Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 14 to April 17, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test conducted according to internationally accepted Guideline and in according to the GLP Principles

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: Internal Procedure SOP 118 008 40
Deviations:
no
GLP compliance:
yes

Test material

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

Test system

Amount / concentration applied:
23.5 mg (Tissue 1)
23.1 mg (Tissue 2)
23.1 mg (Tissue 3)
Duration of treatment / exposure:
60 ± 5 min. after the first application and the incubation period the inserts were rinsed
Details on study design:
TEST SYSTEM
Specification: Commercially available EpiDermTMKit.
The EpiDermTM tissues are cultured on specially prepared cell cultures inserts.
Origin: EpiDermTMKit from MatTek In Vitro Life Science Laboratories (Batch n. 21654).

CHEMICALS AND MEDIA
MTT Reagent
Contains 1 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide as concentrate in DBPS buffer (5 mg/ml, stored at –20 ± 5 °C).
Origin: prepared by LAUS GmbH
A MTT stock solution of 5 mg/mL in DPBS buffer was prepared and stored in aliquots of 2 ml at – 20 ± 5 °C. 2 ml of the stock solution were thawed and diluted with 8 mL of me-dium (resulting in 1 mg/ml).

DPBS-Buffer
Dulbecco’s Phosphate Buffered Saline (DPBS buffer without CaCl2 and MgCl2).
Origin: MatTek In Vitro Life Science Laboratories and prepared by LAUS GmbH

Composition of the subset from MatTek In Vitro Life Science Laboratories (batch n.: 020315MHA):
Used as negative control and for rinsing the test item from the tissues
KCl 0.2 g
KH2PO4 0.2 g
NaCl 8.0 g
Na2HPO4 * 7H2O 2.16 g
H2O ad 1 l
Composition of the subset from LAUS GmbH (batch: 20150226):
Solvent for MTT concentrate and for rinsing the outside of the inserts at the end of the in-cubation time with MTT.
KCl 0.2 g
KH2PO4 0.2 g
NaCl 8.0 g
Na2HPO4 * 7H2O 2.16 g
H2O ad 1 l

Positive Control
Sodium dodecyl sulphate (SDS): solution in deionised H2O containing 5 % SDS
Origin: MatTek In Vitro Life Science Laboratories (batch no.: 020615TMH).

MEM Medium with Phenol Red for Pre-Test
Serum-free MEM (Minimum Essential Medium).
Origin: Gibco/Invitrogen (Batch no.: 1613667)

Assay Medium
Serum-free DMEM (Dulbecco’s Modified Eagle’s Medium).
Origin: MatTek In Vitro Life Science Laboratories (batch no.: 040915TMA)

Isopropanol:
Purity 99.9 %,
Batch no.: 310060213050,
used as extracting solvent for formazan.

TEST VESSELS
Vessels materials: glass or sterilisable plastic.
Sterilised before use by rinsing with ethanol 70% or autoclaving.
Vessels used: 6, 24 and 96 well-plates

INSTRUMENT AND DEVICES used during the test:
Autoclave, 3870 ELV-B
Stop-watch
96-well-plate photometer, Anthos Reader
Precision scales, Mettler Toledo PB 5001-SO2 Labostyle
Analytical scales, Mettler Toledo XS 205 DU
Incubation chamber Binder
Table water bath, neoLabAdjustable pipettes with sterile tips
Clean bench, category 2 (Axo Safe, MARS 12000)Orbital shaker, GFL 3005
Bulb-headed pipettes, sterile
Single-use pipettes
Nylon mesh circles 8 mm diameter (200 µm pore (EPI-MESH))
Pipetting device, Accujet
Refrigerator and freezer
Weighing funnels
Microscope
Standard Laboratory material (glassware)

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other:
Value:
2.089
Remarks on result:
other:
Remarks:
Basis: mean of the three tissues (%). Remarks: relative standard deviation of the three tissues = 2.1 %. (migrated information)
Irritation / corrosion parameter:
other:
Value:
112.2
Remarks on result:
other:
Remarks:
Basis: mean. Remarks: percentage values of formazan production calculated in comparison to the negative control. (migrated information)

Any other information on results incl. tables

The photometric absorbance of the negative controls is considered as 100 %. For each replicate of test item and positive control, formazan production is calculated as % photometric absorbance compared with the mean of the negative controls:

% Formazan production = [OD mean of negative controls/ODreplicate test item resp. positive control]*100%

where OD is the Optical Density

Absorbance values blank isopropanol (OD at 570 nm)

Replicate

1

2

3

4

5

6

7

8

Mean

Absorbance

0.041

0.038

0.038

0.038

0.037

0.037

0.038

0.037

0.038

Absorbance Values of Negative control, Positive Control and Test item

Designation

Measurement

Negative Control

AP 1300 S

Positive Control

Tissue 1 

1

2.023

2.148

0.097

2

1.919

2.206

0.089

Tissue 2 

1

1.701

2.131

0.088

2

1.726

2.069

0.085

Tissue 3 

1

2.002

2.129

0.089

2

2.025

2.081

0.087

Mean absorbance Values

Designation

Negative Control

AP 1300 S

Positive Control

Mean – blank (tissue 1)

1.933

2.139

0.055

Mean – blank (tissue 2)

1.676

2.062

0.049

Mean – blank (tissue 3) 

1.976

2.067

0.050

Mean of the three tissues

1.862

2.089

0.051

Relative standard deviation
of the three tissues

8.7 %

2.1 %

6.3 %

% Formazan Production

Designation

AP 1300 S

Positive Control

% Formazan production (tissue 1)

114.9 %

3.0 %

% Formazan production (tissue 2)

110.7 %

2.6 %

% Formazan production (tissue 3)

111.0 %

2.7 %

% Formazan production (mean)

112.2 %

2.8 %

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information According to the CLP Regulation Criteria used for interpretation of results: EU
Conclusions:
112.2 % of the relative absorbance
Executive summary:

Method

The substance has been tested in order to evaluate the skin irritation potential according to the EU method B.46 and OECD 439.

During the test three tissues of the human skin model EpiDermTM has been treated with on average 25 mg of test item for 60 minutes. Both positive and negative control have been prepared.

Observations

After the treatment with the test item, the relative absorbance values were increased, compared to the negative control, to 112.2 %.

The Optical Density (OD) of the negative control (1.9) was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8. The positive control induced a decrease in the relative absorbance as compared to the negative control to 2.8 % (required: ≤ 20 %). For these reasons the validity criteria of the test system has been fulfilled.

Conclusion

The 112.2 % value of the relative absorbance is above the threshold for irritation potential set out in the OECD guideline, therefore the substance is considered as not irritant in the Human Skin Model Test.