Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 June 2017 - 02 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
21 September 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France.
- Age: at the beginning of the treatment period, the animals were approximately 5 weeks old
- Mean body weight: the males had a mean body weight of 201 g (range: 184 g to 220 g) and the females had a mean body weight of 171 g (range: 151 g to 192 g)
- Fasting period before study: no
- Housing: the animals were housed in twos or threes (same sex and group) in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²) containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for 8 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 21 June 2017 to 02 November 2017.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING FORMULATIONS:
- Solution in the vehicle
- Concentration in vehicle: 20, 60 and 120 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: High Performance Liquid Chromatography with tandem Mass Spectrometry detection (LC/MS-MS)
Test item concentrations: Once in Weeks 1, 4, 8 and 13. A sample was taken from control and test item dose formulations and analyzed using the validated method.
Stability: The stability of dose formulation preparations was demonstrated for up to 23 days when stored in plastic vial at room temperature and protected from light.
Dose formulations ranging from 5 mg/mL to 200 mg/mL are therefore considered to be suitable for routine administration in GLP Toxicological studies within the stability period validated.
Duration of treatment / exposure:
13 weeks followed by a 6-week treatment-free period
Frequency of treatment:
Daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
15 animals per sex (for control and high dose groups)
10 animals per sex (for low and intermediate dose groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for dose selection:
The dose levels were selected in agreement with the Sponsor, based on the results of a previous OECD 407 study performed in Wistar rats.
In this previous study, rats received the test item daily by gavage at dose levels of 150, 550 or 1000 mg/kg/day for 28 days.
Instances of abnormal systemic signs were minimal. There were no significant differences from controls in any of the parameters (i.e. functional observation battery and motor activity, body weight, food consumption, hematology, blood biochemistry and organ weights). Test item-related microscopic changes were observed from 550 mg/kg/day in the kidneys of males (hyaline droplet formation unique to the male rat) and in the stomach of males and females. Based on the microscopic changes, the No Observed Effect Level (NOEL) was estimated to be 150 mg/kg/day.
Thus, based on these available data, the dose levels selected for the present study were 100, 300 and 600 mg/kg/day.

- Rationale for animal assignment: computerized randomization procedure.
Positive control:
no (not required)
Observations and examinations performed and frequency:
MORBIDITY/MORTALITY:
- Time schedule: each animal was checked for mortality and morbidity once a day during the acclimation period and at least twice a day during the treatment and observation periods, including weekends and public holidays.

CLINICAL OBSERVATIONS:
- Time schedule: each animal was observed once a day, at approximately the same time on the days of treatment

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then at least once a week until the end of the study.

FUNCTIONAL OBSERVATION BATTERY (FOB):
- Time schedule: all main animals (animals euthanized at the end of the treatment period) were evaluated once in Week 12 (before the daily treatment). The evaluation included a detailed clinical examination, the assessment of reactivity to manipulation and different stimuli, and motor activity.

MOTOR ACTIVITY:
- Time schedule: for each animal, motor activity was measured by automated infra-red sensor equipment over a 60-minute period.

BODY WEIGHT:
- Time schedule: the body weight of each animal was recorded once before the beginning of the treatment period, on the first day of treatment and at least once a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by the animals in each cage was recorded at least once a week, over a 7 day period, during the study.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule: on all animals, before the beginning of the treatment period and on all main (animals euthanized at the end of the treatment period) control and high-dose animals on one occasion at the end of the treatment period (Week 13).

HAEMATOLOGY:
- Time schedule for peripheral blood: the parameters were determined for all surviving animals euthanized at the end of the treatment period (Week 13). In view of the findings observed at the end of the treatment period, these examinations were carried out in females at the end of the treatment-free period.
- Time schedule for bone marrow: two bone marrow smears were prepared from the femoral bone (at necropsy) of all animals euthanized on completion of the treatment or treatment-free period. As no relevant abnormalities were observed during the hematological investigations, the bone marrow differential cell count was not determined and smears were archived.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: All
- Parameters checked in tables [1 and 2] were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: the parameters were determined for all surviving animals euthanized at the end of the treatment period (Week 13). In view of the findings observed at the end of the treatment period, these examinations were carried out in both sexes at the end of the treatment-free period.
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table [3] were examined.

