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Administrative data

Description of key information

The substance is considered to be corrosive to the eyes, but not a skin irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June - December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
AMPD ULTRA PC (ANGUS Chemical Company)
- CAS name: 2-amino-2-methyl-1,3-propanediol
- CAS number: 115-69-5
- appearance: solid, white crystals
- molecular weight: 105.1 g/mol
- batch #VK014801I1
- purity: 99.5%
- sum of impurities: 0.48%
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
other: human
Justification for test system used:
The purpose of this study was to assess the potential skin corrosivity of the test substance in the EpiDerm Kit (MatTek Corporation). The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after test article exposure .

The method utilizes a 3-minute exposure for a corrosive classification and a 60-minute confirmatory exposure for materials found to be non-corrosive at the 3-minute exposure. Viable cells reduce the yellow, soluble, oxidized form of the MTT to the blue-black, insoluble, reduced form. The reduced dye is extracted from the tissue with isopropanol, and the amount of reduced dye is determined spectrophotometrically. The relative viability of the treated tissues is calculated as a percentage of the negative control viability from the absorbance data by dividing the corrected test article-treated tissue absorbance by the corrected control tissue absorbance, and multiplying by 100. 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.
Vehicle:
unchanged (no vehicle)
Details on test system:
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.
Control samples:
yes, concurrent no treatment
yes, concurrent positive control
Amount/concentration applied:
For the test article, AMPD, approximately 25 mg per tissue were administered using a 25 mg sharp spoon (Aesculap, Cat. No. FK 623R). The sharp spoon was filled with the test article, and leveled by gently stroking away excess test article using a rod-shaped instrument. Care was taken to avoid packing the material into the spoon. The content of the spoon was poured over the tissue surface. Each EpiDerm™ tissue treated with the solid test article received 25 µL of sterile, deionized water applied directly onto the test article. The test article was gently mixed, and spread over the tissue surface using a sterile bulb-headed rod if needed.
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. Twenty-five mg of the solid (powdered) test article was similarly applied. Each EpiDerm™ tissue treated with a solid test article also received 25 µL of sterile, deionized water applied directly onto the test article. 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):
A 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.

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.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value after 3 minutes
Value:
83.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value after 60 minutes
Value:
50.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
GHS criteria not met
Conclusions:
The test substance is considered to be non-corrosive in this system.
Executive summary:

AMPD was tested in the EpiDerm™ Corrosivity Assay according to the OECD Test Guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test”. Two tissues per test article were used to assess viability after a 3-minute exposure, and two tissues per test article were used to assess viability after a 60-minute exposure. Negative and positive controls were tested in parallel. AMPD appeared to be non-corrosive in this assay. The mean % viability was 83.2% and 50.7% after 3 minutes and 60 minutes, respectively. The classification of the positive control, 8N KOH, was determined to be corrosive, thereby meeting the acceptance criterion.

.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June - December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
AMPD ULTRA PC (ANGUS Chemical Company)
- CAS name: 2-amino-2-methyl-1,3-propanediol
- CAS number: 115-69-5
- appearance: solid, white crystals
- molecular weight: 105.1 g/mol
- batch #VK014801I1
- purity: 99.5%
- sum of Impurities: 0.48%
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
other: human
Vehicle:
unchanged (no vehicle)
Details on test system:
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.

Twenty-five mg of the solid test article (AMPD) was added to 1 mL of the MTT solution and the mixture was 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. 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)”. As AMPD reduced 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 article alone.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
The test article AMPD was tested in one valid definitive trial. The definitive assay included a negative control and a positive control. The negative control was 30 µL of sterile, CMF-DPBS and the positive control was 30 µL of 5% Sodium Dodecyl Sulfate (SDS). Both the positive and negative controls were tested in triplicate, and at the same exposure time as the test articles (60±1 minutes).

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 the test article for 60±1 minutes. Since 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.

