Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Feb - 16 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

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:
adopted in 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP-Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
Dipotassium dihydrogen 4-[5-[3-carboxylato-5-hydroxy-1-(4-sulphonatophenyl)-1H-pyrazol-4-yl]penta-2,4-dienylidene]-4,5-dihydro-5-oxo-1-(4-sulphonatophenyl)-1H-pyrazole-3-carboxylate
EC Number:
257-479-6
EC Name:
Dipotassium dihydrogen 4-[5-[3-carboxylato-5-hydroxy-1-(4-sulphonatophenyl)-1H-pyrazol-4-yl]penta-2,4-dienylidene]-4,5-dihydro-5-oxo-1-(4-sulphonatophenyl)-1H-pyrazole-3-carboxylate
Cas Number:
51858-17-4
Molecular formula:
C25H16N4O12S2.2K
IUPAC Name:
dipotassium dihydrogen 4-[(1E,3E)-5-[(4E)-3-carboxy-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazol-4-ylidene]penta-1,3-dien-1-yl]-5-hydroxy-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylate
Test material form:
solid: crystalline

Test animals / tissue source

Species:
human
Strain:
other: EpiOcular™
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe from 2008 to 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS (i.e. “No Category”), and the test method was recommended as scientifically valid for this purpose.

- Description of the cell system used, incl. certificate of authenticity: This test uses the three-dimensional RhCE EpiOcular™ (MatTek). It consists of normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The MatTek EpiOcular™ model has been widely used as a research and testing model for many years. Certificates of authentity of the used tissue batches were provided by MatTek and are attached to the report.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: approximately 50 mg (83.3 mg/cm2)

NEGATIVE CONTROL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL
Duration of treatment / exposure:
6 ± 0.25 h
Duration of post- treatment incubation (in vitro):
post-exposure immersion: 25 ± 2 min
post-exposure incubation: 18 ± 0.25 h
Number of animals or in vitro replicates:
in duplicates for each treatment and contol group
Details on study design:
- RhCE tissue construct used, including batch number : EpiOcular™ (MatTek Corporation, Bratislava, Slovakia), Lot Nos.: 27021 and 27027
- Tissue viability: The quality of the final product was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.427 ± 0.04 and 1.784 ± 0.023 (acceptance criteria: 1.1 - 3.0) for tissues of the two used batches, respectively.
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 μL of 0.3% Triton X-100. The ET-50 value was determined to be 23.64 and 26.15 min (acceptance criteria: 12.2 - 37.5 min) for tissues of the two used batches.
- Contamination: The cells used to produce the EpiOcular tissue were screened for the presence of viruses, bacteria, yeast and other fungi.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 6 h exposure (37 °C), 25 min post-exposure immersion (room temperature), 18 h post-exposure (37 °C)
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: Optical evaluation of the non-specific MTT-reducing capacity of the test item with MTT-reagent showed blue colour. Therefore, an additional test with killed tissue controls was performed for quantitative correction of results.The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water and isopropanol led to a change in colour. Therefore, an additional test with coloured tissue controls were performed for quantitative correction of result. Since the test item showed non-specific MTT-reduction and non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSCkilled) was performed to avoid a possible double-correction for colour interference.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled: 2
- Description of the method used to quantify MTT formazan : For each tissue 2 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.
- Description of evaluation criteria used: If the test item-treated mean tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant.
- Acceptance criteria : The test meets acceptance criteria if: mean absolute OD570 nm of the negative control is > 0.8 and < 2.5; mean relative tissue viability of the positive control is < 50%; relative tissue viability difference of replicate tissues is < 20%.

