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Description of key information

The test substance is not irritating to the skin.

The test substance is not irritating to the eyes.

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:
22 Mar 2016 to 03 Aug 2016
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:
28 July 2015
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
6 July 2012
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No. of test material: 003-142715
- CAS: 85029-58-9
- Purity test date: ca. 98 area-% (LC)
- Content: 98.4 g/100 g (titration)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Storage stability of the test material: The stability under storage conditions over the study period was guaranteed by the sponsor
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
- Homogeneity: The test substance was homogeneous by visual inspection

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Test substance applied minimally moistened with PBS

OTHER SPECIFICS:
- pH-value: Ca. 5 (undiluted test substance, moistened with de-ionized water; determined in the lab prior to start of the GLP study)
- Physical state / color: Solid / brown

Test system:
human skin model
Source species:
human
Cell type:
other: EpiDermTM tissues
Cell source:
other: Tissue derived from a single donor
Source strain:
other: Epiderm tissue (24 EPI-200 tissues - reconstructed epidermis)
Details on animal used as source of test system:
- EpiDermTM tissues: human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis

SOURCE
- All cells used to produce EpiDermTM are purchased or derived from tissue obtained by MatTek Corporation from accredited instructions. In all cases consent was obtained by these institutions from the donor or the donor's legal next of kin, for the use of the tissues or derivatives of the tissue for research purposes.
- Tissue lot number: 23328
Vehicle:
unchanged (no vehicle)
Details on test system:
SKIN DISC PREPARATION
- Test system: Three dimensional human epidermis model (EpiDermTM)
- Tissue model: EPI-200
- Tissue Lon Number: 23328
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Procedure used: Test item tested with EpiDermTM using PBS sterile as negative control and 5% (w/v) sodium dodecyl sulfate (SDS) in water as positive control
- Quality control for skin discs: Time of exposure to reduce tissue viability to 50% (ET50; acceptable range: 4.77-8.72 h): ET50 = 5.81 h (Pass)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (25°C)
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: The tissues were washed with sterile PBS once (1 hour after start of application)
- Observable damage in the tissue due to washing: No damage observed

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
For skin irritation at least 3 tissue replicates should be sufficient for a test. In case of borderline results, a second test should be performed. In case of discordant results between the first 2 tests, a third test should be performed.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.
- A chemical is considered as "irritant", if the mean relative tissue viability with a test material is ≤ 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- Amount of test item: 25 µL ( approx. 9 mg)
- Amount of negative control: 30 µL
- Amount of positive control: 30 µL
Duration of treatment / exposure:
The conditioned, treated tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
Duration of post-treatment incubation (if applicable):
Tissues were washed 1 h after start of application and then placed into the incubator at 37°C for 24 ± 2 hours. After that tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. Afterwards, the assay mediu was replaced by 0.3 mL MTT solution and tissues were incubated in incubator for 3 hours.
Number of replicates:
3 tissue replicates
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
103.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
101.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
100.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
101.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The test substance is not able to reduce MTT directly.
In a pretest it was demonstrated that the color of the test substance did not interfere with the colorimetric test.
Acceptance criteria for negaite control, positive control and variability of the tissues were met.
Interpretation of results:
GHS criteria not met
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:
08 Mar 2016 to 03 Aug 2016
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:
28 July 2015
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
8 December 2010
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test substance: Orasol Yellow 157
- Test-substance No.: 16/0017-1
- Source and lot/batch No. of test material: 003-142715
- Purity test date: c 98 area-% (LC)
- Content: 98.4 g/100 g

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Homogeneity: The test substance was homogeneous by visual inspection.
- Storage condition of test material: Room temperature
- Solubility and stability of the test substance in the solvent/vehicle: no vehicle
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no reactivity

OTHER SPECIFICS:
- pH: Ca. 5
- Physical state / color: Solid / brown
Species:
human
Strain:
other: EpiOcularTM model (OCL-200)
Details on test animals or tissues and environmental conditions:
- EpiOcularTM model (OCL-200): A three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinocytes used to model the human corneal epithelium
- Cell course: All cells used to produce EpiOcularTM are purchased or derived from tissue obtained by MatTek Corporation from accredited institutions.In all cases consent was obtained by these institutions from the donor or the donor's legal next of kin, for the use of the tissues or derivatives of the tissue for research purposes.
- Tissue model: OCL-200
- Tissue Lot Number: 23700 (Certificate of Analysis see appendix)
- Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount of test item: 50 µL
- Amount of positive control: 50 µL
- Amount of negative control: 50 µL
Duration of treatment / exposure:
Total exposure time: 6 h
Duration of post- treatment incubation (in vitro):
Postincubation period: 18 h
Number of animals or in vitro replicates:
2 tissue replicates for a single test is sufficient. In case of borderline results a second test is performed. In case of discordant test results between the 2 tests, a third is performed.
Details on study design:
MATERIAL AND TECHNICAL EQUIPMENT
- Laminar flow bench: HERAsafe KS 18 (Thermo ELECTRON CORPORATION)
- CO2 incubator: Heraeus BBD 6220
- Incubation conditions: 37°C ± 1°C, 5% ± 1% CO2, 90% ± 5% humidity
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm without reference filter
- Assay medium: OCL-200-ASY
- Detection agent: 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT)
- Extracting agent: Isopropanol p.a

