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Diss Factsheets

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:
2015-07-28 to 2015-08-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015-07-28
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) for use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200).
Version / remarks:
Rev. 3/26/2012
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-09-14
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: keep dry in closed container at room temperature
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 prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ 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:
other: Dulbecco's phosphate buffered saline (DPBS)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ skin model (source: MatTek Corporation, 82105 Bratislava, Slovakia)
- Tissue lot number: 21684
- Delivery date: 2015-08-11

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37 ± 1.5 °C (23 hours)
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 minutes, room temperature for 25 minutes
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
Tissues were rinsed with DPBS at least 15 times in order to remove any residual test material.

After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were again rinsed with DPBS. The tissues were then transferred into plates with fresh assay medium. Tissues were incubated for nearly 23 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation the inserts were transferred into new plates containing fresh medium. Thereafter tissues were incubated for another 19 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was about 42 hours. Following incubation, the tissue viablity was measured.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT/ EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/ well)
- 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 three times with DPBS. Inserts were transferred onto new plates and immersed into extractant solution (isopropanol). Tissues were completely covered from both sides and the plate was sealed to inhibit the isopropanol evaporation. The formazan salt was extracted for about 22 hours without shaking in the refrigerator in the dark.
After the extraction period was completed, the inserts were pierced to allow the extract to run into the well from which the insert was taken and the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. The optical density was determined with a spectrophotometer. Mean values were calculated from the 3 wells per tissue.
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm
- Filter bandwidth: 1 nm

TEST FOR COLOUR INTERFERENCE
Prior to the start of the test, the test item’s colour interference potential was evaluated. About 25 mg of the test item were mixed with 300 µL of deionised water. This mixture was incubated for 60 minutes at 37 ± 1.5 °C. Deionized water was used as negative control.

TEST FOR DIRECT MTT REDUCTION
For correct interpretation of results, the ability of the test item to directly reduce MTT was assessed. For this purpose, approx. 25 mg of the test item were added to 1 mL of MTT solution (resulting: 1 mg/mL). This mixture was incubated in the dark at 37 ± 1.5 °C for 60 minutes. MTT/MTT diluent was used as control. If the colour of the MTT turned blue/purple, the test item is persumed to have reduced MTT.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1- virus, Hepatitis B virus, and Hepatitis C virus
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 after blank correction. 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(%) = mean OD test item or positive control/ mean OD negative control) x 100
For the test item and the positive control the mean relative viability ± relative 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 or equal to 50% of the negative control, the test item needs to be classified and labeled for its skin irritauion 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. 25 mg of the test item, wetted with vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of a 5% Sodium Lauryl Sulfate (SLS) solution
Duration of treatment / exposure:
1 hour
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:
89.2
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:
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour. An additional control with viable tissues without MTT application was not necessary to be performed.
- Direct-MTT reduction: the colour did not turn blue/purple and the test item was not considered to be a MTT reducer. An additional test with freeze-killed tissues for data correction did not have to be performed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: after treatment with the negative control, the absorbance values (1.869, 1.894, and 1.949 (mean: 1.904)) were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 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 4.9 % (acceptability criterion: positive control is ≤ 20 %).
- Acceptance criteria met for variability between replicate measurements: the relative standard deviations between the % variabilities of the test item, the positive and negative controls in the main test was 18% at the highest (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: < 18%). The acceptance criteria were closely not met for the positive control (18 %) for its very low absorption values. Nevertheless, the study was valid due to the clearly positive result of the positive control.
Please refer to the field "Any other information on results incl. tables" below

HISTORICAL DATA

Positive Control

Negative Control [OD570]

Mean Viability

5.6%

Mean Absorption

1.796

Rel. Standard Deviation

2.4%

Rel. Standard Deviation

0.281%

Range of Viabilities

2.9% - 11.3%

Range of Absorbance

1.397 – 2.651

Mean Absorption

0.098

 

Rel. Standard Deviation

0.038%

Range of Absorbance

0.030 - 0.194

Data of 125 studies performed from November 2011 until May 2014

Table 1: Results after treatment with tin antimony grey cassiterite and controls

Dose Group

Treatment Interval

Absorbance 570 nm
Tissue 1*

Absorbance 570 nm
Tissue 2*

Absorbance 570 nm
Tissue 3*

Mean Absorbance of 3 Tissues

Mean Rel. Absorbance

[% of Negative Control]**

Negative Control

60 min

1.869

1.894

1.949

1.904

100.0

Positive Control

60 min

0.102

0.105

0.074

0.094

4.9

Test Item

60 min

1.660

1.651

1.781

1.697

89.2

* mean of three replicate wells after blank correction

** relative absorbance per treatment group [rounded values]: 100 x (mean absorbancetestitem/positive control)/(mean absorbance negative 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: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-05-26 to 2015-06-20
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:
2012-10-02
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2014-05-14
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: kept dry in closed container at room temperature
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Manfred Bauer Kaninchen, Lohe 7/1, 74632 Neuenstein, Germany
- Age at study initiation: approx. 6.5 - 7.5 months
- Weight at study initiation: 4.5 kg - 4.9 kg
- Housing: during the acclimatisation period and during the complete study period, 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 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 test item were fine grounded and administered 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 female rabbits
Details on study design:
INITIAL AND CONFIRMATORY TEST
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.

USAGE OF ANAESTHETICS AND SYSTEMIC ANALGESICS
Sixty minutes prior to test item administration, 0.01 mg Buprenovet®/kg bw were administered by subcutaneous (s.c.) injection to all animals to provide a therapeutic level of systemic analgesia to avoid or minimize pain and distress.
Five minutes prior to the test item administration, two drops of Conjuncain®EDO, a topical anaesthetic, was applied to each eye of all animals, to the right eye, in which the test item was to be applied, and to the left eye, which served as control.
Eight hours after administration, Buprenovet® 0.01 mg/kg, s.c. and Metacam® 0.5 mg/kg, s.c. were administered to provide a continued therapeutic level of systemic analgesia.

REMOVAL OF TEST SUBSTANCE
- Washing: the eyes were rinsed with 20 mL of 0.9% aqueous NaCl solution.
- Time after start of exposure: one hour after administration

SCORING SYSTEM: according to the Draize scale and a scale scoring the degree of staining (Fluorescein-test)
Any further lesions or adverse systemic effects were listed.

TOOL USED TO ASSESS SCORE: the eyes were examined ophthalmoscopically with a slit lamp.
One day before and 24, 48 and 72 hours after administration, fluorescein was applied to the eyes before being examined to aid evaluation of the cornea for possible lesions. The fluorescein test was repeated on each day of observation.

OBSERVATIONS:
- body weight 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:
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:
The corneae, irises and conjunctivae were not affected by administration of the test item.
Other effects:
- Lesions and clinical observations: 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)

Respiratory irritation

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

Additional information

Skin irritation

One reliable in vitro study described by Roth (2016) (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 Hansen (2015) (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 (2015) 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 (2012) test and does not require classification as eye irritating according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

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.