THYROID HORMONES:
- Time schedule: An additional blood sample was collected from each animal euthanized at the end of the treatment and treatment-free periods into tubes containing K3-EDTA as anticoagulant.

The levels of the thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH) were not determined as there was no indication of an effect on the pituitary-thyroid axis.

URINALYSIS:
- Time schedule for collection of urine: the parameters were determined for all surviving animals euthanized at the end of the treatment period (Week 13).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [4] were examined.
Sacrifice and pathology:
ORGAN WEIGHTS: see table 5
The body weight of each animal was recorded before euthanasia at the end of the treatment or treatment free period. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection.
The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all animals.

PRESERVATION OF TISSUES:
For all study animals, the tissues specified in the Tissue Procedures Table were preserved in 10% buffered formalin (except for the eyes and optic nerves and Harderian glands, and the testes and epididymides which were fixed in Modified Davidson's Fixative).
Tissues intended for immunohistochemistry (kidneys) were kept for no longer than 96 hours in formalin (main and recovery males).
Two bone marrow smears for the potential determination of the bone marrow differential cell count (see § Bone marrow) were prepared from the femur of each animal euthanized on completion of the treatment period or treatment-free period.

PREPARATION OF HISTOLOGICAL SLIDES:
All tissues required for microscopic examination were trimmed according to the RITA guidelines, when applicable, embedded in paraffin wax, sectioned at a thickness of approximately 4 microns and stained with hematoxylin-eosin (except for the testes and epididymides which were stained with hematoxylin/PAS).
Immunostaining of kidneys from the males (main animals euthanized at the end of the treatment period) in groups 1 and 4 with an antibody for a2u-globulin protein was performed due to an indication of hyaline droplet accumulation at examination of the hematoxylin-eosin slides.
The tissue processing was performed at Citoxlab France.

HISTOPATHOLOGY:
A microscopic examination was performed on all tissues listed in the Tissue Procedure Table:
- for the control and high-dose animals (groups 1 and 4) euthanized at the end of the treatment period and for one group 3 main male that died prematurely,
- for all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) euthanized on completion of the treatment period.

This was performed by the Principal Investigator under the responsibility of Citoxlab France.

As required according to kidney histopathology, immunostained kidneys from control and high-dose males (groups 1 and 4) euthanized at the end of the treatment period were examined.

In addition, a detailed examination of the testes was performed for control and high-dose males (groups 1 and 4), using a thorough understanding of tubule development through the different stages of the spermatogenic cycle. The hematoxylin-PAS stained transverse sections of testes allowed the detection of retained spermatids, missing germ cell layers, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.

In agreement with the Sponsor and based on the microscopic results of the high-dose group, other tissues from the low- and intermediate-dose groups were examined. In addition, according to the results obtained at the end of the treatment period, a microscopic examination of selected tissues from animals euthanized on completion of the treatment-free period was performed as follows:
For low- and intermediate-dose animals (groups 2 and 3) and recovery animals (groups 1 and 4):
- stomach with forestomach from males and females,
- kidneys from males,
- vagina.


Other examinations:
SPERM PARAMETERS:
Before euthanasia at the end of the treatment period, each male was anesthetized by an intraperitoneal injection of sodium pentobarbital.
As no relevant changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.

Epididymal sperm
Under deep anesthesia and after weighing the epididymis, sperm from the cauda of the left epididymis was collected for motility and morphology investigations. Animals were then euthanized.
The cauda of the left epididymis was separated from the corpus using a scalpel and subsequently kept at 20°C pending further investigation.

Epididymal sperm motility
The sperm was evaluated on a slide, after appropriate dilution if required. The numbers of motile and immotile spermatozoa in a sample of 200 spermatozoa were evaluated under a microscope using a 40 fold magnification. Results were expressed as the proportions of motile and non-motile spermatozoa.

Epididymal sperm morphology
Morphology was determined from a sperm smear, after eosin staining and counting of 100 spermatozoa per slide. This was evaluated for groups 1 and 4 in the first instance.
In view of the findings observed in these groups at the end of the treatment period, this determination was not extended to groups 2 and 3 or recovery animals.

Results were expressed as the proportion of spermatozoa in each of the following categories:
- normal,
- normally shaped head separated from flagellum,
- abnormal head separated from flagellum,
- abnormal head with normal flagellum,
- abnormal head with abnormal flagellum,
- normally shaped head with abnormal flagellum.

Epididymal sperm count
After thawing, the left cauda epididymis was weighed, minced and homogenized in a saline-triton solution using a Polytron.
An aliquot of the suspension was collected and the number of spermatozoa was counted in a microscope slide counting chamber.
Results were expressed as the numbers of spermatozoa per cauda and per gram of cauda.

Testicular sperm
The left testis was collected and kept at -20°C for further sperm count investigation. After thawing, the left testis was weighed and ground. The resulting preparation was diluted and sperm heads resistant to homogeneization (i.e. elongated spermatids and mature spermatozoa) were counted in a microscope slide counting chamber.
Results were expressed as the number of sperm heads per gram of testis, and the daily sperm production rate was calculated (using a time divisor of 6.10).

MONITORING OF ESTROUS CYCLE:
The estrus cycle stage was determined for each female euthanized at the end of the treatment period, from a fresh vaginal lavage (stained with methylene blue), daily for 21 consecutive days before the end of the treatment period.
In view of the findings observed at the end of the treatment period, this examination was carried out daily for 14 consecutive days before the end of the treatment-free period.
Statistics:
Citox software was used to perform the statistical analyses of body weight, food consumption, motor activity, sperm parameters, hematology, blood biochemistry and urinalysis data.
PathData software was used to perform the statistical analysis of organ weight data.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See Table 1.
Ptyalism was transiently observed in control males and females and in all test item-treated groups in a dose-related manner, from the first week of treatment in any test item-treated group females at the earliest.
Reflux at administration was noted in 3/15 control males, 1/10 females given 100 mg/kg/day, 1/10 males and females given 300 mg/kg/day and in 1/15 males given 600 mg/kg/day on one occasion. This sign is commonly observed when a test item is administered by gavage and was therefore considered to be of no toxicological importance.
The other clinical signs recorded during the study, i.e. alopecia, scabs, thinning of hair, bent tail, soiled neck, chromodacryorrhea, chromorhynorrhea and/or loud breathing were of isolated occurrence, were observed both in control and test item-treated animals, and/or had no dose-relationship. They were therefore considered to be unrelated to the test item treatment.
Test item-related clinical signs were no longer observed over the treatment-free period. Thin appearance transiently noted from Day 126 to Day 133 (the last two weeks of the treatment-free period) in 1/5 females previously given 600 mg/kg/day was considered to be incidental as this was not observed during the treatment period and as it was accompanied by a body weight gain of 8 g over the same period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related deaths occurred during the study.
One male given 300 mg/kg/day was found dead on Day 43 (Week 7). Ptyalism was observed on Days 24 to 26. A slightly lower body weight gain (+10 g vs. +27 g for its group) was recorded between Days 36 and 43. At necropsy, there was a white mass in the cranial cavity, with red discoloration of the ventral part of the brain. Microscopically, these correlated with hemorrhage (large hematoma of the skull, partly surrounded by fibrous tissue). This was considered to be the cause of death and to be incidental, possibly due to trauma, with no relationship to the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See Table 3.
At 600 mg/kg/day in males, when compared with controls, statistically significant, lower mean body weight gain was recorded throughout the treatment period (-10% vs. controls) leading to a statistically significant lower mean body weight from Week 4 (-4% vs. controls) until the end of the treatment period (-8% vs. controls on Day 91, Week 13). The differences in body weight gain were particularly noticeable from the third week. This effect was attributed to the test item treatment. During the recovery period, a statistically significant, higher mean body weight gain was observed in previously treated males, leading to a terminal mean body weight similar to that of the control animals (-1% vs. controls).
In females, the body weight and the body weight evolution were not affected by the test item.

