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Administrative data

Description of key information

Skin irritation: not irritating (equivalent or similar to OECD 404; non-GLP compliant)

Eye irritation: not irritating (OECD 492; GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-10-08 to 1996-10-11
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Purity and stability were missing - Age and weight at the conclusion of testing of the animals were not stated
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
1992-07-17
Deviations:
yes
Remarks:
please refer to "Rationale for reliability incl. deficiencies" above
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hare Marland, Hewitt, New Jersey
- Age at study initiation: young adult
- Weight at study initiation: 2408 to 2779 grams
- Housing: the rabbits were housed singly in suspended, stainless steel, wire-mesh cages.
- Diet (daily): approximately 125 grams of Purina® Certified High Fiber Rabbit Chow® #5325
- Water (ad libitum, except during the exposure period): water
- Acclimation period: rabbits were quarantined, weighed, and observed for general health for approximately 2 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C ± 1°C
- Relative humidity: 50% ± 10%
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
occlusive
Preparation of test site:
shaved
Vehicle:
water
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approximately 0.5 g of the test substance, moistened with deionized water

Duration of treatment / exposure:
approximately 4 hours
Observation period:
approximately 1, 24, 48, and 72 hours after removal of the test substance
Number of animals:
6 male rabbits
Details on study design:
TEST SITE
- Area of exposure and type of wrap if used: on the day prior to treatment, the hair of New Zealand White rabbits was closely shaved to expose the skin from the scapular to the lumbar region of the back. Each rabbit was placed into a stock which had been fitted with a piece of rubber sheeting, approximately 8″ x 18″. The rabbits remained in the stocks throughout the exposure period and during that time did not have access to food or water.
The test substance moistened with the vehicle was applied directly on the test site beneath a 2-inch gauze square that was held in place with non-irritating tape. The rubber sheeting was then wrapped around the animal and secured with clips to retard evaporation and to keep the test substance in contact with the skin without undue pressure. In the absence of visible evidence to the contrary, the test substance was assumed to be stable under the conditions of administration. One other test substance was tested concurrently on a separate, localized test site on these rabbits.

REMOVAL OF TEST SUBSTANCE
- Washing: the rubber sheeting was loosened, and the skin at the corners of the gauze squares was marked with a waterproof pen; wrappings and gauze squares were then removed. The test sites were gently washed with Ivory® soap and warm water to remove excess test substance. The skin was gently patted dry, and the animals were returned to their cages.
- Time after start of exposure: approximately 4 hours after application of the test substance

SCORING SYSTEM: according to the Draize scale
The test sites were evaluated for evidence of dermal effects
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: At the 1 hour observation the compound adhered to the skin, but the test site could be evaluated.
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: Throughout the observation period the compound adhered to the skin, but the test site could be evaluated.
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: At the 1 hour observation the compound adhered to the skin, but the test site could be evaluated.
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: At the 1 hour observation the compound adhered to the skin, but the test site could be evaluated.
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal #5
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: At the 1, 24 and 48-hour observations the compound adhered to the skin, but the test site could be evaluated.
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal #5
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal #6
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: Throughout the observation period the compound adhered to the skin, but the test site could be evaluated.
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal #6
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
The test substance adhered to the skin of all rabbits; however, the test sites could still be evaluated for irritation. No dermal irritation was observed throughout the study.
Interpretation of results:
GHS criteria not met
Conclusions:
The test substance is non-irritating to the skin.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as skin irritant.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-01-19 to 2021-02-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
2019-06-18
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model
Version / remarks:
2015-06-29
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed signed 2019-10-23
Details on test animals or tissues and environmental conditions:
JUSTIFICATION OF THE TEST METHODS AND CONSIDERATIONS REGARDING APPLICABILITY:
This in vitro method is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS “No Category”) without further testing within a tiered testing strategy from those requiring classification and labelling (UN GHS categories 1 and 2). Therefore, it can be used for regulatory purposes as an initial step in the bottom-up approach or as one of the last steps in a top-down approach to test eye irritation/corrosion potential. It is not intended to differentiate between UN GHS “Category 1” (serious eye damage) and UN GHS “Category 2” (eye irritation) which would require additional testing. Ocular irritation potential is predicted by the relative viability of the tissue after a single exposure to the test substance. Relative viability is determined by measuring the MTT dye to formazan conversion by the EpiOcular™ tissue construct after topical exposure to the test substance.

RhCE TISSUE CONSTRUCT USED: EpiOcular™ (Lot No.: 30694; Standard Assay Kit and MTT-100 kit; source: MatTek Corporation (82105 Bratislava, Slovakia))
The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

Please also refer to the field "Attached background material " below.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg (≙ 83.3 mg/cm2)were applied to the tissue surface. The test item was tested neat.
Duration of treatment / exposure:
6 hours
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
about 18 hours
Number of animals or in vitro replicates:
Number of EpiOcular tissues:
Test item: duplicates
Negative control: duplicates
Positive control: duplicates
Details on study design:
PRE-TEST FOR MTT INTERFERENCE
- To test if a test item directly reduces MTT, 1 ml of a MTT solution (1 mg/mL) including 50 ± 2 mg of the test item was incubated for 180 min under (37 ± 1.5°C, 5 ± 0.5% CO2).
- 50 µL deionised water in MTT solution was used as negative control.
- After incubation the change of colour was determined by the unaided eye.

