3-methoxy-N,N-dimethylpropan-1-amine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(3-methoxypropyl)dimethylamine

EC Number:

827-772-9

Cas Number:

20650-07-1

Molecular formula:

C6H15NO

IUPAC Name:

(3-methoxypropyl)dimethylamine

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

Receipt of the EpiDerm™ Skin Model
Upon receipt of the EpiDerm™ Skin Kit (MatTek Corporation), the solutions were stored
as indicated by the manufacturer. The EpiDerm™ tissues were stored at 2-8ºC until use. On the
day prior to testing, EpiDerm™ Maintenance Medium was set to room temperature prior to use.
Nine-tenths mL of Maintenance Medium were aliquotted into the appropriate wells of 6-well
plates. Each 6-well plate was labeled with the test article, positive control, or negative control.
Each EpiDerm™ tissue was inspected for air bubbles between the agarose gel and cell culture
insert prior to opening the sealed package. Tissue inserts 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 at 37±1ºC in a humidified atmosphere of 5±1% CO2 in air (standard culture
conditions) for 60±5 minutes. After 60±5 minutes, the EpiDerm™ tissues were transferred to
appropriate wells containing 0.9 mL of fresh warmed (to 37ºC) Maintenance Medium. The plates
were returned to the incubator for 18±3 hours to acclimate the tissues.

Test Article Preparation
As instructed by the Sponsor, each test article was administered to the test system without
dilution.

Assessment of Test Article/Nylon Mesh Compatibility
Prior to performing the assay, the compatibility of each liquid test article with the nylon mesh was evaluated. Nylon meshes (Elko) were placed on a slide and 30 μL of each test article
were applied. A negative control, 30 μL of sterile, Calcium and Magnesium Free Dulbecco’s
Phosphate Buffered Saline (CMF-DPBS) (Gibco), was tested concurrently. The slides holding
the treated meshes were placed into a covered petri dish and incubated at standard culture
conditions for 60±1 minutes. Using a microscope, each mesh was checked after 60±1 minutes of exposure to assess any interaction between the test articles and the mesh.
The test articles, DMMOPA (aka RS-12803.00) were not observed to interact with the nylon mesh, and therefore a nylon mesh was used to aid in the spreading of the test article after dosing the EpiDerm™ tissues.

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 30 μL of each liquid test article were added to 1 mL of the MTT solution and the mixtures were incubated in the dark at standard
culture conditions for at least one hour. A negative control, 30 μL of sterile, Calcium and
Magnesium Free Dulbecco’s Phosphate Buffered Saline (CMF-DPBS), was tested concurrently.
If the MTT solution color turned blue/purple, the test article was presumed to have reduced the
MTT. Water insoluble test materials may show direct reduction (darkening) only at the interface
between the test article and the medium.
In cases where the test article was 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 articles, DMMOPA (aka RS-18203.00) was observed to reduce MTT directly in the absence of viable cells. A killed control experiment was performed concurrently in the screening assay to determine the extent of the direct MTT reduction (if any) by the test articles alone.

Assessment of Colored or Staining Materials
Prior to conducting any assays with viable tissues, the test article’s ability to interfere
with the photometric MTT measurement was assessed. Each test article was checked for its
colorant properties (i.e., their ability to absorb light significantly at the wavelength used for the
MTT determination. Approximately 30 μL (liquid test articles) or one leveled spoonful
(approximately 25 mg) (solid test articles) were 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 (570 nm). The absorbance of the test article samples was determined by subtracting
the mean isopropanol blank value from the absorbance of the test article samples. If the OD570 of the test article sample was > 0.08, the material has to be considered as possibly interacting with the MTT measurement.
The test articles, DMMOPA (aka RS-12803.00), were not considered to have probable photometric MTT interference.

pH Determination
The pH of each liquid test article was measured using pH paper (EMD Millipore Corporation). Initially, each liquid 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, each liquid test article was added to pH paper with a narrower range of 0-6 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 articles, DMMOPA (aka RS-12803.00), were tested in one valid definitive trial.
After the overnight incubation for 18±3 hours, the 6-well plates containing the EpiDerm™ tissues were removed from the incubator and placed at room temperature for at least 5 minutes prior to dosing.
The EpiDerm™ tissues were treated in triplicate with each test article for 60±1 minutes.
Since the test article, AMPD, was a powder, immediately before application of the solid test
article, each tissue surface was moistened with 25 μL of sterile CMF-DPBS to improve contact
of the tissue surface with the test chemical. After adding the CMF-DPBS, 25 mg of the test
article was added to each of three tissues at 1 minute intervals per tissue using a 25 mg sharp
spoon (Aesculap #FK 623R). The sharp spoon was filled with the test article and then the spoon
was leveled. After the three tissues were dosed with the test article, the test article was gently
mixed and spread over the tissue surface using a sterile bulb-headed rod. Thirty microliters of
each liquid test article were applied to each of three tissues at 1 minute intervals per tissue. A
nylon mesh was placed gently over the dose to spread the test article. If necessary, the mesh was gently pressed down to assure even spreading. The EpiDerm™ tissues were tested in triplicate with the positive or negative control for 60±1 minutes. Thirty microliters of each control were applied to each of three tissues at 1 minute intervals per tissue. Immediately after control
administration onto the tissue, a nylon mesh was placed gently over the dose to spread the
negative and positive controls. The plates with dosed tissues were kept in the laminar flow hood
until the last tissue was dosed. After the last tissue was dosed, all of plates were transferred to the incubator for 35±1 minutes at standard culture conditions. After 35±1 minutes, all of the plates were removed from the incubator, placed into the laminar flow hood and kept at room
temperature until the exposure period was completed for the first dosed tissue.

Duration of treatment / exposure:

After 60±1 minutes of test or control article exposure, the tissues were rinsed with sterile,
CMF-DPBS by filling and emptying the tissue insert 15 times. A stream of CMF-DPBS was
directed onto the tissue surface. For the test and control articles where a mesh was used, the
mesh was carefully removed with forceps (if necessary) after the 5th rinse. After the removal of
the mesh, the rinsing procedure of the tissue continued for 10 times. After the 15th rinse, each of the 3 inserts per treatment group (test article, positive and negative control) was completely
submerged, gently swirled, and rinse media dumped in a beaker containing approximately 150mL of CMF-DPBS and specifically assigned for each treatment group; this procedure was
repeated three times for each insert of each treatment group. Finally, the tissues were rinsed once more on the inside and outside of the tissue insert with sterile CMF-DPBS from the wash bottle, and the excess CMF-DPBS was decanted. The bottoms of the tissue inserts were blotted on sterile paper towels and the inserts were transferred to new 6-well plates containing 0.9 mL of fresh warmed (to 37ºC) Maintenance Medium. The tissue surface was carefully blotted with
sterile cotton-tipped applicators to remove any excess moisture, and the tissue surface was
visually observed for residual test article using a dissecting scope. The tissues were then placed
into the incubator at standard culture conditions for a post-treatment expression incubation of
42±2 hours. After an initial 24±1 hours of incubation, the 6-well plates were removed from the
incubator and the tissues were transferred into new 6-well plates pre-filled with 0.9 mL fresh
Maintenance Medium warmed to approximately 37ºC. The tissues were placed back into the
incubator at standard culture conditions for an additional 18±1 hours for the remainder of the
42±2 hour post-treatment expression incubation.

Duration of post-treatment incubation (if applicable):

42+-2h

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of triplicates

Value:

2.65

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

During the inspection of the tissues using a dissecting scope, it was observed that all viable tissues treated with the test article, DMMOPA (aka RS-12803.00), possibly had a large blister that spanned the entire surface of the tissue.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

Together with the In vitro skin corrosion assay, this study leads to classification as Skin irrit 2.

Executive summary:

The Skin Irritation Test (SIT) Using the EpiDerm™ Skin Model was used to assess the skin irritation potential of DMMOPA, following OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439). Based on a tissue viability of 2.65%, DMMOPA was predicted to be irritating to the skin.

Together with the In vitro skin corrosion assay, this study leads to classification as Skin irrit 2.