalt concentration in [µg/L]. § according to CoA 39.40% Co as CoO ==> 78.65% cobalt in CoO ==>
(39.40% * 78.65%)/100% = 30.99% Co in test item

 

Table 3. Cobalt zinc aluminate blue spinel (read-across substance):Content (a.i.): 53.7 % Al2O3; 19.4 % CoO; 25.5 % ZnO

(test item) 

 Material 

 Exposure 

 period 

 GMB 

 pH 7,4 

 PBS 

 pH 7,2 

 ASW 

 pH 6,5 

 ALF 

 pH 4,5 

 GST 

 pH 1,7 

 Co release 

 2 h 

 10,6±11,0 

 2,7±2,3 

 7,1±2,5 

 14±4 

 7,6±0,6 

 24 h 

 0,7±0,7 

 1,5±1,2 

 4,5±1,0 

 5,2±0,2 

 10,7±0,5 

Average total concentration of released elements [μg/L] and the standard deviation of triplicate samples in the different media. Blank values for each individual media and exposure period have been subtracted.

Skin sensitisation

Read-across is performed from cobalt zinc aluminate blue spinel to cobalt aluminate blue spinel. Cobalt zinc aluminate blue spinel has been tested negative for skin sensitisation, and does not require classification as a skin sensitiser according to Directive 67/548/EEC and its subsequent amendments, and according to Regulation (EC) No 1272/2008 and subsequent regulations.

Hence, it is considered justified to conclude that cobalt aluminate blue spinel likewise is void of skin sensitising properties, and does not require classification for skin sensitisation.

Respiratory sensitisation

Endpoint conclusion
Additional information:

Read-across argumentation: due to the structural similarity of cobalt aluminate blue spinel and cobalt zinc aluminate blue spinel (both pigments are spinels with Co2+in octahedral lattice position; both pigments consist of the same components: Co, Al, Zn, see Table 1 below), as well as their similar physico-chemical properties (solubility) and their bioaccessibility (tables 2 and 3), read-across is made use of to address this endpoint.

Table 1.

Pigment

CAS

EC

Structure type

Composition

Cobalt zinc aluminate blue spinel

68186878

269-049-5

Spinel

Co: 15.26%, Al: 28.42%, Zn: 20.49%

Cobalt aluminate blue spinel

1345-16-0

310-193-6

 

Spinel

Co: 31%, Al: 30.7%, Zn: 1.12%

 

 

Respiratory sensitisation

There is no specific indication whatsoever to assume any potential for respiratory hypersensitivity of cobalt aluminate blue spinel for the following reasons:

(1) during long-lasting industrial practice in the inorganic pigment industry, no cases of hypersensitivity have been observed until now by workers exposed directly to the substance

(2) in contrast to bioavailable cobalt substance which are known to elicit sensitising properties, cobalt aluminate blue spinel is considered an inert, non-bioaccessible substance chemically and structurally very similar to cobalt zinc aluminate blue spinel which has been tested negative for skin sensitisation (LLNA assay)

(3) bioaccessibility data for both substances confirm that the substance cobalt aluminate blue spinel can be considered as inert (similar to cobalt zinc aluminate blue spinel), and shows very limited solubility in physiological media; in particular with respect to the release of cobalt under physiological conditions, the release is even lower than that documented for cobalt zinc aluminate blue spinel.

Table 2. Cobalt aluminate blue spinel:Concentration of cobalt in artificial media, calculated nominal cobalt concentration and dissolved amount of cobalt.

media and sample

total Co ± SD in method blanks [µg/L]

total Co ±SD in sample vessels [µg/L]

Co ± SD in sample vessels with blank subtraction [µg/L]

calculated nominal Co concentration in [µg/L]#

dissolved amount Co in artificial media [%] normalizedfor measured background in method blank

