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Administrative data

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:
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.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2010-07-22
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to
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. certificate)
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, kept dry in closed container
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 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): approx. 10 mg

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
Value:
92.3
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 silicic acid, zirconium salt, cadmium pigment encapsulated 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

1.057

1.089

1.124

1.090

92.3

* Mean of two replicate wells after blank correction

** relative absorbance per treatment group [rounded values]: 100 x (meanabsorbancetestitem)/(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:
experimental study
Adequacy of study:
key study
Study period:
2012-03-28 to 2012-04-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
2002-04-24
Deviations:
no
GLP compliance:
yes (incl. certificate)
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. 8 - 9 months
- Weight at study initiation: 2.4 - 2.7 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 before and after the exposure period): commercial diet, ssniff® K-H V2333 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitumbefore 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 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 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
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:
Under the present test conditions, a single instillation of 100 mg silicic acid, zirconium salt, cadmium pigment encapsulated per animal into the conjunctival sac of the right eye of three rabbits did not cause any changes.
The corneae, irises and conjunctivae 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:
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

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 = 92.3%

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, irises and conjunctivae were not affected by instillation of the test item. 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.