At 100 and 300 mg/kg/day, instances of lower mean body weight gain were observed throughout the treatment period (statistically significant in Week 4 for females given 100 mg/kg/day, in Weeks 8, 9 and/or 12 for animals given 300 mg/kg/day), whereas statistically significant, higher mean body weight gain was noted in Week 11 for males and females given 100 mg/kg/day.
These sporadic variations without relevant effects at 100 and 300 mg/kg/day on the terminal mean body weight and of opposite trends were considered to be of no toxicological importance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No relevant effects were observed on food consumption in test item-treated animals during the treatment or treatment-free period.
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No findings were observed at the end of the treatment period.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period, hematology parameters were considered to be unaffected by the test item.

At 600 mg/kg/day in females, when compared with mean control values, higher red blood cell parameters (statistically significant), including mean red blood cell count (+6%) and mean hemoglobin concentration (+3%), were noted. A minimal decrease in mean cell volume (-2%) and mean cell hemoglobin (-3%) was also observed at the end of the treatment period.
These effects were considered to be of no toxicological importance, as they were poorly dose-related, of minimal magnitude, did not correlate with any histopathological findings and as values remained within the range of historical control values.

The other statistically significant differences between control and test item-treated animals at the end of the treatment period, namely in large unstained cell count (males given 300 or 600 mg/kg/day) and platelet count (females given 300 mg/kg/day) were considered to be of no toxicological importance as they were isolated, of low magnitude and/or noted with no dose-relationship.

At the end of the treatment-free period, shortened prothrombin time (25.1 s vs. 27.2 s in controls, p<0.05) was recorded in females previously given 600 mg/kg/day. This difference was considered not to be test item-related as no variations had been observed at the end of the treatment period, and the value was close to the control values recorded at the end of the treatment period (26.6 s) and within the range of physiological values.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period, blood biochemistry parameters were considered to be unaffected by the test item.

When compared with mean control values, statistically significant blood biochemistry changes were noted:
- lower mean chloride level in males and females at 600 mg/kg/day (-1%),
- lower mean glucose level in males at 100, 300 and 600 mg/kg/day (-12%, -11% and -14%, respectively)
- higher mean urea level in males at 300 and 600 mg/kg/day (+15%),
- lower mean total bilirubin level in females at 100, 300 and 600 mg/kg/day (-61%, -74% and -55%, respectively).
All the above statistically significant changes were considered to be of no toxicological importance as they were poorly dose-related, of minimal magnitude, with values similar to control recovery values, reversible and/or within the range of historical control values.

The other statistically significant differences observed between control and test item-treated animals, namely in the potassium (males at 100 mg/kg/day), inorganic phosphorus (females at 100 mg/kg/day), glucose (females at 100 and 300 mg/kg/day) and triglyceride (females at 1000 mg/kg/day) levels were considered to be incidental and not test item-related as they were noted with no dose-relationship or without any link to microscopic findings.
At the end of the treatment-free period, no relevant differences from controls were observed in the previous test item-treated groups.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
See Table 5.
At urinary investigations, no test item-related effects were observed at the end of the treatment period.

At 300 and 600 mg/kg/day in males, when compared with controls, statistically significantly lower mean pH values were observed (-11% and -14%, respectively). These differences were of low magnitude, within the range of the HCD, and did not correlate with any other urinary changes, therefore they were considered to be of no toxicological importance.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
See Table 2.
There were no test item-related effects on functional observation battery tests or motor activity data in any group.

Higher mean numbers of horizontal movements and rearing were noted in males at 300 and 600 mg/kg/day; they were considered to be of no toxicological importance as they were of minor magnitude (not statistically significant), poorly dose-related and/or as values remained within the standard deviation of control values.
No differences from controls were noted in the motor activity of test item-treated females.
Differences from controls in landing foot splay were noted in males and females at 600 mg/kg/day (69 mm and 88 mm vs. 79 mm and 99 mm in control males and females, respectively). In view of the very slight magnitude, and in the absence of correlating clinical signs during the study, this finding was considered to be unrelated to the test item treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
See Table 7.
End of treatment period
Mean final body weight was decreased in the high-dose male group (-10%, p=0.01).

