Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Rat / CD® / Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
60 animals (30 males and 30 females)
40 animals (20 male and 20 female rats);
5 animals/sex/group.
Recovery animals:
20 animals (10 male and 10 female rats);
5 animals/sex/group for groups 1 and 4.
Additionally, 6 (3 male and 3 female) spare animals were available for possible replacement during the adaptation period.
Body weight (at 1st administration)
Males: 331.7 g - 367.1 g
Females: 198.4 g - 233.4 g
Age (at 1st administration)
Males: 56 days
Females: 56 days
Selection of species The rat was selected because of its proven
suitability in toxicology and reproduction studies
and to comply with regulatory requirements for
testing in a rodent animal species.
The test item was diluted in the vehicle to the appropriate concentrations and was
administered orally at a constant volume twice daily. The application formulation
was continuously agitated by stirring throughout the entire administration
procedure.
The amount of the test item was adjusted daily to the current body weight of the
animal and was administered orally at a constant volume twice daily.
The control animals received the vehicle at the same administration volume daily in
the same way.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Route of administration: Oral, via gavage
Frequency of administration: Twice daily at 8.00 a.m. and 3.00 p.m. for 28
consecutive days
Vehicle: Tap water
Dose levels: 2 x 10 / 2 x 30 / 2 x 100 mg/kg b.w./day
Administration volume: 5 mL/kg b.w./day; the doses were split into 2 administrations of 2.5 mL/kg b.w.each
Selection of route of administration: According to international guidelines.
As the test item PU-2017-766 causes eye damage, allergic reactions, and is toxic to skin and causing burns on skin, handling of the test item, e.g. aliquoting the test item and preparation of application formulations, were prepared under a hood.
Personnel handling the test item wore appropriate protective gear. Due to the corrosive nature of the test item, necessary precaution measures were taken.
Extra care during administration like wiping of the syringe etc. due to corrosive nature of the test substance was needed.
The test item formulations were freshly prepared every day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For each test item that was mixed with the vehicle, tests by appropriate analytical methods were conducted to determine the concentration and stability of the test item in the formulations.
For the analysis of the administration formulations, samples of approximately 8 mL were taken (divided into 2 aliquots of 4 mL) at the following times and stored at -20°C ±10% until shipment for analysis.
The samples were labelled with the LPT study number, test species, type of sample, aliquot number, concentration, test day, sampling time and date.
Following advance notice the formulation samples obtained during the study were dispatched on dry ice.
The analysis of the samples for PU-2017-766 levels was performed under the responsibility of the analytical laboratory and was covered by the GLP program of Prolytic GmbH.
The samples were analysed according to a method validated by Prolytic GmbH (Prolytic Report no. 18062_BA ‘Validation of an HPLC-MS/MS method for the quantitative determination of DABCO NE 1550 in formulation samples’, issued 06 June 2019).
Duration of treatment / exposure:
28 days
Frequency of treatment:
twice a day
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
split into two doses per day
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
split into two doses per day
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
split into two doses per day
No. of animals per sex per dose:
5 male and 5 female
Control animals:
yes

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No changes in behaviour, external appearance or the faeces were noted for the male and female animals dosed with 2 x 10 or 2 x 30 mg PU-2017-766/kg b.w./day.
However, at 2 x 30 mg PU-2017-766/kg b.w./day, a haemorrhagic nose/snout was noted for 1 of 5 male animals (no. 34 m). This single occurrence is considered to be spontaneous.
At 2 x 100 mg PU-2017-766/kg b.w./day, breathing sounds were noted for 1 of 10 male animals (no. 44 m) and for 2 of 10 female animals (nos. 55 f and 58 f) on individual test days. This finding was also noted in the dose range finding study 36494 for the animals treated with 300 and 1000 mg PU-2017-766/kg b.w./day.
Therefore, breathing sounds were considered to be adverse reactions.
At 2 x 100 mg PU-2017-766/kg b.w./day, breathing sounds were noted for 1 of 10 male animals (no. 44 m) and for 2 of 10 female animals (nos. 55 f and 58 f) on individual test days. This finding was also noted in the dose range finding study 36494 for the animals treated with 300 and 1000 mg PU-2017-766/kg b.w./day.
Therefore, breathing sounds were considered to be adverse reactions.
Mortality:
no mortality observed
Description (incidence):
No mortality was noted for the male and female animals treated with 2 x 10, 2 x 30 or 2 x 100 mg PU-2017-766/kg b.w./day by oral administration for 28 days.
None of the previously high-dosed animals died or had to be sacrificed prematurely during the recovery period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related influence was observed on the mean body weight and the mean body weight gain of the male and female animals treated with 2 x 10, 2 x 30 or 2 x 100 mg PU-2017-766/kg b.w./day for 28 days compared to the control animals, neither during the 4-week treatment period nor during the 14-day treatment-free recovery period. No test item-related differences were noted for the body weight at autopsy between the test item-treated animals and the control animals.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related influence was observed on the food consumption of the male and female animals treated with 2 x 10, 2 x 30 or 2 x 100 mg PU-2017-766/kg b.w./day for 28 days compared to the control animals, neither during the 4-week
treatment period nor during the 14-day treatment-free recovery period.
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related influence was observed on the haematological parameters for the male and female animals treated with 2 x 10, 2 x 30 or 2 x 100 mg PU-2017-766/kg b.w./day by oral administration for 28 days compared to the control animals at the end of the treatment period (test day 29) and at the end of the recovery period (test day 45).
However, the male animal no. 43 treated with the high dose of 2 x 100 mg PU-2017-766/kg b.w./day revealed slight changes in the differential blood count compared to the group mean value of the control group: decreased number of white blood cells (-46%), decreased number of lymphocytes (-52%) and monocytes (-62%).
Similar effects were also noted for the male animals no. 34 and 35 treated with the intermediate dose of 2 x 30 mg PU-2017-766/kg b.w./day.

