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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Nov 2020 - 06 May 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
25 June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-[(4-methylphenyl)imino]bisethanol
EC Number:
221-359-1
EC Name:
2,2'-[(4-methylphenyl)imino]bisethanol
Cas Number:
3077-12-1
Molecular formula:
C11H17NO2
IUPAC Name:
2-[(2-hydroxyethyl)(4-methylphenyl)amino]ethan-1-ol
Constituent 2
Reference substance name:
Ethanol 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-
Cas Number:
878391-30-1
IUPAC Name:
Ethanol 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-
Constituent 3
Reference substance name:
2-[2-(2-hydroxyethoxy)ethyl-(2-hydroxyethyl)amino]ethanol
IUPAC Name:
2-[2-(2-hydroxyethoxy)ethyl-(2-hydroxyethyl)amino]ethanol
Constituent 4
Reference substance name:
2-[bis[2-(2-hydroxyethoxy)ethyl]amino]ethanol
IUPAC Name:
2-[bis[2-(2-hydroxyethoxy)ethyl]amino]ethanol
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models that do not use live animals currently do not exist.
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity test by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: 184 – 243 g males; 115 – 155 g females
- Fasting period before study: no
- Housing: Up to 5 animals of the same sex and same dosing group together in polycarbonate cages (Makrolon type IV, height 18 cm or Makrolon type 2000P, height 21.5 cm Type 2000P) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). Animals will be socially housed for psychological/environmental enrichment and may be provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities; During locomotor activity monitoring, animals are housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: Pellets SM R/M-Z (SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Municipal tap water, ad libitum
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY:
Results of analysis for nutritional components and environmental contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS SET TO MAINTAIN:
- Temperature (°C): 18 to 21
- Humidity (%): 39 to 57
- Air changes (per hr): ten or more
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 10 Dec 2020 To: 12 Mar 2021

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dose formulations (w/w) were homogenized to visually acceptable levels. Formulations were prepared at least weakly, divided into aliquots where required to allow to be dispensed on each dosing occasion and stored in the refrigerator (2-8°C). Test item dosing formulations were kept at room temperature until dosing. An adjustment was made for specific gravity of the test item and the vehicle. No correction was made for the purity/composition of the test item. Trial preparation representative of the dosing concentrations and volumes were carried out prior to the start of the study to assess the suitability of the formulation procedure.

VEHICLE
- Concentration in vehicle: 6, 20, 80 mg/mL
- Amount of vehicle: 5 mL/kg
- Lot/batch no.: PROGLY12, PROGLY14
- Specific Gravity: 1.036
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses was performed by using a validated analytical procedure. Accuracy and homogeneity were determined for formulations prepared for use in Week 1, Week 2, Week 6, and Week 12.
Samples and remaining formulation were stored in normal glassware causing the samples stored at room temperature to be exposed to normal laboratory light conditions.
Duplicate samples (approximately 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 10 or 20 mL. For determination of accuracy, samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations.
For concentration mean sample concentration results were acceptable when they were within or equal to ± 10% solutions of theoretical concentration. For homogeneity, the results were acceptable when relative standard deviation (RSD) of concentrations was <= 10% for each group.

Stability analyses performed previously in conjunction with the method development and
validation (Charles River Study No. 511925) demonstrated that the test item is stable in the
vehicle when prepared and stored under the same conditions at concentrations bracketing
those used in the present study.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily, 7 days a week
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
low dose - group 2
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
mid dose - group 3
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Remarks:
high dose - group 4
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels and vehicle were selected based on results of a (28-day repeated dose toxicity study with oral exposure of Accelerator PT25/E2 in rats, Test Facility Study No. 501370), and in an attempt to produce graded responses to the test item. In the 28-day repeated dose toxicity study (Test Facility Study No. 501370), Wistar Han rats were orally administered with Accelerator PT25/E2 at dose levels of 100, 300 and 600 mg/kg bw/day (the initial high-dose of 1000 mg/kg/day was lowered due to mortality on Day 1 of treatment).: At 600 mg/kg bw/day, one male and one female died on Day 1 of treatment. These animals showed lethargy, spasms, flat posture, quick breathing, erected fur, salivation and/or chromodacryorrhoea. At necropsy, foci on the thymus and/or lungs, and a thickened thymus were seen. Clinical signs noted in remaining animals were comparable. The males at 300 mg/kg bw/day showed incidentally flat posture, quick breathing, rales and/or chromodacryorrhoea. The females showed lethargy, tremor, hunched posture, uncoordinated movements, laboured or shallow respiration, rales, piloerection, salivation and/or ptosis during the study period, predominantly in the first three days. No relevant clinical signs were noted in animals treated at 100 mg/kg bw/day. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
- Fasting period before blood sampling for clinical biochemistry: at the end of treatment, all animals were fasted overnight with a maximum of 24 hours

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily; from Day 1 at 0 to 1 hour during dosing.
- Mortality checks: At least twice daily beginning upon arrival through termination/release. Except on days of receipt and necropsy where frequency was at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Pretreatment and weekly; from Week 1 and throughout the study, and on the day of necropsy. Animals are removed from the cage.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly, prior to dosing; from at least Day 1 and throughout the study. A fasted weight on the day of necropsy was recorded.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was measured quantitively weekly per cage; from at least Day 1 and throughout the study

WATER CONSUMPTION: Yes
- Time schedule for examinations: Regular basis throughout the study.
- Water consumption was monitored by visual inspection of the water bottle. In inter group differences were noted, consumption was assessed by weight.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pretreatment and during week 13
- Dose groups that were examined: all animals during pretreatment period and all group 1 and 4 animals during week 13

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of treatment
- Anaesthetic used for blood collection: Yes (isoflurane) from the retro-orbital sinus
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: White Blood Cell Count (WBC), Neutrophils (absolute and relative), Lymphocytes (absolute and relative), Monocytes (absolute and relative), Eosinophils (absolute and relative), Basophils (absolute and relative), Large unstained cells (LUC) (absolute and relative), Red Blood Cell Count, Reticulocytes (absolute and relative), Red Blood Cell Distribution Width (RDW), Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelets, Prothrombin time (PT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of treatment
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: Alanine aminotransferase (ALT), Triglycerides, Aspartate aminotransferase (AST), HDL and LDL Cholesterol, Alkaline Phosphatase (ALP), Sodium, Total protein, Potassium, Albumin, Chloride, Total Bilirubin, Calcium, Urea, Inorganic Phosphate (Inorg. Phos), Creatinine, Glucose, Cholesterol

PLASMA/SERUM HORMONES: Yes
- Time of blood sample collection: At the end of treatment
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: Triiodothyronine (T3), Thyroxine (T4), Thyroid-Stimulating Hormone (TSH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during the dosing period; Week 12-13
- Dose groups that were examined: The first 5 animals per sex per group in Week 12-13
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex and static righting reflex), grip strength and locomotor activity
- Arena Observations: Once before the first administration of the test item and weekly during the Treatment Period. Animals were observed for clinical signs outside the home cage in a standard arena.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Animals surviving until scheduled euthanasia had a terminal body weight recorded and were deeply anesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy.
- Animals that were euthanized prematurely were deeply anesthetized using isoflurane and subsequently exsanguinated
- All animals were subjected to a complete necropsy examination, which will include 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.

ORGAN WEIGHTS: Yes
- Organ weights were recorded at necropsy only for animals surviving scheduled euthanasia.

HISTOPATHOLOGY: Yes
- Representative samples of tissues were collected and preserved in 10% neutral buffered formalin.
- Tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.
- Microscopic examination of routinely prepared hematoxylin-eosin stained paraffin sections was performed on all tissues collected at necropsy (with exceptions as indicated on the tissue list table 2 below) from all control group and 400 mg/kg bw/day treated animals, slides of the kidney of males and liver of males and females from all 30 and 100 mg/kg bw/day treated rats. Gross lesions were examined from all animals and correlated to microscopic findings if possible. Tissues as detailed evaluated histopathologically by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.

For more details on tissue weighing, collection, processing and evaluation see table 2 presented below in section "Any other information on materials and methods incl. tables".
Other examinations:
ESTROUS STAGE DETERMINATION
- On the day of necropsy, a vaginal smear was taken to determine the stage of estrus from all females, except for females that have to be euthanized in extremis or die spontaneously.
- Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal smear procedures.
Statistics:
All statistical analyses were performed within the respective study phase, unless otherwise noted. Numerical data collected on scheduled occasions will be summarized and statistically analyzed as indicated below according to sex and occasion.

Body Weight Gains: Calculated between each scheduled interval.
Food Consumption: Calculated between each scheduled interval.
Organ Weight Relative to Body Weight: Calculated against the terminal body weight.

All statistical tests will be conducted at the 5% significance level. All pairwise comparisons will be conducted using two sided tests and will be reported at the 1% and 5% levels, unless otherwise noted.
The pairwise comparisons of interest are listed below:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Analyses will be performed according to the matrix below when possible, but will exclude any group with less than 3 observations.

For more details see table 1 Statistical matrix presented below in section "Any other information on materials and methods incl. tables"

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Hunched posture was observed in one female at 100 mg/kg bw/day, and in two males and one female at 400 mg/kg bw/day on one or several occasions. At 400 mg/kg bw/day, prostrate was observed in four males on one or several occasions in the first week, and for all females on Day 1. Finally, erected fur was observed in one male on two days. At the low incidence observed, these findings were considered not toxicologically relevant.
Incidental abnormal breathing sounds, labored breathing and/or vocalization were observed in individual males and/or females of all groups. These clinical signs were considered to be related to the method of dosing and are not test item-related.
Salivation and/or ploughing after dosing were observed in all groups (including the control group) at a dose-related incidence and was not considered toxicologically relevant, taking into account the nature of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.
Any other clinical signs noted during the Dosing Period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend or occurred in the control group. At the incidence observed, these were considered to be unrelated to treatment with the test item.
Mortality:
mortality observed, treatment-related
Description (incidence):
Mortality occurred in one female at 30 mg/kg bw/day, and in one male and four females at 400 mg/kg bw/day.

At 30 mg/kg bw/day, one female was found dead on Day 92 of dosing. With exception of slight salivation on Day 43, no clinical signs were observed in this animal during the dosing period. A slight body weight loss was observed between Days 85 and 91 (-3%). Macroscopic evaluation showed general autolysis, multifocal dark-red foci on the thymus and a gaseous distended gastro-intestinal tract without clear microscopic correlate. Microscopic findings of note consisted of a marked ulceration with necrosis and inflammation in the trachea. This strongly suggests a gavage-related accident and was therefore considered unrelated to the test item.

At 400 mg/kg bw/day, one male and four females were prematurely euthanized or were found dead after dosing.

One male at 400 mg/kg bw/day was prematurely euthanized directly after dosing on Day 1 of treatment due to severe clinical signs, consisting of convulsions. Macroscopic evaluation showed multifocal red foci in the glandular stomach (correlating with glandular hemorrhages at microscopy) and abnormal white mucoid content in the stomach. The cause of moribundity was unclear. Although it would be unlikely that the test item was responsible for the moribund condition after one day of treatment, this cannot be fully excluded.

The first female at 400 mg/kg bw/day was found dead on Day 4. The only clinical signs observed was prostrate on Day 1. Macroscopic and microscopic evaluation showed a few relevant findings that consisted of lungs failed to collapse and multifocal red foci in the lungs, which corresponding microscopically with congestion and abnormal white frothy content in the trachea without microscopic correlate. The macroscopic findings in the trachea, together with the short time-period of dosing (4 days), is suggestive for a gavage-related accident and therefore considered not a direct test item-related effect.

