Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-) (1:1) and Amines,C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-)

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

02 Aug 2016 to 28 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

02 Aug 2016 to 28 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

6 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Specific details on test material used for the study:

Name as cited in study report: Orasol Red 395
Test substance No.: 16/0124-1
Batch identification: 001-152202
EC No.: 943-145-3
Purity: 97.1% (HPLC, 242 nm) or 98.3% (HPLC, 272 nm) main component
Content: 96.5 g/100 g
Homogeneity: The test substance was homogeneous by visual inspection.
Physical state: Red to brown solid
Storage conditions: Room temperature

Test system:

human skin model

Remarks:

Three dimensional human epidermis model: EpiDerm model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: cultured

Justification for test system used:

The present test is based on the experience that corrosive and irritant chemicals produce cytotoxicity in human reconstructed epidermis after a short term topical exposure.

Vehicle:

unchanged (no vehicle)

Details on test system:

MATERIALS 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 for the determination of the optical density of colored extracts. Measurement using a filter wavelength 570 nm without reference filter
- EpiDermTM 200 kit: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia; containing: 24 EPI-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells® ø 1 cm
- Tissue for MTT reduction control: EPI-200 tissue that is killed by freezing at –20°C
- Assay medium: EPI-100-NMM assay medium
- MTT diluent: Dulbecco's modified eagle's medium (DMEM) based medium used for diluting MTT (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma, Germany)
- Wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca2+, Mg2+ (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Biochrom, Germany)
- Detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma, Germany), 1.0 mg / mL MTT diluent
- Extracting agent: Isopropanol p.a.

TEST SYSTEM
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, 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 EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ø) and commercially available as kits (EpiDermTM 200), containing 24 tissues on shipping agarose.
- Tissue model: EPI-200
- Tissue Lot Number: 23348
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

DIRECT MTT REDUCTION
- The direct reduction of MTT by a test substance interferes with the colour density produced by metabolic capacity of the tissue and would falsify the test results.
- To assess the ability of the test material to directly reduce MTT a pretest (experimental conduct in accordance with GLP but without a GLP status) was performed as described below.
- The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 3 hours. A negative control (de-ionized water) was tested concurrently. If the colour of the MTT solution or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.
- In case that direct MTT reduction occurred, three freeze-killed control tissue (KC) were treated with the test article and the negative control.
- Due to the intense colour of the test substance it was not possible to evaluate whether or not the test substance is able to reduce MTT directly, therefore freeze-killed control tissues (KC) were treated with the test article and the negative control in the same way

COLOUR CONTROL
- The colour of a test substance may interfere with the colour density produced by metabolic capacity of the tissue and would falsify the test results when residues of the test substance remain on the tissues after washing and are extracted by the isopropanol.
- Due to the colour of the test substance a pretest (experimental conduct in accordance with GLP but without a GLP status) was performed as follows: the test substance was applied to a KC tissue, incubated for 1 hour and removed by washing in the same way as in the main experiment. Thereafter extraction in isopropanol was performed and the OD570 of the extract was determined spectrophotometrically.
- Based on the result of the pretest it was judged that application of colour control tissues is not necessary.

BASIC PROCEDURE
- Several test substances were tested in parallel within the present test using the same control tissues (negative control, NC and positive control, PC).
- On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.
- Three tissues were treated with the test substance, the PC and NC, respectively. In addition three killed control tissues were used for the test substance and NC, respectively, in order to detect direct MTT reduction.
- A nylon mesh was placed carefully onto the tissue surface of the NC, NC KC and PC after application.
- The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
- The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab.
- Subsequently, the tissues were placed into the incubator at 37°C for 24 ± 2 hours.
- After 24 ± 2 hours the 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.
- 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. The tissues used as colour control were placed into assay medium without MTT.
- 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. 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.

DATA EVALUATION
- Principle: The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material is irritant.
- Calculation of individual and mean optical densities: The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of three tissues treated in the same way is calculated.
- Application of measurements using killed control tissues: In case of direct reduction of MTT by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the test-substance treated tissues (mean OD570 KC corrected). Since killed tissue might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than zero it is subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.
- Tissue viability: The quantification of tissue viability is presented as the quotient of the mean OD570 (or mean OD570 KC corrected, if applicable) divided by the respective OD570 NC value in percent for each exposure time.

