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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:
From July 10, 2017 to September 07, 2017
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:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
solid: nanomaterial, surface-treated

In vitro test system

Test system:
human skin model
Source species:
other: Reconstructed human epidermis (Epiderm™)
Source strain:
other: 00267
Details on animal used as source of test system:
Not applicable
Justification for test system used:
The method followed, described in XcellR8 SOP L0048 'Epiderm™ In Vitro Reconstructed Human Epidermis Skin Corrosion Test', is based on the current MatTek Protocol (MK-24-007-0024, 2014-11-07) using the MatTek Corporation Epiderm™ reconstructed tissue model: EPI-200. This method was endorsed in 2002 by National Co-ordinators of OECD Test Guideline Programme (WNT), originally adopted in 2004 and updated in 2013 and 2015. The Epiderm™ skin model and assay for skin corrosion testing is endorsed by OECD TG 431.
Vehicle:
water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epiderm™ (EPI-200) Reconstructed Human Epidermis
- Tissue batch number(s): Lot 25834
- Delivery date: August 01, 2017 (tissues kept at 4°C until use)
- Date of initiation of testing: July 11, 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during pre-incubation: 37°C (5 % CO2, ≥95 % RH)
- Temperature used during treatment / exposure: 37°C (5 % CO2, ≥95 % RH)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Spectrophotometer: BMG LabTech FluoStar Optima (calibrated July 24, 2017)
- Wavelength: 570 nm
- Filter: No

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Optical density = 1.866 ± 0.143 (mean and SD of MTT value of three tissues exposed to H2O). PASS: Test guideline acceptance range = ≥0.8 and ≤2.8
- Barrier function: ET 50% of tissues exposed to 100 μl Triton X-100 1%, n = 3 = 6.02 h. PASS: Test guideline acceptance range = ≥4.0 and ≤8.7 h
- Morphology: Histological examination demonstrated human epidermis-like structure: including multiple layers (at least 4) of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) which are present under multi-layered stratum corneum (PASS). Tissue thickness = 129.1 μm (11 layers). PASS. Acceptance range = >70 and <130 μm
- Contamination: No evidence of contamination during long term antibiotic and antimycotic free culture. PASS: No contamination reported

NUMBER OF REPLICATE TISSUES: Triplicate

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: A preliminary test was undertaken to check the potential of the test item to interfere with the MTT viability test endpoint. This was followed by the main experiment

DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean OD570 of the negative control tissues is <0.8 after 3 min and 1 h; the mean of the positive control relative percentage viability after 1 h exposure is <15 % of the mean of the negative control; and the coefficient of variation (CV) is <0.3 (30 %)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: Nominal 25 mg-neat to the surface of the Epiderm™ skin model

VEHICLE
- Amount applied: 25 μl

NEGATIVE CONTROL
- Concentration: Neat
- Lot/batch no.: RNBF8521

POSITIVE CONTROL
- Concentration: Single topical application in ultrapure water of 8N
- Lot/batch no.: SLBD3295V
- Purity: ≥85 %
Duration of treatment / exposure:
3 and 60 min
Number of replicates:
Triplicate

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
(% to negative control)
Run / experiment:
3 minutes
Value:
101.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
(% to negative control)
Run / experiment:
60 minutes
Value:
106.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: The test item was found not to interfere
- Colour interference with MTT: The test item was found not to interfere

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the mean OD570 was >0.8: 1.668 after 3 minutes and 1.546 after 1 hour
- Acceptance criteria met for positive control: The mean relative percentage viability after 1 hour exposure was >15 % (23 %) of the mean negative control, suggesting a failed criteria. However, it is still possible to classify the positive control as Corrosive (Category 1A) as the mean viability value after 3 minutes was <25 % (19.3 %). Additionally, KOH is an MTT interfering compound and the subtraction of interfering values would have led to a viability percentage below 15 %. Subsequently, the results are considered to be valid
- Acceptance criteria met for variability between replicate measurements: Yes, the coefficient of variation (CV) for the triplicate measurements did not exceed 0.3 (i.e. 30 %) in any circumstance

Any other information on results incl. tables

Mean and SD of Cell Viability Measurements after 3 Minutes and 1 Hour of Application:

Material 3 Minutes 1 Hour Prediction (OECD TG 431)
Mean of Viability (%) SD of Viability CV (%) Mean of Viability (%) SD of Viability CV (%)
Negative Control 100 9.3 9.3 100 3.8 3.8 Non-Corrosive

Positive Control

19.3 5.6 29.2 23 4.5 19.4 Corrosive (Category 1A)

Test Item

101.9 20.8 20.4 106.4 3.1 2.9 Non-Corrosive

The test item did not reduce the viability below 50 % after 3 minutes nor below 15 % after 1 hour and should be considered as non-corrosive.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro skin corrosion experiment using reconstructed human epidermis (RHE), calcium titanium trioxide resulted in a mean tissue viability (% of the negative control) of 101.9 % after 3 minutes and 106.4 % after 1 hour. Subsequently, calcium titanium trioxide cannot be classified as corrosive to human skin (CLP Regulation (EC) No. 1272/2008).
Executive summary:

To determine the corrosion potential of calcium titanium trioxide to human skin, an in vitro experiment was performed according to Good Laboratory Practise (GLP) and OECD Guideline 431 (Reconstructed Human Epidermis (RHE) Test Method) without deviation. MatTek Corporation Epiderm™ reconstructed tissue model (EPI-200) was used, which comprises normal human-derived epidermal keratinocytes cultured to form a multi-layered highly differential model of the human epidermis. As skin corrosion is related to cytotoxicity in the Epiderm™ tissue, determined by the reduction of MTT to formazan by viable cells relative to a negative control, the results offer a robust foundation from which true corrosivity can be predicted. 

A preliminary test was undertaken to establish the potential of calcium titanium trioxide to interfere with the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) viability endpoint. No interference (water colouration or MTT interference) from calcium titanium trioxide was detected. Following pre-incubation for 1 hour, the surface of Epiderm™ was exposed to a single topical application of 25 µl H20 plus nominal 25 mg / 50 µl neat test or reference items for a time period of 3 minutes or 1 hour (n = 3). The negative control consisted of sterile water (tissue grade) and the positive control of potassium hydroxide (KOH) in ultrapure water. After 3 minutes and 1 hour, tissues were tested for MTT viability. Tissue viability was assessed as a percentage and compared to the negative control.

The acceptance criteria for the negative and positive control were satisfied and all results were deemed to be valid. The OD570 of the negative control was >0.8 after 3 minutes (1.668) and 1 hour (1.546) and the mean viability of the positive control was <15 % (when interfering values have been removed). The percentage viability of the reconstructed human tissue was 101.9 and 106.4 % after 3 minutes and 1 hour of exposure, respectively. As the test item did not reduce viability below 50 % after 3 minutes, nor below 15 % after 1 hour, it was concluded that calcium titanium trioxide is not corrosive to human skin and cannot be classified as such according to CLP Regulation (EC) No. 1272/2008.