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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
version 18 June 2019
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Cadmium
EC Number:
231-152-8
EC Name:
Cadmium
Cas Number:
7440-43-9
Molecular formula:
Cd
IUPAC Name:
cadmium
Test material form:
solid: particulate/powder
Details on test material:
- Product name: Cadmium
- Physical state: solid, crystalline (in the powder form), Grey
- Composition regarding the element of interest : 99.67 % Cd (contains 2.68 % CdO)
- Batch/Lot number: CDPMC190316
- Expiry date: 16 September 2020
- Storage condition of test material: Controlled room temperature (15-25°C, ≤70% relative humidity)
Specific details on test material used for the study:
Name: Cadmium
Batch/Lot number: CDPMC190316
CAS number: 7440-43-9
Description: Grey solid (in the powdered form)
Purity: 99.67%
Expiry date: 16 September 2020
Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity)
Safety precautions: Enhanced safety precautions (half mask at least with P3 filter cartridge, nitrile gloves, lab coat) were applied considering the supplied safety datasheet to assure personnel health and safety.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM (SM) (Manufacturer: SkinEthic, France
- Tissue batch number(s):respectively 20-EKIN-003 (for Experiment I) and 20-EKIN-004 (for Experiment II)
- Expiry Date: respectively 20 January 2020 and 27 January 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 22.2-22.8°C in Experiment I and 23.1-23.5°C in Experiment II
- Temperature of post-treatment incubation (if applicable): not applicable

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1washing step: rinsing thoroughly with PBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT per well
- Incubation time: 3h
- Spectrophotometer: 96-well plate spectrophotometer
- Wavelength: 570nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE (see any other info on mat and meth)




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): 20 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): na; no formulation was required

NEGATIVE CONTROL: NaCI (9 g/l saline)
- Concentration (if solution): 50µl

POSITIVE CONTROL: glacial acetic acid
- Concentration (if solution): 50µl
Duration of treatment / exposure:
4h
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
29.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: suggestion of a pos effect but the variability between the 2 test item treated skin units was 59.9%(>50% OECD 431) and may indicate a technical error with the delicate epidermal membrane test system. An additional experiment (Experiment II) was performed
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
46.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: no indication of corrosion

Any other information on results incl. tables

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented below: 

Table: Optical Density (OD) and the calculated relative viability % of the samples (Corrosivity test) in Experiment I

Substance

Optical Density (OD)

 

Viability 

(% RV)

 

Measured

Blank corrected

Negative Control:

1

0.987

0.942

97.7

Physiological saline

2

1.031

0.986

102.3

 (0.9% w/v NaCl)

mean

--

0.964

100.0

Positive Control:

1

0.060

0.014

 

1.5

glacial acetic acid

 

 

2

0.054

0.008

 

0.9

 

mean

--

0.011

 

1.2

Test Item:

1

0.247

0.202

 

20.9

cadmium

2

0.420

0.374

 

38.8

 

mean

--

0.288

 

29.9

Notes:

1. Mean blank value was 0.045.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places)

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented below: 

Table: Optical Density (OD) and the calculated relative viability % of the samples (Corrosivity test) in Experiment II

Substance

Optical Density (OD)

 

Viability 

(% RV)

 

Measured

Blank corrected

Negative Control:

1

1.096

1.050

113.3

Physiological saline

2

0.882

0.836

88.7

 (0.9% w/v NaCl)

mean

--

0.943

100.0

Positive Control:

1

0.053

0.007

 

0.7

glacial acetic acid

 

 

2

0.060

0.014

 

1.4

 

mean

--

0.010

 

1.1

Test Item:

1

0.587

0.541

 

57.4

cadmium

2

0.431

0.385

 

40.8

 

3

 

0.429

 

0.383

 

40.6

 

 mean

 --

 0.384

46.2

Notes:

1. Mean blank value was 0.046

2. Optical density means the mean value of the duplicate (or triplicate) wells for each sample (rounded to three decimal places)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
in this in vitro EPISKINTM (SM) model test with Cadmium, the results indicate that the test item is not corrosive and not irritant to the skin, UN GHS Classification: No Category.
Executive summary:

An in vitro skin corrosivity and irritation test of Cadmium was performed in a reconstructed human epidermis model.EPISKINTM(SM) is designed to predict and classify the corrosivity and irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,Thiazolyl blue; CAS number 298-93-1) assay. The corrosivity and irritation potential of the test item was evaluated according to the OECD No. 431 and No. 439 guidelines.

Disks of EPISKINTM(SM) were treated with the test item and incubated for 15 minutes (three units forirritation testing) and 4 hours (at least two units for corrosivity testing) at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2,in a > 95% humidified atmosphere(irritation testing). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light,in a > 95% humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control) in case of the corrosivity testing in both experiments. PBS treated epidermis were used as negative control and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive control (three units/control) in case of the irritation testing. Two additional disks were used to provide in each case an estimate of colour contribution (NSCliving) from the test item in Experiment. For each treated tissue, the viability was expressed as a % relative to the negative control. For corrosivity, if the mean relative viability is <35% the test item is considered to be corrosive to skin. For irritation, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

 

In this study two experiments were performed to be sure of scientifically valid data.In Experiment I, three test item treated skin units and two NSClivingcontrol tissues were used in part of irritation test; and two test item treated skin units and two NSClivingcontrol tissues were used in part of corrosion test. In Experiment II, three test item treated skin units were used in part of corrosion testbut NSClivingcontrol was not used.Irritation testing was not required in the Experiment II.

Corrosivity testing:

In the Experiment I, the individual cell viabilities were 20.9% (corrosive) and 38.8% (non-corrosive) compared to the negative control value; the mean cell viability was

29.9%. Although this mean value suggests a positive effect, the variability between the two test item treated skin units was 59.9%, which is larger than permitted under the OECD guideline, and may indicate a technical error with the delicate epidermal membrane test system. Due to this equivocal result, an additional experiment (Experiment II) was performed to clarify for this specific result and to provide proper information for classification. Experiment II confirmed a negative result with a mean cell viability of 46.2% compared to the negative control. The result of Experiment II is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

Irritation testing:

Following exposure withCadmium, themean cell viability was 103.3% compared to the negative control. This is above the threshold of 50%, thereforethe test item was considered as being non-irritant to skin.

The experiment met thevalidity criteria, therefore the study was considered to be valid.

In conclusion, in thisin vitroEPISKINTM(SM) model test with Cadmium, the results indicate that the test item is not corrosive and not irritant to the skin, UN GHS Classification: No Category.


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