pentasodium 4-amino-3-[{4-[(2,4-disulfonatophenyl)diazenyl]-3-methylphenyl}diazenyl]-5-hydroxy-6-[(4-nitro-2-sulfonatophenyl)diazenyl]naphthalene-1,7-disulfonate

Endpoint summary

Description of key information

in vitro skin irritation: not irritating

in vitro eye irritation: not irritating

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:

weight of evidence

Study period:

2020

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:

18 June 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Human full thickness skin models and reconstituted epidermal equivalents are in vitro engineered tissue cultures that provide a three dimensional architecture which is biochemically, morphologically and functionally comparable to human epidermal tissue/skin in vivo. In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and EpiSkin™ 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.
MatTek Corporation Protocol: In vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT); Version 11 July 2017

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™; Epi-200-SIT Kit and MTT-100 kit (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number(s): Lot No. 30869
- Date of initiation of testing: 05 May 2020
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: incubator (37 ± 1.5° C) and 5 ± 5% CO2 in DMEM Medium
INCUBATION TIME
main experiment: treatment time 60 min; complete incubation time about 43 h
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm
NUMBER OF REPLICATE TISSUES:
test item and controls were tested in triplicate
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
the optical density of the isopropanol-extracts of the triplicate cultures was determined in duplicate per insert = 6 OD values.
PREDICTION MODEL / DECISION CRITERIA:
- The mean optical density (OD) value obtained with the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- A category of UN GHS (Category 2 or Category 1) is predicted if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤ ) to 50 %.

Pre-check for potential optical interferences of the test item
Optical properties of the test item or its chemical action on MTT may interfere with the measurement of MTT formazan leading to a false estimate of tissue viability.
The test item was therefore tested in advance for a potential direct influence on the test results not related to cytotoxic effects on tissue cells. For this pre-check the following parameters were tested:
1. Assessment of potential direct MTT-reduction of the test item
In case of a direct MTT-reduction of the test item a killed tissue control (inserts, which were killed by freezing) was used in the main assay.
2. Assessment of potential interference of colored or staining test items, which become colored after application to the tissues, with OD read out
2.1. Assessment of the color reaction with water
2.2. Assessment of the color reaction with isopropanol
In case of an influence of test item color on OD measurement, a color control was used in the main assay.
The evaluation criteria for the pre-check were:
- For MTT reduction (visual assessment): If the MTT solution color turns blue/purple, the test item is presumed to have reduced the MTT. A killed control (PG KC) must be conducted.
- For color reaction (measurement of the OD): If, after subtraction of the OD for water or isopropanol the OD of the test item solution is >0.08 a Color Control (PG CC) must be conducted.
If a test item is identified as producing both, color interference and direct MTT reduction, a third set of controls (Non-specific killed control NsKC) will be require, additionally from the PG KC and PG CC controls. This is usually the case with darkly coloured test items interfering with the MTT assay (e.g., blue, purple, black) because their intrinsic colour impedes the assessment of their capacity to directly reduce MTT.

Procedure color control
Color control (CC): The test item was applied to two additional inserts which were in principle treated as described for the main assay.
However the further processing for the determination of cell viability differs in that these color control inserts were placed in MTT-Assay medium instead of placing them in MTT solution.

The irritating potential of the test item is assessed by determination of its cytotoxic effect on an in vitro reconstructed human epidermis. The test principle is based on the MTT assay reflecting the cell viability after exposure to the topically applied test item.
All tests were performed in triplets for each time point. The test item was applied at a 100% concentration, i.e. 25 mg per insert for 60 min. Cell viability was measured by the amount of MTT reduction (calculated on the basis of optical density of the negative control).

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg (39.7 mg/cm² according to guideline) wetted with 25 µL DPBS (buffer)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μl
- Concentration (if solution): Dulbecco's Phosphate Buffered Saline (DPBS)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 5% Sodium Dodecyl Sulfate (SDS) solution in deionised water

Duration of treatment / exposure:

The incubation of the treated Epi-200 STI inserts was carried out for 60 min.

