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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:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

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:
july 2016
Deviations:
no
Principles of method if other than guideline:
Experimental Procedure
Upon receipt of the EpiDerm TM, the tissues were inspected visually and transferred into 6-well plates containing 0.9 mL pre-warmed assay medium per well. The 6-well plates were pre-incubated in a humidified incubator at 37  1°C, 5.0% CO2 / 95% air for at least 1 h. Then the medium was replaced by 0.9 mL fresh assay medium and the surface was dried using a sterile cotton tip. The 6-well plate used for the 3 min experiment was placed back into the incubator. The other plate was used for the 60 min treatment. About 1 h before the end of the first treatment period the MTT solution was prepared and pre-warmed in the incubator.
60 min experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of e.g. 20 sec. was kept between dosing. Then the 6-well plate was incubated at 37  1°C, 5.0% CO2 / 95% air.

3 min experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of 20 sec. was kept between dosing.
After 3 min of application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS (phosphate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate“ containing 300 µL pre-warmed assay medium per well. All inserts were treated in the same manner.
Then the inserts were dried again and transferred into a prepared 24-well “MTT assay plate“ containing 300 µL pre-warmed MTT solution. The plate was incubated for 3 h at 37  1°C, 5.0% CO2 / 95% air.

60 min experiment: after 60 min application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate“ containing 300 µL pre-warmed assay medium per well. All inserts were treated in the same manner.
Then the inserts were dried again and transferred into a prepared 24-well “MTT assay plate“ containing 300 µL pre-warmed MTT solution. The plate was incubated for 3 h at 37  1°C, 5.0% CO2 / 95% air.

3 min and 60 min experiment: after the 3 h MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried. Then the inserts were transferred into 12-well “extraction plates“. 2 mL of isopropanol were pipetted into each insert, thus the insert was covered from both sides. The extraction plates were sealed in zip-bags to inhibit isopropanol evaporation. Extraction was carried out either over night without shaking at room temperature or, alternatively, at least 2 h with shaking at room temperature.

After the extraction period the inserts were pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. Then the inserts were discarded and the extraction plates were placed on a shaker for 15 min.
Per each tissue 3 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Hydrocotyle asiatica, ext.
EC Number:
283-640-5
EC Name:
Hydrocotyle asiatica, ext.
Cas Number:
84696-21-9
Molecular formula:
not applicable
IUPAC Name:
Hydrocotyle asiatica, ext.
Test material form:
solid

In vitro test system

Test system:
human skin model
Source species:
other: stratum corneum of the epidermis the cytotoxic effects of the test item on EpiDerm, a reconstituted three-dimensional human epidermis model
Vehicle:
other: water and isopropanol (1:1 v/v)
Control samples:
yes, concurrent vehicle
Amount/concentration applied:
25mg in 25 µL of water
Duration of treatment / exposure:
3 min and 60 min
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: NSC living (%)
Run / experiment:
3 min
Value:
ca. 0.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: NSC living (%)
Run / experiment:
60 min
Value:
ca. 0.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Results of 3 min Experiment

Name

Negative Control

Positive Control

Test Item

Replicate Tissue

1

2

1

2

1

2

Absolute OD570

1.995

2.067

0.344

0.357

1.862

1.870

1.891

2.057

0.361

0.340

1.852

1.887

2.008

2.080

0.341

0.359

1.839

1.897

Mean Absolute OD570

2.016****

0.350

1.868

OD570-
Blank Corrected

1.951

2.023

0.300

0.314

1.818

1.827

1.847

2.013

0.317

0.297

1.809

1.844

1.965

2.037

0.297

0.316

1.795

1.854

Mean OD570of 3 Aliquots (Blank Corrected)

1.921

2.024

0.305

0.309

1.807

1.841

SD OD570 of 3 Aliquots

0.064

0.012

0.011

0.010

0.011

0.014

Total Mean OD570of 2 Replicate Tissues
(Blank Corrected)

1.973*

0.307

1.824

SD OD570 of 2 Replicate Tissues

0.073

0.003

0.024

Mean Relative Tissue Viability [%]

100.0

15.6

92.5

Coefficient Of Variation [%]***

3.7

0.9

1.3

*              corrected mean OD570of the negative control corresponds to 100% absolute tissue viability.

***            coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is£ 30%.

****          The mean absolute OD570of the negative control is ≥ 0.8 and ≤ 2.8

Results of 60 min Experiment

Name

Negative Control

Positive Control

Test Item

Replicate Tissue

1

2

1

2

1

2

Absolute OD570

1.871

1.889

0.155

0.162

1.502

1.423

1.904

1.888

0.158

0.154

1.497

1.423

1.845

1.900

0.155

0.163

1.490

1.443

Mean Absolute OD570

1.883****

0.158

1.463

OD570-
Blank Corrected

1.827

1.846

0.112

0.118

1.459

1.379

1.860

1.844

0.115

0.110

1.454

1.380

1.802

1.857

0.111

0.120

1.446

1.400

Mean OD570of 3 Aliquots (Blank Corrected)

1.830

1.849

0.113

0.116

1.453

1.386

SD OD570 of 3 Aliquots

0.029

0.007

0.002

0.005

0.006

0.012

Total Mean OD570of 2 Replicate Tissues (Blank Corrected)

1.839*

0.114

1.420

SD OD570 of 2 Replicate Tissues

0.014

0.002

0.047

Mean Relative Tissue Viability [%]

100.0

6.2**

77.2

Coefficient Of Variation [%]***

0.7

2.1

3.3

*              corrected mean OD570of the negative control corresponds to 100% absolute tissue viability.

**             mean relative tissue viability of the 60 min positive control < 15%,

***            coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is£ 30%.

****          The mean absolute OD570of the negative control is ≥ 0.8 and ≤ 2.8

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item showed no corrosive effects.
The mean relative tissue viability (% negative control) was <= 50% (92.5%) after 3 min treatment and >=15% (77.2%) after 60 min treatment.
The controls confirmed the validity of the study. The mean OD570nm of the two negative control tissues was >= 0.8 and ≤ 2.8 for each exposure period (2.016, 1.883). The mean relative tissue viability (% negative control) of the positive control was < 15% (6.2%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was <= 30% (0.7% – 3.7%).

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