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria was not met, repetition of the test was considered.
- Barrier function and Quality control (QC): The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDermTM batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD guidelines. Lower acceptance limit: ET50 = 4.0 hours; Upper acceptance limit: ET50 = 8.7 hours
- Acceptance criteria for the negative control (NC): The absolute OD570 of the negative control tissues in the MTT test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 0.8. The mean OD570 of the NC should not exceed 2.8.
- Acceptance criteria for the positive control (PC): 5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.
- Acceptance criteria for the variability of the tissues: For every treatment three tissues are treated in parallel. The inter-tissue variability is considered to be acceptable if the SD of %-viability is ≤ 18%.
- Acceptance criteria for the killed controls (KC): The OD570 of the tissues for the KC of the NC should be ≤ 0.35. The OD570 value for direct MTT-reduction of a test substance should be ≤ 30% of the OD570 of the NC.
- Acceptance criteria for the colour controls (CC): The OD570 value for the colour control of a test substance should be ≤ 30% of the OD570 of the NC.

EVALUATION OF RESULTS
- Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.
- A single test composed of at least three tissue replicates should be sufficient for a test chemical when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to ±5% of the cut-off value, a second test should be considered, as well as a third one in case of discordant results between the first two tests.

DECISION CRITERIA
See table in 'Any other information on materials and methods incl. tables'

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
25 µL sterile PBS was applied first. Thereafter, a bulk volume of ca. 25 µL (about 7 mg) of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid.

CONTROLS
Control tissues were concurrently treated with 30 µL of sterile PBS (NC, NC KC) or with 30 µL of 5% SDS (PC) or test substance (KC). A nylon mesh was placed carefully onto the tissue surface of the NC, NC KC and PC afterwards.

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

42 hour post-incubation period

Number of replicates:

Three tissues were treated with the test substance, the PC and NC, respectively. In addition three killed control tissues were used for the test substance and NC.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

99.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: the final mean viability is given after KC correction

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: Due to the intense colour of the test substance, the ability of the test substance to reduce MTT directly could not be determined in a pretest. Therefore KC tissues were applied in parallel.
The results of the KC tissues indicate an increased MTT reduction (mean viability 1.1% of NC).
- Colour interference with MTT: Based on the result of the pretest it was judged that application of colour control tissues is not necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria for negative control are met.
- Acceptance criteria for positive control are met.
- Acceptance criteria for variability between replicate measurements are met.

Interpretation of results:

GHS criteria not met

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: B.40 BIS.: Methods for the determination of toxicity and other health effects: In Vitro Skin Corrosion: Human Skin Model Test

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-) (1:1) and Amines,C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-)

EC Number:

943-145-3

IUPAC Name:

Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-) (1:1) and Amines,C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-)

Specific details on test material used for the study:

Name as cited in study report: Orasol Red 395
Test substance No.: 16/0124-1
Batch identification: 001-152202
EC No.:943-145-3
Purity: 97.1% (HPLC, 242 nm) or 98.3% (HPLC, 272 nm) main component
Content: 96.5 g/100 g
Homogeneity: The test substance was homogeneous by visual inspection.
Physical state: Red to brown solid
Storage conditions: Room temperature

In vitro test system

Test system:

human skin model

Remarks:

Three dimensional human epidermis model: EpiDerm model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: cultured

Justification for test system used:

The present test is based on the experience that corrosive and irritant chemicals produce cytotoxicity in human reconstructed epidermis after a short term topical exposure.

Vehicle:

unchanged (no vehicle)

Details on test system:

MATERIALS 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 for the determination of the optical density of colored extracts. Measurement using a filter wavelength 570 nm without reference filter
- EpiDermTM 200 kit: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia; containing: 24 EPI-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells® ø 1 cm
- Tissue for MTT reduction control: EPI-200 tissue that is killed by freezing at –20°C
- Assay medium: EPI-100-NMM assay medium
- MTT diluent: Dulbecco's modified eagle's medium (DMEM) based medium used for diluting MTT (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma, Germany)
- Wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca2+, Mg2+ (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Biochrom, Germany)
- Detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma, Germany), 1.0 mg / mL MTT diluent
- Extracting agent: Isopropanol p.a.

TEST SYSTEM
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, 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 EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ø) and commercially available as kits (EpiDermTM 200), containing 24 tissues on shipping agarose.
- Tissue model: EPI-200
- Tissue Lot Number: 23348
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

DIRECT MTT REDUCTION
- The direct reduction of MTT by a test substance interferes with the colour density produced by metabolic capacity of the tissue and would falsify the test results.
- To assess the ability of the test material to directly reduce MTT a pretest (experimental conduct in accordance with GLP but without a GLP status) was performed as described below.
- The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 3 hours. A negative control (de-ionized water) was tested concurrently. If the color of the MTT solution or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.
- In case that direct MTT reduction occurred, three freeze-killed control tissue (KC) were treated with the test article and the negative control.
- Due to the intense colour of the test substance it was not possible to evaluate whether or not the test substance is able to reduce MTT directly, therefore freeze-killed control tissues (KC) were treated with the test article and the negative control in the same way

COLOUR CONTROL
- The colour of a test substance may interfere with the colour density produced by metabolic capacity of the tissue and would falsify the test results when residues of the test substance remain on the tissues after washing and are extracted by the isopropanol.
- Due to the colour of the test substance a pretest (experimental conduct in accordance with GLP but without a GLP status) was performed as follows: the test substance was applied to a KC tissue, incubated for 1 hour and removed by washing in the same way as in the main experiment. Thereafter extraction in isopropanol was performed and the OD570 of the extract was determined spectrophotometrically.
- Based on the result of the pretest it was judged that application of colour control tissues is not necessary.