The changes are not considered to be test item-related but spontaneous findings.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item-related influence was observed on the clinical chemistry parameters for the male and female animals treated with 2 x 10, 2 x 30 or 2 x 100 mg PU-2017-766/kg b.w./day by oral administration for 28 days compared to the control animals at the end of the treatment period (test day 29) and at the end of the recovery period (test day 45).
No test item-related effects were noted on the plasma levels of albumin and globulin and on the albumin/globulin ratio, the BUN/creatinine ratio, on the plasma levels of bile acids, total bilirubin, total cholesterol, creatinine, glucose, total protein, potassium, sodium and urea on test day 29 and on test day 45. No test item-related influence was noted on the plasma enzyme activities of the alanine aminotransferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), gamma glutamyl-transferase (Gamma-GT) and lactate dehydrogenase (LDH). All data are within the limits of normal biological variability.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
None of the animals treated with 2 x 10, 2 x 30 or 2 x 100 mg PU-2017-766/kg b.w./day revealed any test item-related changes in external appearance, body posture, movement and coordination capabilities and behaviour in test weeks 1 to 4. No changes occurred during the 14 day recovery period.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item related changes were noted for the relative and absolute organ weights of the animals treated with 2 x 10, 2 x 30 or 2 x 100 mg PU-2017-766/kg b.w./day by repeated oral administration at terminal sacrifice on test day 29 or at recovery sacrifice on test day 45.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic inspection at necropsy did not reveal any test item-related changes in the organs and tissues of the animals treated with 2 x 10, 2 x 30 or 2 x 100 mg PU-2017-766/kg b.w./day by repeated oral administration after terminal sacrifice at the end of the treatment period (test day 29).
The following incidental not test item-related findings were noted:
In one animal treated with 2 x 10 mg PU-2017-766/kg b.w./day the urinary bladder was filled with white gravel-like content. One male animal treated with 2 x 30 mg PU-2017-766/kg b.w./day revealed a deformed and reduced in size left kidney and an enlarged right kidney.

No pathological changes were observed in the organs and tissues of the male and female rats previously treated with 2 x 100 mg PU-2017-766/kg b.w./day for 28 days at the end of the 14-days treatment-free recovery period (test day 45).
Neuropathological findings:
no effects observed
Description (incidence and severity):
No test item-related influence was noted on any of the parameters examined during the functional observation tests, on the fore- and hind limb grip strength, or on the spontaneous motility for any of the male and female animals after repeated oral treatment with 2 x 10, 2 x 30 or 2 x 100 mg PU-2017-766/kg b.w./day in test week 4.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The histopathological examination of a variety of organs and tissues was restricted to the main study animals of control group 1 and the high dose group 4 treated with 2 x 100 mg PU-2017-766/kg b.w./day.
The organs of males and females observed at microscopy did not reveal any changes that could be related to the administration of the test item when dosed up to 2 x 100 mg PU-2017-766/kg b.w./day for 4 weeks neither at terminal sacrifice on test day 29 nor on recovery sacrifice on test day 45.
There was no evidence for local irritation in the upper digestive tract.
A few minor microscopic changes were recorded for the organs examined in this study. The type, incidence and severity of all microscopic findings observed did not indicate any relationship to the treatment with the test item. All changes are regarded to be spontaneous in nature being within the normal background pathology commonly seen in rats of this strain and age.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
System:
respiratory system: upper respiratory tract
Organ:
bronchi
bronchioles
lungs
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

At 2 x 100 mg PU-2017-766/kg b.w./day, breathing sounds were noted for 1 of 10 male animals (no. 44 m) and for 2 of 10 female animals (nos. 55 f and 58 f) on individual test days. This finding was also noted in the dose range finding study 36494 for the animals treated with 300 and 1000 mg PU-2017-766/kg b.w./day.

Therefore, breathing sounds were considered to be adverse reactions.

Applicant's summary and conclusion

Conclusions:
The experimental no-observed-effect level (NOEL) was 60 mg/kg b.w./day, by daily oral administration.
Executive summary:

The aim of this repeated dose toxicity study was to obtain information an the toxicity of PU-2017-766 administered daily by oral administration to rats for 28 consecutive days and to assess the reversibility of any effects at the end of a 14-day recovery period. The rats were treated with 2 x 10, 2 x 30 or 2 x 100 mg/kg b.w./day. The control animals received the vehicle {tap water).

None of the animals died or had to be sacrificed prematurely.

At 2 x 100 mg/kg b.w./day, breathing sounds were noted for 1 of 10 male animals (no. 44 m) and for 2 of 10 female animals (nos. 55 f and 58 f) an individual test days. This finding was also noted in the dose range finding study for the animals treated with 300 and 1000 mg/kg b.w./day.

Therefore, breathing sounds were considered to be adverse reactions.

No test item-related changes were observed for the behaviour or external appearance of the animals, the detailed clinical observations, the neurological screening, the body weight, body weight gain and body weight at autopsy, the food and drinking water consumption, for any of the haematological and clinical chemistry parameters, the relative and absolute organ weights, and at macroscopic inspection at necropsy at any dose level.

The histopathological examination did not reveal any test item-related morphological changes. There was no evidence for local irritation in the upper digestive tract.

No test item-related changes were noted during or at the end of the 14-day treatment-free recovery period.

The experimental no-observed-effect level (NOEL) was 60 mg/kg b.w./day, by daily oral administration.