Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Sep - 05 Oct 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
human
Source strain:
other: EpiDermTM; reconstructed three-dimensional human epidermis (EPI-200)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200 SIT kits, EpiDerm™ tissue, purchased from MatTek Corporation (Bratislava, Slovakia)
- Tissue batch number(s): 21697
- Delivery date: 29 Sep 2015
- Date of initiation of testing: 29 Sep 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: tissue samples were rinsed with DPBS at least 15 times, then the tissues were carefully dried using sterile cotton tipped swabs and transferred to new plates

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Filter: 570 ± 1 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD (540 - 570 nm) = 1.798 ± 0.070
- Barrier function: 6 h
- Contamination: No, sterile

NUMBER OF REPLICATE TISSUES: triplicates each in 3 independent tissues

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues:
Test item’s colour interference potential: The test substance was assessed for its potential to interfere with the MTT assay prior to the main test. The colour of test item/water mixture did not change during the incubation period compared with the colour of the pure test item. Therefore, the test item passed the colour interference test and an additional test with a viable tissue without MTT addition was not necessary.
Ability of the test item to directly reduce MTT: Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer, and an additional test with freeze-killed tissues was not necessary.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability is less than or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability is greater than 50%
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 ± 2 mg (~ 39 mg/cm² according to guideline) of the solid test item were applied directly onto the EpiDerm™ tissue, after the surface had been wetted with 25 μL of DPBS, respectively.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL Dulbecco`s phosphate buffered saline was used as negative control in each independent tissue.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL of a 5% sodium lauryl sulphate solution in deionised water was used a positive control in each independent tissue.
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
triplicates each in 3 independent tissues

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
>= 94.6
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system:
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes (mean absorbance of the negative control = 1.660)
- Acceptance criteria met for positive control: Yes (mean absorbance of the positive control = 0.083)
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: Data of 13 studies performed from Jul 2015 - Oct 2015
Positive Control:
Mean Viability: 4.77%; RSD: 14.9%; Range of Viabilities: 4.00 - 5.90%; Mean Absorption: 0.084; RSD: 16.6%; Range of Absorbance: 0.069 - 0.097
Negative Control:
Mean Absorption: 1.77; RSD: 8.55%; Range of Absorbance: 1.66 - 1.98

Any other information on results incl. tables

Table 1: Results after treatment with the test item and the controls

Dose Group

Treatment
Interval

Absorbance
570 nm
Tissue
1*

Absorbance
570 nm
Tissue
2*

Absorbance
570 nm
Tissue
3*

Mean
Absorbance
of
3 Tissues

Rel. Absorbance
[%]
Tissue 1, 2
+ 3**

Relative
Standard
Deviation
[%]

Mean Rel.
Absorbance
[% of Negative
Control]***

Negative
Control

60 min

1.685

1.610

1.685

1.660

101.5
97.0
101.5

2.6

100.0

Positive
Control

60 min

0.091

0.083

0.076

0.083

5.5
5.0
4.6

9.0

5.0

Test Item

60 min

1.575

1.531

1.607

1.571

94.9
92.2
96.8

2.4

94.6

* Mean of three replicate wells after blank correction

** Relative absorbance per tissue [rounded values]: 100 x (absorbance of tissue) / (mean absorbance negative control)

*** Relative absorbance per treatment group [rounded values]: (100 x mean absorbance (test item/positive control)) / (mean absorbance negative control)

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:
Under the conditions of the RHE test method, the test substance did not show irritant properties.