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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:
key study
Study period:
23-28 April 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted according to OECD test Guideline No. 431. However, functional model conditions and references to historical control data are not included in the report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
Adopted April 13, 2004
Deviations:
yes
Remarks:
functional model conditions and references to historical control data are not included in the report.
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
yes
Remarks:
functional model conditions and references to historical control data are not included in the report.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
UK GLP Compliance Programme (inspected on 05 July 2016 / signed on 28 October 2016)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Storage conditions: In the refrigerator at 2 – 8 °C, protected from light in N2 atmosphere

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Following the REACH bottom-up strategy, the EpiDerm™ Human Skin Model method was used to assess skin corrosion as recommended in the OECD test guideline No. 431.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-Kit, MatTek Corporation (Ashland, MA 01721, USA).
- Lot number: 10504 Kit N
- Production Date: no data
- Shipping date: no data
- Delivery date: no data
- Date received: April 24, 2008
- Date of initiation of testing: April 25, 2008

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 and 60 minutes at 37 °C in a humidified atmosphere of 5% CO2
- Temperature of post-treatment incubation: 3 h at 37 °C in a humidified atmosphere of 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: At the end of the exposure period the first insert was removed from the 6-well plate. Using a wash bottle the tissue was gently rinsed with PBS Rinse Solution to remove any residual test material. Excess PBS Rinse Solution was removed by gently shaking the insert and
blotting the lower surface with blotting paper.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT solution
- Incubation time: 3 hours at 37 °C, 5% CO2 in air
- Spectrophotometer: Versamax® Molecular Devices, D-85737 Ismaning
- Wavelength: 570 nm
- Filter: without reference filter
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Not reported

NUMBER OF REPLICATE TISSUES: Duplicate tissues for test item, negative and positive controls

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not needed

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- 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
- Amount(s) applied (volume or weight with unit): 50 µL of the undiluted test item.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of sterile distilled water was used as supplied

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of 8.0 N Potassium Hydroxide was used as supplied
Duration of treatment / exposure:
3 and 60 minutes.
Duration of post-treatment incubation (if applicable):
not applicable
Number of replicates:
Duplicate tissues for test item, negative and positive controls

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes exposure
Value:
92.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
26.4%
Remarks on result:
other: No indication of corrosion
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes exposure
Value:
85.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
12.4%
Remarks on result:
other: No indication of corrosion
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: none.

DEMONSTRATION OF TECHNICAL PROFICIENCY: not included in the report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.637 for the 3 Minute exposure period and 1.703 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied (≥ 0.8).
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 26.4% and 12.4% relative to the negative control following the 3 and 60 Minute exposure period, respectively. The positive control acceptance criterion was therefore satisfied (≤ 30%).

Any other information on results incl. tables

Table 7.3.1/1: Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item


Dose group

Treatment

Interval

Absorbance

570 nm

Tissue 1*

Absorbance

570 nm

Tissue 2*

Mean

Absorbance

of 2 Tissues

Rel. Absorbance

[% of Negative

Control]**

Negative Control

3 min

1.628

1.647

1.637

100.0

 

Positive Control

3 min

0.494

0.369

0.432

26.4

Pink Pepper (ST

08 C 08)

3 min

1.547

1.486

1.517

92.6

 

Negative Control

1 hour

1.755

1.650

1.703

100.0

 

Positive Control

1 hour

0.191

0.231

0.211

12.4

 

Pink Pepper (ST

08 C 08)

1 hour

1.436

1.477

1.456

85.5

 

* Mean of three replicate wells after blank correction

** relative absorbance [rounded values]: 100 x (absorbance test item) / (absorbance negative control)

Applicant's summary and conclusion

Interpretation of results:
other: Non-corrosive base don GHS criteria
Conclusions:
Under the experimental conditions of this study, the test substance is not classified as corrosive, according to EU CLP Regulation (EC) No 1272/2008 and to the GHS.
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 431 and in compliance with GLP, using the EpiDerm™ Human Skin Model.

 

Duplicate tissues were treated with 50 µL of the undiluted test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 92.6% and 85.5% after 3 and 60 minutes exposure, respectively. The relative mean viability of the positive control treated tissues was 26.4% and 12.4% after 3 and 60 minutes exposure. The quality criteria required for acceptance of results in the test were satisfied.

Under the experimental conditions of this study, the test substance is not classified as corrosive according to EU CLP Regulation (EC) No 1272/2008 and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin corrosion endpoint.