Fatty acids, tall-oil, reaction products with triethylenetetramine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 24, 2015 to March 06, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline 439 and EU Method B.46, in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, reaction products with triethylenetetramine

EC Number:

272-905-0

EC Name:

Fatty acids, tall-oil, reaction products with triethylenetetramine

Cas Number:

68919-79-9

Molecular formula:

Not applicable for this UVCB.

IUPAC Name:

Fatty acids, tall-oil, reaction products with triethylenetetramine

Test material form:

semi-solid (amorphous): gel

Remarks:

migrated information: paste

Test animals

Species:

other: EpiDerm-Kit consisting of human-derived epidermal keratinocytes, Batch no.: 21630

Test system

Type of coverage:

open

Vehicle:

other: Dulbecco’s Phosphate Buffered Saline (DPBS buffer without CaCl2 and MgCl2)

Controls:

other: in vitro study: Dulbecco’s Phosphate Buffered Saline (DPBS buffer without CaCl2 and MgCl2) was used as negative control. Sodium dodecyl sulphate (SDS) solution in deionised H2O containing 5% SDS was used as positive control.

Amount / concentration applied:

24.1, 24.2, 24.4 mg test substance (Main test); 26.3, 26.9 mg test substance (Additional test with frozen tissues).
On average, 25 mg of the solid test substance (wetted with 25 μL DPBS-buffer) were applied to each tissue and spread to match the tissue size of 0.63 cm2

Duration of treatment / exposure:

60 minutes

Details on study design:

Test System: 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. Tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.

Pre-Incubation of Tissues:
Eight 6-well-plates were prepared with 0.9 mL assay medium in three of the six wells (upper row). The tissues were inspected for viability. Viable tissues were transferred (three per plate) in the wells with the medium using sterile forceps under the clean bench and placed into the incubator at 37±1°C and 5.0±0.5% CO2 for 1 h.

After the pre-incubation (1 h), the other three wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37±1°C and 5.0±0.5% CO2 for 18 h.

Treatment with test substance:
The pre-incubated tissues were placed into fresh 6-well-plates containing 0.9 mL assay medium per well, using the upper row only. One plate (three tissues) was used as negative control; each tissue was treated with 30 μL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
One plate was used as positive control; each tissue was treated with 30 μL SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface. One plate was used for treatment with the test substance:
The tissues were wetted with 25 μL DPBS buffer before applying the test substance and spreading it to match the tissue size.
Tissues were dosed in 1 min-intervals. After dosing the last tissue, all plates are transferred into the incubator for 35 min. 37±1°C and 5±0.5% CO2. 60 min. after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1 min-intervals.
After rinsing, each tissue was dried with a sterile cotton tip and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). Then, the tissues were set in the incubator for 24 h.

Medium Renewal:
For three incubated tissues each, a new 6-well-plate with 0.9 mL assay medium in the upper row was prepared. The tissues were removed from the incubator and shaken for ten min (500 rpm). Then the inserts were transferred into the new 6-well-plate and set into the incubator for 18 h for post-incubation.

MTT Assay
After a total incubation time of 42 h, a 24-well-plate was prepared with 300 μL freshly prepared MTT-reagent in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 h. After this time, the MTT reagent was aspirated and replaced by DPBS buffer. 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 h at room temperature. After 2 h, the inserts in which formazan had been produced 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, two replicates with 200 μL solution (each) were pipetted into a 96-wellplate which was read in a plate spectral photometer at 570 nm.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: Relative absorbance value (percent)

Value:

97.9

Remarks on result:

other:

Remarks:

Basis: mean (in comparison to the negative control). Time point: 60 min. Remarks: Value was well above the threshold value for irritation potential (50%) . (migrated information)

In vivo

Irritant / corrosive response data:

After the treatment, the relative absorbance values were reduced to 97.9%. This value is well above the threshold value for skin irritation (50%).
The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8 with an OD value of 2.3. The positive control induced a decrease in the relative absorbance as compared to the negative control to 2.8% (required: ≤20%) for thus ensuring the validity of the test system. Variation within replicates was within the accepted range for negative control, positive control and test substance (< 18%). For these reasons, the result of the test is considered valid.

Any other information on results incl. tables

Table 1: Corrected Mean Absorption Values of Test Substance

Designation	Test substance
Mean – blank (tissue 1)	2.256
Mean – blank (tissue 2)	2.121
Mean – blank (tissue 3)	2.230
Mean of the three tissues	2.202
Relative standard deviation of the three tissues	3.3%

Comparison of Formazan Production

For the test substance and the positive control, the following percentage values of formazan production were calculated in comparison to the negative control:

Table 2: Percent Formazan Production

Designation	Test substance	Positive control
Percent formazan production (tissue 1)	100.3%	2.8%
Percent formazan production (tissue 2)	94.3%	2.8%
Percent formazan production (tissue 3)	99.1%	2.9%
Percent formazan production (mean)	97.9%	2.8%

Applicant's summary and conclusion

Conclusions:

The test substance was considered as not irritating to the skin.

Executive summary:

An in vitro study was conducted to assess the skin irritation potential of the test substance according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. Three tissues of a human skin model EpiDermTM were exposed for 60 minutes. On average, 25 mg of the test substance (wetted with 25 μL DPBS-buffer) were applied to each tissue and spread to match the tissue size (0.63 cm²). DPBS buffer was used as negative control and 5% SDS solution as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, with an OD value of 2.3. The positive control showed clear irritating effects with a relative absorbance value reduced to 2.8%. Variation within tissues was acceptable (< 18%). The relative absorbance value representing the test substance was reduced to 97.9%. This value is well above the threshold for irritation potential (50%). Therefore, under the study conditions, the test substance is considered as not irritating to the skin in a Human Epidermis Skin Model Test (Andres I, 2015a).