The second female at 400 mg/kw bw/day was found dead on Day 25 without excessive clinical signs prior to its death (prostrate on Day 1 and incidentally slight salivation). No effect on body weight was observed. Minor macroscopic findings (lungs failed to collapse) were observed, without relevant microscopic findings that could explain the cause of death. A possible relation to treatment with the test item could not be excluded.

The third female at 400 mg/kw bw/day was prematurely euthanized on Day 37 of dosing (died just before performing the euthanasia procedure) as it presented with severe clinical signs (sustained convulsions and labored breathing) after dosing. In addition, prostrate on Day 1 and slight salivation between Days 2-37 were observed. No effect on body weight was observed. Minor macroscopic findings (lungs failed to collapse) were observed, without relevant microscopic findings that could explain the cause of the moribundity. A possible relation to treatment with the test item could not be excluded.

The fourth female at 400 mg/kw bw/day was found dead on Day 60 of dosing after it had shown sustained convulsions, moderate salivation and labored breathing post-dosing. In addition, prostrate on Day 1, (mostly slight) salivation between Days 2-60 and a slight body weight loss between days 50-57 (-2%) were observed. No relevant macroscopic or microscopic findings were observed that could explain the cause of death. A possible relation to treatment with the test item could not be excluded.

For all unscheduled sacrifices/deaths, the nasal cavities were examined microscopically. No relevant findings were observed, which excluded reflux as a possible cause of death/moribundity.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain were considered to be unaffected by treatment with the test item in males and females treated up to 400 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effect on food consumption was observed in males and females up to 400 mg/kg bw/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No ophthalmology findings were noted that were considered to be related to treatment with the test item.
The nature and incidence of ophthalmology findings noted during the Pretreatment Period and in Week 13 was similar among the groups and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in hematological parameters were observed in males and females up to 400 mg/kg bw/day.

Increased platelet count was observed in females treated at 400 mg/kg bw/day (1.12x control). As values remained within the normal range observed for female rats of this age and strain these findings were not considered test item-related.

Other differences in hematology parameters in males and females, regardless of statistical significance, were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values and/or were of a magnitude of change commonly observed in rats under similar study conditions.
(Historical Control Data for Wistar Han rats (2015-2020) Platelet count (109/L) – females: mean = 738; P5-P95 = 565-924 (n=131)).
No test item-related changes in coagulation parameters were observed in males and females up to 400 mg/kg bw/day.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No test item-related changes in clinical chemistry parameters were observed in males and females up to 100 mg/kg bw/day.
An increase in total protein, albumin, total bilirubin, cholesterol, HDL cholesterol, LDL cholesterol and/or calcium were observed in males and females at 400 mg/kg bw/day (see Table 3 for the fold change below under "Any other information on results incl. tables" section).

Other differences in clinical chemistry parameters in males and females, regardless of statistical significance, were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
Thyroid hormones showed decreased thyroid stimulating hormone (TSH) in females at 400 mg/kg bw/day. The decrease in TSH concentration in males at 30 and 400 mg/kg bw/day lacked a clear dose related response and remained within the historical control data and were therefore considered to be not toxicologically relevant (see Table 3 for the fold change below under "Any other information on results incl. tables" section).

(Historical Control Data for Wistar Han rats (2015-2021) TSH (mU/L) – females: mean = 0.089; P5-P95 = 0.010-0.269 (n=174), – males: mean = 0.184; P5-P95 = 0.035-0.541 (n=182))

Other differences in thyroid hormones T3 and T4 in males and females, regardless of statistical significance, were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were considered to be normal in all examined animals. Grip strength was similar between control and treated groups, except for males at 100 mg/kg bw/day and females at 30 mg/kg bw/day, which showed lower grip strength compared to the control in the fore and hind leg, respectively. In absence of a dose-related response, these findings were considered not test item-related.
Motor activity was similar between treated and control groups. Higher mean values for total movements and ambulations were observed in males and females treated at 400 mg/kg bw/day (not statistically significant), which was mainly caused by interval 6 and 11 (statistically significant, included in the raw data), respectively. As the overall results remained within historical control data, these changes were therefore considered to be not toxicologically relevant. All other groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher liver weights (absolute and relative to body weights) were noted in the 400 mg/kg bw/day groups males and females (see Table 4 for the fold change below under "Any other information on results incl. tables" section).