Duration of treatment / exposure:
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 plates were transferred to the incubator for 35±1 minutes at standard culture conditions. After 35±1 minutes, all 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 post-treatment incubation (if applicable):
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 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 150 mL 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.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
74.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
GHS criteria not met
Executive summary:

The purpose of this study was to evaluate the skin irritation potential of AMPD, in the context of identification and classification of skin irritation hazard according to the UN GHS and EU classification system (Category 2/Category 1 and No Category).The skin irritation potential was evaluated based upon measuring the relative conversion of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) in the test article-treated tissues after exposure to the test article for a 60-minute exposure period, followed by a 42-hour post-exposure expression period. The skin irritation potential of the test article was predicted if the relative viability was less than or equal to 50%. The study was conducted according to the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG439). The mean OD570of the negative control, CMF-DPBS, was 1.899. The mean viability of the positive control, 5% SDS, was 2.49%. The standard deviation calculated from individual percent tissue viabilities of the 3 identically treated replicates was < 18% for the positive control and negative control. Since the acceptance criteria were met, the assay was considered valid. AMPD was determined to directly reduce MTT. Therefore, a killed-control experiment was performed. Since the final corrected OD570 values pertaining to the killed control tissues associated with thes test article were negative, they were equaled to zero for calculation purposes; therefore, no additional calculations were performed to check for the amount of MTT reduced directly by test article residues. AMPD was not determined to be a colorant and was not considered to have potential interference with the MTT measurement. The mean % vialbility measured with AMPD in this assay was 74.8%. Based upon the results of this assay, AMPD was predicted to be non-irritating to the skin, and thus would not require classification and labelling for skin irritation according to the UN GHS classification system (No Category).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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
Study period:
July-November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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. certificate)
Specific details on test material used for the study:
AMPD ULTRA PC (ANGUS Chemical Company)
- CAS name: 2-amino-2-methyl-1,3-propanediol
- CAS number: 115-69-5
- appearance: solid, white crystals
- molecular weight: 105.1 g/mol
- batch #VK014801I1
- purity: 99.5%
- sum of impurities: 0.48%
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.

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.
Vehicle:
water
Controls:
yes, concurrent positive control
Amount / concentration applied:
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 OP-KIT 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 article 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 article, positive control, or negative control. The solid test article, AMPD, was tested as a 20% (w/v) dilution in sterile, deionized water. An aliquot of 750 µL of the test article, positive control, or negative control was introduced into the anterior chamber while slightly rotating the holder to ensure uniform distribution over the cornea.
Duration of treatment / exposure:
Three corneas were incubated in the presence of the negative or positive control at 32 ± 1ºC for approximately 4 hours. Five corneas were incubated in the presence of the test article at 32 ± 1ºC for approximately 4 hours. After the 4-hour exposure period, the control or test article treatments were removed. The epithelial side of each cornea was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of the control or test articles. The corneas were then given a final rinse with Complete MEM (without phenol red). The anterior and the posterior chambers were refilled with fresh Complete MEM (without phenol red) and a final opacity measurement was performed immediately (with no further post-exposure incubation).
Duration of post- treatment incubation (in vitro):
After the final opacity measurement was performed, the medium was removed from the anterior chamber of the holder and replaced with 1 mL of a 5 mg/mL fluorescein solution. 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 sample was 1.500 or above, a 1:5 dilution of the sample was prepared in Complete MEM (without phenol red). 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.

After the medium was removed for the permeability determination, each cornea was carefully separated from its corneal holder and transferred to an individual prelabeled tissue cassette containing a biopsy sponge. The endothelial surface of each cornea was placed on the sponge to protect it. The cassettes were placed in 10% neutral buffered formalin to fix the corneal tissue for at least 24 hours. The fixed corneas will be stored up to one year.
Irritation parameter:
cornea opacity score
Value:
63.9
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The BCOP assay was accepted as the positive control (imidazole) caused an in vitro score that fell within two standard deviations of the historical mean.
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The test substance is considered to be corrosive in the in vitro assay with isolated bovine eyes.
Executive summary:

The purpose of this study was to evaluate the potential ocular irritancy of AMPD as measured by changes in opacity and permeability (to fluorescein) in isolated bovine corneas. Four to five corneas were treated with the test article. Based on changes in corneal opacity and permeability (relative to the control corneas), an in vitro score was determined for the test article. After a 4 hour exposure, the in vitro score for AMPD was 63.9. Therefore, the test substance is considered to be corrosive in the in vitro assay with isolated bovine eyes.


Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The substance is consider to be corrosive to the eye based on the results of an in vitro assay with isolated bovine eyes. Therefore, classification for eye damage is required according to EU/CLP. AMPD is not considered to be a skin irritant, based on the results of in vitro corrosion and irritation assays and on read-across from in vivo studies with AEPD and APD.