Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 - 28 April 2017
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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
06 July 2012
Deviations:
no
Qualifier:
according to
Guideline:
other: EpiSkin SOP, ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.
Version / remarks:
Version 1.8 (February 2009)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid: crystalline
Remarks:
crystalline powder
Details on test material:
Batch No: 11679700
Storage: at room temperature, protected from light

In vitro test system

Test system:
human skin model
Remarks:
EpiSkin Small Model (three-dimensional human epidermis model) manufactured by EPISKIN SNC Lyon, France
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EpiSkin Model has been validated for irritation testing in an international trial. The EpiSkin method is a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404) for the purposes of distinguishing between skin irritating and no-skin irritating test substances.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model
- Supplier: SKINETHIC Laboratories 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
- Tissue batch number(s): 17-EKIN-017
- Expiry date: 01 May 2017
- Date of initiation of testing: 26 April 2017

PRE-INCUBATION
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of an assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5 % CO2, >= 95% humidified atmosphere.

EXPOSURE
- Test Item: The epidermal surface was first moistened with 5 µL deionised water (prepared by Direct Q5 water purification system in Toxi-Coop ZRT) in order to improve further contact between powder and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.
- Positive and negative control: A volume of 10 µL positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette tip in order to cover evenly all the epidermal surface if necessary.
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.

NUMBER OF REPLICATE TISSUES FOR TEST ITEMS AND CONTROLS
Triplicates

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 mL PBS 1x solution, once. rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source
- Observable damage in the tissue due to washing: none

POST-INCUBATION
After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5 % CO2, >=95% humidified atmosphere.

MTT TEST
After the 42 hours incubation the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37°C in an incubator with 5 % CO2 protected from light, >=95% humidified atmosphere.

FORMAZAN EXTRACTION
A disk of epidermis was cut from the unit using a biopsy punch, placed into a tube of 500 µL acidified isopropanol (0.04N HCl), mixed by using a vortex mixer and incubated for 4 hours at room temperature protected from light with gentle agitation (~150 rpm). At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

CELL VIAVILITY MEASUREMENTS
Following the formazan extraction, 2×200 µL sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at 570 nm using acidified isopropanol solution as the blank (6×200 µL).

PREDICTION MODEL / DECISION CRITERIA:
The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal to 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: The epidermal surface was first moistened with 5 µL deionised water in order to improve further contact between powder and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface.

NEGATIVE CONTROL
- Amount(s) applied:10 µL
- Concentration: 1x PBS (Phosphate Buffered Saline)

POSITIVE CONTROL
- Amount(s) applied:10 µL
- Concentration: Sodium Dodecyl Sulphate (SDS) 5% aq. solution
Duration of treatment / exposure:
15 minutes (± 0.5 min)
Duration of post-treatment incubation (if applicable):
42 hours (± 1h)
Number of replicates:
Three replicates were used for the test item and controls, respectively.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
105
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: mean viability value of three tissues
Other effects / acceptance of results:
OTHER EFFECTS:
- Possible direct MTT reduction with test item
No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colouring potential of test item
The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is colourless and therefore considered to be not able to significantly stain the tissues and lead false estimate of viability. Furthermore, the test item was completely removed from the epidermal surface at rinsing period. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Mean OD value 0.697 and standard deviation value (SD) for the % viability of 13.32 (The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be = or < 18)
- Acceptance criteria met for positive control: 7% mean viability range standard deviation value (SD) for the % viability of 0.74 (The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be = or < 18.)
- For test chemicals, the standard deviation value (SD) of the % viability should be = or < 18 : 5.31 % SD

Any other information on results incl. tables

Cell viability

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells is presented below:

Negative Control (1xPBS):

 Replicate  Optical Density (OD)  Viability (%)
 1 0.705 101
 2 0.785 113
 3 0.600 86
 Mean 0.697 100

Standard Deviation (SD)

  13.32

Positive Control (SDS 5% aq.):

 Replicate  Optical Density (OD)  Viability (%)
 1 0.049 7
 2 0.044 6
 3 0.054 8
 Mean 0.049 7
 Standard Deviation (SD)   0.74

Test item:

 Replicate  Optical Density (OD)  Viability (%)
 1 0.690 99
 2 0.760 109
 3 0.747 107 
 Mean 0.732 105
 Standard Deviation (SD)   5.31

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item Adenosine-5’-monophosphate (AMP) (CAS No. 61-19-8) is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category / CLP not classified).
Executive summary:

EpiSkinTM SM test of Adenosine-5’-monophosphate (AMP) (CAS No. 61-19-8) has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5 % CO2, =95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light, =95% humidified atmosphere. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

No colour change was observed after three hours of incubation during the check-test for possible direct MTT reduction with test item. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary.

The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is white and therefore considered not to be able to significantly stain the tissues and lead false estimate of viability. Furthermore, the test item was completely removed from the epidermal surface at rinsing period. Additional controls and data calculations were not necessary.

SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (=) to 50% of the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item Adenosine-5’-monophosphate (AMP) did not show significantly reduced cell viability in comparison to the negative control (mean value: 105 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. Positive and negative control values were within the corresponding historical control data ranges.

All assay acceptance criteria were met, the experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilized testing conditions. The test item Adenosine-5’-monophosphate (AMP) (CAS No. 61-19-8)is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category / CLP not classified).