(6-amino-2-pyridyl)-(1-methyl-4-piperidyl)methanone;dihydrate;dihydrochloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6th October to 18th December 2017

Reliability:

1 (reliable without restriction)

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: Human Skin Model Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

6-(1-methylpiperidine-4-carbonyl)pyridin-2-amine dihydrate dihydrochloride

EC Number:

842-055-0

Cas Number:

2356133-42-9

Molecular formula:

C12H17N3O.2HCl.2H2O

IUPAC Name:

6-(1-methylpiperidine-4-carbonyl)pyridin-2-amine dihydrate dihydrochloride

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Source strain:

not specified

Details on animal used as source of test system:

The EpiDerTM Kit (MatTek Corporation) was used in this study. 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 protocol is consistent with the OECD Test Guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test”

Justification for test system used:

The protocol is consistent with the OECD Test Guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test”

Vehicle:

unchanged (no vehicle)

Details on test system:

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 protocol is consistent with the OECD Test Guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test”.
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 3minute 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%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

25mg of test article (100% concentration)

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

The tissues exposed for 3 minutes were held at room temperature.
The tissues exposed for 60 minutes were incubated at standard culture conditions until the completion of the exposure time.

Number of replicates:

2 for 3minutes and 2 for 60 minutes

Test system

Details on study design:

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 each liquid control were applied topically on the EpiDerm™ tissue. Twenty-five mg of solid (powdered) test article were 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 test article was gently mixed, and spread over the tissue surface using a sterile bulb-headed rod if needed. The three-minuteexposure 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 tissues exposed for 3 minutes were held at room temperature during dosing, while the tissues exposed for the 60 minutes were incubated at standard culture conditions until the completion of the exposure time.

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:

LSN584368 3 minute assay

Value:

ca. 95.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

LSN584368 60 minutes

Value:

ca. 91.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Non corrosive