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Description of key information

Violuric acid was tested for skin and eye irritation in the SkinEthic RHE Human Epidermis SinEthic Human Corneal Epithelial Model, respictevely. Violuric acid was found to possess irriation potential in both test systems and should thus be classified. However, the present test systems cannot distinguish between category 1 and 2 classification and follow-up studies will be initiated.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
no
Remarks:
The study has been performed in House according to current guidelines by trained technicians. The study is commonly performed at the lab and leaves up to the validty criteria.Thus data from the study can be considered reliable.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Control samples:
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg



NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16µl PBS


POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µl SDS
- Concentration (if solution): 50mg/mL
Duration of treatment / exposure:
42 min +/- 2 min
Duration of post-treatment incubation (if applicable):
42h +/- 1h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
1.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Interpretation of results:
study cannot be used for classification
Conclusions:
Violuric was found to reduce the cell viabilty more than 50% in the present test system and must thus be considered as an irritant. However, the present test system cannot distinguish between category 1 and 2 classification.
Executive summary:

Violuric acid has been tested for skin irritation according to OECD with the ' SkinEthicTMReconstructed Human Epidermis kit RHE/S/17'.

The irritation potential was assessed indirectly by assessing the uptake of MTT in living cells, which results in conversion to MTT in a blue dye, which can be measured spectophotometrically and by visual assessment of the tissue after 42 min exposure to the test substance followed by 42 h post treatment period. Viability of cells exposed to violuric acid was 1% compared to control cells and thus indicates irritaion potential of the test substance.

According to the test system violuric acid should be classified as irritant, but the test system cannot distinguish between category 1 and 2 classification.

In conclusion, violuric acid was found to be irritaing in the present test system.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-07-25 to 2018-09-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
no
Remarks:
The study has been performed in House according to current guidelines by trained technicians. The study is commonly performed at the lab and leaves up to the validty criteria.Thus data from the study can be considered reliable.
Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability : The SkinEthic Human Corneal Epithelial Model (SkinEthicTM HCE) in order to assess eye irritation potential of violuric acid. The test method has been chosen in order to investigate whether or not the test material should be classified. Based on prvious QSAR assessment, which did not indicate yirritation potential, this method was chosen.
- Description of the cell system used
The assay is a cytotoxicity test performed with SkinEthic Human Corneal Epithelium kit. The model consists of fully differentiated epithelium of normal human keratinocytes in a three-dimensional structure. The cells are grown on a microporous polycarbonate filter, which mimics the structure of the outer layer of the cornea of the eye. Batch No 18-HCE-029 was used for the present assay.
The absence of mycoplasma, bacteria and fungus has been verified.
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg


Duration of treatment / exposure:
Exposure: 4h +/- 5 min
Duration of post- treatment incubation (in vitro):
18h +/- 30 min
Number of animals or in vitro replicates:
2 replicates
Details on study design:
- RhCE tissue construct used, including batch number : Immortalized Human Corneal Epithelim Cells batch no. 18-HCE-029
- Doses of test chemical and control substances used : 30 mg violuric acid, 30 µL Methyl acetate, 30 µl PBS
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) : Treatment period: 4 hours ± 5min (37°C, 5% CO2, ≥ 95% humidity) , post exposure immersion 30 minutes ± 2min at room temperature; post-incubation period: 18 hours ± 30min incubation at 37°C, 5% CO2
- Justification for the use of a different negative control than ultrapure H2O (if applicable) : PBS was used to premoisten the tissue before application of violuric acid
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable) 2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
- Acceptable variability between tissue replicates for positive and negative controls : Viability of PC expressed as % of neg control <=30%
- Acceptable variability between tissue replicates for the test chemical: Difference in viability between 2 replica is <=20%
Irritation parameter:
other: percent cell viability compared to control tissue, which is set to 100%
Value:
0.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
study cannot be used for classification
Conclusions:
Violuric acid was found be irritating to the eye and should be classified. However, the present system cannot distinguish between UN GHS Category 1 from Category 2.
Executive summary:

The in vitro eye irritation potential of violuric acid was investigated with SkinEthic Human Corneal Epithelium model. The test was performed in accordance with OECD 492.

The irritation potential was assessed indirectly by assessing the uptake of MTT in living cells, which results in conversion to MTT in a blue dye, which can be measured spectophotometrically and by visual assessment of the tissue after exposure of 4h followed by immersion period of 30 min and post treatment period of 18h at 37°C and 5% CO2. Viability of cells exposed to violuric acid was 0.6 % compared to control cells and thus indicates eye irritation potential of the test substance.

According to the test system violuric acid should be classified as irritant, but the test system cannot distinguish between category 1 and 2 classification.

In conclusion, violuric acid was found to be irritaing in the present test system

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification