Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 27 April 2016 to 29 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: provided by Sponsor
- Expiration date of the lot/batch: 15 March 2017
- Purity: 45.4% active ingredient in water solution
- Colour: light yellow

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator (2-8°C).
The substance is deemed to be stable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Preliminary purification step (if any): Water solution provided by Sponsor was Freeze-dried to produce the dry Test Item used in this study. Considered as 100%

FORM AS APPLIED IN THE TEST: the dry Test Item was used in the study (solid)

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Source strain:
other: adult donors
Details on animal used as source of test system:
Not applicable
Justification for test system used:
The EPISKINTM (SM) model has been validated for irritation testing in an international validation study [9] 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: EPISKINTM(SM) Model- Tissue batch number(s): 15-EKIN-047 and 16-EKIN-017- Expiration date: 15-EKIN-047 November 30, 2015, 16-EKIN-017 May 2, 2016- Date of initiation of testing: 27 April 2016TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 23.8-27.5°C- Temperature of post-treatment incubation (if applicable): 37°CREMOVAL OF TEST MATERIAL AND CONTROLS- Number of washing steps: 3 steps two with PBS thoroughly and one with pipette- Observable damage in the tissue due to washing: washing was conducted without touching the epidermis in order to avoid damage- Modifications to validated SOP: noMTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 0.3 mg/mL MTT- Incubation time: 3 hours (± 5 min)- Spectrophotometer: hermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14. Date of calibration: 02 September 2014, calibration is valid until September 2016- Wavelength: 570 nmFUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA- Viability: Historical data for negative control is an OD of 0.827 with a range of 0.573-1.362. - Barrier function: historical data IC 50: specification 1.5mg/ml <= IC50 <= 3 mg/ml, result 2.8 mg/ml (16-EKIN-017) and 2.1 mg/ml (15-EKIN-017)- Morphology: well- differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum- Contamination: on blood, it was verified the absence of HIV1 and 2 antibodies, the absence of hepatitis C antibodies, the absence of hepatitis B antigen HBs, on epidermal cells of the donors it was verified the absence of bacteria, fungus and mycoplasma.- Reproducibility: historical control dataNegative control:Mean optical density (OD): 0.827, SD 0.182, range 0.573- 1.362, n= 67Positive control: Mean optical density (OD): 0.109 , SD 0.054, range 0.032-0.354, n= 63NUMBER OF REPLICATE TISSUES: 3CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE- Killed tissues- Procedure used to prepare the killed tissues (if applicable): For killed epidermis, living epidermis units (Manufacturer: SkinEthic, France, Batch No.:15-EKIN-047, Expiry Date: 30 November 2015) were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 48 hours (±1 hour). At the end of the incubation the water was discarded and the dead epidermis units were frozen on 27 November 2015 (the frozen units can be used up to 6 months). Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Assay Medium). Further use of killed tissues was similar to living tissues.- N. of replicates : 2- Method of calculation used: These tissues followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.- Check-method for possible direct MTT reduction with test item: 10 mg of test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in a shaking water bath for 3 hours.Purple colour of the mixture was detected in the test tube Thus, the test item reacted with MTT and therefore the use of additional controls was necessary.PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)- The test item considered to be irritant to skin (Category 2), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.- The test substance is considered to be non-corrosive to skin if the mean relative viability after 15 minutes exposure and 42 hours post incubation is more than (>) 50% of the negative control.OTHER:After receipt of the test system, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.Additional controls: As the test item was showed being an MTT-interacting substance and the test item had an intrinsic colour, two additional test item-treated killed tissues were used for the non specific OD evaluation. As colour change (purple) was observed after three hours of incubation of the test item in MTT working solution, thus the test material might interact with MTT. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit): 20 mg of the test itemNEGATIVE CONTROL- Amount(s) applied (volume or weight): 50 μL of PBS- Concentration (if solution): not diluted, at the comercial concentration, Sigma-Aldrich Co. Batch number: BCBQ2925V, Expiry date: January 2020POSITIVE CONTROL- Amount(s) applied (volume or weight): 50 μL- Concentration (if solution): (5% (w/v) SDS solution
Duration of treatment / exposure:
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (23.8-27.5°C).
Duration of post-treatment incubation (if applicable):
The rinsed units were incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2.
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
ca. 84.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
ca. 91.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
ca. 94.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
ca. 90.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:- Visible damage on test system: No damage was observed.- Direct-MTT reduction: Yes, the test item was showed being an MTT-interacting substance (see Table 2 on Any other information on results inc. tables”field)- Colour interference with MTT: Yes, colour change (purple) was observed after three hours of incubation of the test item(See Table 3 and 4 on “Any other information on results inc. tables” field).ACCEPTANCE OF RESULTS: - Acceptance criteria met for negative control: yes. The mean OD value of the three negative control tissues was in the recommended range (0.831). Standard deviation of the viability results for negative control samples was 4.3. The mean OD value of the blank samples (acidified isopropanol) was 0.046.- Acceptance criteria met for positive control: yes. The positive control treated tissues showed 6.7% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 2.0.- Acceptance criteria met for variability between replicate measurements: yes. The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 4.9.

Any other information on results incl. tables

Table 1: Optical Density (OD) and calculated relative viability % of the samples

 

Optical Density (OD)

Viability

Substance

 

Measured

Blank corrected

(% RV)

Negative Control:

1

0.858

0.812

97.6

Phosphate buffered saline

2

0.919

0.873

105.0

 

3

0.856

0.810

97.4

 

mean

0.831

100.0

Positive Control:

1

0.118

0.072

8.6

5% (w/v) SDS solution

2

0.084

0.038

4.6

 

3

0.103

0.057

6.9

 

mean

--

0.056

6.7

Test Item:

1

0.750

0.704

84.7

Nopcote 1661

2

0.809

0.763

91.8

 

3

0.828

0.782

94.1

 

mean

--

0.750

90.2

Table 2: Optical Density (OD) and the calculated Non-Specific MTT Reduction (NSMTT) of the Additional Control Tissues (Killed Epidermis)

Additional control

Optical Density (OD)

NSMTT

NSMTT%

(killed epidermis)

 

Measured

Blank corrected

T reated with

1

0.084

0.038

-0.013

 

Nopcote 1661

2

0.085

0.039

-0.012

-1.5

 

mean

0.038

*-0.012

 

T reated with

1

0.118

0.072

 

 

Physiological saline

2

0.075

0.029

 

 

(0.9% (w/v) NaCl)

mean

--

0.050

 

 

Table 3: Optical Density (OD) and the calculated Non Specific Colour % (NSCliving%) of the Additional Control Tissue

Additional control

Optical Density (OD)

NSC%

(living)

 

Measured

Blank corrected

T reated with

1

0.053

0.007

 

Nopcote 1661

2

0.055

0.009

0.9

 

mean

-

0.008

 

Mean blank value was 0.046

Table 4: Optical Density (OD) and the calculated Non Specific Colour % (NSCkilled%) of the Additional Control Tissues (Killed Epidermis)

Additional control

(killed epidermis)

Optical Density (OD)

NSC%

(killed)

 

Measured

ank corrected

T reated with

1

0.056

0.010

 

Nopcote 1661

2

0.066

0.020

1.7

 

mean

--

0.015

 


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
based on UN GHS/ CLP classification
Conclusions:
The test item was considered as being non-irritant to skin under the test conditions.
Executive summary:

The skin irritation potential of the test substance was determined in accordance with the OECD nº 439 with GLP. This experiment was conducted in a reconstructed human epidermis model; EPISKIN (SM). Disks of EPISKINTM (SM) (three units) were treated with the test item (20 mg) and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSC living) from the test item. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units. Furthermore to avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSC killed), two additional disks were used. Following exposure with the test item, the mean cell viability was 90.2% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid. In conclusion, in this in vitro EPISKIN model test with freeze-dried test item, the results indicate that the test item is non-irritant to skin.