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

Endpoint summary

Description of key information

The results from the skin irritation study indicated a positive result for skin sensitization by the test material. The in vitro skin corrosion study indicated a negative result for skin sensitization by the test material. Therefore, based on the results from the two studies, the test material was classified as a category 2 skin irritant according to the GHS criteria.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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 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

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

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%.

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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 6 - November 30, 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

no

Remarks:

The protocol was based upon the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439)

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 meets the requirements of the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439)

Vehicle:

unchanged (no vehicle)

Details on test system:

The EpiDerm™ Skin Model (MatTek Corporation, MA, USA) was used to assess the potential dermal irritation of the test articles. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium 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 the test aticle.

The test article is a solid and was not evaluated in the mesh compatibility test.

The test article, the positive control (5% Sodium Dodecyl Sulfate (SDS)), and the negative control (Calcium & Magnesium Free-Dulbecco’s Phosphate Buffered Saline (CMF-DPBS)), were treated in triplicate EpiDermTM tissues for a 60±1 minute exposure period, followed by a 42-hour post-exposure expression period. 25 µLof sterile CMF-DPBS were added to the tissue surface prior to the addition of the solid test article (which was added using a 25 mg dosing spoon). The test article was mixed on the surface of the tissues using a sterile glass rod.

The MTT assay is performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/mL). After a 3-hour incubation, the blue formazan salt formed by cellular mitochondria is extracted with 2ml isopropanol per tissue and the optical density of the extracted formazan is determined with a spectrometer at 570nm. Relative cell viability is calculated for each tissue as % of the mean of the negative control tissues.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Since the test article is a solid, 25 μL of sterile CMF-DPBS were added to the tissue surface prior to the addition of the solid test article (which was added using a 25 mg dosing spoon). The test article was mixed on the surface of the tissues using a sterile glass rod.

Duration of treatment / exposure:

The test articles, the positive control (5% Sodium Dodecyl Sulfate (SDS)), and the negative control (Calcium & Magnesium Free-Dulbecco’s Phosphate Buffered Saline (CMF-DPBS)), were treated in triplicate EpiDermTM tissues for a 60±1 minute exposure period, followed by a 42-hour post-exposure expression period.

Duration of post-treatment incubation (if applicable):

42-hour post-exposure expression period.

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Value:

12.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The assay was accepted when the following criteria were met:

1) if the positive control (5% SDS) resulted in a mean tissue viability
2) if the mean OD570 value of the negative control tissues was >/= 0.8 and < 2.8, and

3) if the standard deviation calculated from the individual % tissue viabilities of the 3 identically treated replicated of the negative or positive control were < 18%.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

A test article was predicted to be an irritant (GHS Category 1 or 2) when the mean relative viability of the triplicate-treated tissues is ≤50% of the mean viability of the negative control. A test article was not predicted to be a skin irritant (GHS No Category) when the mean relative viability of the triplicate tissues was >50%.
The test article had a value of 12.4% ± 2.17, and therefore is classed as an irritant.

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

Study period:

September 6 - November 30, 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

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:

no

Remarks:

The methods and procedures used in this assay were consistent with OECD Test Guideline 437.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Bovine corneas, obtained as a byproduct from freshly slaughtered animals, were mounted in special holders and exposed to the test articles.
Four corneas were incubated in the presence of the test article at 32 ± 1ºC. Three corneas were incubated in the presence of each control at 32 ± 1ºC.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The solid test article was administered to the test system as 20% (w/v) (200 mg/mL) dilutions in sterile, deionized water.

Duration of treatment / exposure:

The solid test article and the concurrent assay controls (20% (w/v) dilution of imidazole and sterile, deionized water) were exposed to the corneas for 4 hours. After removal of the test or control article from the corneas, a final opacity was determined (i.e., the corneas did not receive a post-exposure incubation).

Number of animals or in vitro replicates:

Four corneas were incubated in the presence of the test article. Three corneas were incubated in the presence of each control.

Details on study design:

The Bovine Corneal Opacity and Permeability Assay (BCOP) was used to assess the potential ocular irritancy of the test article to isolated bovine corneas. Bovine corneas, obtained as a byproduct from freshly slaughtered animals, were mounted in special holders and exposed to the test article. An In Vitro Score was determined for the test article based on the induction of opacity and permeability (to fluorescein) in the isolated bovine corneas.

The test article was administered to the test system as 20% (w/v) (200 mg/mL) dilutions in sterile, deionized water. The test article dilution was prepared by weighing the test article into a conical tube, adding sterile, deionized water, until a 20% (w/v) dilution was achieved, and then vortexing the dilution for approximately 1 minute prior to application. The positive control (a 20% (w/v) dilution of imidazole prepared in Complete Minimal Essential Medium (without phenol red)) and the negative control (sterile, deionized water) were tested concurrently.

Four corneas were incubated in the presence of the test article and three in the presence of the control at 32 ± 1ºC .for four hours. After removal of the test or control article from
the corneas, the corneas were returned to the incubator at 32 ± 1ºC for a post-exposure incubation period of 2 hours, after which a final opacity value was determined.

Irritation parameter:

in vitro irritation score

Run / experiment:

13 September 2018

Value:

112.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The positive control in vitro irritancy score was 97.8.

Interpretation of results:

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

Conclusions:

The in vitro score was calculated as 112.1 and as it is > 55, the test material is classified as Category 1, according to OECD TG 437.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The results from the skin irritation study indicated a positive result for skin sensitization by the test material. The in vitro skin corrosion study indicated a negative result for skin sensitization by the test material. Therefore, based on the results from the two studies, the test material was classified as a category 2 skin irritant according to the GHS criteria. The results from the BCOP eye irritation study were postive for eye irritation. Therefore, based on the results from this study, the test material was classified as a category 1 eye irritant according to the GHS criteria.