8-((3R,4S)-4-ethylpyrrolidin-3-yl)-3h-imidazo[1,2-a]pyrrolo[2,3-e]pyrazine dihydrocloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 21, 2018 - February 6, 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

no

Remarks:

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

Test material

Test material information

Constituent 1

Reference substance name:

12-[(3R,4S)-4-ethylpyrrolidin-3-yl]-1,5,7,10-tetraazatricyclo[7.3.0.0²,⁶]dodeca-2(6),3,7,9,11-pentaene dihydrochloride

EC Number:

845-622-0

Cas Number:

2050038-84-9

Molecular formula:

C14H19Cl2N5

IUPAC Name:

12-[(3R,4S)-4-ethylpyrrolidin-3-yl]-1,5,7,10-tetraazatricyclo[7.3.0.0²,⁶]dodeca-2(6),3,7,9,11-pentaene 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

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 EpiDermTM Model (EPI-200) used in the study consists of normal, human-derived epidermal keratinocytes that are cultured to form a multilayered, highly differentiated model of human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in Vivo.

The EpiDerm™ Skin Model was used to assess the potential skin corrosivity of the test article. 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 exposure to a test article.

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. Additionally, two killed control tissues per exposure time were treated with the negative control and positive control to correct for direct reduction of MTT by KOH (positive control).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg/tissue

Duration of treatment / 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. 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. Furthermore, sub-classification of corrosive materials is possible using the 3 minute exposure time as follows: a sub-categoryclassification of 1A is assigned if the viability is <25%, and 1B/1C if the viability is ≥ 25%.

Number of replicates:

3 minute and 60 minute exposure time

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 Minutes

Value:

70

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 Minutes

Value:

58.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The assay was accepted if: the positive control resulted in a corrosive classification (i.e., <50% cell viability compared to negative controls, after a 3-minute exposure and/or <15% cell viability compared to negative controls after a 60-minute exposure); if the mean OD550 value of the negative control tissues was ≥ 0.8 and < 2.8; and if the standard deviation values calculated from the individual % tissue viabilities of the 2 identically treated replicates of the negative or positive control per each exposure time were < 30%.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test article, A-1335930.3, turned the MTT solution yellow but was not observed to reduce MTT directly in the absence of viable cells. Therefore, a killed control experiment was not conducted.

The test article, A-1335930.3, was not considered to have probable photometric MTT interference.


Test materials which resulted in tissue viability ≥ 50% after 3-minute exposure and ≥ 15% after 60-minute exposure would be classified as non-corrosive. Since this test material has a % viability of 70 after 3 minutes and 58.4 after 60 minutes, it was determined to be non-corrosive.