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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:
2018-03-21 to 2018-03-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015-07-28
Deviations:
no
Qualifier:
according to
Guideline:
other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)
Version / remarks:
2014-11-07
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2015-06-05

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: solid, white powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, in a tightly closed container

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: normal, human-derived epidermal keratinocytes
Cell source:
other: humans
Source strain:
other: not applicable
Details on animal used as source of test system:
not applicable
Justification for test system used:
This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human-derived epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely resembles the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Vehicle:
other: Dulbecco's phosphate buffered saline
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200-SIT; MatTek)
- Tissue lot number: 25888

TEST FOR DIRECT MTT REDUCTION
- to check the non-specific MTT-reducing capability of the test item 25 mg of the test item were mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator (5 % CO2, 95 % RH).
- untreated MTT medium was used as control.
- if the mixture turns blue/purple, the test item is presumed to have reduced MTT and the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using freeze-killed tissues.

TEST FOR COLOUR INTERFERENCE
- to check the colouring potential of the test item 25 mg of the test item were mixed per 300 µL aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min (5 % CO2, 95 % RH).
- the mixture showed no colouring detected by unaided eye assessment. So the additional functional test with viable tissues and the quantitative corrections were not necessary.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 ± 1 minutes followed by incubation at room temperature until the 60 ± 1 minute treatment period was completed
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- after the end of the treatment period the tissues were washed 15 times with DPBS.
- subsequently, the inserts were submerged three times in DPBS and shaken to remove rests of the test item.
- then inserts were rinsed once from the inside and the outside with sterile DPBS.
- inserts were placed in prepared 6-well plates containing pre-warmed fresh assay medium per well.
- plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing fresh assay medium and incubated for additional 18 ± 2 h.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/well)
- Incubation time: 3 hours ± 5 minutes
- Extraction of formazan: after the MTT incubation period, the tissues were rinsed three times with DPBS and allowed to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature at least for 2 hours with shaking on a plate shaker.
Before using the extracts, the plate had been shaken for at least 15 minutes on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 µL aliquots per each tissue were transferred into a 96-well plate. Optical density (OD) was measured with a filter band without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
- Wavelength: 570 nm
- Filter bandwidth: maximum ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1-virus, Hepatitis B virus and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponds to 100 % tissue viability in the current test. For each individual tissue treated with the test item or the positive control, the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [(mean ODtest item / positive control) / ODmean of negative control] * 100
For the test item and the positive control the mean relative viability ± relative standard deviation of the three individual tissues were calculated and used for classification according to the following prediction model:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification of the test substance The test substance may be considered as non-irritant to skin in accordance with UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more 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): 25 mg (39 mg/cm²) of the test item
Firstly, 25 µL of the vehicle were applied to the epidermal surface in order to improve the contact between the powder and the epidermis. Afterwards, 25 mg (39 mg/cm²) of the test item were applied directly atop the EpiDermTM tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue by using a bulb-headed Pasteur pipette.

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL of the vehicle

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5 % SDS solution
Duration of treatment / exposure:
60 ± 1 minute
Duration of post-treatment incubation (if applicable):
approx. 42 hours
Number of replicates:
triplicates

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
(mean)
Value:
2.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, the test item did not directly reduce MTT (NSMTT equalled 0%) and a functional test with freeze-killed tissue was not necessary.
- Colour interference with MTT: mixture 25 mg of the test item per 300 µL aqua dest. and/or per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, an additional test with viable tissues (without MTT addition) was not necessary (NSC equalled 0%).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (value: 1.385).
- Acceptance criteria met for positive control: mean relative tissue viability (% negative control) of the positive control was ≤ 20% (2.5 %)
- Acceptance criteria met for variability between replicate measurements: standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2 % - 15.4 %).
- The absorbance values were not below historically established boundaries.

Please also refer to the field "An other information on results incl. tables" below.

Any other information on results incl. tables

Table 1: Result of the test item magnesium 2-ethylhexanoate

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

3

1

2

3

1

2

3

Absolute OD570

1.156

1.505

1.496

0.057

0.092

0.070

0.080

0.081

0.084

1.136

1.498

1.521

0.060

0.100

0.081

0.082

0.079

0.086

Mean Absolute OD570

1.385****

0.077

0.082

OD570(Blank Corrected)

1.114

1.463

1.454

0.014

0.049

0.028

0.037

0.039

0.042

1.094

1.456

1.479

0.018

0.057

0.038

0.039

0.036

0.043

Mean OD570of the Duplicates (Blank Corrected)

1.104

1.459

1.466

0.016

0.053

0.033

0.038

0.038

0.043

Total Mean OD570of 3 Replicate Tissues (Blank Corrected)

1.343*

0.034

0.040

SD of Mean OD570 of the Duplicates (Blank Corrected)

0.207

0.019

0.003

Relative Tissue Viability [%]

82.2

108.6

109.2

1.2

4.0

2.5

2.9

2.8

3.2

Mean Relative Tissue Viability [%]

100.0

2.5**

2.9

SD of Relative Tissue Viability [%]***

15.4

1.4

0.2

CV [% Viabilities]

15.4

54.4

6.7

* Blank-corrected mean OD570 nmof the negative control corresponds to 100% absolute tissue viability.

** Mean relative tissue viability of the three positive control tissues is  20%.

*** Standard deviation (SD)of relative tissue viabilityobtained from the three concurrently tested tissues for test item, positive and negative controlis  18%

**** Mean absolute OD570of the negative control tissues is 0.8 and 2.8.

Table 2: Historical data:

 

Mean Absolute

OD570±30nmNK

MeanAbsolute

OD570±30nmPC

Mean Relative

Viability [%] PC

SD Viability

[%]
NK, PC, TI

Mean

1.861

0.114

3.7

4.4

SD

0.247

0.033

1.5

4.1

Range of
LCL –

UCL

1.367 – 2.355

0.048 – 0.181

0.7 – 6.8

0.0 – 12.5

n

25

25

25

117

Historical data were generated from 2015 to 2017.

Applicant's summary and conclusion

Interpretation of results:
other: Category 1 (corrosive) or Category 2 (irritant) based on GHS criteria
Conclusions:
The test item, magnesium 2-ethylhexanoate, is either corrosive or irritating to the skin. Since the RhE test methods covered by OECD TG 439 cannot resolve between UN GHS Categories 1 or 2, further information on skin corrosion is required to decide on its final classification.