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

Endpoint summary

Description of key information

DMMOPA was corrosive to eyes in a BCOP assay. It was irritant and non-corrosive in vitro on human skin.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

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

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.

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.

Reference 2

Endpoint:

skin corrosion: 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

GLP compliance:

yes (incl. QA statement)

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 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.

Preparation and Delivery of Test Article
The test articles were handled in a fully vented hood. The test articles were tested neat. Fifty (50) μL of the test articles, DMMOPA (aka RS-12803.00) were each 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. All
exposure conditions were documented in the study workbook.

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), were each 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 articles were 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 of each test article, DMMOPA (aka RS-12803.00) 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 (550 nm). The absorbance of the test article samples were 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 articles were not considered to have probable photometric MTT interference.

pH Determination
The pH of the test articles, DMMOPA (aka RS-12803.00) were measured using pH paper (EMD Millipore Corporation). Initially, the test articles were 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 articles were 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:

Fifty (50) microliters of each (liquid) test article were applied topically on the EpiDerm™ tissue.
The test and control articles were tested by treating four EpiDerm™ tissues per material, neat.

Duration of treatment / exposure:

Two tissues were used to assess viability after the 3-minute exposure, and two were used to assess viability after the 60-minute exposure. 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.

Duration of post-treatment incubation (if applicable):

The EpiDerm™ tissues were transferred to the appropriate wells after rinsing. The plates were incubated at standard culture conditions for 3 ± 0.1 hours.

Number of replicates:

Four per test item

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

60.9

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

16.6

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

DMMOPA classification is driven by the in vitro skin irritation study.

Executive summary:

In the in vitro Skin Corrosion Assay (Epiderm™, OECC 431), DMMOPA induced tissue viability of 60.9% and 16.6% over 3- and 60-minute exposure, resp.

Based on this result, DMMOPA was not corrosive to the skin.

DMMOPA classification is driven by the in vitro skin irritation study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes were obtained from a local abattoir as a by-product from freshly slaughtered animals (J.W. TREUTH & SONS, Inc., Baltimore, MD). The eyes were excised and then placed in Hanks' Balanced Salt Solution, containing Penicillin/Streptomycin (HBSS), and transported to the laboratory on ice packs. Immediately upon receipt of the eyes into the laboratory, preparation of the corneas was initiated.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Neat, 750 μL

Details on study design:

Preparation of Corneas
The eyes were grossly examined for damage and those exhibiting defects were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised such that a 2 to 3 mm rim of sclera was present around the cornea. The isolated corneas were then stored in a petri dish containing HBSS until they were mounted in a corneal holder. The corneas were mounted in the holders with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and the screws were tightened. Starting with the posterior chamber, the two chambers were then filled with Minimum
Essential Medium (EMEM) without phenol red, containing 1% fetal bovine serum and 2 mM L-glutamine (Complete MEM (without phenol red)). Each corneal holder was uniquely identified with a number written in permanent marker, on both the anterior and posterior chambers. The corneal holders were incubated at 32 ± 1ºC for a minimum of 1 hour.

Controls:
The positive controls used in this study were ethanol (CAS# 64-17-5, Purity = 100%) for the liquid test substances (Pharmco; Brookfield, CT). The negative control used in this study was sterile, deionized water (Quality Biological; Gaithersburg, MD).

Test Substance Preparation
As instructed by the Sponsor, the test substances, DMMOPA (aka RS-12803.00) were administered to the test system without dilution.

Test Substance pH Determination
The pH of each test substance was determined using pH paper (EMD Millipore Corporation; Billerica, MA). Initially, each test substance was added to 0-14 pH paper with 1.0 pH unit increments to approximate a narrow pH range. Next, the test substances were added to 0-6 and 5-10, or 7.5-14.0 pH paper with 0.5 pH unit increments, to obtain more accurate pH
values.

Bovine Corneal Opacity and Permeability Assay
After a minimum of 1 hour of incubation, the corneas were removed from the incubator. The medium was removed from both chambers and replaced with fresh Complete MEM (without phenol red). The initial opacity was determined for each cornea using an Electro Designs OPKIT opacitometer. Any cornea with an initial opacity greater than 7 was not used in the assay.
The treatment of each cornea was identified with the test substance number written in permanent marker on colored tape, affixed to each holder. The medium was then removed from the anterior chamber and replaced with the test substance, positive control, or negative control.

Method for Testing Liquid or Surfactant Materials
The liquid test substances, DMMOPA (aka RS-12803.00) were tested
neat. An aliquot of 750 μL of the test substance, positive control, or negative control was introduced into the anterior chamber while slightly rotating the holder to ensure uniform distribution over the cornea. Each treated cornea was completely covered with the test substance.
Three corneas were incubated in the presence of the negative or positive control at 32 ± 1ºC for 10 minutes. Four corneas were incubated in the presence of each test substance at 32 ± 1ºC for 10 minutes. After the 10-minute exposure times, the control or test substance treatments were removed. The epithelial side of the corneas was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of the control or test substances. The corneas were then given a final rinse with Complete MEM (without phenol red). The anterior chambers were refilled with fresh Complete MEM (without phenol red) and an opacity
measurement was performed. The corneas were returned to the incubator for approximately 2 hours after which a final measure of opacity was obtained.
After the final opacity measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was filled with fresh Complete MEM (without phenol red) and 1 mL of a 4 mg/mL fluorescein solution was added to the anterior chamber. The
corneas were then incubated in a horizontal position (anterior side up) for approximately 90 minutes at 32 ± 1ºC. At the end of the 90-minute incubation period, the medium was removed from the posterior chamber and placed into tubes numbered corresponding to chamber number. Aliquots of 360 μL from the numbered tubes were placed into their designated wells on a 96-well plate. The optical density at 490 nm (OD490) was determined using a Molecular Devices Vmax kinetic microplate reader. If the OD490 value of a control or test substance sample was 1.500 or above, a 1:5 dilution of the sample was prepared in Complete MEM (without
phenol red) (to bring the OD490 value within the linear range of the platereader). A 360 μL sample of each 1:5 dilution was transferred to its specified well on the 96-well plate. The plate was read again and the final reading was saved to a designated print file.

Irritation parameter:

in vitro irritation score

Run / experiment:

10 min exposure

Value:

130.2

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Executive summary:

In the Bovine Corneal Opacity and Permeability Assay, DMMOPA (aka RS-12803.00), induced an irritation score of 130.2 over 10-minute exposure. Thus, it was predicted to cause serious eye damage (GHS category I).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on results of standard in vitro assays, DMMOPA is classified as Skin irritation 2 and Eye Damage 1.