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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Sep - 27 Nov 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
Qualifier:
according to
Guideline:
other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SCT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SCT).
Version / remarks:
Version 07 -Nov- 2014
GLP compliance:
yes (incl. certificate)
Remarks:
GLP-Landesleitstelle Bayer, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The test was carried out with the reconstituted three-dimensional human skin model EpiDermTM (MatTek). This skin model consists of normal (non-cancerous), human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDermTM skin model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous, granular and cornified layers analogous to those found in vivo.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm (TM): reconstructed three-dimensional human epidermis model
- Tissue Lot number(s): 25840
- Date of initiation of testing: 06 Sep 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed with DPBS to remove any residual test item; excess DPBS was removed by blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 42 h at 37 ± 1 °C, 5.0% CO2
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: MTT QC assay. 4 hours, n=3; the OD value (540 – 570 nm) locate in 1.0 – 3.0.
- Barrier function: ET-50 assay. 100 μL 1% Triton X- 100, 4 time – points. N=3, MTT assay; the ET-50 time locate in 4.77 - 8.72.

NUMBER OF REPLICATE TISSUES: 3 each, for the test substance, positive and negative control.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: not applicable.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTI ON: single experiment.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after 60 minutes exposure and 42 h post-incubation is less or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 60 minutes exposure and 42 h post-incubation is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 μL test item (undiluted) per tissue (47 μL/cm²)

NEGATIVE CONTROL
- Amount applied: 30 μL DPBS

POSITIVE CONTROL
- Amount applied: 30 μL SDS solution
- Concentration: 5%
Duration of treatment / exposure:
60 ± 1 min
Duration of post-treatment incubation (if applicable):
42 ± 4 h
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
60 ± 1 min
Value:
3
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The mixture of 30 μL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple.
- Colour interference with MTT: The mixture of 30 μL test item per 300 μL aqua dest. and per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean absolute OD570 of the three negative control tissues was >= 0.8 and <= 2.8.
- Acceptance criteria met for positive control: the mean relative tissue viability (% negative control) of the three positive control tissues was <= 20% (3.3 %).
- Acceptance criteria met for variability between replicate measurements: the standard deviation (SD) of viability of replicate tissues of all dose groups was <= 18% (0.4% - 4.3%).

Any other information on results incl. tables

Table 1. MTT assay after 60 min exposure

 Name        NK        PC        TM
 Tissue  1  2  3  1  2  3  1  2  3

Absolute OD570

2.378

2.329

2.488

2.472

2.632

2.491

0.147

0.155

0.105

0.107 

0.108

0.110 

0.124

0.120

0.105

0.106

0.120

0.121

OD570 (blank - corrected)

2.335

2.286

2.446

2.429

2.590

2.448

0.105

0.112

0.062

0.065

0.065

0.067

0.082

0.078

0.062

0.063

0.077

0.078

Mean OD570 of the duplicates (blank - corrected)

2.311 2.437  2.519 0.108  0.063 0.066 0.080 0.063 0.078

Total mean OD570 of 3 replicate tissues (blank - corrected)

2.422*    0.079

0.073

 SD OD570

0.105 0.025 0.009 

 Relative tissue viability [%]

95.4 100.6 104.0  4.5 2.6 2.7   3.3 2.6    3.2

 Mean relative tissue viability [%]

100.0 3.3** 3.0

 SD tissue viability [%]***

4.3 1.0 0.4

 CV [% viabilities]

4.3 31.7 12.4

* Blank-corrected mean OD570 nm of the negative control corresponds to 100% absolute tissue viability.

** Mean relative tissue viaability of the three positive control tissues is ≦ 20%.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≦ 18%.

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS Category 2 according to Regulation (EC) No 1272/2008
Conclusions:
CLP: Category 2
The test item showed irritant effects. The test item is therefore classified as "irritant" in accordance with CLP/EU GHS "Category 2".