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-)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 Jul 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:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 Jul 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 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 July 2013

Deviations:

no

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

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

Species:

cattle

Strain:

other: Bovine

Details on test animals or tissues and environmental conditions:

TISSUE MODEL
- Test system: Isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).
- Supplier: Schlachthof Mannheim, Schlachthofstr. 21, 68165, Mannheim, Germany

MATERIALS AND TECHNICAL EQUIPMENT
- Corneal holder: Supplier: BASF SE, Germany
- Incubator: Temperature 32 ± 1 °C
- Opacitometer: Kit BASF-OP3.0, BASF SE, Germany
- Spectrophotometer: SunriseTM Absorbance Reader, Measurement using wavelength of 490 nm

REAGENTS
- Hanks' Balanced Salt Solution with Ca++ and Mg++ (HBSS) (Biochrom, Germany) containing Fetal Bovine Serum (FBS) and/or Penicillin/Streptomycin (P/S)
- Eagle’s MEM without phenol red (Biochrom, Germany) containing FBS and P/S
- Eagle's MEM with phenol red (Biochrom, Germany)
- Sodium fluorescein diluted in DPBS

Vehicle:

water

Remarks:

De-ionized

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- 750 μL of the 20% (w/v) test-substance preparation

CONTROLS
For the control tissues 750 μL of de-ionized water (negative control) or 750 μL of 20% (w/v) solution of imidazole in de-ionized water (positive control)

Duration of treatment / exposure:

4 hours

Number of animals or in vitro replicates:

Triplicates of substance and controls

Details on study design:

TEST-SUBSTANCE PREPARATION AND HOMOGENIZATION
The test-substance preparation was produced on a weight per volume (w/v) basis shortly before application by stirring with a magnetic stirrer. The homogeneity of the test-substance preparation during application was provided by stirring with a magnetic stirrer.

SELECTION AND PREPARATION OF CORNEAS
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 548 opacity units were discarded. According to OECD TG 437 corneas that have an opacity value >7 or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period are to be discarded. In the used opacitometer in combination with the used cornea holder set 2013-22 and 2013-24, the maximal initial opacity of >7 arises from I= 548 lux with Io= 639 lux. The remaining corneas were then distributed into negative control, positive control and treatment groups. Each corneal holder was uniquely identified with a number on the chambers.

APPLICATION DOSE AND EXPOSURE TIME
- Several test substances were tested in parallel within the present test using the same control corneas (NC and PC).
- Before application, the medium in the anterior chamber was removed using a syringe.
- The 20% (w/v) test-substance preparation could not be applied with a pipette. Therefore 750 µL of the 20% (w/v) test-substance preparation (non-surfactant) was applied directly to the epithelial surface of the cornea using a syringe (open chamber method).
- For the control tissues 750 µL of de-ionized water (negative control, NC) or 750 µL of 20% (w/v) solution of imidazole in de-ionized water (positive control, PC), were applied into the anterior chamber using a pipette.
- The corneas were incubated in a horizontal position at about 32 °C for approximately 4 hours (non-surfactant solids).

TREATMENT METHOD
- Controls: Closed chamber
- Test material: Open chamber

POST-INCUBATION PERIOD: Not applicable

REMOVAL OF TEST SUBSTANCE
- The NC and PC were removed after the exposure period from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).
- Because the test substance could not be removed using a syringe, the epithelium was rinsed with the open chamber method.

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
- Corneal permeability: For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (5 mg/mL for solid test substances) and incubated for 90 ± 5 min in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined. An aliquot was diluted 1:5 in Eagle’s MEM (without phenol red) and measured analogously (PC, only).

SCORING SYSTEM
The mean corneal opacity and permeability values of each treatment group were used to calculate an In Vitro Irritancy Score (IVIS). When negative values for corneal opacity or permeability were obtained, the values were set to zero for calculation of the means for corneal opacity or permeability.

ACCEPTANCE CRITERIA
- A study is considered acceptable if the PC gives an IVIS that falls within two standard deviations of the current historic mean.
- The NC responses should result in opacity and permeability values that are not higher than the established upper limits.
- Since the IVIS per treatment group is determined from the mean of three single corneas, the variability between the corneas treated per test substance should be acceptably low. In cases of borderline results in the first testing run, a second testing run should be considered (but not necessarily required), as well as a third one in case of discordant mean IVIS results between the first two testing runs. In this context, a result in the first testing run is considered borderline if the predictions from the 3 corneas were non-concordant, such that:
* 2 of the 3 corneas gave discordant predictions from the mean of all 3 corneas, OR,
* 1 of the 3 corneas gave a discordant prediction from the mean of all 3 corneas, AND the discordant result was >10 IVIS units from the cut-off threshold of 55.

