Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-10-20 to 2017-01-17
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:
revised in 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
amendment July 6th, 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Justification for test system used:
This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM
- Tissue batch number(s): Lot No.: 16-EKIN-048; Lot No.: 16-MAIN3-079; Lot No.: 16-ESSC-051
- Date of initiation of testing: 2016-10-28

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: One washing step, volume not specified
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h ± 5 min
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm
- Filter: Filter band pass
- Filter bandwidth: maximum 30 nm


FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 11.7 ± 8.2 % of negative control
- Barrier function: IC50 determination (SDS concentration, MTT test, n = 14): Specification ≥ 1.5 mg/mL Result: 1.7 mg/mL
- Morphology: Histology scoring (HES stained vertical paraffin sections, n = 6): Specification ≥ 19.5
Result: 22.3 ± 0.3, CV = 1.2%
Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
NUMBER OF REPLICATE TISSUES:
triplicate
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- N. of replicates : 3
- Method of calculation used: non-specific reduction of MTT was calculated relative to the negative control of living tissues (NK) per treatment period according to the following formula:
NSMTT [%] = [(ODKT-ODKU)/ODNK]* 100
If non-specific MTT reduction was ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues (TM) was corrected according to the following formula:
TODTT = OD TM- (ODKT-ODKU)
PREDICTION MODEL
- The test substance is considered to be irritant to skin if the viability after 3 minutes exposure is less than 50%, and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-irritant to skin if the viability after 3 minutes exposure is greater 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg + 5 µl aqua dest.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL DPBS medium, no treatment

POSITIVE CONTROL
- Concentration (if solution): 5% sodium dodecyl sulfate in 10 µl aqua dest.
Duration of treatment / exposure:
15 ± 0.5 min
Duration of post-treatment incubation (if applicable):
42 ± 1 h
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value
Value:
98.8
Vehicle controls validity:
not examined
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:
mean absolute OD570 nm of the three negative control tissues is ≥ 0.6 and ≤ 1.5; mean value absolute OD570 nm: 0.687
- Acceptance criteria met for positive control:
mean relative tissue viability of the three positive control tissues is ≤ 40%; mean relative viability [%]: 7.2
- Acceptance criteria met for variability between replicate measurements:
standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%; Max. SD of % Viability [%]: 7.6

Any other information on results incl. tables

Table 1: Results of the Test Item L-Asparagine

 

Negative control

Positive control

Test item

Total Mean OD510 Of 3 Replicate Tissues (Blank-Corrected)

0.644*

0.047

0.636

SD OD570

0.049

0.010

0.046

,Mean Relative Tissue Viability [%]

100.00

7.2**

98.8

SD Tissue Viability [%]***

7.6

1.5

7.2

cv [% Viability]

7.6

21.2

7.3

*Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

**Mean relative tissue viability of the three positive control tissues is40%.

***Standard deviation (SD) obtained from the three concurrently tested tissues is18%.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In the present study conducted according to OECD Test guideline 439 L-Asparagine was tested for oits irritating potential to the skin. After performance of the test the mean relative viability of the cells was 98.8%. Thus, L-Asparagine is not classified as irritating to the skin.