Although a statistically increase in kidney weight was observed in males (relative to body weight at 30 and 400 mg/kg bw/day) and females (absolute at 400 mg/kg bw/day), this increase was considered not to be related to the test item. This was based on the absence of a dose relationship and the small decrease in final body weight in males and the small increase in final body weight in females.
There were no other test item-related organ weight changes.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related gross observations.
All the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the liver and kidney of males treated at 100 and 400 mg/kg bw/day and in the liver of females treated at 400 mg/kg bw/day (see Table 5 and 6 for the fold change below under "Any other information on results incl. tables" section).

In the liver, centrilobular hypertrophy was observed in males treated at 100 and 400 mg/kg bw/day, with a dose relationship response, and in a single female treated at 400 mg/kg bw/day.

In the kidney, an increased incidence and/or severity of hyaline droplet accumulation and tubular basophilia was observed in males treated at 100 and 400 mg/kg bw/day.

There were no other test item-related histologic changes. Remaining histologic changes 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

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
organ weights and organ / body weight ratios
serum/plasma hormone analyses
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
serum/plasma hormone analyses

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
no

Any other information on results incl. tables

DOSE FORMULATION ANALYSIS


The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%), except for the formulation of Group 2 prepared for use in Week 1. The mean accuracy was below the target concentration (i.e. 86% of target).


Re-analysis of the Group 2 formulation prepared for use in Week 1 resulted in the same mean accuracy (i.e. 86% of target concentration). Therefore, it was decided to do an extra analysis on Group 2 formulation prepared for use in Week 2. This resulted in a mean accuracy of 96% (i.e. mean accuracy between 90% and 110%). Based on these results it can be concluded that Group 2 received the accurate dose level throughout the study with exception of Week 1 where the dose level was approximately 25.8 mg/kg bw/day. This has no effect on the study integrity.


No test item was detected in the Group 1 formulations and the formulations of Groups 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).


 


Table 3
Accelerator PT25/E2‑Related Clinical Chemistry Changes – Fold Change






































































































Groups



4



Dose (mg/kg bw/day/day)



400



400



Sex



M



F



Total protein



 



 



    End of Treatment



1.05x



1.04x



Albumin



 



 



    End of Treatment



1.06x



1.05x



Total bilirubin



 



 



    End of Treatment



1.16x



1.18x



Cholesterol



 



 



    End of Treatment



1.20x



1.34x



HDL cholesterol



 



 



    End of Treatment



1.23x



1.39x



LDL cholesterol



 



 



    End of Treatment



-



1.16x



Calcium



 



 



End of Treatment



1.10x



1.04x



TSH



 



 



End of Treatment



0.72x



0.38x



 


Bolded values indicate the mean value was statistically different from controls at P ≤ 0.05 or P ≤ 0.01.


 


 


Table 4
Mean Percent Liver Weight Differences from Control Groups
























































 



Males



Females



Dose level (mg/kg bw/day):



30



100



400



30



100



400



 



 



 



 



 



 



 



LIVER



 



 



 



 



 



 



               Absolute



1



-3



19**



6



4



21**



               Relative to body weight



4



2



23**



3



3



16**



*: P<0.05, **: P<0.01


 


 


Table 5
Summary Test Item-Related Liver Findings – Scheduled Euthanasia Animals













































































 



Males



Females



Dose level (mg/kg bw/day):



0



30



100



400



0



30



100



400



 



 



 



 



 



 



 



 



 



LIVER a



10



10



10



10



10



10



10



10



        Hypertrophy, centrilobular



 



 



 



 



 



 



 



 



           Minimal



-



-



2



5



-



-



-



1



           Mild



-



-



-



5



-



-



-



-



a  =  Number of tissues examined from each group.


