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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:
12 - 16 Nov 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test (Altern Lab Anim. 2007 Dec.; 35 (6): 559-601)
Deviations:
no
Qualifier:
according to
Guideline:
other: Protocol for: IN VITRO EpiDerm™ SKIN IRRITATION TEST (EPI-200-SIT), Rev. 3/23/2009, MatTek Corporation, Ashland, USA
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, 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: EpidermTM, reconstructed three-dimensional human epidermis model (EPI-200)
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek, In Vitro Life Science Laboratories, Bratislava, Slovakia
- Tissue batch number(s): 16854
- Delivery date: 13 Nov 2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 min at 37 °C, 25 min at room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were rinsed in one-minute intervals.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL
- Incubation time: 3 h
- Spectrophotometer: Photometer MRX
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Contamination: No biological contamination ofHIV-1 virus, Hepatitis B virus, Hepatitis C virus or bacteria, yeast and fungi were detected.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 1 h exposure is less than 50%.
- The test substance is considered to be non-irritant to skin if the viability after 1 h exposure is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 30 µL

NEGATIVE CONTROL
- Amount(s) applied: 30 µL

POSITIVE CONTROL
- Amount(s) applied: 30 µL
- Concentration: 5% solution in deionised water
Duration of treatment / exposure:
35 min at 37 °C, 25 min at room temperature
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
Triplicate tissues; from each tissue, 2 absorbance measurements after MTT incubation were performed

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of 3 tissues
Value:
99.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.329, 1.057 and 1.049) was in the range of ≥ 1.0 and ≤ 2.5 for all three tissues. As the negative control result fell within the range defined in the acceptance criteria, the result is considered to be valid.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control to 14.7% and thus, lower than threshold of 20%.
- Acceptance criteria met for variability between replicate measurements: The standard deviations between the three tissue replicates of the treatment group, the negative control and the positive control were 4.4%, 13.9% and 0.0%, respectively, which is ≤ 18%.

Any other information on results incl. tables

Table 2. Results of MTT assay after 1 h exposure

 

Negative control

Test item

Positive control

Tissue sample

1

2

3

1

2

3

1

2

3

OD570

1.466

1.277

1.113

1.087

1.096

1.088

1.298

1.179

1.164

1.161

1.140

1.146

0.212

0.209

0.210

0.212

0.211

0.210

OD570(mean)

1.329

1.057

1.049

1.196

1.120

1.100

0.168

0.168

0.168

OD570(mean values of replicates±%RSD)

1.145 ± 13.9

1.139 ± 4.4

0.168 ± 0.0

Viability (%)

100

99.4

14.7

OD = optical density; RSD = relative standard deviation

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.
CLP: not classified