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

Irritation parameter:

cornea opacity score

Run / experiment:

Mean opacity value

Value:

14.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein leakage

Run / experiment:

Mean permeability value

Value:

0.002

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

mean IVIS score

Value:

14.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria for negative control are met.
- Acceptance criteria for positive control are met.

Interpretation of results:

GHS criteria not met

Remarks:

In combination with EpiOcular test (OECD 492)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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

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

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

TISSUE MODEL
The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinocytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs®, 10 mm diameter) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.
Tissue model: OCL-200
Tissue Lot Number: 23724
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

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 coloured extracts. Measurement using a filter wavelength 570 nm without reference filter
- EpiOcular™ OCL-200 kit: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia containing: 24 OCL-200 tissues (reconstructed cornea): surface 0.6 cm² cultured in Millicells® (1 cm diameter)
- Tissue for MTT-reduction control: OCL-200 tissue that is killed by freezing at –20°C
- Assay medium: OCL-200-ASY 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)
- Pre-treatment / 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 and Sigma, Germany), 1.0 mg / mL MTT diluent
- Extracting agent: Isopropanol p.a.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

other: MTT reduction control (KC)

Amount / concentration applied:

Bulk volume of ca. 50 µL (about 14 mg) of the test material was applied covering the whole tissue surface.
Control tissues were concurrently applied with 50 µL of sterile de-ionized water or with 50 µL of methyl acetate or test substance

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

duplicates

Details on study design:

DIRECT MTT REDUCTION
- 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. 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 MTT solution colour 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.
- 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.
- In case where direct MTT reduction occurred, two freeze-killed control tissues each were treated with the test article and the negative control, in the same way as described in the following section.
- 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 6 hours and removed by washing. 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).
- Two tissues were treated with each, the test substance, the PC and the NC. In addition two killed tissues were used for each, the test substance and the NC, in order to detect direct MTT reduction.
- There are two separate protocols for liquids and solids, differing in exposure time and postincubation period. Due to the physical state of the test substance the protocol for solids was applied.

PRE-INCUBATION OF THE TISSUES
- 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 hour the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.

PRETREATMENT OF THE TISSUES
- 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 SUBSTANCE
- Using a sharp spoon, a bulk volume of ca. 50 μL of the test material was applied covering the whole tissue surface.
- Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC, NC KC) or with 50 μL of methyl acetate (PC) or test substance (KC).
- After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed.

REMOVAL OF THE TEST SUBSTANCE AND POSTINCUBATION PERIOD
- To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, 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.
- Subsequently, the tissues were incubated at standard culture conditions for 18 hours (postincubation period).

MTT INCUBATION
- After the postincubation 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.

DATA EVALUATION
- Principle: The OD570 values determined for the various tissues are measures of their viability. The ratio 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 was an irritant.
- Calculation of individual and mean optical densities: The corrected measured OD570 value for each individual tissue was 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 two tissues treated in the same way was 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 ratio of the mean OD570 (or mean OD570 KC corrected, if applicable) divided by the respective OD570 NC value in percent.

ACCEPTANCE CRITERIA
- 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 (min) value following exposure to 100 μL of 0.3% Triton X-100 for each EpiOcular™ EIT (OCL-200) 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 Guideline. Lower acceptance limit: ET50 = 12.2 min; Upper acceptance limit: ET50 = 37.5 min
- Acceptance criteria for the NC: The absolute OD570 of the NC-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 2.5.
- Acceptance criteria for the PC: Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.
- Acceptance criteria for tissue variability: Two tissues were treated under the same conditions. A variability between the the two tissues is considered to be acceptable if the relative difference of the viability is < 20%.
- Acceptance criteria for the KC: The OD570 of the killed control tissues treated as negative control should be ≤ 0.35. The value for direct MTTreduction of a test substance should be ≤ 30% of the NC.

EVALUATION OF RESULTS
- The irritation potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile water. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 60%.
- 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 nonconcordant 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 'Any other information on materials and methods incl. tables'

Results and discussion

In vitro

Results

Irritation parameter:

other: Tissue viability

Run / experiment:

mean viability

Value:

112.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: final mean viability is given after KC correction.

Other effects / acceptance of results:

OTHER EFFECTS
- Colour control: Based on the result of the pretest it was judged that application of colour control tissues is not necessary.
- 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 9.9% of NC). Thus for the test substance the final mean viability is given after KC correction.

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.
- Acceptance criteria for killed control tissues are met.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

In combination with BCOP test (OECD 437)