Duration of post-treatment incubation (if applicable):

about 43 hours

Number of replicates:

3 per test item

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of 3 replicate cultures

Value:

101.07

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation

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

OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: yes
- Colour interference with MTT: yes

All acceptance criteria were met.

The test item proved to be an MTT reducer in the MTT interference pre-experiment. Also, it changed colour when mixed with isopropanol. Therefore, additional tests with freeze-killed tissues, viable tissues and Non-specific Killed Control (NSKC) tissues (without MTT addition) had to be performed, see following table:

 

color of test item	MTT reduction	Color reaction in isopropanol	Killed control test to be conducted	Color control test to be conducted
black	+	+	yes	yes

+  positive reaction, -  negative reaction

 

As can be seen from the information in the table below the test item did not show a significant impact on cell viability and is thus identified as non-irritant substance in this test model.

 

Summary of results:

Test item	OD mean	% Cell Viability	Evaluation of result
test item	1.807	101.07*	non-irritant
negative control (NaCl 0.9%)	1.757	100	non-irritant
positive control (SDS 5%)	0.064	3.64	irritant

* Corrected mean viability = TI viability – TI_CC viability – (TI_KC viability – NC_KC viability) + TI_NSKC viability

 

Thus the test item does not require classification according to UN GHS (Category 2 or Category 1) under the experimental conditions described in this report.

Interpretation of results:

GHS criteria not met

Executive summary:

This in vitro study was performed to assess the irritation potential of the registered substance by means of the Human Skin Model Test (OECD TG 439).

The test item proved to be an MTT reducer in the MTT interference pre-experiment. Also, it changed colour when mixed with isopropanol. Therefore, additional tests with freeze-killed tissues, viable tissues and Non-specific Killed Control (NSKC) tissues (without MTT addition) had to be performed.

Each three tissues of the human skin model EpiDerm™ were treated with the solid test item (wetted with phosphate buffer), the negative or the positive control for 60 minutes followed by a post-treatment incubation period of about 43 hours. The results obtained with the positive and negative controls ensured the validity of the test system

After treatment with the test item the mean relative viability value was 101.07% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, in this study and under the experimental conditions reported, the registered substance is non-irritant to skin according to UN GHS and EU CLP regulation.

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:

2020

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:

Adopted 18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: in vitro test with human cornea cells

Strain:

other: three-dimensional human cornea model tissue model

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability:
Eye irritation is generally defined as "the production of reversible changes in the eye". The potential for chemical induced eye irritation is an important consideration in establishing procedures for the safe handling, packing and transport of chemicals. It was usually determined in vivo in the Draize rabbit eye irritation test as described in OECD guideline 405. In a pre-validation study performed by Avon Products Inc. and MatTek Corporation, the in vitro eye test using the human cornea model EpiOcular™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for eye irritancy potential.
A limitation of the Test Guideline OECD 492 is that it does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B), as defined by UN GHS. For these purposes further testing with other suitable test methods is required.
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.
The test consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazol 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.
The technical proficiency of the test system according to OECD Guideline 492 guideline recommended proficiency substances was demonstrated.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg (corresponds to according to Guideline a minimum of 83.3 mg/cm²)
- positive control: 50 µL
- negative control: 50 µL

Duration of treatment / exposure:

6 hours at standard culture conditions (5% CO2, 37°C, 95% humidity) followed by a post-soak immersion period of about 25 min in fresh medium

Duration of post- treatment incubation (in vitro):

18 hours (37°C, 5% CO2, 95% humidity)

Number of animals or in vitro replicates:

each 2 tissues for the test item, positive and negative controls

Details on study design:

- RhCE tissue construct used, including batch number: The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a non-keratinized surface as recommended by the test guidelines. The cell viability and barrier function as well as sterility of each batch of the RhCE tissue construct used is adequate, as has been demonstrated by the supplier.
RhCE tissue viability in EpiOcular™ EIT is measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.

