Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-04-27 to 2017-05-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2018

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 29 July 2016)
Deviations:
yes
Remarks:
The coefficient of variation for the 10% v/v 60 minute exposure period was 30.6% with the acceptance criteria being 30%. This deviation was considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
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: Room temperature in the dark
- Solubility and stability of the test substance in the solvent/vehicle: The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a liquid: The test item was used as supplied and also prepared as 33% v/v and 10% v/v aqueous solutions.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
recommended by guideline
Vehicle:
unchanged (no vehicle)
Remarks:
water for the 33% v/v and 10% v/v solutions
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 25810
- Production date:
- Shipping date:
- Delivery date: 03 May 2017
- Date of initiation of testing:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation (if applicable): 37 °C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper and the tissue surface was swabbed. The rinsing procedure was then repeated once more to ensure the tissues were completely decontaminated.
- Observable damage in the tissue due to washing: no


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function:
- Morphology:
- Contamination:
- Reproducibility:

NUMBER OF REPLICATE TISSUES: duplicates

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable): freeze killed
- N. of replicates : duplicates
- Method of calculation used: True viability = mean OD tvt-(OD tkt-OD ukt)

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
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL
- Concentration (if solution): neat, 33% v/v and 10% v/v aqueous solutions

NEGATIVE CONTROL
- Sterile distilled water
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL
- Potassium Hydroxide
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 8.0 N
Duration of treatment / exposure:
3-Minutes and 60-Minutes
Duration of post-treatment incubation (if applicable):
The plates stood overnight at room temperature, to allow MTT extraction to proceed
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
neat test item
Run / experiment:
3 minutes exposure period
Value:
59.7
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
4.0% tissue viability
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes exposure period
Value:
31.1
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
3.9% tissue viability
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: yes; The test item was shown to directly reduce MTT and freeze-killed tissues were employed, the results of the MTT assay were therefore corrected as follows: True viability = mean OD tvt-(OD tkt-OD ukt)
- Colour interference with MTT: test item did not have the potential to cause color interference

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group were satisfied with the exception that the 10% v/v concentration of the test item after a 60 minute exposure period was borderline, this is reported as a deviation.

Applicant's summary and conclusion

Interpretation of results:
other: not corrosive
Conclusions:
All three concentrations of the test item, 10%, 33% and neat, were considered to be non-corrosive to the skin.
Executive summary:

In a skin corrosion study performed in accordance with OECD Guideline 431 (In Vitro Skin Corrosion) (adopted July 29, 2016), CGE-PMDA adduct (100%, 33% and 10% a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 3 and 60 minutes respecitvely in triplicates.

Each approximately 50 µL of the test item were applied to the tissues. After 3 or 60 minutes exposure at room temperature (3 min) or 37°C (60 min exposure time), the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 3 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (8.0 N KOH) and negative (deionised water) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after treatment with neat CGE-PMDA adduct compared to the negative control tissues was 59.7% for the 3 minute exposure period and 31.1% for the 60 minute exposure period. The relative mean tissue viability obtained after treatment with 33% CGE-PMDA adduct compared to the negative control tissues was 75.3% for the 3 minute exposure period and 15.8% for the 60 minute exposure period. The relative mean tissue viability obtained after treatment with 10% CGE-PMDA adduct compared to the negative control tissues was 94.4% for the 3 minute exposure period and 39.2% for the 60 minute exposure period.

Since the mean relative tissue viability for the neat test substance and both dilutions was ≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure, the prediction to be considered according to EU CLP Regulation (EC) No 1272/2008 is non-corrosive.