Decanoic acid, mixed esters with heptanoic acid, octanoic acid and pentaerythritol

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Nov. 2016 to 01 Dec. 2016

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

OECD Guidelines for the Testing of Chemicals Part 431, adopted 29. Jul. 2016 “In vitro Skin Corrosion: reconstructed human epidermis (RHE) test method”

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU-Method B.40 BIS.

Version / remarks:

Commission Regulation (EU) No.440/2008, EU-Method B.40 BIS. “IN VITRO SKIN CORROSION: HUMAN SKIN MODEL TEST“ dated 30. May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Decanoic acid, mixed esters with heptanoic acid, octanoic acid and pentaerythritol

EC Number:

270-471-7

EC Name:

Decanoic acid, mixed esters with heptanoic acid, octanoic acid and pentaerythritol

Cas Number:

68441-67-8

Molecular formula:

Not applicable - UVCB

IUPAC Name:

Decanoic acid, mixed esters with heptanoic acid, octanoic acid and pentaerythritol

Test material form:

liquid

Details on test material:

Designation in Test Facility: 16102602G
Date of Receipt: 26. Oct. 2016
Condition at Receipt: Room temperature, in proper conditions

Specification
Name Hatcol 3178
Batch no. 2011280182
Appearance: Clear liquid
CAS No.: 68441-67-8
EINECS-No.: 270-471-7
Molecular formula: C33H60O8 to C41H76O8 (in CH2 unit increments)
Molecular weight: 692 daltons
Purity: 99.6 %
Homogeneity: homogeneous liquid
Stability: H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility: H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Production date: 18. Jul. 2011
Expiry date: 01. Nov. 2019
Storage: Room Temperature (20 ± 5°C)

Storage in Test Facility
The test item was stored in a closed flask at 15.8 – 22.0 °C, protected from humidity.

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

Remarks:

EpiDerm TM

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

other: Not applicable

Details on animal used as source of test system:

The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis.
It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cultures inserts.

Origin
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-212-SCT
Day of delivery: 29. Nov.

Justification for test system used:

The skin corrosion test refers to the production of irreversible tissue damage following the application of a test material on a reconstructed human skin model. It allows the identification of corrosive chemical substances and mixtures.
The test item is applied topically to a three-dimensional human skin model, comprised of non-transformed, human-derived epidermal keratinocytes, which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, as well as a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo.
The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion and are cytotoxic to the underlying cell layers.
Corrosive chemicals are identified by their ability to decrease cell viability. The viability is measured by enzymatic conversion of the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) into a blue formazan salt, that is quantitatively measured after extraction from tissues.

Vehicle:

unchanged (no vehicle)

Details on test system:

Additional Tests
Nylon mesh compatibility
First, Hatcol 3178 was tested for possible reaction with the nylon mesh which is used to ensure sufficient contact with the tissue surface. 50 μL of the liquid test item was pipetted onto a sample of nylon mesh, and then placed on a pre-cleaned glass microscope slide for analysis. No reaction with the mesh was visible after 1 hour incubation at room temperature.

Assessment of Coloured or Staining Test Items
A preliminary examination was performed to determine whether the test item independently reacts, and develops a colour without the addition of MTT. 50 μL of the test item was aliquoted into a test tube along with 0.3 mL demineralised water, and incubated at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour.
The results showed that the resulting solution was colourless, therefore binding capacity did not have to be tested.

Assessment of Direct Reduction of MTT by the Test Item
The test item Hatcol 3178 was also tested for the ability to reduce formazan directly. To test for this ability, 50 μL of the test item was added to 1 mL of MTT reagent and incubated under dark room conditions at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour. An additional sample of untreated MTT medium was used as control.
The MTT solution did not change its colour within 1 hour. It was therefore concluded that direct MTT reduction had not taken place, and no data correction was necessary in the final analysis.

Preparations
On the day of the start of the experiment, the MTT concentrate was removed from the freezer and allowed to thaw at room temperature. The concentrate was diluted using the assay medium supplied, and the solution was stored at 2-8°C under dark room conditions.
The tissue plate was removed from the refrigerator 1 hour before the treatment.
The assay medium was warmed in the water bath to 37 ± 1°C.

