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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:
From 17 December 2017 to 29 January 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
30 mg of test item was used instead of 25 mg and the post incubation period was 24h instead of 42h,as required in the current OECD method. No details were given on MTT interference or colou interference assessment, no certificate on tissue was given
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
supplied by the sponsor, lot No. 160405
- Expiration date of the lot/batch:
30June 2018
- Purity test date:not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not specified
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: prepared as slurry with sterile water

FORM AS APPLIED IN THE TEST (if different from that of starting material)
the test item was applied with 25 µL of DPBS

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: EpiDermTM
Cell source:
other: not applicable
Source strain:
other: not applicable
Justification for test system used:
The test system is given by the sponsor
Vehicle:
other: DPBS
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpidermTM SIT
- Tissue batch number(s): not detailed
- Production date: not detailed
- Shipping date: not detailed
- Delivery date: not detailed
- Date of initiation of testing: 12 December 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: washing was performed 15 times with sterile PBS
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP:not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: 96 well plate reader spectrophotometer/fluorimeter/bioluminometer (TECAN infinite 2000)
- Wavelength:570nm
- Filter: no filter was used
- Filter bandwidth:no filter was used
- Linear OD range of spectrophotometer:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
No deviation from test system or model condition was observed. No historical data was mentionned

NUMBER OF REPLICATE TISSUES: triplicates were used

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : not detailed
- Procedure used to prepare the killed tissues (if applicable): not detailed
- N. of replicates : not detailed
- Method of calculation used: not detailed

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
Only MTT assay was described in the report

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if tthe viability after 60 minutes and 24 hours post incubation period is equal to or less than 50
- The test substance is considered to be non-irritant to skin if the viability after 60 minutes and 24 hours post incubation period is greater than 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 29 , 30.2, 31.2 mg test item powder with 25 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): pure

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5%
Duration of treatment / exposure:
60 minutes (including 35 minutes at 37°C)
Duration of post-treatment incubation (if applicable):
24 hours
Number of replicates:
triplicates were used

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test item
Value:
107.98
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Negative control
Value:
100
Negative controls validity:
not valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Positive control
Value:
2.3
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: not specified
- Colour interference with MTT: not specified

DEMONSTRATION OF TECHNICAL PROFICIENCY: positive control indicated proficiency of the test system to measured tissue viability

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: conform
- Acceptance criteria met for positive control: conform
- Acceptance criteria met for variability between replicate measurements: conform

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the experimental condition of the study, the test item TMAO did not induced decrease of tissue viability after exposure. Hence, according to CLP criteria, the TMAO did not required skin irritation classification.
Executive summary:

The aim of this GLP compliant study was the detection of the skin irritation potency of TMAO tested on Human skin model EpiDermTM SIT according to OECD 439 method.

30 mg of the test item was applied on the tissue (EpiDermTM) used in triplicate for 60 minutes (including 35 minutes period at 37°C).5% SDS was used as positive condition and PBS as negative condition. Thereafter, the test item was removed from the tissue by rincing step. The cells were incubated for 24 hours. MTT assay was performed at the end of the post incubation period. The tissue viability was measured at 570 nm using a spectrophotometer.

The viability of the tissue exposed to test item was measured at 107,98%

Under the experimental condition of the study, the test item TMAO did not induced decrease of tissue viability after exposure. Hence, according to CLP criteria, the TMAO did not required skin irritation classification.