Condensation products of fatty acids, tall oil with 2-amino-2-ethylpropanediol

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2017 - March 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: Human Skin Model Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Condensation products of fatty acids, tall oil with 2-amino-2-ethylpropanediol

EC Number:

946-010-7

Molecular formula:

None

IUPAC Name:

Condensation products of fatty acids, tall oil with 2-amino-2-ethylpropanediol

Test material form:

liquid: viscous

Specific details on test material used for the study:

Test Material Name: Alkaterge E
CAS Number: 68140-98-7
Batch nr.: D598F47BC1
Appearance (physical state, color): Liquid, brown, viscous
Molecular Weight: 365.60 g/mol

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Details on test system:

The EpiDerm™ Skin Model
Upon receipt of the EpiDerm™ Skin Bioassay Kit, the solutions were stored as indicated by the manufacturer. The EpiDerm™ tissues were stored at 2-8ºC until used. On the day of dosing, an appropriate volume of EpiDerm™ assay medium was removed and warmed to approximately 37ºC. Nine-tenths (0.9) mL of assay medium were aliquotted into the wells of each 6-well plate. The six-well plates were labeled to indicate test article and exposure time. The EpiDerm™ tissues were inspected for air bubbles between the agarose gel and cell culture insert prior to opening the sealed package. Tissues with air bubbles covering greater than 50% of the cell culture insert area were not used. The 24-well shipping containers were removed from the plastic bag and their surfaces were disinfected with 70% ethanol. The EpiDerm™ tissues were transferred aseptically into the 6-well plates. The EpiDerm™ tissues were then incubated in the dark at 37±1ºC in a humidified atmosphere of 5±1% CO2 in air (standard culture conditions) for at least one hour. The medium was then aspirated and 0.9 mL of fresh medium were added to each assay well below the EpiDerm™ tissues. The plates were returned to the incubator until treatment was initiated. Upon opening the bag, any remaining unused tissues were briefly gassed with an atmosphere of 5% CO2/95% air and placed back at 2-8ºC for later use.

Assessment of Direct Test Article Reduction of MTT
Each test article was added to a 1.0 mg/mL MTT (Sigma) solution in warm Dulbecco’s Modified Eagle’s Medium (DMEM) containing 2 mM L-glutamine (MTT Addition Medium) to assess its ability to directly reduce MTT. Approximately 50 μL of the test articles, DMMOPA (aka RS-12803.00) and Alkaterge E, were each added to 1 mL of the MTT solution, and the mixture was incubated at standard culture conditions for at least one hour. Approximately 25 mg of the test article, AMPD, were added to 1 mL of the MTT solution, and the mixture was incubated at standard culture conditions for at least one hour. A negative control, 50 μL of sterile, deionized water (Quality Biological), was tested concurrently. If the MTT solution color turned blue/purple, the test articles were presumed to have reduced the MTT.

In cases where the test articles were shown to reduce MTT, only those test articles that remained bound to the tissue after rinsing, resulting in a false MTT reduction signal, could present a problem. To evaluate whether residual test article was binding to the tissue and leading to a false MTT reduction signal, a functional check (using freeze-killed control tissue) was performed as described in the section labeled “Killed Controls (KC)”. The test article was observed to directly reduce MTT in the absence of viable cells. The positive control, 8N potassium hydroxide (8N KOH), is known to directly reduce MTT in the absence of viable cells. Therefore, a killed control experiment was performed concurrently in the definitive assay to determine the extent of the direct MTT reduction (if any) by the test articles and the positive control in non-viable freeze-killed tissues.

Assessment of Colored or Staining Materials
Approximately 50 μL o Alkaterge E was added to 2.0 mL isopropanol in 6-well plates and placed on an orbital place shaker for 2-3 hours at room temperature. After shaking, 200 μL aliquots of the isopropanol solutions and two blank samples of isopropanol were transferred to a 96-well plate and the absorbance was measured with a plate reader at the MTT measurement wavelength (550 nm).The absorbance of the test article sample was determined by subtracting the mean isopropanol blank value from the absorbance of the test article samples. If the OD550 of the test article samples were > 0.08, the materials had to be considered as possibly interacting with the MTT measurement. The test article was not considered to have probable photometric MTT interference.

pH Determination
The pH of Alkaterge E was measured using pH paper (EMD Millipore Corporation). Initially, the test article was added to pH paper with a 0-14 pH range in 1.0 pH unit increments to approximate a narrow pH range. Next, the test article was added to pH paper with a narrower range of 0-6.0 pH units with 0.5 pH unit increments, to obtain a more accurate pH value.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test article was tested neat. Fifty (50) μL of Alkaterge E was applied directly on the tissue so as to cover the upper surface (epithelial side). The tissues designated to the negative control were treated with 50 μL of sterile, deionized water. The tissues designated to the positive control, 8N KOH (Sigma), were tested using the same method.

Duration of treatment / exposure:

The test and control articles were tested by treating four EpiDerm™ tissues per material. Two tissues were used to assess viability after the 3-minute exposure, and two were used to assess viability after the 60-minute exposure. Fifty (50) microliters of the test article was applied topically on the EpiDerm™ tissue. The negative and positive controls were treated as described above. The three-minute exposure time began as soon as the material was spread onto the tissue. This short exposure time precluded treating more than a small number of tissues at once. The cultures exposed for 3 minutes were held at room temperature during dosing, while the cultures exposed for the 60 minutes were incubated at standard culture conditions until the completion of the exposure time.

1.0 mg/mL solution of MTT in warm MTT Addition Medium was prepared no more than 2 hours before use. Three hundred (300) μL of MTT reagent solution were added to designated wells in a pre-labeled 24-well plate. The plate was held in the incubator until tissues were added. After the appropriate exposure time, the EpiDerm™ tissues were extensively rinsed with warm (approximately 37ºC) Calcium and Magnesium-Free Dulbecco's Phosphate Buffered Saline (Ca++Mg++-Free DPBS) and the wash medium was decanted. The EpiDerm™ tissues were transferred to the appropriate wells after rinsing. The plates were incubated at standard culture conditions for 3 ± 0.1 hours.

Duration of post-treatment incubation (if applicable):

After the incubation period with MTT solution, the EpiDerm™ tissues were blotted on absorbent paper, cleared of excess liquid, and transferred to a pre-labeled 24-well plate containing 2.0 mL of isopropanol in each designated well. The plates were covered with paraffin film and stored in the refrigerator (2-8ºC) until the last exposure time was harvested. Then the plates were shaken for 2 - 3 hours at room temperature. At the end of the extraction period, the liquid within the cell culture inserts was decanted into the well from which the cell culture insert was taken. The extract solution was mixed and 200 μL were transferred to the appropriate wells of a 96-well plate. Two hundred (200) μL of isopropanol were placed in the two wells designated as the blanks. The absorbance at 550 nm (OD550) of each well was measured with a Molecular Devices Vmax plate reader.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

97.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

97.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Alkaterge E was predicted to be non-corrosive to the skin in the in vitro Skin Corrosion Assay Using Epiderm™ Skin Model (Epi-200), and thus would not be classified or labeled as corrosive according to the UN GHS classification system.

Executive summary:

The in vitro Skin Corrosion Assay Using Epiderm™ Skin Model (Epi-200): 3- And 60-Minute Exposure Protocol was used to assess the potential skin corrosivity of Alkaterge E. The skin corrosion potential was evaluated using the protocol that is consistent with the OECD guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test. Test materials which reduce tissue viability to <50% within 3 minutes are considered corrosive by this method. In addition, test materials which result in tissue viability of ≥50% after a 3-minute exposure, but result in tissue viability of <15% after a 60-minute exposure are also classified corrosive. Test materials which result in tissue viabilities of ≥50% after a 3-minute exposure and ≥15% after a 60-minute exposure are classified non-corrosive. Furthermore, sub-classification of corrosive materials is possible using the 3 minute exposure time as follows: a sub-category classification of 1A is assigned if the viability is <25%, and 1B/1C if the viability is ≥ 25%. According to the current prediction model, Alkaterge E was predicted to be non-corrosive to the skin, and thus would not be classified or labeled as corrosive according to the UN GHS classification system.