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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:
04 Sep - 08 Sep 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
GLP compliance:
yes (incl. certificate)
Remarks:
GLP-Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm™ SIT (Epi-200)
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 multi layered, highly differentiated model of the human epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ SIT (EPI-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue Lot number(s): 25840
- Date of initiation of testing: 31 Aug 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- 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 (Dulbecco’s Phosphate-Buffered Sallines) 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: 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.504 ± 0.03 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 7.65 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

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 than 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: 25 mg test item + 25 µL DPBS

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:
87.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT (non-specific reduction of MTT) equaled 0%.
- Colour interference with MTT: The mixture of 25 mg of the test item per 300 µl aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC (non-specific colour) equaled 0%.

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% (1.0% - 4.3%).

Any other information on results incl. tables

Table 2: Result of the Test Item Frits

Name

Negative control

Positive control

Test item

Tissue

1

2

3

1

2

3

1

2

3

Absolute OD570

2.378

2.488

2.632

0.147

0.105

0.108

2.183

2.089

2.304

2.329

2.472

2.491

0.155

0.107

0.110

2.126

2.069

2.231

OD570

(blank-corrected)

2.335

2.446

2.590

0.105

0.062

0.065

2.140

2.046

2.262

2.286

2.429

2.448

0.112

0.065

0.067

2.083

2.027

2.188

Mean OD570

of the duplicates (blank-corrected)

 

2.311

 

2.437

 

2.519

 

0.108

 

0.063

 

0.066

 

2.112

 

2.037

 

2.225

Total mean OD570 of 3 replicate tissues

(blank-corrected)

 

2.422*

 

0.079

 

2.125

SD OD570

0.105

0.025

0.095

relative tissue viability [%]

95.4

100.6

104.0

4.5

2.6

2.7

87.2

84.1

91.9

mean relative tissue viability [%]

100.0

3.3**

87.7

SD tissue viability [%]***

4.3

1.0

3.9

CV [% viabilities]

4.3

31.7

4.5

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

**    Mean relative tissue viability 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 criteria not met, no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
In this study under the given conditions the test item showed no irritant effects.