Hydrogen [3-[[1-[anilinocarbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzene-1-sulphonato(3-)]hydroxychromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Endpoint summary

Description of key information

Skin:

In an in vitro Reconstructed Human like Epithelium (RhE) skin irritation study according to OECD Guideline 439, a tissue viability of 86.6 % was observed (BASF Colors&Effects, 2017).

In an in vitro Reconstructed Human like Epithelium (RhE) skin corrosion study according to OECD Guideline 431, a tissue viability of 90.9 % (3 min incubation) and 96.3 % (1 h incubation) was observed (BASF Colors&Effects, 2017).

Eye:

In an in vitro eye irritation study according to OECD Guideline 437 an In Vitro Irritancy score (IVIS) of 17.2 was determined (BASF Colors&Effects, 2017).

In an in vitro Reconstructed Human Cornea like Epithelium (RhCE) eye irritation study according to OECD Guideline 492, a tissue viability of 106.0 % was observed (BASF Colors&Effects, 2017).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-12-09 to 2017-03-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No of test material: 002-152503

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

OTHER SPECIFICS: Solid / orange to brown; pH ca. 5 (undiluted test substance moistened with de-ionized water, determined in the lab prior to start of the GLP study)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Demanded by the corrosponding OECD Guideline.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue lot number: 23385
- Date of initiation of testing: 2016-12-13

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min at room temperature or 1 h at 37 °C.
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
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 value following exposure to Triton X-100 (1 %) for each EpiDerm 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 guidelines. Lower acceptance limit: ET50 = 4.0 hours, Upper acceptance limit: ET50 = 8.7 hours

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues: freezing
- No of replicates: 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50 %, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 µL (bulk volume)

NEGATIVE CONTROL
- Amount applied: 50 µL


POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

3 min or 1 h

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

90.9

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h

Value:

96.3

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Minimal yellowish discoloration of the test-substance treated tissues was observed after the washing procedure.
- Direct-MTT reduction: No
- Colour interference with MTT: Based on the result 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

Table 3: Exposure period 3 min: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

		Tissue 1	Tissue 2	Mean	SD	CV [%]
NC	Mean OD570	1.914	1.662	1.788
	Viability [% of NC]	107.1	92.9	100.0	10.0	10.0
Test Substance	Mean OD570	1.662	1.589	1.626
	Viability [% of NC]	93.0	88.9	90.9	2.9	3.2
PC	Mean OD570	0.358	0.235	0.297
	Viability [% of NC]	20.0	13.2	16.6	4.8	29.2

 

Table 4: Exposure period 1 h: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

		Tissue 1	Tissue 2	Mean	SD	CV [%]
NC	Mean OD570	1.649	1.711	1.680
	Viability [% of NC]	98.1	101.9	100.0	2.6	2.6
Test Substance	Mean OD570	1.658	1.576	1.617
	Viability [% of NC]	98.7	93.8	96.3	3.5	3.6
PC	Mean OD570	0.085	0.081	0.083
	Viability [% of NC]	5.1	4.8	5.0	0.2	3.4

Interpretation of results:

GHS criteria not met

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-12-09 to 2017-03-22

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:

2015-07-28

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012-07-06

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No of test material: 002-152503

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

OTHER SPECIFICS: Solid / orange to brown; pH ca. 5 (undiluted test substance moistened with de-ionized water, determined in the lab prior to start of the GLP study)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue lot number: 23385
- Date of initiation of testing: 2016-12-13

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25 °C at rroom temperature followed by 35 min at 37 °C
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
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 value following exposure to Triton X-100 (1 %) for each EpiDerm 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 guidelines. Lower acceptance limit: ET50 = 4.0 hours, Upper acceptance limit: ET50 = 8.7 hours

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues: freezing
- No of replicates: 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 µL (bulk volume)

NEGATIVE CONTROL
- Amount applied: 30 µL


POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration (if solution): 5 %

Duration of treatment / exposure:

1 h

Duration of post-treatment incubation (if applicable):