Description of the Method
Four 6-well-plates were prepared with 0.9 mL assay medium in each well. The inserts containing the tissues were transferred to the wells using sterile forceps and the 6-well-plates were set into the incubator at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour (pre-incubation).
For each experiment (“3 minutes” and “1 hour”), one 24-well-plate was prepared as holding plate. 12 wells of each plate were filled with 300 μL assay medium, the other 12 with 300 μL MTT reagent. One additional plate was left empty. The plates were stored in the incubator at 37 ± 1°C and 5.0 ± 0.5% CO2.
For each experiment (“3 minutes” and “1 hour”), two 6-well-plates for the assay were used.
After pre-incubation, the assay medium was replaced by fresh assay medium and the test was started, using two wells as negative control with 50 μL demineralised water, two wells as positive controls with 50 μL potassium hydroxide solution and two other wells for the test item.
The liquid test item was applied without preparation (50 μL).
After the respective incubation time (“3 minutes” and “1 hour”) at 37 ± 1°C and 5.0 ± 0.5% CO2, the inserts were removed from the plates using sterile forceps. The inserts were thoroughly rinsed with DPBS, blotted with sterile cellulose tissue and set into the respective holding plate, using the wells containing assay medium. After transfer of all inserts, they were immediately moved to the wells containing MTT medium, blotting the bottom with cellulose tissue again before setting the insert into the MTT well. The tissues were incubated with MTT medium for 3 hours at 37 ± 1°C and 5.0 ± 0.5% CO2.
Next, the MTT medium was aspirated and replaced by DPBS. This washing step was repeated several times. Finally, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. 2 mL isopropanol was pipetted into each well, taking care to ensure the solution reached the upper rim of the insert. The plate was then covered with Parafilm® and shaken for 2 hours on an orbital shaker at room temperature.
The inserts were then pierced with an injection needle to extract all traces of coloured solution.
The inserts were then discarded and the content of each well was thoroughly mixed.
From each well, three replicates of 200 μL solution (each) was pipetted into a 96-well-plate which was read in a plate spectrophotometer at a wavelength of 570 nm.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The liquid test item was applied without preparation (50 μL).

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

For each experiment (“3 minutes” and “1 hour”), two 6-well-plates for the assay were used.
After pre-incubation, the assay medium was replaced by fresh assay medium and the test was started, using two wells as negative control with 50 μL demineralised water, two wells as positive controls with 50 μL potassium hydroxide solution and two other wells for the test item.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

96.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h

Value:

105.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Corrosivity of the Test Item
The relative absorbance values were reduced to 96.7% after 3 minutes of treatment. This value is above the threshold for corrosivity (50%). After 1 hour of treatment, the relative absorbance values were increased to 105.8%, a value that is above the threshold for corrosivity (15%). Therefore, the test item is considered to be non-corrosive to skin.

Validity
The criterion for optical density of the negative control (≥ 0.8 and ≤ 2.8) was fulfilled: optical density was measured and found to be 1.9 (3 minutes) and 1.7 (1 hour).
The positive control showed clear corrosive effects. The criterion for the viability of the 1 hour experiment, expressed as % of the negative control (< 15%), was also fulfilled. The measured viability was observed to be 10.1%.
Values for negative control and positive control were within the range of historical data of the testing facility.
All measures obtained, and validated procedures conducted lead to the conclusion that the results obtained in relation to the corrosivity of Hatcol 3178 are to be considered as valid.

Any other information on results incl. tables

Absorbance Values

Negative Control		Test Item		Positive Control		Incubation
Tissue 1	Tissue 2	Tissue 1	Tissue 2	Tissue 1	Tissue 2
1.987	1,900	1.896	1.847	0.390	0.359	3 min
1.907	1.887	1.842	1.836	0.390	0.385
1.900	1.868	1.852	1.801	0.389	0.390
1.726	1.687	1.791	1.802	0.175	0.168	1 h
1.703	1.698	1.791	.1802	0.175	0.168
1.707	1.662	1.779	1.808	0.174	0.167
Mean		Mean		Mean
1.908		1.846		0.384		3 min
1.697		1.795		0.171		1 h

 

% Tissue Viability

Test Item	Positive Control	Incubation
96.7%	20.1%	3 min
105.8%	10.1%	1 h

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Hatcol 3178 is considered to be non-corrosive to skin when tested in accordance with OECD guideline 431.
After 3 minutes of treatment, the relative absorbance values were decreased to 96.7%.
This value is well above the threshold for corrosivity (50%). After 1 hour treatment relative absorbance values were increased to 105.8%. This value is well above the threshold for corrosivity (15%). In OECD guideline 431, values greater or equal to the threshold are to be considered as “non-corrosive to skin”.

Executive summary:

Title of Study: Determination of Skin Corrosion Potential of Hatcol 3178 in the Reconstructed Human Epidermis (RHE) Test Method following OECD Guideline 431 and EU Method B.40-BIS

 

Findings and Results:

One valid experiment was performed.

Two tissues of the human skin model EpiDermTM were treated with Hatcol 3178 for 3 minutes and 1 hour, respectively. The test item was applied to each tissue and spread to match the tissue size.

Demineralised water was used as negative control, 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. The OD was 1.9 (3 minutes experiment) and 1.7 (1 hour experiment). The positive control showed clear corrosive effects for both treatment intervals. The relative absorbance value was reduced to 10.1 % for the 1 hour treatment.

After 3 minutes treatment with the test item, the relative absorbance values were reduced to 96.7 %. This value is above the threshold for corrosion potential (50%). After 1 hour treatment, relative absorbance values were increased to 105.8%. This value, too, is above the threshold for corrosion potential (15%). In the guideline, values greater or equal to the threshold are considered as “non-corrosive to skin”.

 

Therefore, Hatcol 3178 is considered as non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test Method.