Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 September 2019 - 17 September 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

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:
yes
Remarks:
Standard deviation between the 2 NSCkilled replicates treated with the test item was 35.4% instead of 18% at the maximum as defined in OECD test guideline. This deviation is considered as without impact on the conclusion of the study (see reasoning below)
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis (3‐(diethylamino)‐7‐hydroxy‐5‐phenylphenazinium) sulphate
EC Number:
950-123-7
Cas Number:
97849-65-5
Molecular formula:
C44H44N6O6S
IUPAC Name:
Bis (3‐(diethylamino)‐7‐hydroxy‐5‐phenylphenazinium) sulphate
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Remarks:
(SkinEthic RHE® model)
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: foreskin (number of donors not specified)
Source strain:
not specified
Justification for test system used:
The SkinEthic RHE® model has been validated for irritation testing (Validation study based on the original ECVAM Performance Standards (21) in 2008) and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model
- Tissue batch number(s): 19-RHE-156 (living epidermis); 17-RHE-124, 18-RHE-002, 18-RHE-142 and 19-RHE-096 (killed epidermis).
- Production date: N/A
- Shipping date: 17/09/2019
- Delivery date: 17/09/2019
- Expiration date: 23/09/2019
- Date of initiation of testing: 17/09/2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- 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: 300 µL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours at 37ºC, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: O.D. = 1.4 (CV = 2.7%) specification OD > 0.7. Historical negative control mean OD range = 0.489-1.217 (OD measured after 1:2 dilution in isopropanol; acceptability criteria should be in the range ≥ 0.4 and ≤1.5)
- Barrier function: 5.8 h (Specification 4.0h < ET50< 10.0h)
- Morphology: 8 Cell layers (specification > 4.), highly differentiated epidermis consisting of organized basal, spinous and granular layers and a multilayered stratum corneum.
- Contamination: No.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
- Fresh tissues / killed tissues: 2 killed tissues ( NSMTT control); 2 living tissues ((NSCliving control) and 2 additional killed tissues (NSCkilled control).
- N. of replicates : 2
- Method of calculation used: True viability % = [(OD of living tissues exposed to test item - OD of killed tissues exposed to test item - OD of living tissues exposed to test item incubated with medium without MTT + OD of killed tissues exposed to test item incubated with medium without MTT) / OD of living tissues exposed to negative control] x 100

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

PREDICTION MODEL / DECISION CRITERIA
- The test item is considered as non-irritant to skin if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2) if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is “non corrosive”.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and in absence of information on a skin corrosion test.




Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg (32mg/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% SDS
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
41 hours and 15 minutes
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
112.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
(distilled water)
Positive controls validity:
valid
Remarks:
1.3% viability (5% SDS)
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: assessed by NSMTT controls (see table below).
- Colour interference with MTT: assessed by NSCliving controls and NSCkilled controls (see table below).

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A full demonstration of proficiency was performed for the EpiSkin-SM model, plus a reduced validation with the SkinEthic RHE model. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, mean OD = 0.999 (OD measured after 1:2 dilution in isopropanol; acceptability criteria should be in the range ≥ 0.4 and ≤1.5).
- Acceptance criteria met for positive control: yes, mean viability = 1.3% (acceptability criteria should be < 40%).
- Acceptance criteria met for variability between replicate measurements: Standard deviation between the 2 NSCkilled control replicates treated with the test item was 35.4% instead of 18% at the maximum as defined in OECD test guideline. This deviation is considered as without impact on the conclusion of the study because regardless of the addition of the viability obtained by the NSCkilled control, the viability obtained with the test item remains higher than 50% and does not change the classification of the product.

Any other information on results incl. tables

Table 1. Table of results

Well ID

OD

Mean OD /
disc (#)

Mean OD /
product

Viability
%

Mean viability
%

SD

Conclusion

Negative
control

SPL 1

0.964

0.955

0.999

95.6

100.0

4.5

 

0.945

0.956

SPL 2

1.044

1.045

104.6

1.046

1.045

SPL 3

0.981

0.997

99.8

1.017

0.992

Positive
control

SPL 4

0.012

0.013

0.013

1.3

1.3

0.1

Irritant

0.012

0.013

SPL 5

0.013

0.013

1.3

0.013

0.013

SPL 6

0.011

0.012

1.2

0.011

0.012

Test item
PH-19/0483

SPL 13

1.080

1.087

0.995

108.8

99.6

9.4

 

1.080

1.100

SPL 14

0.985

1.000

100.1

1.017

0.998

SPL 15

0.888

0.899

90.0

0.919

0.889

Test item
PH-19/0483
NSC Control
living tissues

SPL 18

0.054

0.055

0.069

5.5

6.9

1.9

0.057

0.052

SPL 19

0.083

0.082

8.2

0.083

0.078

Test item
PH-19/0483
NSC Control
killed tissues

SPL 20

0.021

0.020

0.270

2.0

27.0

35.4

0.020

0.019

SPL 21

0.514

0.520

52.1

0.513

0.531

Test item
PH-19/0483
killed tissues
MTT control

SPL 16

0.060

0.062

0.072

6.2

7.2

1.4

0.062

0.062

SPL 17

0.082

0.082

8.2

0.082

0.080

Test item
PH-19/0483
corrected

 

112.6

 

Non irritant

# mean of 3 values (triplicate of the same extract).

Applicant's summary and conclusion

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
In the vitro human reconstructed epidermis test the mean corrected percent viability of the treated tissues was 112.6%. Therefore, the test item can be considered as not irritant to skin.
Executive summary:

An in vitro skin irritation test of the test item was performed in a reconstructed human SkinEthic™ RHE epidermis model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 16 mg test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated at 37°C for 41 hours and 15 minutes in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol during 2 hours under gentle agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol. Additionally, 2 killed Human skin model surfaces were treated (SkinEthic RHE® model) in the same manner in order to generate non-specific MTT reduction. Moreover, 2 living and 2 killed Human skin model surfaces were treated in the same manner but they were incubated in culture medium instead of MTT solution in order to generate non-specific living and killed colour controls. Under the test conditions, the mean corrected percent viability of the treated tissues was 112.6%, versus 1.3% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is considered as not irritant to the skin.