42 h ± 4 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

86.6

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Minimal yellowish discoloration of the test-substance treated tissues was observed after the washing procedure. The lower value of the third tissue resulted from mechanical damage of the supporting membrane below the epidermis, which was noticed before formazan extraction of the tissue. Thus, the lower viability value cannot be attributed to the test substance application alone. As all other acceptance criteria were met and due to the unambiguous result of the test substance the study is considered valid despite this deviation.
- Direct-MTT reduction: No
- Colour interference with MTT: No

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

Table 3: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

		Tissue 1	Tissue 2	Tissue 3	Mean	SD	CV [%]
NC	Mean OD570	1.980	1.997	1.873	1.950
	Viability [% of NC]	101.5	102.4	961	100.0	3.4	3.4
Test Substance	Mean OD570	1.830	2.005	1.230	1.689
	Viability [% of NC]	93.9	102.8	63.1	86.6	20.8	24.1
PC	Mean OD570	0.047	0.046	0.052	0.049
	Viability [% of NC]	2.4	2.4	2.7	2.5	0.2	6.6

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

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-01-03 to 2017-03-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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:

2013-07

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: 002-152503

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: The test-substance preparation was produced on a weight per volume (w/v) basis shortly before application by stirring with a high-speed homogenizer (Ultra-Turrax) and a magnetic stirrer. The homogeneity of the test-substance preparation during application was provided by stirring with a magnetic stirrer.

FORM AS APPLIED IN THE TEST
20 % (w/v) suspension in deionized water

OTHER SPECIFICS: Solid / orange to brown, pH ca. 5 (undiluted test substance moistened with de-ionized water or 20 % aqueous preparation, determined in the lab prior to start of the GLP study)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany
- Characteristics of donor animals: Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).
- indication of any existing defects or lesions in ocular tissue samples: free of defects

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 20 % (w/v) test-substance preparation

Duration of treatment / exposure:

4 h

Number of animals or in vitro replicates:

3

Details on study design:

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. After the equilibration period the medium in both chambers was replaced with fresh pre-warmed medium.

QUALITY CHECK OF THE ISOLATED CORNEAS
Initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 550 opacity units were discarded.

NUMBER OF REPLICATES
3

NEGATIVE/SOLVENT CONTROL USED
de-ionized water

POSITIVE CONTROL USED
20 % (w/v) solution of imidazole in deionized water

APPLICATION DOSE AND EXPOSURE TIME
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.

TREATMENT METHOD:
open chamber

POST-INCUBATION PERIOD:
no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period. 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: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of an microtiter plate reader] (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
see under "Any other information on materials and methods"

Irritation parameter:

in vitro irritation score

Value:

17.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Table 2: In Vitro Irritancy score (IVIS) of the test substance, NC and PC

				IVIS
	Cornea No	Opacity per cornea	Permeability per cornea	Per cornea	Per group
					Mean	SD
Test substance	13 14 15	13.3 22.7 14.6	0.003 0.018 0.051	13.4 23.0 15.4	17.2	5.1
NC	1 2 3	1.8 9.6 7.3	0.002 0.002 0.004	1.9 9.6 7.3	6.3	4.0
PC	4 5 6	90.1 87.9 91.0	2.719 2.266 2.467	130.8 121.9 128.0	126.9	4.6

Interpretation of results:

GHS criteria not met

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-01-03 to 2017-03-22

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:

2015-07-28

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 002-152503

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: The test-substance preparation was produced on a weight per volume (w/v) basis shortly before application by stirring with a high-speed homogenizer (Ultra-Turrax) and a magnetic stirrer. The homogeneity of the test-substance preparation during application was provided by stirring with a magnetic stirrer.

FORM AS APPLIED IN THE TEST
20 % (w/v) suspension in de-ionized water

OTHER SPECIFICS: Solid / orange to brown, pH ca. 5 (undiluted test substance moistened with deionized water or 20 % aqueous preparation, determined in the lab prior to start of the GLP study)

Species:

human

Details on test animals or tissues and environmental conditions:

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
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 (bulk volume)

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used:
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 pre-incubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours. 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.

- RhCE tissue construct used, including batch number:
OCL-200, Lot No 23760, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

- Doses of test chemical and control substances used:
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) or with 50 µL of methyl acetate (PC).