BASIC PROCEDURE
- Several test substances were tested in parallel within the present test using the same control tissues (negative control, NC and positive control, PC).
- From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test-substance application, tissues were transferred to 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The pre-incubation medium was replaced with fresh medium immediately before application.
- Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used. In addition, two killed control tissues per exposure time were treated with the test substance and NC, respectively, in order to detect direct MTT reduction.
- The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment. Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were incubated 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. 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.

DATA EVALUATION
- Principle: The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material is corrosive
- Calculation of individual and mean optical densities: The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of three tissues treated in the same way is calculated.
- Application of measurements using killed control tissues: In case of direct reduction of MTT by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the test-substance treated tissues (mean OD570 KC corrected). Since killed tissue might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than zero it is subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.
- Tissue viability: The quantification of tissue viability is presented as the quotient of the mean OD570 (or mean OD570 KC corrected, if applicable) divided by the respective OD570 NC value in percent for each exposure time.

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria was not met, repetition of the test was considered.
- Barrier function and Quality control (QC): The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDermTM batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD guidelines. Lower acceptance limit: ET50 = 4.0 hours; Upper acceptance limit: ET50 = 8.7 hours
- Acceptance criteria for the negative control (NC): The absolute OD570 of the negative control tissues in the MTT test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 0.8. The mean OD570 of the NC should not exceed 2.8.
- Acceptance criteria for the positive control (PC): Potassium hydroxide as 8.0 normal ready-made solution is used as positive reference. A tissue viability of ≤ 30% is acceptable for the 3-minute exposure. Mean viability of the tissues exposed for 1 hour should be <15%.
- Acceptance criteria for the variability of the tissues: For every treatment two tissues are treated in parallel. In the range of 20% and 100% viability, variability between the tissues is considered to be acceptable if the coefficient of variation (CV) of %-viability is ≤ 30%.
- Acceptance criteria for the killed controls (KC): The OD570 of the tissues for the KC of the NC should be equal to or less than 0.35. The OD570 value for direct MTT-reduction of a test substance should be ≤ 30% of the OD570 of the NC.

EVALUATION OF RESULTS
- Corrosive potential of the test materials is predicted from the mean relative tissue viabilities obtained after 3 min treatment compared to the negative control tissues concurrently treated with de-ionized water. A chemical is considered as "corrosive", if the mean relative tissue viability after 3 min treatment with a test material is decreased below 50%. In addition, those materials with a viability of ≥ 50% after 3 min treatment are considered as "corrosive" if the mean relative tissue viability after 1 hour treatment with a test material is decreased below 15%.
- A single test composed of at least two tissue replicates should be sufficient for a test chemical when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to ±5% of the cut-off values cited above, a second test should be considered, as well as a third one in case of discordant results between the first two tests.

DECISION CRITERIA
See table in 'Any other information on materials and methods incl. tables'

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
25 µL de-ionized water was applied first. Thereafter, a bulk volume of ca. 25 µL (about 7 mg) of the solid test material was applied with a sharp spoon and homogeneously distributed with the water.

CONTROLS
Control tissues were concurrently treated with 50 µL of de-ionized water (NC, NC KC) or with 50 µL of 8 N potassium hydroxide (PC) or test substance (KC).

Duration of treatment / exposure:

3 minutes (room temperature) or 1 hour (incubator)

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

99.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: the final mean viability is given after KC correction

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

95

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: the final mean viability is given after KC correction

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: Due to the intense colour of the test substance, the ability of the test substance to reduce MTT directly could not be determined in a pretest. Therefore KC tissues were applied in parallel. The results of the KC tissues indicate an increased MTT reduction (mean viability of 2.1% 1.7 % of NC after 3 min and 1 hour, respectively). Thus for the test substance the final mean viability is given after KC correction.
- Colour interference with MTT: Based on the result of the pretest it was judged that application of colour control tissues is not necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria for negative control are met.
- Acceptance criteria for positive control are met.
- Acceptance criteria for variability between replicate measurements are met.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met