Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Reaction products of diazotized 4-amino-6-[(4-aminophenyl)diazenyl]-3-[(4-aminophenyl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid, coupled with benzene-1,3-diamine, sodium salts
EC Number:
812-037-7
Cas Number:
1793011-72-9
Molecular formula:
Unknown for all components
IUPAC Name:
Reaction products of diazotized 4-amino-6-[(4-aminophenyl)diazenyl]-3-[(4-aminophenyl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid, coupled with benzene-1,3-diamine, sodium salts
Test material form:
solid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EPISKIN Small model
Source strain:
not specified
Details on animal used as source of test system:
Not applicable. EPISKIN small model was used which is a a three-dimensional human epidermis model, which consists of adult human derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN small model
- Tissue batch number(s): 17EKIN008
- Production date: Not specified
- Shipping date: Not specified
- Delivery date: Not specified

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): Not applicable

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Washed with phosphate buffered saline (volume and number of steps not specified)
- Observable damage in the tissue due to washing: Not specified
- Modifications to validated SOP: None noted

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in PBS
- Incubation time: 3 hours at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm
- Filter: Not specified
- Filter bandwidth: Not specified
- Linear OD range of spectrophotometer: Not specified

NUMBER OF REPLICATE TISSUES: 3 tissues per test item alongside negative and positive controls

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- The substance was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the colour interference, at least 10 mg of the substance was added to 90 μl Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μl Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed. The substance was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, at least 10 mg of the test item was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the incubation period a colour check was performed.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 15 minutes exposure and 42 hours of post incubation is less than or equal to 50%.
- The test substance is considered to be non-corrosive to skin if the viability after 15 minutes exposure and 42 hours of post incubation is greater than 50%.
Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 15.7 to 19.9 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): Test substance applied directly.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL PBS (3 tissues)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL 5% SDS (3 tissues)
Duration of treatment / exposure:
Exposure period of 15 minutes after which the tissues were rinsed.
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3 tissues per test item, negative control and positive control.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
112
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None noted
- Direct-MTT reduction and colour interference: The substance was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that the substance did not interact with the MTT endpoint.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The control values were within the range of historical controls.

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

Any other information on results incl. tables

Mean absorption in the in vitro skin irritation test with the test substance

 

A (OD570)

B (OD570)

C (OD570)

Mean (OD570)

SD

Negative control

0.826

0.872

0.808

0.835

0.033

Test substance

0.988

0.817

1.011

0.938

0.106

Positive control

0.193

0.224

0.139

0.186

0.043

 

Mean tissue viability in the in vitro skin irritation test with the test substance

 

Mean tissue viability (% of control)

Standard deviation (percentage)

Negative control

100

3.9

Test substance

112

13

Positive control

22

5.2

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
It is concluded that this test is valid and that the substance is non-irritant in the in vitro skin irritation test under the experimental conditions used and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and EU CLP (EC No. 1272/2008, as amended).