Results and discussion

In vitro

Results
Irritation parameter:
other: mean tissue viability of 2 tissues
Remarks:
corrected for NSMTT and NSCliving
Run / experiment:
6 h
Value:
94.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY: Laboratory technical proficiency with the test system according to OECD 492 was demonstrated at Eurofins Biopharma Product Testing Munich GmbH.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes: the negative control OD (1.480) was in the range of > 0.8 and < 2.5
- Acceptance criteria met for positive control: yes: mean relative viability of the positive control is below 50% of the negative control viability (22.3%).
- Acceptance criteria for difference in viability: yes: the maximum difference of viability between the two relating tissues of a single item is < 20% (16.9%)

Any other information on results incl. tables

Table 3. Results of the main experiment

Name

Negative Control

Positive Control

Test item

Tissue

1

2

1

2

1

2

OD570values

1.352

1.606

0.316

0.413

1.342

1.341

1.366

1.598

0.319

0.405

1.400

1.312

OD570values
(blank-corrected)

1.308

1.563

0.273

0.369

1.299

1.297

1.323

1.554

0.276

0.362

1.356

1.268

mean of the duplicates

1.316

1.559

0.274

0.365

1.328

1.283

mean OD

1.437*

0.320

1.305

TODTT- NSMTT

 -

 -

1.309

TODTTNSMTT und NSCliving

 -

 -

1.308

mean sd OD

0.172

0.064

0.032

tissue viability [%]

91.5

108.5

19.1

25.4

92.4

89.3

relative tissue viability difference [%]***

16.9

6.3

3.1

mean tissue viability [%]

100.0

22.3**

90.8

mean tissue viability [%]
- NSMTT corrected

 -

 -

91.1

mean tissue viability [%]
- NSMTT and NSClivingcorrected

 -

 -

91.0

True Tissue Viability

 -

 -

94.1

*              Corrected mean OD570of the negative control corresponds to 100% absolute tissue viability

**             Mean relative tissue viability of the positive control is < 50%

***            Relative tissue viability difference of replicate tissues is < 20%

Table 4. Results of the NSMTT control

NSMTT

KU

KT

Negative Control

Tissue

1

2

1

2

1

2

absolute OD570 -values

0.074

0.075

0.069

0.072

1.352

1.606

0.075

0.077

0.070

0.072

1.366

1.598

OD570(Blank Corrected)

0.030

0.032

0.026

0.029

1.308

1.563

0.032

0.033

0.027

0.029

1.323

1.554

mean OD570
(mean of 2 aliquots)

0.031

0.033

0.026

0.029

1.316

1.559

total mean OD570
(mean of the replicate tissues)

0.032

0.028

1.437

SD OD570(of the replicate tissues)

0.001

0.002

0.172

NSMTT [%]

-0.3

 -

Relative Tissue Viability [%]

 -

100.0

118.5

Mean Relative Tissue Viability [%]

 -

109.2

SD Tissue Viability [%]

 -

13.1

CV [% Viabilities]

 -

12.0

Table 5. Results of the NSClivingcontrol

NSCliving

TVT

Negative Control

Tissue

1

2

1

2

absolute OD570 -values

0.044

0.044

1.352

1.606

0.044

0.045

1.366

1.598

absolute OD570 -
Blank corrected values

0.001

0.001

1.308

1.563

0.001

0.002

1.323

1.554

mean OD570
(mean of 2 aliquots)

0.001

0.001

1.316

1.559

total mean OD570
(mean of replicate tissues)

0.001

1.437

SD OD570
(of the 2 replicate tissues)

0.000

0.172

NSCliving[%]

0.1

 -

Relative Tissue Viability [%]

 -

91.5

108.5

Mean Relative Tissue Viability [%]

 -

100.0

SD Tissue Viability [%]

 -

12.0

CV [% Viabilities]

 -

12.0

Table 6. Results of the NSCkilledcontrol

NSCkilled

TKT

Negative Control

Tissue

1

2

1

2

absolute OD570 -values

0.045

0.046

1.352

1.606

0.047

0.045

1.366

1.598

absolute OD570 -
Blank corrected values

0.045

0.046

1.352

1.606

0.047

0.045

1.366

1.598

mean OD570
(mean of 3 aliquots)

0.046

0.045

1.359

1.602

total mean OD570
(mean of replicate tissues)

0.046

1.480

SD OD570
(of the 2 replicate tissues)

0.001

0.172

NSCkilled[%]

3.1

 -

Relative Tissue Viability [%]

 -

91.8

108.2

Mean Relative Tissue Viability [%]

 -

100.0

SD Tissue Viability [%]

 -

11.6

CV [% Viabilities]

 -

11.6

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
Under the conditions of the conducted test, the test substance does not possess irritating properties towards human-derived epidermal keratinocytes in the EpiOcular™ model.