Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Endpoint summary

Description of key information

Skin irritation

The test substance does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Eye irritation

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

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:

2016-11-30 to 2016-01-05

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

Specific details on test material used for the study:

Batch identification: 13298465
pH-value: ca. 5
Storage conditions: Room temperature

Test system:

human skin model

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm™ 200 kit: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
containing: 24 EPI-200 tissues (reconstructed epidermis): surface 0.6 cm²
cultured in Millicells® diameter 1 cm

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C ± 1°C
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1x after 1 hour application
- Observable damage in the tissue due to washing: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm
- Filter: without reference filter

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 μL sterile PBS was applied first. Thereafter, a bulk volume of ca. 25 μL of the solid test
material was applied with a sharp spoon and homogeneously distributed together with the fluid.

NEGATIVE CONTROL
- Amount applied: 30 μL of sterile Dulbecco's phosphate buffered saline (PBS)

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

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

After 24 ± 2 hours for additional 18 ± 2 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 1

Value:

97.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 2

Value:

125.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 3

Value:

90.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: Based on the results of the pretest it was judged, that application of color control tissues is not necessary.


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
- Range of historical values if different from the ones specified in the test guideline: no

Test substance
identification Tissue 1 Tissue 2 Tissue3 mean SD CV [%]
NC mean OD570 2.128 2.338 1.997 2.152
viability
[% of NC] 98.8 108.5 92.7 100.0 8.0 8.0
Test substance mean OD570 2.102 2.698 1.956 2.252
viability
[% of NC] 97.6 125.2 90.8 104.5 18.3 17.5
PC mean OD570 0.071 0.076 0.069 0.072
viability
[% of NC] 3.3 3.5 3.2 3.3 0.2 4.7

Table 1: Decision criteria for evaluation of results of irritation test

Mean tissue viability (% of negative control)	Prediction
< 45	Irritant
45 - 55	Borderline
> 55	Non-irritant

The „borderline“-evaluation (50 ± 5%) was determined statistically using historic BASF data and hence considers the variance of the test method. This evaluation is confirming the borderline range provided in OECD Guideline 439.

Table 2: Results

Test substance identification		Tissue 1	Tissue 2	Tissue 3	mean	SD	CV [%]
NC	mean OD570	2.128	2.338	1.997	2.152
	viability [% of NC]	98.8	108.5	92.7	100.0	8.0	8.0
Test substance	mean OD570	2.102	2.698	1.956	2.252
	viability [% of NC]	97.6	125.2	90.8	104.5	18.3	17.5
PC	mean OD570	0.071	0.076	0.069	0.072
	viability [% of NC]	3.3	3.5	3.2	3.3	0.2	4.7

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:

2 Dec 2015 to 05 Jan 2016

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch identification: #13298465

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 μL

Details on study design:

- Details of the test procedure used:
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:
Tissue model: EpiOcularTM model (OCL-200)
Tissue Lot Number: 21588
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

- Duration:
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.
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.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: No

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

- Wavelength used for quantifying MTT formazan, and linearity range of measuring device (spectrophotometer): 570 nm (OD570), blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

- Description of the method used to quantify MTT formazan
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 isopropanolextraction of the formazan from the tissues, the optical density of the extract is determined spectrophotometrically.
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.
Tissue viability
The quantification of tissue viability is presented as the ratio of the mean OD570 divided by the respective OD570 NC value in percent.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
see in section "any other information"

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

- Acceptable variability between tissue replicates for positive (PC) and negative controls (NC)
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.
PC: Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.

- Acceptable variability between tissue replicates for the test chemical
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%.

- Color control: Based on the results of the pretest it was judged, that application of color control tissues is not necessary.

Irritation parameter:

other:

Remarks:

viability [% of NC]

Run / experiment:

tissue 1

Value:

66.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: viability [% of NC]

Run / experiment:

tissue 2

Value:

68.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Individual and mean OD570 values, individual and mean viability values and inter-tissue variability

 

Test substance identification		tissue 1	tissue 2	mean	Inter-tissue variability [%]
NC	mean OD570	1.652	1.514	1.583
	viability [% of NC]	104.4	95.6	100.0	8.7
Test substance	mean OD570	1.059	1.086	1.072
	viability [% of NC]	66.9	68.6	67.7	1.7
PC	mean OD570	0.479	0.584	0.532
	viability [% of NC]	30.2	36.9	33.6	6.7

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

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 study - in vitro

The test substance was tested in vitro in line with GLP for skin corrosion according OECD 431 and EU guideline B.40 and for skin irritation according OECD 439and EU guideline B.46.

However, in the current case the results derived with Skin Irritation Test (SIT) alone were sufficient for a final assessment. Therefore further testing in Skin Corrosion Test (SCT) SCT was waived.

 

The potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of ca. 25 μL bulk volume (ca. 26 mg) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™).

The irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-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 quotient of the values indicates the relative tissue viability.

The following results were obtained in the EpiDerm™ skin irritation test:

The test substance is not able to reduce MTT.

The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 104.5%.

The test-substance treated tissues showed a slightly yellow discoloration after the washing procedure. However, this did not interfere with the colorimetric test as was demonstrated in a pretest prior to start of the GLP study.

The inter-tissue variability of the test-substance treated tissues (SD of %-viability) is minimally out of the acceptance range. Since the single viability values are well above the cut off for skin irritation and all other quality criteria of the test were met, this deviation is not considered to adversely affect the result of this study.

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

Eye irritation study - in vitro

In the GLP and OECD 437 guideline compliant study the test substance was assessed for eye irritating potential with an EpiOcular Eye Irritation Test.

The potential of the test substance to cause ocular irritation was assessed by a single topical application of ca. 50 μL bulk volume (ca. 36 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.

The following results were obtained in the EpiOcular™ eye irritation assay:

The test substance is not able to reduce MTT directly.

The mean viability of the test-substance treated tissues was 67.7%.

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

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

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 under Regulation 1272/2008 (CLP). As a result the test substance is not considered to be classified for skin irritation and eye irritation under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776