- Doses of test chemical and control substances used: 50 mg of the solid test item, 50 µL negative control (deionized water) and positive control (neat methyl acetate), respectively, were applied to the EpiOcular™ tissue surface in duplicate.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods for solid test items: 37 ± 2°C; 6 hours exposure, 25 min. post-soak immersion, 18 hours post-treatment incubation

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): The optical pre-experiment (color interference pre-experiment) to investigate the test item’s color change potential in water or isopropanol led to a change in color. Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent showed purple color. Therefore, an additional test with freeze-killed tissues was necessary.
Since the OD of the test item in deionised water or isopropanol at 570 nm after blank correction was > 0.08 and the test item interfered with MTT, Non-Specific Killed Controls (NSKC) were necessary in the main experiment additionally.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): each 2 tissues for the test item, positive and negative controls

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): The concentration of formazan was measured by determination of the OD of the isopropanol-extracts in duplicate at 570 nm in an automatic reader of a spectrophotometer.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: For interpretation of cell viability results the cut-off value distinguishing classified (irritant) from non-classified substances as given in OECD TG 492 was used:
- The test chemical is identified as not irritant and not requiring classification according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%.
- The test chemical is identified as irritant and potentially requiring classification according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post exposure incubation is less than or equal (≤) to 60%.

- Positive and negative control means and acceptance ranges based on historical data: The following acceptance criteria determine the validity of an assay:
- mean OD 570 nm negative control (NC) is > 0.8 and < 2.8
- mean relative viability of the positive control (PC) is < 50 % (relative to negative control)
- the difference of viability between the two replicates is < 20 %.

Irritation parameter:

other: mean percent tissue viability

Run / experiment:

mean of two tissue cultures

Value:

88.77

Vehicle controls validity:

not examined

Remarks:

no vehicle used

Negative controls validity:

valid

Remarks:

100 % final cell viability

Positive controls validity:

valid

Remarks:

5.30 % final cell viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

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

According to the conducted pre-check the test items were demonstrated to exert optical interferences directly affecting the test results that are not related to cytotoxic effects on tissue cells. Therefore killed controls and colour controls had to be conducted.

Colour of test item	MTT Reduction	Colour reaction in isopropanol	Colour reaction in water	Killed control test to be conducted	Colour control test to be conducted	Non specific Killed control test to be conducted
black	+	+	+	yes	yes	yes

+  positive reaction, -  negative reaction

 

As the final test item-treated tissue viability was > 60% relative to negative control, the test item was characterized as NOT having eye irritating properties (UN GHS No Category):

Compound	Final cell viability [%]	Category
Test item	88.77	No category
Positive control	5.30	Cat. 1 / 2
Negative control	100.00	No category

 

All assay acceptance criteria were met.

Interpretation of results:

GHS criteria not met

Remarks:

not irritant

Executive summary:

This in vitro study was performed to assess the eye irritation potential of the registered substance by means of the Human Cornea Model Test following OECD TG 492. The test item proved to be an MTT reducer and to dye water and isopropanol in the color interference pre-experiment. The OD of the test item in isopropanol at 570 nm after blank correction was > 0.08. Therefore, additional tests with freeze-killed tissues, viable tissues and Non-specific Killed Control (NSKC) tissues (without MTT addition) had to be performed.

Each 50 mg of the solid test item or 50 µL of the negative control (deionised water) and of the positive control (methyl acetate), were applied to each duplicate tissue for 6 hours. Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system.

After treatment with the test item the mean relative viability value was 88.77% compared to the mean value of the negative control. This value is above the threshold for irritancy of ≤ 60%. Therefore, the test item does not need to be classified according UN GHS.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the registered substance does not need to be classified for eye irritation according UN GHS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Based on in vitro tests for skin and eye irritation no classification is warranted.