ALF 2h

0.07 ± 0.01

6.78 ± 0.22

6.70 ± 0.22

31469

0.02 ± <0.01

ALF 24h

0.08 ± 0.01

12.9 ± 0.37

12.9 ± 0.37

31469

0.04 ± <0.01

ASW 2h

<LOD

<LOD

<LOD

31123

-

ASW 24h

<LOD

1.56 ± 0.08

1.56 ± 0.08

31123

0.01 ± <0.01

GMB 2h

0.02 ± <0.01

0.08 ± 0.01

0.06 ± 0.01

31396

<0.001

GMB 24h

0.03 ± 0.01

0.40 ± 0.02

0.37 ± 0.02

31396

0.001 ± <0.001

GST 2h

<LOD

4.96 ± 0.16

4.96 ± 0.16

31517

0.02 ± <0.01

GST 24h

<LOD

9.18 ± 0.08

9.18 ± 0.08

31517

0.03± <0.01

PBS 2h

<LOD

0.05 ± 0.02

0.05 ± 0.02

31399

<0.001

PBS 24h

<LOD

0.46 ± 0.14

0.46 ± 0.14

31399

0.002 ± <0.001

# (initial weight (e.g. 50mg)*30.99 § (percentage cobalt in test item)/100)*2 (multiplication to calculate cobalt amount in one litre --> 100 mg/L) = nominal cobalt concentration in [mg/L]/1000 = nominal cobalt concentration in [µg/L]. § according to CoA 39.40% Co as CoO ==> 78.65% cobalt in CoO ==>
(39.40% * 78.65%)/100% = 30.99% Co in test item

 

Table 3. Cobalt zinc aluminate blue spinel (read-across substance):Content (a.i.): 53.7 % Al2O3; 19.4 % CoO; 25.5 % ZnO

(test item) 

 Material 

 Exposure 

 period 

 GMB 

 pH 7,4 

 PBS 

 pH 7,2 

 ASW 

 pH 6,5 

 ALF 

 pH 4,5 

 GST 

 pH 1,7 

 Co release 

 2 h 

 10,6±11,0 

 2,7±2,3 

 7,1±2,5 

 14±4 

 7,6±0,6 

 24 h 

 0,7±0,7 

 1,5±1,2 

 4,5±1,0 

 5,2±0,2 

 10,7±0,5 

Average total concentration of released elements [μg/L] and the standard deviation of triplicate samples in the different media. Blank values for each individual media and exposure period have been subtracted.

 

(4)  Assuming that both effects (skin and respiratory sensitisation) for cobalt are based on similar immunological mechanisms, the absence of skin sensitisation properties supports the assumption of an absence of respiratory sensitisation.

In conclusion, the classification criteria acc. to regulation (EC) 1272/2008 as respiratory sensitiser are not met for the pigment cobalt aluminate blue spinel.

No cases of hypersensitivity have been observed until now by workers exposed directly to the substance and bioaccessibility data for both substances (cobalt zinc aluminate blue spinel and cobalt aluminate blue spinel) confirm that the substance cobalt aluminate blue spinel can be considered as inert (similar to cobalt zinc aluminate blue spinel), and shows very limited solubility in physiological media; in particular with respect to the release of cobalt under physiological conditions, the release is even lower than that documented for cobalt zinc aluminate blue spinel. Assuming that both effects (skin and respiratory sensitisation) for cobalt are based on similar immunological mechanisms, the absence of skin sensitisation properties supports the assumption of an absence of respiratory sensitisation.

Hence,  cobalt aluminate blue spinel does not meet the criteria as respiratory sensitiser and should not be classified as such.

Justification for classification or non-classification

Skin sensitisation

Read across is made to cobalt zinc aluminate blue spinel. Cobalt zinc aluminate blue spinel has no skin sensitisation potential and does not require classification as skin sensitiser according to Regulation (EC) No 1272/2008 and subsequent regulations. Hence, cobalt aluminate blue spinel will also not be classified.

Respiratory sensitisation

During long-lasting industrial practice in the inorganic pigment industry, no cases of hypersensitivity have been observed until now by workers exposed directly to the substance. In contrast to bioavailable cobalt substance which are known to elicit sensitising properties, cobalt aluminate blue spinel is considered an inert, non-bioaccessible substance chemically and structurally very similar to cobalt zinc aluminate blue spinel which has been tested negative for skin sensitisation (LLNA assay). Bioaccessibility data for both substances confirm that the substance cobalt aluminate blue spinel can be considered as inert (similar to cobalt zinc aluminate blue spinel), and shows very limited solubility in physiological media; in particular with respect to the release of cobalt under physiological conditions, the release is even lower than that documented for cobalt zinc aluminate blue spinel.

Assuming that both effects (skin and respiratory sensitisation) for cobalt are based on similar immunological mechanisms, the absence of skin sensitisation properties supports the assumption of an absence of respiratory sensitisation.

In conclusion, the classification criteria acc. to regulation (EC) 1272/2008 as respiratory sensitiser are not met for the pigment cobalt aluminate blue spinel.