Glycerides, tall-oil mono-

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19. Feb. 2018 to 22. Feb. 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

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:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

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:

Glycerides, tall-oil mono-

EC Number:

263-034-7

EC Name:

Glycerides, tall-oil mono-

Cas Number:

61789-12-6

Molecular formula:

C19H38O4 to C21H42O4 * (mono-glycerides) # C37H66O5 to C39H76O4 * (di-glycerides) # C55H96O6 to C57H108O4 * (tri-glycerides) # * in H2 increments (i,e, decreasing # of double bonds)

IUPAC Name:

Glycerides, tall-oil mono-

Test material form:

liquid

Details on test material:

Name: LUMULSE GMT-K
Batch no.: 385932
Appearance: opaque amber liquid
Purity: 100%
Homogeneity: homogeneous
Expiry date: 26. Oct. 2019
Storage: Room Temperature: (20 ± 5°C)

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: Not specified

Source strain:

not specified

Details on animal used as source of test system:

Specification
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-200-SCT
Day of delivery: 20. Feb. 2018
Batch: 25882

Justification for test system used:

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.

Vehicle:

unchanged (no vehicle)

Details on test system:

Pre-Tests
Nylon mesh compatibility
First, the test item 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 were brought onto a nylon mesh on a microscope slide. No reaction with the mesh was visible after 1 hour incubation at room temperature.

Assessment of Coloured or Staining Test Items
It was tested whether the test item develops a colour without MTT addition. 50 μL of the test item were given in a test tube with 0.3 mL demineralised water and incubated at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour.
The resulting solution was colourless, therefore no binding capacity had to be tested.

Assessment of Direct Reduction of MTT by the Test Item
The test item was also tested for the ability of direct MTT reduction. To test for this ability, 50 μL of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour. Untreated MTT solution was used as control.
The MTT solution did not change its colour within 1 hour. Therefore, direct MTT reduction had not taken place and no data correction was necessary.

Preparations
On the day of the start of the experiment, the MTT concentrate was thawed. The concentrate was diluted with the assay medium directly before use.
The tissue plate was brought out of the fridge 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 solution. 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 testing the test item.
The liquid test item was applied without preparation (50 μL).
At the start of each experiment (application of negative controls), a stop watch was started.
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 solution for 3 hours at 37 ± 1°C and 5.0 ± 0.5% CO2.
After this time, the MTT solution was aspirated and replaced by DPBS. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken for 2 hours at room temperature.
Afterwards, the inserts were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, three replicates with 200 μL solution (each) were pipetted into a 96-wellplate which was read in a plate spectrophotometer at 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):

The tissues were incubated with MTT solution for 3 hours

Number of replicates:

For each experiment, two 6-well-plates for the assay were used.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

99.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

87.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Assessment and Validity
Corrosivity of the Test Item
The mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 99.6% and 87.8% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item LUMULSE GMT-K is considered to be not corrosive.

Validity
The criterion for optical density of the negative control (≥ 0.8 and ≤ 2.8) was fulfilled: optical density was 1.8 (3 minutes) resp. 1.9 (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 fulfilled, too. The mean value of relative tissue viability was 6.6%
Values for negative control and for positive control were within the range of historical data of the test facility.
Therefore the experiment is considered valid.

Any other information on results incl. tables

Measured Values

As blank, the optical density of isopropanol was measured in 12 wells of the 96-well-plate.

The measured values and their mean are given in the following table:

Absorbance values blank isopropanol (OD 570 nm)

Replicate	1	2	3	4	5	6	Mean 0.038
Absorbance	0.040	0.038	0.040	0.040	0.037	0.038
Replicate	7	8	9	10	11	12
Absorbance	0.039	0.038	0.037	0.038	0.038	0.037

 

The absorbance values of negative control, test item and positive control are given in the following table:

Absorbance Values (OD 570 nm)

Incubation	Negative Control		Test item		Positive Control
	Tissue 1	Tissue 2	Tissue 1	Tissue 2	Tissue 1	Tissue 2
3 min	1.800	1.787	1.758	1.802	0.486	0.475
	1.780	1.800	1.756	1.814	0.492	0.473
	1.785	1.769	1.760	1.790	0.487	0.473
1 h	1.884	1.961	1.635	1.698	0.161	0.156
	1.860	1.916	1.631	1.743	0.160	0.165
	1.864	1.927	1.641	1.700	0.161	0.166

 

From the measured absorbances, the mean absorbance of isopropanol was subtracted. The corrected mean and relative standard deviation (RSD) of the two tissues were also calculated.

Mean Absorbance Values of the 3 Minutes Experiment

Designation	Negative Control	Test Item	Positive Control
Mean – blank (tissue 1)	1.750	1.720	0.450
Mean – blank (tissue 2)	1.747	1.764	0.435
Mean of the two tissues	1.749	1.742	0.443
RSD	0.1%	1.8%	2.3%

 

Mean Absorbance Values of the 1 h Experiment

Designation	Negative Control	Test Item	Positive Control
Mean – blank (tissue 1)	1.831	1.597	0.122
Mean – blank (tissue 2)	1.896	1.675	0.124
Mean of the two tissues	1.864	1.636	0.123
RSD	2.5%	3.4%	1.0%

 

Comparison of Tissue Viability

For the test item and the positive control, the following percentage values of mean tissue viability were calculated in comparison to the mean of the negative controls:

% Tissue Viability

Test Item	Positive Control	Incubation
99.6%	25.3%	3 min
87.8%	6.6%	1 h

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

LUMULSE GMT-K is considered non-corrosive to skin.
The mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 99.6% and 87.8% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item LUMULSE GMT-K is considered to be not corrosive.
The values of the negative control met the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals, thus showing the quality of the tissues.
The positive control has met the validity criterion too, thus ensuring the validity of the test system.
For these reasons, the result of the test is considered valid.

Executive summary:

Title of Study: Determination of Skin Corrosion Potential of LUMULSE GMT-K 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 the test item LUMULSE GMT-K 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 requiredacceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. The OD was 1.8 (3 minutes experiment) and 1.9 (1 hour experiment).

The positive control showed clear corrosive effects for both treatment intervals. The mean relative tissue viability value was reduced to 6.6% for the 1 hour treatment.

The mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 99.6% and 87.8% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item LUMULSE GMT-K is considered to be not corrosive.

 

Therefore, LUMULSE GMT-K is considered non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test Method.