Reaction mass of (2S)-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl]butanamide and (2S)-2-[(4S)-2-oxo-4-propylpyrrolidin-1-yl]butanamide

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl]butanamide; (2S)-2-[(4S)-2-oxo-4-propylpyrrolidin-1-yl]butanamide

EC Number:

700-041-0

Molecular formula:

C11 H20 N2 O2

IUPAC Name:

(2S)-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl]butanamide; (2S)-2-[(4S)-2-oxo-4-propylpyrrolidin-1-yl]butanamide

Test material form:

solid: particulate/powder

Remarks:

white powder

Details on test material:

- Name of test material (as cited in study report): ucb108628-1
- Stability under test conditions: not indicated
- Storage condition of test material: room temperature, in the dark

Test animals

Species:

rat

Strain:

other: Crl: WI (Han)

Details on species / strain selection:

Untreated females males were used at the initiation of the study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- The animals were 9 weeks old at initiation of dosing and females weighed between 176 and 203 g, males weighed between 244 and 282 g.
- Fasting period before study: no
- Housing: On arrival and following randomization, animals were single housed in polycarbonate cages (Makrolon type III, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study.
- Water: Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: for 9 days prior to start of treatment;

DETAILS OF FOOD AND WATER QUALITY:
Diet and water evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
(set to maintain)
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: .... January 2018 To: 16 Feb 2018

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

water

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
Prior to the start of the study, test formulation were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. These test formulations were not used for dosing and were discarded after the assessment was complete. These test formulations have a non-GLP status and were carried out in the quality assured environment of the Test Facility.
The dosing formulations were prepared in daily portions and dosed within 6 hours after completion of the preparation of the test item.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. No adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

- PREPARATION OF THE SKIN:
At least 24 hours before first treatment, an area of approximately 6x10 cm on the back of the animals was clipped. Whenever necessary (during the course of the study) the skin-area was re-clipped at least 3 hours before a next application. Care was taken to avoid abrading the skin.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of samples of formulations taken in week 1 during the treatment phase were analysed according to a validated method.
Duplicate sets of samples (approximately 500 mg) for Groups 1 and 3 (concentration analysis only) and duplicate top, middle, and bottom samples for Groups 2 and 4 (concentration and homogeneity analysis) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to 10% for solutions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ±10%. At one occasion during the treatment phase, stability of the prepared formulation was determined at 6 hours at room temperature.

Duplicate sets of samples (approximately 500 mg) for Groups 1 and 3 (concentration analysis only) and duplicate top, middle, and bottom samples for Groups 2 and 4 (concentration and homogeneity analysis) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to 10% for solutions of target concentration.

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals dermally once daily (for at least 6 hours) for 7 days a week for a minimum of 28 days. Application was performed approximately the same time each day. Animals were treated up to the day prior to necropsy. The dose volume for each animal was based on the most recent body weight measurement. The test item formulation was applied onto a surgical gauze patch (Surgy 1D; less than or equal to 8 ply), with an area of approximately 10% of the total body surface (i.e. approx. 40 cm² for males and approx. 30 cm² for females). The gauze patch was mounted on Medical tape or Microfoam and held in contact with the skin with Tegaderm flexible bandage. To protect the application Lomir jackets were used. (Manufacturers: Laboratoires Stella s.a., Liege, Belgium (surgical gauze) and 3M, St. Paul, Minnesota, U.S.A. (Medical Tape and Microfoam) and Lomir Biomedical, Hull, UK (Jackets).)
After the dressing was removed, the skin was cleaned of residual test item (formulation) using water or an appropriate vehicle.

During the exposure period the dressing was checked regularly, at least every 2 hours (i.e. after application, and approximately 1, 3 and 5 hours after application). In case the application was found to be dislodged or improperly secured on the animal before or on the 3-hour bandage check time point, a new dressing with formulation was applied. The maximum time during which the animal was subsequently not exposed was estimated retrospectively, and reported in the results section.

Frequency of treatment:

Once daily.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dermal route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The dose levels were selected based on information provided by the results of the 8-day dose range finder study with dermal exposure of PPB-2 in rats, and in an attempt to produce graded responses to the test item. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level was expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy

ARENA OBSERVATIONS: Yes
- Animals were observed for specific clinical signs outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded. These observations were conducted once before the first administration of the test item and at weekly intervals.

BODY WEIGHT: Yes
- Animals were weighed individually weekly, starting on Day 1 (prior to dosing). A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION: Yes
- Food consumption was quantitatively measured weekly starting on Day 1 and continuing weekly throughout the Dosing Period.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected, between 7.00 and 10.30 a.m. at the day of necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Animals were fasted (overnight with a maximum of 24 hours) before blood sampling, but water was available.
- How many animals: all
- Parameters checked according to Guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected, between 7.00 and 10.30 a.m. at the day of necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Animals were fasted (overnight with a maximum of 24 hours) before blood sampling, but water was available.
- How many animals: all
- Parameters checked according to Guidelines. In addition, coagulation parameters (Prothrombin Time and Activated Partial Thromboplastin Time) were included.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: No

Sacrifice and pathology:

All animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

GROSS PATHOLOGY: Yes, according to Guidelines
HISTOPATHOLOGY: Yes, according to Guoidelines

Other examinations:

The following organs were weighed at necropsy for all scheduled euthanasia animals: adrenal gland, kidney, liver, testis.
Organ to body weight ratio (using the terminal body weight) were calculated.

Statistics:

All statistical tests was conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.

Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No toxicologically significant clinical signs were noted during daily detailed clinical observations. No findings were noted during the arena observations in this study.

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

Slight erythema maculate or scales were noted on the treated skin on one occasion in one female treated at 300 mg/kg or one male treated at 1000 mg/kg, respectively. At the incidence observed, these clinical signs were considered not to be toxicologically significant.
Skin effects outside the treated area (including erythema, scabs and/or wounds) were noted at all dose levels, including controls. These were considered to have occurred due to the application method. As these findings were slight, they were considered not to be toxicologically significant.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight was similar to the control level over the study period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Description (incidence and severity):

No toxicologically relevant changes were noted in haematological parameters.
Treatment related statistically significant decreases were observed in white blood cell and red blood cell counts in males treated at 1000 mg/kg. This was accompanied by a slight trend of reduced neutrophil, monocytes and reticulocytes counts and reduced haemoglobin and haematocrit concentrations. Because of the minimal magnitude of the changes, these changes were not considered as toxicologically relevant.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in organ weights in the weighed organs.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain or were related to the experimental procedure (i.e. clipping, jackets, and/or adhesive bandages).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in the examined tissues.
The few findings noted in the treated skin were of low severity, generally comparable to the control group and/or or the nature associated with the experimental procedure.
Remaining histologic changes were considered to be incidental findings or were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Unscheduled removal of (part of) the bandage from the treated skin area by the animals was observed among all dose groups, including the control group (details specified in the raw data). This may relate to both the study procedure and a response of the animals to irritating substances in these types of studies. The incidence of bandage removal in this study was not related to the administered dose. Therefore, this occurrence was considered to be related to the study procedures. Since the total time of removal of bandage per group was generally less than 5% of the total exposure time period, except for females at control and low dose group, it was considered that the incidences of bandage removal did not adversely affect the outcome or the integrity of the study.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, daily dermal PPB-2 exposure was well tolerated in rats at levels up to 1000 mg/kg. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg.

Executive summary:

Wistar rats were treated with PPB-2 for 28 consecutive days by daily dermal administration at dose levels of 0, 100, 300 and 1000 mg/kg. 

Test formulations preparedwere considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels. Test formulations preparedwere consideredstable, for at least 6 hours at room temperature.

No toxicologically significant changes were noted in any of the following parameters investigated in this study: clinical appearance, body weight, food consumption, coagulation parameters, macroscopic examination, organ weights, and microscopic examination.

Clinical pathology investigations revealed dose dependent higher chloride and lower inorganic phosphate concentration in treated males. In absence of related macroscopic or microscopic findings and related changes in clinical biochemistry measurements and due to the minimal magnitude of the changes, these changes were considered as non-adverse.