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Description of key information

Skin:

In an in vitro skin irritation test according to OECD Guideline 439 (BASF Colors&Effects, 2017), a cell viablility of 103.5 % was determined.

In an in vitro skin corrosion test according to OECD Guideline 431 (BASF Colors/Effects, 2017), a cell viability of 97.0 % after 3 min and 102.7 % after 1 h incubation was determined.

Eye:

In an in vitro eye irritation study according to OECD Guideline 492 (BASF Colors&Effects, 2017), a cell viability of 104.4 % was determined.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016-05-09 to 2017-08-15
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:
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: 002-151203

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

OTHER SPECIFICS: Solid / orange, pH ~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: EpiDerm - model (EPI-200)
- Tissue lot number: 23339
- Date of initiation of testing: 2016-05-31

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (3 min) or 37 °C incubator (1 h)

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
-
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 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
see Any other Information on Materials and Methods, Table 1 and 2.

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
- 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, ~ 7mg)

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
Duration of treatment / exposure:
3 min or 1 h
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min
Value:
97
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 h
Value:
102.7
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- 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
Interpretation of results:
GHS criteria not met
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016-05-09 to 2017-08-15
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: 002-151203

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

OTHER SPECIFICS: Solid / orange, pH ~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:
As demanded by the OECD Guideline 439.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm - model (EPI-200)
- Tissue lot number: 23339
- Date of initiation of testing: 2016-05-31

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Romm temperature (25 min), 37 °C in incubator (35 min)
- Temperature of post-treatment incubation: 37 °C in incubator (24 ± 2 h)

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: 1.0 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
see Any other Information on Materials and Methods, Table 1 and 2.

NUMBER OF REPLICATE TISSUES:3

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
- A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50 % after 3 h incubation and a post-treatment-period of 24 h.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 µL (bulk volume, ~ 7 mg)

NEGATIVE CONTROL
- Amount applied: 30 µL


POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5 %
Duration of treatment / exposure:
1 h
Duration of post-treatment incubation (if applicable):
24 ± 2 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
97
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: Due to the results of a pretest with a comparable test system 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 4: Result of the irritation test, individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Test substance identification

 

Tissue 1

Tissue 2

Tissue 3

Mean

SD

CV [%]

NC

Mean OD570

1.835

1.859

1.787

1.827

 

 

 

Viability [% of NC]

100.4

101.8

97.8

100.0

2.0

2.0

Test substance

Mean OD570

2.026

1.894

1.753

1.891

 

 

 

Viability [% of NC]

110.9

103.7

95.9

103.5

7.5

7.2

PC

Mean OD570

0.064

0.053

0.072

0.063

 

 

 

Viability [% of NC]

3.5

2.9

3.9

3.4

0.5

14.8

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:
2016-05-09 to 2017-08-15
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: 002-151203, Test-substance No.: 16/0082-1

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

OTHER SPECIFICS: Solid / orange, pH ~ 5 (undiluted test substance, moistened with de-ionized water; determined in the lab prior to start of the GLP study)
Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: The EpiOcular tissue system is demanded by the OECD Guideline 492.
- 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, ~ 13 mg)

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
- RhCE tissue construct used, including batch number: EpiOcularTM model (OCL-200), Tissue Lot No 23713, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Doses of test chemical and control substances used:
Test substance: 50µL
Negative Control: 50 µL
Positive Control: 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods.
Exposure: 6 h at 37 °C in the incubator
post-exposure immersion: 25 min
post -exposure incubation: 18 h at 37 °C in the incubator
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: 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 was not necessary.
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength used for quantifying MTT formazan: Spectrophotometer, measurement using a filter wavelength 570 nm without reference filter
- 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:
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 (min) value following exposure to 100 µL of 0.3 % Triton X-100 for each EpiOcular™ EIT (OCL-200) 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 Guideline. Lower acceptance limit: ET50 = 12.2 min; Upper acceptance limit: ET50 = 37.5 min
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. The 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.
Acceptance criteria for tissue variability: 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 %.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: see Any other information on materials and methods, Table 1.
- Analysis for tissue functionality and quality: see Any other information on materials and methods, Table 2.








Irritation parameter:
other: Cell viability [mean, %]
Value:
104.4
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

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

1.818

1.638

1.728

 

 

Viability [% of NC]

105.2

94.8

100.0

10.4

Test substance

Mean OD570

1.652

1.957

1.805

 

 

Viability [% of NC]

95.6

113.2

104.4

17.6

PC

Mean OD570

0.392

0.364

0.378

 

 

Viability [% of NC]

22.7

21.1

21.8

1.

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/corrosion:

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. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test according to OECD Guideline 431 and Skin Irritation Test according to OECD Guideline 439 (BASF Colors&Effects, 2017).

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 7 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 test substance is not able to reduce MTT directly.

Results of the corrosion test:

The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 97.0 % (single values: 98.9 and 95.0 %), and it was 102.7 % (single values: 100.2 and 150.2 %) after an exposure period of 1 hour.

Results of the irritation test:

The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours’ post-incubation was 103.5 % (single values: 110.9 %, 103.7 %, and 95.9 %).

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

In conclusion, based on the result of the integrated testing strategy, 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:

In an in vitro eye irritation study according to OECD Guideline 492 (BASF Colors&Effects, 2017), the potential of the test substance to cause ocular irritation was assessed by a single topical application of ca. 50 µL bulk volume (ca. 13 mg) of the undiluted test substance to a reconstructed three-dimensional human cornea model (EpiOcular™). Tissue destruction was determined by measuring the metabolic activity of the tissue after 3 h exposure and 18 h 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 test substance was not able to reduce MTT directly. Appropriate negative and positive controls were valid. The mean viability of the test-substance treated tissues was 104.4 %. Minimal discoloration of the test-substance treated tissues was observed after the washing procedure. However, this did not interfere with the colorimetric test as demonstrated in the pretest prior to start of the study. 

Based on the observed results for the EpiOcular Test it was concluded, that the test substance does not show an eye irritating potential 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.

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