There were some minimal but noteworthy differences between treated and control groups.

The absolute and relative kidney weights were increased in a dose related manner in male treated groups from the dose of 300 mg/kg/day. At the high-dose, it was associated with increased hyaline droplets, focal/multifocal tubular basophilia and hyaline casts at histological examination. In females, a minimal but statistically significant increase in relative value was observed at the high-dose only. Given the absence of histological correlate this difference of low magnitude was considered fortuitous.
Higher adrenal weights were recorded in females at all doses of test item. These increases were unrelated to dose, not statistically significant and with no histological correlate. They were considered unrelated to treatment with the test item.
Liver weights were minimally increased in high-dose females, reaching statistical significance in relative values. There was no histological correlate. This minor difference is considered unrelated to test item administration.

End of recovery period
Kidney weights in males previously treated at the dose of 600 mg/kg/day were increased compared to controls, +9 and +10%, p=0.05, in absolute and relative to body weight ratio, respectively. Like at the end of treatment, it was associated with renal histological changes.
All other differences between treated and controls were considered fortuitous and bore no relationship to treatment.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See Table 8.
End of treatment period
At the end of treatment period, white discolorations of the forestomach were observed from the dose of 300 mg/kg/day in females and at 600 mg/kg/day in males.

These white discolorations generally correlated histologically with acanthosis/hyperkeratosis, on occasion accompanied by inflammatory infiltrates in the submucosa and/or ulceration.

All other macroscopic observations belonged to the spectrum of spontaneous findings in rats of this age and strain.

End of recovery period
At the end of recovery, one female previously treated at the high-dose had white discoloration of the stomach, correlating to a focal ulceration of the non-glandular stomach. Although such an observation is on occasion made in control rats, given the context in this study, a relationship to treatment cannot be excluded.

All other macroscopic observations were spontaneous and bore no relationship to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See Tables 9 and 10.
End of treatment period
There were test item-related changes in the stomach, kidneys (males only), and possibly vagina.

Minimal to moderate increases in hyaline droplets in the kidney was diagnosed in males at all dose levels. The severity was dose-related. Hyaline droplets are composed of alpha 2u-globulin, and this was confirmed by IHC. The average score for IHC positivity in high-dose rats was 2.6 vs. 1.8 in controls, confirming the increase noted in hematoxylin and eosin-stained slides. Hyaline droplets were associated with granular casts (from 300 mg/kg/day) and tubular basophilia (all doses). Together, these findings, which were directly related to the test item, are part of the so-called alpha 2u-globulin nephropathy, which is specific to male rats and irrelevant to human risk assessment. Nevertheless, in the context of this study, given the severity and the presence of hyaline casts, it was considered adverse from the dose of 300 mg/kg/day.

Acanthosis/hyperkeratosis was observed in the forestomach from the dose of 100 mg/kg/day in both sexes. On rare occasions, it was associated with ulceration and/or inflammatory cell infiltration in the submucosa. A direct irritant effect of the test item on the forestomach (a structure absent in humans) cannot be excluded. However, such an observation is also observed under stressful conditions. In any case, given the low severity, these gastric changes are not considered as adverse.

Mucification of vaginal mucosa was observed in 3/10 high-dose, 2/10 mid-dose and 1/10 low-dose females. Other females in these groups displayed a normal distribution of estrous cycle assessed histologically. With the exception of one high-dose female, all these rats had prolonged diestrus. However, prolonged diestrus was also observed in two control rats without vaginal mucification. The mean duration of diestrus was only marginally increased at the high-dose, without statistical significance. Overall, vaginal mucification is possibly related to treatment at the high-dose only, and could represent an indirect non-adverse effect of the test item.

All other changes belonged to the spectrum of spontaneous pathology for this strain and age, and bore no relationship to treatment.