PRE-TEST FOR COLOUR INTERFERENCE
- To check the colouring potential of the test item, 50 ± 2 mg of the test item was added to 1 mL of deionised water and mixed. 1 mL of deionised water was used as control (blank). Both were incubated for 60 min (37 ± 1.5°C, 5 ± 0.5% CO2).
- In parallel, 50 ± 2mg of the test item was added to 2 mL of isopropanol and mixed. A control (2 mL of isopropanol, blank) was run concurrently. Both were incubated for 3 hours at room temperature.
- After incubation the presence of the staining was evaluated by OD measurement.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE AND COLOUR INTERFERENCE
Since the test item interfered with MTT and reduced it to formazan by itself in the second pre-experiment two additional controls in duplicates run with the main experiment – the freeze-killed tissue controls (killed controls = KC):
- Deionised water treated freeze-killed tissues (NC_KC)
- Test item treated freeze-killed tissues (TI_KC)
At the end, the following data correction procedure for Viability plus killed controls were performed:
Corrected viability (%) = TI viability – (TI_KC viability – NC_KC viability)

DETAILS OF THE TEST PROCEDURE USED
Pre-warming:
- EpiOcularTM tissues were equilibrated at room temperature for 15 min. The inserts with the tissues were transferred into 6-well-plates containing 1.0 ml assay medium and incubated for 60 minutes (37 ± 1.5°C, 5 ± 0.5% CO2). Afterwards, the medium was changed and a further pre-incubation for 17 h 53 min (37 ± 1.5°C, 5 ± 0.5% CO2) follows.
- Treatment:
After pre-warming and prior to application of the test item respectively the controls, all tissues (the freeze killed tissues also) were pre-wetted with 20 µL Ca2+Mg2+free-DPBS and incubated for 30 min (37 ± 1.5°C, 5 ± 0.5% CO2).
Concurrent negative and positive control were applied at a volume of 50 µL and for the test item 50 mg to the tissue surface and incubated for 6 h in assay medium (37 ± 1.5°C, 5 ± 0.5% CO2)
Afterwards all tissues were rinsed 3 times in 100 mL DPBS and incubated for 25 min in 5 ml assay medium at room temperature in a 12-well plate (post-exposure immersion). At the end of this incubation the tissues were transferred into a 6-well plate with 1 ml assay medium and were incubated for a post exposure incubation for 18 h (37 ± 1.5°C, 5 ± 0.5% CO2).
- MTT Assay:
The tissues were placed into the 24-well plate containing 300 µL of MTT solution and were incubated for 180 min (37 ± 1.5°C, 5 ± 0.5% CO2).
At the end of this incubation the tissues were transferred into new 24-well plates containing 2 mL isopropanol. The formazan salt was extracted with isopropanol within 2 h 10 min while shaking at room temperature.
Then, the extracts were mixed and two 200 µL aliquots formazan solution were transferred to a 96-well plate for OD measurement. 200 µL of isopropanol were added to the wells designated as blanks for 96-well plate.
-Measurement:
The optical density (OD570nm) was determined spectrophotometrically in duplicates by a microplate reader (Versamax® Molecular Devices). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

REMOVAL OF TEST MATERIAL
Afterwards all tissues were rinsed several times in 100 ml of Ca2+/Mg2+-free PBS

TEST ACCEPTANCE CRITERIA
The test meets acceptance criteria:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.5
- mean relative tissue viability of the positive control is < 50%
- relative tissue viability difference of replicate tissues is < 20%
- OD control values are not below historically established boundaries/ positive and negative control mean values and acceptance ranges based on historical data.

DEMOSTRATING OF PROFICIENCY IN PERFORMING THE TEST METHOD BEFORE ROUTINE USE BY TESTING OF THE PROFICIENCY CHEMICALS
Prior to routine use of EpiOcularTM EIT for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the eye irritation potential of fifteen proficiency chemicals listed in Table 1 of OECD TG 492. The respective proficiency certificate given by MatTek is attached in the field "Attached background material" below.

