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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
05 Aug - 02 Sep 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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted Sep 2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Cool and dark place- Stability: 1 year

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm™ (EPI-200)
Source strain:
other: Keratinocyte strain 00267
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE- Model used: EpiDerm™ (EPI-200-SCT) (MatTek Corporation, Bratislava, Slovakia)- Tissue batch number(s): 21691- Shipping date: 2 Sept 2015 - Delivery date: 2 Sept 2015- Date of initiation of testing: 2 Sep 2015TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 37 °CREMOVAL OF TEST MATERIAL AND CONTROLS-Volume and number of washing steps: The test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 1 mg/mL- Incubation time: 3 h- Spectrophotometer: Tecan Sunrise Magellan Version 6.4 (Tecan Deutschland GmbH, Crailsheim, Germany)- Wavelength: 540 nmFUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA- Viability: The quality of the EpiDerm™ tissue was assessed by an MTT cell viability test.- 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 4.91 h.- Contamination: All biological components of the epidermis and the culture medium were tested by the manufacturer for viral, bacterial, fungal and mycoplasma contamination.NUMBER OF REPLICATE TISSUES: Duplicate tissues; from each tissue, 3 absorbance measurements after MTT incubation were performed.CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCEThe test substance did not directly reduce the MTT solution.PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL- Amounts applied: 50 µLNEGATIVE CONTROL- Amounts applied: 50 µLPOSITIVE CONTROL- Amounts applied: 50 µL- Concentration: 8.0 N
Duration of treatment / exposure:
3 and 60 min
Number of replicates:
Duplicate tissues; from each tissue, 3 absorbance measurements after MTT incubation were performed.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 2 tissues
Run / experiment:
3 min exposure
Value:
79.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 2 tissues
Run / experiment:
60 min exposure
Value:
73.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:- Direct-MTT reduction: The test substance did not interfere with the MTT assay (no reducing capacity).ACCEPTANCE OF RESULTS: - Acceptance criteria met for negative control: The mean negative control OD, both for the 3 and 60 min exposure period, was in the range of ≥ 0.8 and ≤ 2.8.- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative cell viability as compared to the negative control, both for the 3 min exposure period (7.1%) and for the 60 min exposure period (5.4%) thus the values were below the cut-off value at 60 min exposure confirming the validity of the test system and the specific batch of tissue models.- Acceptance criteria met for variability between replicate measurements: The SD of all replicates determined was below the limit of acceptance of 30%.- Range of historical values if different from the ones specified in the test guideline: the results of the negative and positive control fell within the historical control range (see Table 1 under "Any other information on results incl. tables").

Any other information on results incl. tables

Table 1: Historical data of negative and positive controls

Material Criteria Average viability (%) OD (mean±SD) Range of OD Coefficient of variation (%)
3 min 60 min 3 min 60 min 3 min 60 min 3 min 60 min
Negative control Non-corrosive 100 100 1.78± 0.40 1.82± 0.52 1.10 - 2.29 1.02 - 2.42 22.2 28.7
8 N KOH Corrosive 10.0 5.0 0.18± 0.09 0.09± 0.03 0.03 - 0.33 0.04 - 0.13 50.5 38.6

OD = Optical Density

Table 2: Results after treatment with the test substance and controls

Test group OD (n = 2 tissues) SD % OD540compared to the control
% viability
OD (n = 2 tissues) SD % OD540compared to the control
% viability
  3 min 60 min
Negative control 1.573 0.185 100 1.507 0.035 100
Test substance 1.251 0.061 79.5 1.112 0.252 73.8
Positive control 0.111 0.10 7.1 0.082 0.006 5.4

Applicant's summary and conclusion

Interpretation of results:
other: the results of this study as a stand-alone study are not suitable for classification according to CLP/EU GHS criteria; the results may be used for classification purposes in a weight of evidence approach
Conclusions:
Under the conditions of the RHE test method the test substance showed no corrosive properties.