End of recovery period
The same spectrum of renal and gastric changes were observed in recovery animals, with the exception of increased hyaline droplets, which was no longer present in high dose males, indicating full recovery.
Other renal changes (tubular basophilia and hyaline casts), as well as acanthosis/hyperkeratosis of the non-glandular part of the stomach, were present with a similar incidence compared to the end of treatment period, but a lower severity, indicating ongoing recovery.
None of the treated females had modification of the vagina.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Details on results:
See Table 4.
Estrus cycle
There were no statistically significant test item-related effects on the mean length of the estrus cycle or the mean number of cycles. A trend towards an increase in the mean length of diestrus was observed in females given 600 mg/kg/day at the end of the treatment period. This was due to two high-dose females for which the diestrus period represented 13 to 14 days.
This was considered to be of minor importance in the absence of statistical significance and as microscopic findings (marked mucification of vaginal mucosa for the same two females) were considered to be an indirect, non-adverse effect of the test item and as these variations were no longer observed at the end of the treatment-free period.
Other differences observed in the diestrus duration for females given 100 or 300 mg/kg/day (i.e. 8.4 and 8.5 days vs. 7.0 days in controls) were considered to be of no toxicological importance as there was no clear relationship between the cycle length and the severity of the vaginal mucosa mucification.

Seminology
See table 6.
No test item-related effects were noted on testicular sperm count, or on epididymal sperm count, motility or morphology.

The lower values in the epididymal (only statistically significant at 300 mg/kg/day) and/or testicular sperm counts recorded in males given 300 or 600 mg/kg/day were considered to be of no toxicological importance as they resulted from slightly higher mean control values (due to one male: 162.4 10E6/g of testis and 26.6 10E6/g of testis/day), as most of individual values were within the standard deviation of the control group, and/or as differences were of minor magnitude and/or not dose-related.
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Table 1: Clinical signs

Sex

Male

Female

Dose level (mg/kg/day)

0

100

300

600

0

100

300

600

Ptyalism

3

6

9

15

1

2

3

14

Reflux at administration

3

-

1

1

-

1

1

-

Total affected animals

5/15

6/10

9/9

15/15

1/15

3/10

4/10

14/15

-: no clinical signs.

Table 2: Motor activity

 

Sex

Male

Female

Dose level (mg/kg/day)

0

100

300

600

0

100

300

600

Horizontal movements

399

397

508

502

722

672

573

722

% from controls

-

-1

+27

+26

-

-7

-21

0

Rearing

98

99

132

137

165

171

145

167

% from controls

-

+1

+35

+40

-

+4

-12

+1

-: not applicable.

Table 3: Body weight

 

 

Sex

Male

Female

Dose level (mg/kg/day)

0

100

300

600

0

100

300

600

 

Treatment period

 

Mean BW gain Weeks 1/3

+141

+136

+141

+137

+53

+48

+52

+53

 

Mean BW gain Weeks 3/13

+274

+275

+250

+235**

+90

+91

+89

+89

 

Mean BW gain Weeks 1/13

+415

+412

+391

+372**

+143

+140

+141

+142

 

% from controls

-

-1

-6

-10

-

-2

-1

-1

 

Mean body weight Week 1

204

202

200

199

172

169

172

169

 

Mean body weight Week 4

401

392

393

385*

239

233

236

232

 

Mean body weight Week 13

619

613

591

571**

315

308

313

311

 

% from controls

-

-1

-5

-8

-

-2

-1

-1

 

Treatment-free period

 

Mean BW gain Weeks 13/19

+33

-

-

+58*

+15

-

-

+14

 

Mean BW Week 19

635

-

-

628

326

-

-

318

 

% from controls

-

-

-

-1

-

-

-

-2

 

Statistically significant from controls: *: p<0.05; **: p<0.01; -: not applicable.

Table 4: Estrus cycle

 

Dose level (mg/kg/day)

0

100

300

600

Treatment period

 

 

 

 

Number of cycles

3.8

3.4

3.7

2.9

Cycle length (days)

4.3

4.2

4.6

5.1

Number of females having a mean
average cycle of 4-5 days

8

8

6

7

Number of days of diestrus

7.0

(2)

8.4

(3)

8.5

(3)

9.2

(4)

Treatment-free period

 

 

 

 

Number of cycles

2.8

/

/

2.6

Cycle length (days)

3.9

/

/

3.8

Number of females having a mean
average cycle of 4-5 days

3

/

/

3

Number of days of diestrus

3.8

/

/

3.4

/: not applicable.