DESCRIPTION OF DATA EVALUATION
1) The mean OD value of the two wells for each tissue and the blank control (ODBlk) was calculated (Mean [OD570] (well 1 and well 2).
2) The mean ODBlk was subtracted from each mean OD value of the two wells.
(Mean [OD570] blank corr. (well 1 and well 2)). These values were used for all further calculations below.
3) The mean OD of the two relating tissues for each test group (negative control (NC), positive control (PC)) and the test item (TI) was calculated with the blank corrected mean OD (Mean [OD570] of T1 and T2)
4) The percent viability of each test group relative to the negative control (= 100%) was calculated:
Viability (%) =100 × (mean OD_TI/PC/NC) / mean OD_NC)
5) The relative OD of each tissue per test group was calculated. 100 divided by the mean ODNC T1 and T2 x mean OD of each test group.
6) The difference of the viability values between duplicate tissues was calculated: The relative OD of T2 was subtracted from T1.

PREDICTION MODEL
If the test item-treated tissue viability is > 60% after exposure and post-exposure incubation relative to the negative control treated tissue viability, the test item is identified as not requiring classification and labelling according to UN GHS (No Category).
If the test item-treated tissue viability is ≤ 60% after exposure and post-exposure incubation relative to negative control treated tissue viability, no prediction can be made for this test item.

Irritation parameter:
other: mean percent tissue viability  (migrated information)
Value:
81.94
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:
TEST FOR MTT INTERFERENCE
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent showed purple colour. Therefore, an additional test with freeze-killed tissues was necessary in the main experiment.

TEST FOR COLOUR INTERFERENCE
In the colour interference the test item not changed colour when mixed with deionised water or isopropanol, the mean OD of the test item in deionised water or isopropanol was < 0.08 . Therefore, no additional viable tissues (without MTT addition) were necessary.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of EpiOcularTM EIT for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the fifteen proficiency chemicals listed in Table 1 of OECD TG 492. The respective proficiency certificate given by MatTek is annexed to this endpoint study record.

ACCEPTANCE OF RESULTS:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.8 (1.720)
- mean relative tissue viability of the positive control is < 50% (18.32%)
- relative tissue viability difference of replicate tissues is < 20% (0.1 p.p. to 1.23 p.p.)
- The OD control values of the positive control were within the historically established boundaries.
- positive and negative control mean values and acceptance ranges based on historical data.
The acceptance criteria were met.

Please also refer to the field "Another information on results incl. tables" below.

Results after treatment with Cobalt aluminate blue spinel and controls

Treatment Group

Tissue No.

Well 1 [OD570]

Well 2 [OD570]

Mean [OD570] (Well 1 and well 2)

Mean [OD570] blank corr. (Well 1 and well 2)

Mean [OD570] of T1 and T2

Mean tissue viabil. [%]

Viabil. of T1 and T2 [%]

Diff. of viabil. between T1 and T2 [p.p.]

Blank

-

0.037

0.036

0.037

-

-

-

-

-

Negative Control

1

1.770

1.742

1.756

1.719

1.720

100.0

100.0

0.10

2

1.794

1.721

1.758

1.721

100.0

Positive Control

1

0.360

0.350

0.355

0.319

0.315

18.32

18.5

0.40

2

0.345

0.351

0.348

0.312

18.1

Test Item

1

1.492

1.423

1.458

1.421

1.411

81.94*

82.6

1.23

2

1.436

1.438

1.437

1.400

81.4

Negative Control
Freeze killed Tissues

1

0.072

0.070

0.071

0.034

0.038

2.19

2.0

0.41

2

0.079

0.077

0.078

0.041

2.4

Test Item Freeze killed Tissues

1

0.069

0.069

0.069

0.032

0.039

2.26

1.9

0.75

2

0.082

0.082

0.082

0.045

2.6

* Corrected viability (%) = TI viability – (TI_KC viability – NC_KC viability)

Historical Control Data

Positive Control; OD at 570 nm after

exposition to Methyl acetate (MatTek)

Negative Control OD at 570 nm

DPBS (MatTek)

Mean Viability

27.13%

Mean Absorption

1.70

Standard Deviation

9.54 p.p.

Standard Deviation

0.29

Range of Viabilities

6.73%–42.54%

Range of Absorbance*

1.02–2.32

Mean Absorption

0.46

* should be 0.8 - 2.8 (OECD 439)

or 1.0 - 2.5 (MatTek)

Standard Deviation

0.17

Range of Absorbance

0.08–0.79

Data of 33 sets of controls shared between 75 studies performed from June 2016 until May 2020. (p.p. – percentage points)

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, in this study and under the experimental conditions reported, Cobalt aluminate blue spinel does not require classification and labelling for eye irritation or serious eye damage according to UN GHS and EU CLP.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

One reliable in vivo study described by Sarver (1996) (equivalent or similar to OECD 404; non-GLP compliant) is considered to be reliable with restrictions. The substance was determined to be not irritating to the skin.

Eye irritation

The substance was not observed to be an eye irritant in a reliable in vitro eye irritation study according to OECD 492.

Justification for classification or non-classification

Skin irritation

The substance does not possess a skin irritation potential based on an in vivo study similar to OECD 404 and does not require classification as skin irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

 

Eye irritation

The substance does not possess an eye irritation potential based on an in vitro OECD 492 study and does not require classification as eye irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.