2-amino-2-methylpropane-1,3-diol

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 - December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-amino-2-methylpropane-1,3-diol

EC Number:

204-100-7

EC Name:

2-amino-2-methylpropane-1,3-diol

Cas Number:

115-69-5

Molecular formula:

C4H11NO2

IUPAC Name:

2-amino-2-methylpropane-1,3-diol

Details on test material:

- Name of test material (as cited in study report): Aminomethyl propanediol, AMPD

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%

In vitro test system

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.

Results and discussion

In vitro

Results

Results 1

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

Results 2

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

Applicant's summary and conclusion

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.

.