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
exposure: 6 h, 37 °C
post exposure immersion: 25 min, room temperature
post-exposure incubation: 18 h, 37 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals
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 color 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.
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 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 color control tissues is not necessary.


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

- Wavelength used for quantifying MTT formazan, and linearity range of measuring device: SunriseTM Absorbance Reader, 570 nm

- 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
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.


- Complete supporting information for the specific RhCE tissue construct used
see under "Any other information on materials and methods"

Irritation parameter:

other: cell viability (%)

Value:

106

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No, ninimal yellowish discoloration of the test-substance treated tissues was observed after the washing procedure

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

Table 4: 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	1.490	1.523	1.506
	Viability [% of NC]	98.9	101.1	100.0	2.2
Test substance	Mean OD570	1.794	1.401	1.597
	Viability [% of NC]	119.1	93.0	106.0	26.1
PC	Mean OD570	0.345	0.222	0.283
	Viability [% of NC]	22.9	14.7	18.8	8.2

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:

The objective was to assess the skin irritation and corrosion potential of the test substance. Using the currently available methods a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential (BASF Colors&Effects, 2017).Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) according to OECD Guideline 431 and Skin Irritation Test (SIT) according to OECD Guideline 439. The potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of ca. 25 µL bulk volume (about 10 mg) of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm).

For the corrosion test two EpiDerm tissues were incubated with the test substance for 3 minutes and 1 hour, each. 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 corrosion/irritation test:

The 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.

Results of the Corrosion Test (SCT):

Minimal compound residues or yellowish discoloration of the test-substance treated tissues of the 1-hour exposure were observed after the washing procedure The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 90.9 %, and it was 96.3 % after an exposure period of 1 hour.

Results of the Irritation Test (SIT):

Minimal yellowish discoloration of the test-substance treated tissues was observed after the washing procedure. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours’ post-incubation was 86.6 %. The values for single tissues of the test substance showed higher variability due to the result of the third tissue (single values 93.9 %, 102.8 % and 63.1 %). The lower value of the third tissue resulted from mechanical damage of the supporting membrane below the epidermis, which was noticed before formazan extraction of the tissue. Thus, the lower viability value cannot be attributed to the test substance application alone. As all other acceptance criteria were and due to the unambiguous result of the test substance the study is considered valid despite this deviation.

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 EpiDermin vitro skin irritation and corrosion test strategy under the test conditions chosen.

Eye:

The objective was to determine a possible eye irritating potential of the test substance by in vitro studies (BASF Colors&Effects, 2017). A single in vitro assay may not always be sufficient, using the currently available methods, to cover the full range of eye irritating potential. Consequently, two in vitro assays were performed: The Bovine Corneal Opacity and Permeability Test (BCOP Test) according to OECD guideline 437 and the EpiOcular Eye Irritation Test according to OECD guideline 492.

 

BCOP

The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 µL of a 20 % test-substance preparation to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test-substance preparation for an exposure period of 4 hours. In addition to the test substance, a negative control (NC; de-ionized water) and a positive control (PC; 20 % imidazole in de-ionized water) were applied to three corneas each. Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance. The following results were obtained in the BCOP Test:

 

	Mean Opacity Value	Mean Permeability Value	Mean In vitro Irritancy Score
Test Substance	16.9	0.024	17.2
NC	6.2	0.003	6.3
PC	89.6	2.484	126.9

 

EpiOcular

The potential of test substance to cause ocular irritation was assessed by a single topical application of ca. 50 µL bulk volume (about 14 mg) of the undiluted test substance to a reconstructed three-dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissue samples were incubated with the test substance for 6 hours followed by a 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 EpiOcular™ eye irritation test showed the following results:

The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues was 106.0 % (values for single tissues: 119.1% and 93.0%).

 

Summary of the individual test results of the in vitro eye irritation turnkey testing strategy:

Test method	Test Result	Test Evaluation	Evaluation Test Strategy
BCOP Test	The mean IVIS of the test-substance treated corneas was 17.2	not identified as corrosive or severe irritant	non-irritant
EpiOcular	Mean viability of the test-substance treated tissues was 106 %	non-irritant

 

Based on the results for BCOP and EpiOcular Test and considering the evaluation criteria, 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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

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