Cobalt chromite green spinel

Endpoint summary

Description of key information

Skin irritation: not irritating (OECD 439; GLP compliant)

Eye irritation: not irritating (OECD 405; GLP compliant)

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:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2012-03-28 to 2012-04-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

During the MTT assay isopropanol / 2 N HCl 49:1 (v/v) should have been used as extractant solution. Due to inattention isopropanol without HCl was used as extractant solution. Due to Harlan CCR’s experimental experience concerning formazan salt extraction with and without acidifying of isopropanol, and due to the long extraction period of nearly three days, the risk of inadequate extraction is minimized. Therefore, the described deviation did not have a detrimental impact on the outcome of the 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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2010-07-22

Deviations:

no

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)

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Specific details on test material used for the study:

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

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human-derived epidermal keratinocytes

Cell source:

other: not specified

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:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin™ tissues (purchased from SkinEthic Laboratories)
- Tissue batch number: 12-EKIN-013
- Experiation date: 2012-04-02
- Delivery date: 2012-03-28
- Date of initiation of testing:2012-03-28

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 the tissues were rinsed with PBS to remove any residual test material. Then the inserts were placed in the plates with maintenance medium. The tissues were incubated for about 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time with MTT: 3 hours
- Extraction of formazan: after the incubation period, MTT solution was aspirated from the wells and the wells were rinsed with PBS. Tissue samples were cut out of the
inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by pipetting 0.5 mL extractant solution (isopropanol) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for about 69 hours in the refrigerator.
Per each tissue sample 2 x 200 μL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. Optical density (OD) was read with a spectrophotometer. Mean values were calculated from the 2 wells per tissue sample.
- Spectrophotometer: microplate reader (Versamax® Molecular Devices)
- Wavelength: 570 nm
- Filter: 1 nm

TEST FOR DIRECT MTT REDUCTION
To test for the ability of the test item to directly reduce MTT approx. 10 mg of the test item were added to 1 mL of MTT solution and 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 persumed to have reduced the MTT.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: tissues passed 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 HIV 1 and 2 antibodies, hepatitis C antibodies and absence of hepatitis B antigen
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density 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 was calculated according to the following formula: relative viability(%) = (OD test item/ OD mean of negative control) x 100
For the test item and the positive control the mean relative viability ± standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model: if the mean relative tissue viability of three individual tissues is less than or equal to 50% of the negative control, the test item needs to be classified and labeled for its skin irritation potential: Category 2 – irritant, H315 according to Regulation (EC) No 1272/2008.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approximately 10 mg of the test item was applied to each tissue replicate

VEHICLE
- Amount(s) applied (volume or weight with unit): 20 μL of deionised water

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

Duration of treatment / exposure:

15 ± 1 minutes

Duration of post-treatment incubation (if applicable):

about 42 hours

Number of replicates:

Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Value:

94.8

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 (1.233, 1.178, and 1.129, respectively) were well within 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 21.4 % (acceptability criterion: positive control is ≤ 40 %).
- Acceptance criteria met for variability between replicate measurements: the standard between the % variabilities of the test item, positive and negative controls were below 5 % (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%).
Please refer to the field "Any other information on results incl. tables" below

HISTORICAL DATA

Positive Control		Negative Control
Number of Studies	163	Number of Studies	163
Period	July 2007 – March 2012	Period	July 2007 – March 2012
Mean Viability	19.7%	Mean OD	1.012
Standard Deviation	10.0%	Standard Deviation	0.215
Range of Viabilities	0.7% - 39.7%	Range of ODs	0.590 – 1.680

Table 1: Results after treatment with cobalt chromite blue green spinel (Pigment Blue 36) and controls

Dose group	Treatment Interval	Absor-bance 570 nm Tissue 1*	Absor-bance 570 nm Tissue 2*	Absor-bance 570 nm Tissue 3*	Mean Absor-bance of 3 Tissues	Rel. Absor-bance [% of Negative Control]**
Negative Control	15 min	1.233	1.178	1.129	1.180	100.0
Positive Control	15 min	0.254	0.254	0.250	0.253	21.4
Test item	15 min	1.109	1.139	1.110	1.119	94.8

* Mean of two replicate wells after blank correction

** relative absorbance per treatment group [rounded values]: 100 x (meanabsorbance_testitem)/(mean absorbancenegative control)

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, other

Remarks:

in vivo study

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2012-04-05 to 2012-04-22

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.

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

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

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. 4.5 - 5.5 months
- Weight at study initiation: 2.7 to 3.0 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 libitumbefore and after the exposure period ): commercial diet, ssniff® K-H V2333 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum before and after the exposure period): tap water
- Acclimation period: at least 20 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C ± 3°C (maximum range)
- Relative humidity: 30% - 70% (maximum range)
- 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 volume of particulates was measured after gently compacting them, e.g. by tapping the measuring container.
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

Observation period (in vivo):

1, 24, 48 and 72 hours after the administration

Number of animals or in vitro replicates:

3 male rabbits

Details on study design:

The test was performed 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
- Washing: the eyes were rinsed with portions of 20 mL 0.9% aqueous NaCl solution, each.
- Time after start of exposure: 1 hour after administration

SCORING SYSTEM: according to the Draize scale
Any further lesions are listed.

TOOL USED TO ASSESS SCORE:
The eyes were examined ophthalmoscopically with a slit lamp prior to the administration and 1, 24, 48 and 72 hours after the administration. The eye reactions were observed and registered.
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.

OBSERVATIONS:
Body weight of all animals was measured at the beginning and the end of the study. Behaviour and food consumption were monitored.

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:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

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:

Under the present test conditions, a single instillation of 100 mg cobalt chromite blue green spinel (Pigment Blue 36) per animal into the conjunctival sac of the right eye of three rabbits caused following change:
Conjunctival redness (grade 1) was observed in one animal 24 hours after instillation.
The corneae and irises were not affected by instillation of the test item.
24 hours fluoresscein test: all animals: no pathological findings.

Other effects:

There were no systemic intolerance reactions.

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not irritating to the eyes.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does not require classification as eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Read-across approach:

 

Cobalt chromite green spinel is a spinel-type oxide/pigment with the general structural element AB₂O₄which 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 substanceCobalt 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 17.2µg/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 0.264µg/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 pigmentCobalt chromite blue green spinelin physiological media was in a range of below LODand 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 pigmentCobalt chromite green spinelafforded the following solubility at 1mg loading after 28 days:

Cobalt: 2.038 ± 0.008 and 0.790 ± 0.039μg/L at pH 8 and pH 6, respectively

Chromium: 1.489 ± 0.008μg/L and <LOD (<0.7829μg/L) at pH 8 and pH 6, respectively

Zinc: 1.840 ± 0.378μg/L and <LOD (<0.0961μg/L) at pH 8 and pH 6, respectively

 

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 substanceCobalt 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.

Read-across information:

Skin irritation

One reliable in vitro study described by Heppenheimer (2012) (OECD 439; GLP compliant) is considered to be reliable without restrictions. The substance was determined to be not irritating to the skin.

Eye irritation

One reliable in vivo study described by Leuschner (2012) (OECD 405; GLP compliant) is considered to be reliable without restrictions. The substance was determined to be not irritating to the eyes.

Justification for classification or non-classification

Skin irritation

The substance does not possess an skin irritating potential based on an in vitro OECD 439 (2010) test and does not require classification as skin irritating according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation

The substance does not possess an eye irritating potential based on an in vivo OECD 405 (2002) test and does not require classification as eye irritating according to Regulation (EC) No 1272/2008 and its subsequent adaptations.