TISSUE PREPARATION:
Preincubation: On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 1 h, the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 h.
Pretreatment: After the pre-incubation, the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.

APPLICATION OF THE TEST ITEM:
Using a sharp spoon, approx. 50 μL of the undiluted test material was applied covering the whole tissue surface. Control tissues were applied with 50 μL of negative and of positive control. After application, the tissues were placed into the incubator until the total exposure time of 6 h.

REMOVAL AND POSTINCUBATION PERIOD:
To remove the test item, the tissues were washed with sterile PBS. Tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates and pre-filled with 5 mL/well prewarmed medium (post-soak immersion) in order to remove residual test substance. After 25 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium.
The tissues were incubated at standard culture conditions for 18 hours (postincubation period).

MTT INCUBATION:
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

EVALUATION CRITERIA:
A chemical is considered as "irritant", if the mean relative tissue viability with a test material is ≤ 60%.
However a borderline range is statistically determined. Therefore, mean percent tissue viability equal to ± 5% of the cut-off value is considered to be discordant.
Irritation parameter:
other: tissue viability (%)
Run / experiment:
1
Value:
115.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: tissue viability (%)
Run / experiment:
2
Value:
100.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: tissue viability (%)
Run / experiment:
mean
Value:
108.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Yellowish discoloration of the test substance-treated tissues was observed after the washing procedure.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation: 

In an in-vitro skin irritation study (BASF, 2016) performed according to the OECD guideline No. 439, the EpiDermTM test method was employed. Human skin cells derived from reconstructed human epidermis tissue containing normal human keratinocytes were used for the test system. Sterile PBS was used a negative control and 5% (w/v) sodium dodecyl sulfate (SDS) in water was used as a positive control. Tissues were washed 1 h after start of application and then placed into the incubator at 37°C for 24 ± 2 hours. After that tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. The test methods was performed in at least 3 tissue replicates. In case of equivocal results a second or a third test should be performed. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. Based on the available information, the test item is not determined to cause irritation to skin

 

In a primary dermal irritation study (BASF, 1974) according to BASF internal standard, two male Vienna White rabbits were dermally exposed to 1 g of a 50% aqueous test substance preparation. The test substance was applied in a single dose to the clipped skin of the back of an experimental animal (2.5 x 2.5 cm, occlusive) for 20 hours and was not washed off. Untreated skin areas served as control. The degree of irritation was observed after 24 hours and 8 days. For the evaluation of the results, the scheme was converted to the OECD Draize scheme. Due to test substance coloring, skin reddening was not visible after 24 hours and 8 days. No abnormalities were noted. Based on the available information the test item is not considered to be irritating to the skin.

Eye irritation: 

In an in-vitro eye irritation testing method (BASF, 2016) performed according to the OECD guideline No. 492, the EpiOcularTM test method was employed. The ocular irritation potential was assessed by a single topical application of ca. 50μL bulk volume (about 12 mg) of the undiluted test substance to a reconstructed three dimensional human cornea model. 2 EpiOcular™ tissues were incubated with the test substance for 6 h followed by an 18-h post- incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is ≤ 60%. Based on the available information, the test item is not considered to be irritating to the eye.

 

In a primary irritation study (BASF ,1974) according to BASF-internal standard, 50 mg of the test substance without a vehicle was instilled into the conjunctival sac of the right eye of two Vienna White rabbits. The left eye was treated with talcum powder and served as control. The test substance was not washed out. The animals were observed after 24 hours and 8 days. For the evaluation of the results, the BASF scheme was converted to the OECD Draize scheme. After 24 hours the mean conjunctiva score was 1. No other abnormalities were noted. Effects were fully reversible after 8 days. Based on the available information the test substance is not considered to be irritating to the eye. 

 

Justification for classification or non-classification

Based on the available information classification for skin and eye irritation is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.


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