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Diss Factsheets

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:
01 Feb 2021 to 08 Feb 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
OECD Guideline 439. In vitro Skin Irritation: Reconstructed Human Epidermis Test Method, (corrected 26 June 2020).
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 “In vitro Skin Irritation: Reconstructed Human Epidermis Model Test ". Official Journal of the European Union No. L142; Amended by EC No. 2019/1390 OJ No. L247, 26 September 2019.
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid
EC Number:
270-470-1
EC Name:
Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid
Cas Number:
68441-66-7
Molecular formula:
not available due to complexity of the substance
IUPAC Name:
Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid
Test material form:
liquid
Details on test material:
Identification: Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid
Batch (Lot) Number: 2019194336
Expiry date: 03 June 2022
Physical Description: Clear light yellow liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature

Additional information
Test Facility test item number: 212207/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required

Information about the purity and composition of the test item is not available since the test item is an UVCB (Substance of Unknown or Variable composition, Complex Reaction Products or Biological Materials). Since a sample relevant for the purpose of this study was tested, it was concluded that the study integrity was not affected by the omission of this
information.
Specific details on test material used for the study:
No further details specified in the study report.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: three-dimensional human epidermis model
Source strain:
not specified
Details on animal used as source of test system:
Test System
EPISKIN Small ModelTM (EPISKIN-SMTM), 0.38 cm2.
This model is a three-dimensional human epidermis model, which consists of adult human derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes are cultured for 13-days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Source
SkinEthic Laboratories, Lyon, France.
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
Experimental Design
Test for the Interference of the Test Item with the MTT Endpoint
A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

Test for Color Interference by the Test Item
The test item was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the tissues during the exposure. To assess the color interference, 50 μL of the test item or 50 μL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water. The mixture was incubated for at least 1 hour at 37.0 ± 1.0°C in the dark. Furthermore, 50 μL of the test item or 50 μL sterile Milli-Q water as a negative control was added to 2.0 mL isopropanol.
The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking.
At the end of the exposure time, the mixtures were centrifuged for 30 seconds at 16000 g if needed and the absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
If after subtraction of the negative control, the OD for the test item solution is >0.08, the test item is considered as possibly interacting with the MTT measurement.

Test for Reduction of MTT by the Test Item
The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 μL of the test item was added to 1 mL MTT solution (1 mg/mL MTT in phosphate buffered saline). The mixture was incubated for approximately 3 hours at 37.0 ± 1.0°C in the dark. A negative control, 50 μL sterile Milli-Q water was tested concurrently. If the MTT solution color turned blue / purple or if a blue /purple precipitate was observed the test item interacts with MTT.

Test System Set Up
On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for approximately 22.5 hours at 37°C.
Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

MTT medium
MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 1 mg/mL in PBS) diluted (3x) in Assay medium (final concentration 0.3 mg/mL).

Environmental conditions
All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 68 - 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.6 - 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test Item Preparation
No correction was made for the purity/composition of the test item.
The liquid test item was applied undiluted (25 μL) directly on top of the tissue.

Application/Treatment of the Test Item
The test was performed on a total of 3 tissues per test item together with negative and positive controls. Twenty five μL of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. Negative and positive controls were shared with parallel studies. All information pertaining to shared tissues are archived in the raw data.
After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 ± 1 hours at 37°C.

Cell Viability Measurement
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 hours at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for approximately 68 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
Twenty five μL of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively.
Duration of treatment / exposure:
15 ± 0.5 minutes at room temperature
Duration of post-treatment incubation (if applicable):
42 ± 1 hours at 37°C
Number of replicates:
The test is performed on a total of 3 tissues per test item together with a negative control and positive control.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean - Test item
Value:
101
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: Optical density
Run / experiment:
Mean - Test item
Value:
1.254
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The test item was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of -0.0006 and -0.0011, respectively. Therefore it was concluded that the test item did not induce color interference.
In addition, because no color change was observed in the presence of MTT it was concluded that the test item did not interact with the MTT endpoint.
Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 101%. Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 6.6%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 7.4%, indicating that the test system functioned properly.

Any other information on results incl. tables

Mean Adsorption in the In Vitro Skin irritation Test with Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid

 

A

(OD570)

B

(OD570)

C

(OD570)

Mean

(OD570)

 

SD

Negative control

1.134

1.295

1.289

1.239

±

0.092

Test item

1.323

1.224

1.216

1.254

±

0.059

Positive control

0.033

0.056

0.157

0.082

±

0.066

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background adsorption (0.042). Isopropanol was used to measure the background adsorption.

 

Mean Tissue Viability in the In Vitro Skin irritation Test with Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid

 

Mean tissue viability (percentage of control)

Standard deviation (percentage)

Negative control

100

7.4

Test item

101

4.8

Positive control

6.6

5.3

 

Individual OD (ODraw) Measurements at 570 nm

 

A

(OD570)

B

(OD570)

C

(OD570)

Negative control

OD570measurement 1

OD570measurement 2

 

1.1993

1.1523

 

1.2600

1.4152

 

1.3479

1.3144

Test item

OD570measurement 1

OD570measurement 2

 

1.3476

1.3822

 

1.2272

1.3053

 

1.2457

1.2702

Positive control

OD570measurement 1

OD570measurement 2

 

0.0733

0.0777

 

0.0900

0.1062

 

0.1938

0.2048

OD = Optical density

Triplicate exposure are indicated by A, B and C.

 

Historical Control Data for In Vitro Skin Irritation Studies

 

Negative control

(adsorption; OD570)

Positive control

(adsorption; OD570)

Range

0.507 – 1.426

0.026 – 0.549

Mean

1.020

0.108

SD

0.155

0.087

n

147

147

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2017 to November 2020.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.
Executive summary:

The objective of this study was to evaluate Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid for its ability to induce skin irritation on a human three-dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)). The possible skin irritation potential of the test item was tested through topical application for 15 minutes.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

 

Batch 2019194336 of the test item was a clear light yellow liquid. The test item was applied undiluted (25 μL), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 ± 1 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed.

 

Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

 

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control (phosphate buffered saline) tissues was 101%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.

 

The positive control (5% sodium dodecyl sulfate) had a mean cell viability of 6.6% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 7.4%, indicating that the test system functioned properly.

 

In conclusion, Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations.