Reaction mass of 2-[4-[(Hexahydro-2,4,6-trioxo-5-pyrimidyl) azo]phenyl]-6-methylbenzothiazole-7-sulphonic acid, compound with 2,2',2''-nitrilotris[ethanol] (1:1) and Lithium 2-[4-[(hexahydro-2,4,6-trioxopyrimidin-5-yl)azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Endpoint summary

Description of key information

Skin irritation

The test item does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy.

Eye irritation

The test item does not show an eye irritation potential in the in vitro eye irritation test strategy.

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:

2016-02-25 to 2016-04-27

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EU) No 640/2012 of 6 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch no.: 0013479406

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue batch number: 23317 (test run 1) and 23328 (test run 2)
- Date of initiation of testing: 2016-03-01

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room termperature for 25 minutes and in the incubator (37 °C) for 35 minutes
- Temperature of post-treatment incubation: 37°C ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Once, 1 hour after start of application with sterile PBS
- Observable damage in the tissue due to washing: Yes, due to mechanical damage of tissue 1 during the washing procedure, only two tissues of the negative control could be evaluated.
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Positive control: mean 3 %; range: 2.2-3.9

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Due to the color 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 freeze-killed control tissue (KC), incubated 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 color control tissues is not necessary.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the mean tissue viability is ≤ 50 %.
- The test substance is considered to be non-irritant to skin if the mean tissue viability is > 50 %

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amounts applied: bulk volume of ca. 25 μL of the solid test material
- Concentration: undiluted

NEGATIVE CONTROL
- Amount applied: 30 μL
- Concentration: no data

POSITIVE CONTROL
- Amount applied: 30 μL
- Concentration: 5 % SDS

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 (per independent experiment, 2 test runs)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean value of 3 tissues

Value:

92.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

4 %

Remarks on result:

other: Result refers to 2nd run. Due to the high inter-tissue variability of the test substance (1st run), a 2nd test run was performed to clarify the result.

Other effects / acceptance of results:

Yellowish discoloration of the test-substance treated tissues was observed after the washing procedure.

Values for single tissues
1st test run: 101.1%, 95.3% and 7.6%. Due to the high inter-tissue variability of the test substance, a 2nd test run was performed to clarify the result.
2nd test run: 91.6%, 94.7% and 90.5%.

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, for the 2nd test run.
Due to the non-concordant replicate measurements of the test-substance treated tissues after the 1st test run, the study was repeated.

- Range of historical values:

Historic Range of NC: OD570
Period: Jan 2014 - Jan 2016;
Mean OD: 2.373; SD: 0.263; Mean + 2 SD: 2.899; Mean - 2 SD: 1.847

Historic Range of PC: OD570
Period: Jan 2014 - Jan 2016; Mean OD: 0.071; SD: 0.011; Mean + 2 SD: 0.093 Mean - 2 SD: 0.050

Viability (%)
Period: Jan 2014 - Jan 2016; Mean %: 3.0; SD: 0.4; Mean + 2 SD: 3.9; Mean - 2 SD: 2.2

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:

2016-02-25 to 2016-04-27

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch no.: 0013479406

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The test is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed cornea after a short term topical exposure. The test is designed to predict an eye irritation potential of a chemical by using the three dimensional human cornea model EpiOcularTM.

- Description of the cell system used
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.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 μL

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
The test is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed cornea after a short term topical exposure. The test is designed to predict an eye irritation potential of a chemical by using the three dimensional human cornea model EpiOcularTM. After application of the test material to the surface of the EpiOcularTM tissue the induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanol extraction of the formazan from the tissues, the optical density of the extract is determined spectrophotometrically. Optical density of the extracts of test-substance treated tissues is compared to values from negative control tissues and expressed as relative tissue viability.

- RhCE tissue construct used, including batch number
Tissue model: OCL-200
Tissue Lot Number: 23700
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

- Doses of test chemical and control substances used
Test chemical: Undiluted
Negative control (NC): De-ionized water, sterile
Positive control (PC): neat

- Temperature of exposure/ incubation: 37 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals
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. The test substance is not able to reduce MTT directly.

Color control
Due to the color 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 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 color control tissues is not necessary.

- Number of tissue replicates used per test chemical and controls: 2

- Wavelength used for quantifying MTT formazan
Spectrophotometer: SunriseTM Absorbance Reader
Wavelength: 570 nm (OD570)

- Description of the method used to quantify MTT formazan
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.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model

Decision criteria for evaluation of results
Mean tissue viability (% of negative control)
< 55%: irritant
55 - 65 %: Borderline
> 65%: Non-irritant

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria:
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. Them 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.

