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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:
experimental study
Adequacy of study:
key study
Study period:
2012-03-28 to 2012-04-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study reliable without restrictions 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.
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:
2009-08-24
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
Details on test animals or test system and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.
Vehicle:
water
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approximately 10 mg of the neat test item were applied to each of triplicate tissues (≙ 26.32 mg/cm2).

VEHICLE
- Amount(s) applied (volume or weight with unit): 20 µL of deionised water
Duration of treatment / exposure:
15 ± 1 minutes
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
CELL CULTURE
EpiSkin™ kits (Lot No.: 12-EKIN-013) are purchased from SkinEthic Laboratories (06000 Nice, France). The EpiSkin™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
EpiSkin™ tissues were shipped with ice packs on medium-supplemented agarose gels in a 12-well plate and reached Harlan CCR on March 28, 2012. On day of experiment EpiSkin™ tissues were transferred to 12-well plates with maintenance medium.

TEST FOR DIRECT MTT REDUCTION
For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 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 presumed to have reduced the MTT. A colour change could not be observed.

TREATMENT
- Prewarming of EpiSkin™ tissues: after approximately 3 hours incubation of the EpiSkin™ tissues, they were treated with the test item. Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium.
- The negative control (deionised water (Lot no. 260112); volume 10 µL) and positive control (5% SLS (Sodium lauryl sulphate, Fluka, Sigma-Aldrich 89555 Steinheim) solution in deionised water, prepared freshly prior to the performance of the experiment; volume 10 µL), and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues. Additionally, the test item tissues were wetted with 20 µL of deionised water. The 12-well plates were placed into the incubator for 15 ± 1 min at 37 ± 1.5 °C, 5 ± 0.5% CO2.
- After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.
- The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for about 42 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2.

CELL VIABILITY TEST
Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues (Faller, C., Bracher, M., Dami, N., Roguet, R., 2002. Predictive ability of reconstructed human epidermis equivalents for assessment of skin irritation of cosmetics. Toxicology in vitro 16 (5), 557-552).
The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential (see OECD TG 439) and is used for the purpose of classification as irritating or non-irritating according to chemicals law (EU CLP, UN GHS).
After the treatment procedure was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to plates filled with 2 mL assay medium containing 0.3 mg/mL MTT per well. 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. 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 gently 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. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) with 570 ± 1 nm filter. Mean values were calculated from the 2 wells per tissue sample.

EVALUATION OF RESULTS
The mean 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 (%) = (ODtest item/ODnegative 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 R38 (according to directive 67/548/EEC), H315 (according to regulation (EC) 1272/2008) is recommended if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control.

ACCEPTABILITY OF THE ASSAY
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is ≥ 0.6 till ≤ 1.5.
The standard deviations in between tissues of the same treatment group should be ≤ 18%.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 40%.
The acceptance limit of the IC50 should be between 1.0 and 3.0 mg/mL after 18 hours treatment with SLS (please also refer to "Attached background material" below) .
Irritation / corrosion parameter:
other: other: relative viability (%)
Value:
97.6
Remarks on result:
other:
Remarks:
Basis: mean. Time point: after 15 min incubation. Reversibility: no data. (migrated information)
Irritant / corrosive response data:
After treatment with the test item copper chromite black spinel (Pigment Black 28) the mean relative absorbance value decreased irrelevantly to 97.6%. This value is well above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

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 copper chromite black spinel (Pigment Black 28) 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

1.079

1.268

1.107

1.151

97.6

* Mean of two replicate wells after blank correction

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

- after treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD

≥ 0.6 till ≤ 1.5 for the 15 minutes treatment interval thus showing the quality of the tissues.

- treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 21.4% thus ensuring the validity of the test system.

- The standard deviations between the % variabilities of the test item, the positive and negative controls were below 9% (threshold of the "OECD TG 439 Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.

- optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item is not irritating to the skin.
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is not a skin irritant.
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 vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-04-05 to 2012-04-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
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. 3.5 - 5.5 months
- Weight at study initiation: 2.2 to 2.5 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)
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
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 at the end of the study. Behaviour and food consumption were monitored.
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #1
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #1
Time point:
other: 24,48, and 72 hours
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #1
Time point:
other: 24, 48, and 72 hours
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #1
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #2
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #2
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #2
Time point:
other: 24, 48, and 72 hours
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #2
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #3
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #3
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #3
Time point:
other: 24, 48, and 72 hours
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal# 3
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
4
Irritant / corrosive response data:
Under the present test conditions, a single instillation of 100 mg copper chromite black spinel (Pigment Black 28) per animal into the conjunctival sac of the right eye of three rabbits caused following change:
Conjunctival redness (grade 1) was observed in all animals 24 hours after instillation.
The corneae and irises were not affected by instillation of the test item.
24 hours fluorescein test: all animals: no pathological findings
Other effects:
There were no systemic intolerance reactions.
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material is non-irritating to the eyes.
According to 67/548/EC and subsequent regulations, the substance is not classified as an eye irritant.
According to the EC Regulation No. 1272/2008 and subsequent regulations, the substance is not classified as an eye irritant.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional 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 selection of skin irritation / corrosion endpoint:
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.

Justification for selection of eye irritation endpoint:
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

Reference Heppenheimer (2012) will be used for classification. The mean relative absorbance (% of the negative control, correlating with mean tissue viability) after 15 minutes incubation in the in vitro human skin model test (EpiSkin, according to OECD 439) was as follows:

Heppenheimer (2012): relative viability = 97.6%

The classification criteria according to regulation (EC) 1272/2008 as skin irritant were not met, since the mean tissue viability was above the threshold for skin irritants of 50.0%.

Eye irritation

Reference Leuschner (2012) is considered as the key study for in vivo eye irritation and will be used for classification. During the study the test item was applied to one eye of three animals each and the eye irritation was scored according to the Draize scale.The corneae and irises were not affected by instillation of the test item. For all animals a mean score of 0.33 (24, 48 and 72 hours) was recorded for conjunctival redness (fully reversible within 48 hours). Thus, according to Regulation (EC) 1272/2008 and subsequent amendments the substance will not be classified as irritating to the eyes.

Respiratory irritation

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

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