 


 


Table 6
Summary Test Item-Related Kidney Findings – Scheduled Euthanasia Animals
















































































 



Males



Dose level (mg/kg bw/day):



0



30



100



400



 



 



 



 



 



KIDNEY a



10



10



10



10



        Accumulation, hyaline droplets



 



 



 



 



           Minimal



4



6



6



2



           Mild



3



3



4



7



           Moderate



-



-



-



1



       Basophilia, tubular



 



 



 



 



           Minimal



2



3



2



2



           Mild



-



-



1



3



a = Number of tissues examined from each group.

Applicant's summary and conclusion

Conclusions:
In an oral screening study for toxicological effects, performed according to OECD guideline 408 and following GLP principles Accelerator PT25/E2 was administered by daily oral gavage for at least 90 days at 30, 100 and 400 mg/kg bw/day. Based on these results, the No Observed Adverse Effect Level (NOAEL) was established to be at least 400 mg/kg bw/day in males and 100 mg/kg bw/day in females.


Executive summary:

A 90-day screening study for toxicological effects was performed according to OECD/EC guidelines and GLP principles. Accelerator PT25/E2 was administered by daily oral gavage at dose levels of 0 (vehicle), 30, 100 and 400 mg/kg bw/day.


Chemical analyses of formulations were conducted on Weeks 1, 2, 6 and 12 and confirmed that formulations of test item in propylene glycol were prepared accurately and homogenously (expect for accuracy of Group 2 formulations in Week 1) and were stable over at least 8 days in the refrigerator and 24 hours at room temperature.


The following parameters and end points were evaluated in this study: mortality, clinical signs, functional observation tests, body weights, food consumption, ophthalmology, estrus stage determination, clinical pathology parameters (hematology, coagulation, and clinical chemistry), gross necropsy findings, organ weights, and histopathologic examinations.


At 30 mg/kg bw/day, no test item-related findings were observed. One female was found dead on Day 92, which was likely due to a gavage-related accident and therefore not directly related to the treatment with the test item.


At 100 mg/kg bw/day, test item-related, non-adverse liver hypertrophy and an increase in hyalin droplet accumulation (with minor increased tubular basophilia) in the kidney was observed in males.  


At 400 mg/kg bw/day, three females were euthanized (1/3) or found dead (2/3) and one male was euthanized for which a test item-relationship could not be excluded. In addition, one female was found dead on Day 4, which was likely due to a gavage-related accident and therefore considered not test item-related.
Clinical chemistry parameters showed an increase in total protein, albumin, total bilirubin, cholesterol, HDL cholesterol, LDL cholesterol, calcium and concentrations in males and/or females and a decrease in thyroid-stimulating hormone in males. In absence of a microscopic correlate, these findings were considered non adverse. Furthermore, microscopic evaluation showed a test item-related, non-adverse liver hypertrophy with correlating higher liver weights in males and females (about plus 20%), and a small increased incidence and/or severity in tubular hyaline droplet accumulation (with minor increased tubular basophilia) in the kidney in males.


No test item-related or toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, functional observations, ophthalmoscopy, hematology and coagulation parameters, thyroid hormones, and macroscopic examination).


In conclusion, administration of Accelerator PT25/E2 by once daily oral gavage for at least 90 days was well tolerated in Wistar Han rats at a dose level of 100 mg/kg bw/day in females and up to 400 mg/kg bw/day in males. At 400 mg/kg bw/day, mortality occurred in three females for which a test item-relationship could not be excluded. Furthermore, non-adverse changes in clinical chemistry parameters and thyroid stimulating hormone were observed in males and/or females at 400 mg/kg bw/day. Non-adverse morphologic alterations were present in the kidney (increased accumulation of hyaline droplets and tubular basophilia) and liver (centrilobular hypertrophy with correlating increased liver weight) of males treated at 100 and 400 mg/kg bw/day, and in the liver of females (centrilobular hypertrophy with correlating increased liver weight) at 400 mg/kg bw/day.


Based on these results, the No Observed Adverse Effect Level (NOAEL) was established to be at least 400 mg/kg bw/day in males and 100 mg/kg bw/day in females.