- Complete supporting information for the specific RhCE tissue construct used: Certificate of Analysis on test system quality is provided by the supplier (MatTek Corporation)

- Positive and negative control means and acceptance ranges based on historical data
Protocol for solids Jan 2014 - Feb 2016
Negative control: Mean OD: 1.674 +/- 0.164
Positive control: Mean OD: 0.403 +/- 0.092
Mean % viability: 24.0 +/- 4.7

- Acceptable variability between tissue replicates for positive and negative controls: < 20%.
- Acceptable variability between tissue replicates for the test chemical: < 20%.

Irritation parameter:

other: tissue viability [%]

Run / experiment:

Mean value of tissue 1and tissue 2

Value:

68.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

22.3 %

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

Key study

The test item was assessed for skin irritation according OECD 439 and GLP.

The potential of the test item to cause dermal irritation was assessed by a single topical application of ca. 25 μL bulk volume (about 13 mg) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). The irritation test was performed with three EpiDerm™ tissues per test run, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period.

The 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.

It was determined that the test substance is not able to reduce MTT directly. In a pretest it was demonstrated that the colour of the test substance did not interfere with the colorimetric test.

After the 1st test run of the skin irritation test non-concordant replicate measurements of the test substance treated tissues was observed (values for single tissues: 101.1%, 95.3% and 7.6%). Due to the high inter-tissue variability of the test substance, a 2nd test run was performed to clarify the result.

In the 2nd test run the mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 92.3% (values for single tissues: 91.6%, 94.7% and 90.5%).

All acceptance criteria were met.

Yellowish discoloration of the test-substance treated tissues was observed after the washing procedure. However, this did not interfere with the colorimetric assay, as was demonstrated in a pretest prior to start of the study.

Based on the observed results and applying the evaluation criteria it was concluded, that the test item does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

 

Supporting study

As the test material was a preparation containing less than 20 % test substance, the available in vivo study was only considered as supporting information.

However the result is in agreement with the observed effects in the in vitro study and therefore is an appropriate support.

To assess the irritant property of the test substance, a study was carried out using three male rabbits. 0.5 mL of the unchanged test substance were applied to the untreated skin on the upper third of the back or flanks of each animal using a patch. The application site was then covered by a semiocclussive dressing. After 4 hours, the patch was removed and the test substance washed off with Lutrol (R) and Lutrol(R) / water. The untreated skins sites of the animals served as control. Using a scoring table according to OECD Guideline 404, the scores were as follows: The mean scores for edema over the reading times 24 h /48 h/ 72 h were 0 for all animals for both erythema and edema formation. Therefore, the test substance was determined to be non irritating to the rabbits' skin.

Eye irritation

Key study

The test item was assessed for eye irritating potential according to OECD 492 and GLP.

Using the currently available methods a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.

However, in the current case for the test item the results derived with EpiOcular alone were sufficient for a final assessment as it showed that the test item has no eye irritation or corrosion potential. Therefore further testing in BCOP was waived.

The potential of the test item to cause ocular irritation was assessed by a single topical application of ca. 50 μL bulk volume (about 19 mg) of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™).

Two EpiOcular™ tissues were incubated with the test substance for 6 hours followed by an 18-hours 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.

It was determined that 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. The mean viability of the test-substance treated tissues was determined to be 68.2%.

Yellowish discoloration of the test-substance treated tissues was observed after the washing procedure. However, this did not interfere with the colorimetric assay, as was demonstrated in a pretest prior to start of the study.

Based on the observed results for the EpiOcular Test alone and applying the evaluation criteria it was concluded, that the test item does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Supporting study

As the test material was a preparation containing less than 20 % test substance, the available study was only considered as supporting information.

The potential of the test substance to cause eye irritation was assessed in a study conducted on 3 rabbits. Therefore, 0.1 mL of the unchanged material was applied to one eye of each animal and not washed out. As no findings were observable after 48 hours, the study was terminated after 72 hours. The average scores for the time points 24, 48 and 72 hours were 0 for corneal opacity, iris and chemosis (all animals), 0 for conjunctivae redness for animal 1 and 0.3 for conjunctivae redness for animals 2 and 3. Therefore, the substance was considered to be non-irritating to the rabbits’ eyes.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes. The substance does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy and does not show an eye irritation potential in the in vitro eye irritation test strategy. As a result the substance is not considered to be classified for skin irritation/corrosion or eye irritation/ serious eye damage under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.