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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:
16 - 18 Dec 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: ECVAM 2009, Performance Standards for In-Vitro Skin Irritation Test Methods based on Reconstructed Human Epidermis (RHE)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: SkinEthic Skin Irritation Test-42bis Standard Operating Procedure (SOP) (2009): Using the Reconstructed Human Epidermis (RHE) model, INVITTOX Version 2.1
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
Diammonium oxalate hydrate
Cas Number:
6009-70-7
Molecular formula:
C2H8N2O4.H2O
IUPAC Name:
Diammonium oxalate hydrate

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: SkinEthic™ RHE-model RHE/S/17
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model (Episkin/Skin Ethic Laboratories, Lyon, France)
- Tissue batch number: 15-RHE-151
- Expiration Date: Dec 21, 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 42 ± 1 min at room temperature; thereafter at 37 °C for 42 ± 1 h

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with DPBS in order to remove any residual test material. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissues inserts with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min
- Spectrophotometer: microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the RHE model was assessed by undertaking a 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 5.4 h.
- Other: Absence of significant abnormalities after histological observations (HES stained vertical paraffin)

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test substance did not directly reduce MTT.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability is less than 50%
- The test substance is considered to be non-irritant to skin the viability is greater than or equal to 50%

ACCEPTABILITY CRITERA
The negative control OD values for the RHE-model have to be in the range of ≥ 0.8 and ≤ 3.0.
The negative control data meet the acceptance criteria if the mean OD value is higher or equal than a historically established boundary at 570 nm. The boundary is two standard deviations below the current historical mean (1.423). The standard deviation value is considered valid if ≤ 18% of the group mean value.
The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is lower than or equal to a historically established boundary. The boundary is three standard deviations above the current historical mean (3.42%). The standard deviation value is considered valid if ≤ 18% of the group mean value.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 16 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL per tissue

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL per tissue
- Concentration (if solution): 5% in deionised water
Duration of treatment / exposure:
42 ± 1 min
Duration of post-treatment incubation (if applicable):
42 ± 1 h
Number of replicates:
triplicates for each treatment and control group.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
42 min exposure
Value:
99.65
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: MTT was not reduced by the test substance.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD values were 2.111, 2.004 and 2.105 and, thus, in the range of ≥ 0.8 and ≤ 3.0. The mean OD was 2.074 and the Standard Deviation was 2.90% and, thus, higher than the historically established boundary of 1.423.
- Acceptance criteria met for positive control: The mean viability value was 1.10% and the Standard Deviation was 2.22% and, thus, lower than the historically established boundary of 3.42%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of the three tissues treated with the test substance was 0.93% and, thus, ≤ 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:
Under the conditions of the conducted test, the test substance did not show irritating properties towards reconstructed human epidermis tissue. The RhE-based test methods are able to identify Cat. 2 and No Cat. chemicals and can thus serve as stand-alone skin irritation methods for non-corrosives.

CLP: not classified