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Administrative data

Description of key information

Cobalt zinc aluminate blue spinel has been tested in one in vitro skin irritation study (Commision regulation (EC) No. 440/2008 B.46) and one eye irritation study. All tests show a negative response, thus cobalt zinc aluminate blue spinel does not require classification either as skin or as eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-06-23 to 2010-06-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ECVAM international validation study on in vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6):559-601)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline for the testing of chemicals; Draft proposal for a new guideline, in vitro skin irritation: Reconstructed Human Epidermis (RhE) Test method, 11 December 2009, Vers. 4.
Version / remarks:
2009-12-11
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2009-03-30
Test system:
human skin model
Source species:
human
Cell type:
other: normal, human-derived epidermal keratinocytes
Cell source:
other: humans (adult donors)
Source strain:
not specified
Details on animal used as source of test system:
not applicable
Justification for test system used:
In an international validation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiSkin™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin™ (source: Skinethic Laboratories, 06000 Nice, France)
- Tissue lot number: 10-EKIN-023
- Delivery date: 2010-06-22
- Date of initiation of testing: 2010-06-23

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the end of the treatment interval the inserts were removed from the plate and rinsed with PBS to remove any residual test material. The inserts were placed in the plates with 2 mL
maintenance medium. The tissues were incubated for 42 hours ± 5 minutes. Following incubation, the tissue viablity was measured.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL per well
- Incubation time with MTT: 3 hours
- Extraction of Formazan: after a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Then, the tissue samples were immersed into extractant solution (0.5 mL extractant solution (isopropanol)). The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted about 70 hours in the refrigerator.
Per each tissue sample 2 × 200 µL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate. Optical density was determined with a spectrophotometer. Mean values were calculated from the 2 wells per tissue sample.
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm
- Filter: no reference filter was used

TEST FOR DIRECT MTT REDUCTION
For correct interpretation of results, the ability of the test item to directly reduce MTT was assessed. To test for this ability approx. 15 mg of the test item were added to 1 mL of MTT solution. The mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: absence of bacteria, fungus, and mycoplasma as well as absence of HIV1- virus, Hepatitis B antigen HBs, and Hepatitis C antibodies
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula: relative viability(%) = (OD test item/ OD negative control) x 100
For the test item and the positive control the mean relative viability ± standard deviation of the three individual tissues are calculated and used for classification according to the following prediction model: for the current test, an irritation potential of a test item according to EU classification (Regulation 1272/2008/EC; Category 2) is predicted if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 15 mg of the neat test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 15 µL deionised water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 15 µL of a 5% sodium lauryl sulfate (SLS) solution
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours ± 5 minutes
Number of replicates:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates
Vehicle:
unchanged (no vehicle)
Irritation / corrosion parameter:
% tissue viability
Value:
87.2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: after treatment with the negative control the absorbance values (1.131, 1.042, and 1.049 (mean: 1.074)) were well above the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 for the 15 minutes treatment interval.
- Acceptance criteria met for positive control: treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 19.3% (acceptability criterion: positive control is ≤ 40 %).
- Acceptance criteria met for variability between replicate measurements: the standard deviations between the % variabilities of the test item, the positive and negative controls were below 9% (threshold of the "OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation": ≤ 18%),
Please refer to the field "Any other information on results incl. tables" below.

Results after treatment with Pigment Al-Co-Zn

 

Dose group

Treatment Interval

Absorbance 570 nm
Tissue 1*

Absorbance 570 nm
Tissue 2*

Absorbance 570 nm
Tissue 3*

Mean Absorbance of 3 Tissues

Absorbance [%] Tissue 1, 2 + 3

Standard Deviation [%]

Rel. Absorbance

[% of Negative Control]**

Negative Control

15 min

1.131

1.042

1.049

1.074

105.3
97.0
97.7

4.6

100.0

Positive Control

15 min

0.304

0.124

0.193

0.207

28.3
11.5
18.0

8.5

19.3

Test Item

15 min

0.946

0.920

0.943

0.936

88.1
85.7
87.8

1.3

87.2

* Mean of three replicate wells after blank correction

** relative absorbance [rounded values]: (100 * (absorbance test item))/(absorbance negative control)

Historical data:

Positive Control

Negative Control

Number of Studies

85

Number of Studies

85

Period

July 2007 - June 2010

Period

July 2007 - June 2010

Mean Viability

17.8 %

Mean OD

1.051

Standard Deviation

10.7 %

Standard Deviation

0.185

Range of Viabilities

3% - 34%

Range of ODs

0.7 – 1.5

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is not irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not irritating to the skin.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-06-11 to 2010-06-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
2002-04-24
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2009-11-12
Species:
rabbit
Strain:
Himalayan
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Branch Löhndorf, 24601 Löhndorf/Post Wankendorf, Germany
- Age at study initiation: Approx. 2.5 - 3.5 months
- Weight at study initiation: 1.9 kg - 2.4 kg (Animal No. 1: 2.2 kg; Animal No. 2: 2.4 kg; Animal No. 3: 1.9 kg
- Housing: For 8 hours following test item application, the animals were kept singly in restrainers which allowed free movement of the head but prevented a complete body turn, wiping of the eyes with the paws and excluded irritation of the eye by excrements and urine. During the acclimatisation period and after the 8 hour period in restrainers, the animals were kept singly in cages with dimensions of 380 mm X 425 mm X 600 mm (manufacturer: Dipl.Ing. W. EHRET GmbH, 16352 Schönwalde, Germany).
- Diet (ad libitum): Commercial diet, ssniff® K-H V2333 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany
- Water (ad libitum): Tap water
- Acclimation period: At least 20 adaption days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 °C +/- 3 °C (maximum range)
- Relative humidity: 30 % - 70 % (maximum range; aim was 50 % - 60 %)
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg of the test item were administered into one eye each of three animals.The test item was placed into the conjunctival sac of the right eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second in order to prevent loss of the material. The left eye, which remained untreated, served as a control.
Duration of treatment / exposure:
1 hour after administration the eyes were rinsed.
Observation period (in vivo):
1, 24, 48 and 72 hours after administration
Number of animals or in vitro replicates:
3 male rabbits
Details on study design:
The test was perfrmed initially using one animal. As no corrosive or severe irritant effects were observed in this animal, 2 further animals were employed 24 hours after start of the initial test.

REMOVAL OF TEST SUBSTANCE
- Time after start of exposure: 1 hour after administration
- Washing (if done): The eyes were rinsed with portions of 20 ml 0.9 % aqueous NaCl solution, each.


SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: The eyes were examined ophthalmoscopically with a slit lamp prior to administration and 1, 24, 48 and 72 hours after administration.
24 hours after administration, fluorescein (Fluorescein SE Thilo drops (ALCON PHARMA GmbH, 79108 Freiburg, Germany) was applied to the eyes before being examined to aid evaluation of the cornea for possible lesions.

OTHER OBSERVATIONS: Any further lesions not covered by the scoring system were recorded. Body weight of all animals was measured at the beginning of the study and at the end of the study. Behaviour and food consumption were monitored.
No further information on the study design was stated.
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
Conjunctival redness (grade 1: Some blood vessels hyperaemic (injected)) was observed in all animals 1 hour after instillation. This effect was reversible after 24 hours.
The cornea and the irises were not affected by instillation of the test item and the fluorescein test perforemd 24 hours after instillation did not reveal any changes.
Other effects:
No systemic intolerance reactions were observed in all animals.
Interpretation of results:
GHS criteria not met
Conclusions:
According to the EC Regulation 1272/2008 and subsequent regulations, the Pigment Al-Co-Zn is non-irritating.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

One study has been performed on skin irritation (Heppenheimer, 2010, Commision regulation (EC) No. 440/2008 B.46) and one study was performed on eye irritating properties (Leuschner, 2010, OECD guideline 405).

After treatment with the test item pigment Al-Co-Zn the relative absorbance values were irrelevantly decreased to 87.2 %. This value is well above the threshold for irritancy of ≤ 50 %. Therefore, the test item is not considered to possess an irritant potential.

Conjunctival redness (grade 1: Some blood vessels hyperaemic (injected)) was observed in all animals 1 hour after instillation. This effect was reversible after 24 hours. The cornea and the irises were not affected by instillation of the test item and the fluorescein test perforemd 24 hours after instillation did not reveal any changes.

Justification for classification or non-classification

Skin irritation in vitro:

relative viability, mean, after 15 min incubation: score=87.2%

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item pigment Al-Co-Zn is not irritant to skin and therefore, the test item should not be classified and labeled as skin irritant according to regulation (EC) No.: 1272/2008.

Eye irritattion in vivo:

conjunctivae score, mean animal #1, 24 -72h: score=0            

conjunctivae score, mean animal #2, 24 -72h: score=0          

conjunctivae score, mean animal #3, 24 -72h: score=0          

cornea score, mean animal #1, 24 -72h: score=0

cornea score, mean animal #2, 24 -72h: score=0

cornea score, mean animal #3, 24 -72h: score=0

iris score, mean animal #1, 24 -72h: score=0

iris score, mean animal #2, 24 -72h: score=0

iris score, mean animal #3, 24 -72h: score=0

chemosis score, mean animal #1, 24 -72h; score=0

chemosis score, mean animal #2, 24 -72h; score=0

chemosis score, mean animal #3, 24 -72h; score=0

According to the EC Regulation 1272/2008 and subsequent regulations, the test item is non-irritating.

Respiratory irritation

The classification as respiratory irritant is covered under the endpoint specific target organ toxicity- single exposure. Please refer to the endpoint summaries on acute toxicity (endpoint 7.2) for further information.