( ): number of females with diestrus length =10 days.

Table 5: Urinalysis

 

Sex

Male

Female

Dose level (mg/kg/day)

0

100

300

600

0

100

300

600

End of treatment period

pH

7.4
(6.0-8.0)

6.9
-7%

6.6**
-11%

6.4**
-14%

6.5
(6.0-7.0)

6.7
+3%

6.6
+2%

6.2
-5%

Statistically significantfrom controls:**: p<0.01.

( ): minimum-maximum values from Historical Control Data (HCD).

Table 6: Seminology

 

Dose level (mg/kg/day)

0a

100

300

600

% of motile epididymal sperm

87.4

90.8

94.6

96.7

% of morphologically normal epididymal sperm

97.4

/

/

97.4

Mean number of epididymal sperm (106/cauda)

% from controls

185.1

/

180.5

-2

169.0

-9

170.3

-8

Mean number of epididymal sperm (106/g cauda)

% from controls

601.5

/

613.7

+2

561.3

-7

554.0

-8

Mean number of testicular sperm heads (106/g testis)

% from controls

117.6

/

105.5

-10

98.1*

-17

101.3

-14

Daily sperm production rate (106/g testis/day)

% from controls

19.3

/

17.3

-10

16.1*

-17

16.6

-14

/ : not applicable. Statistically significant from controls: *: p<0.05.

Table 7: Organ weights

 

Selected differences in organ weights (% from controls)

(End of treatment)

Gender

Males

Females

Group

2

3

4

2

3

4

Dose (mg/kg/day)

100

300

600

100

300

600

Concentration (mg/mL)

20

60

120

20

60

120

Number of animals

10M

9M

10M

10F

10F

10F

Final body weight

-2

-6

-10##

-3

-2

-2

Adrenal glands

 

. Absolute (%)

-4

-7

-1

+14

+13

+12

. Relative to body weight (%)

-2

-1

+9

+17

+15

+13

Kidney

 

. Absolute (%)

+2

+7

+12*

+5

+4

+8

. Relative to body weight (%)

+4

+14#

+23##

+8

+6

+10#

Liver

 

. Absolute (%)

-8

-10

-5

+4

+7

+10

. Relative to body weight (%)

-6

-4

+5

+8

+9

+12**

               #: p=0.05; ##: p=0.01 (Dunn’s test); *: p=0.05; **: p=0.01 (Dunnett’s test) (based on actual values and not on the percentages presented in the table).

Table 8: Macroscopic examination

 

Incidence of macroscopic findings on the forestomach

(End of treatment)

Gender

Males

Females

Group

1

2

3

4

1

2

3

4

Dose (mg/kg/day)

0

100

300

600

0

100

300

600

Concentration (mg/mL)

0

20

60

120

0

20

60

120

Number of animals

10M

10M

9M

10M

10F

10F

10F

10F

Forestomach

 

. White discoloration

0

0

0

7

0

0

2

1

 

Table 9: Microscopic examination (end of treatment period)

 

Incidence and severity of selected microscopic findings

(End of treatment)

Gender

Males

Females

Group

1

2

3

4

1

2

3

4

Dose (mg/kg)

0

100

300

600

0

100

300

600

Concentration (mg/mL)

0

20

60

120

0

20

60

120

Number of animals

10M

10M

9M

10M

10F

10F

10F

10F

Kidney

 

Tubular basophilia

 

. Minimal

2

4

5

3

1

-

-

0

. Slight

1

0

4

6

0

-

-

0

. Moderate

0

0

0

1

0

-

-

0

Increased hyaline droplets

 

. Minimal

0

6

1

1

0

-

-

0

. Slight

0

3

6

4

0

-

-

0

. Moderate

0

1

2

5

0

-

-

0

Cast, hyaline

 

. Minimal

0

0

3

0

0

-

-

0

. Slight

0

0

1

6

0

-

-

0

. Moderate

0

0

0

2

0

-

-

0

Stomach

 

Acanthosis/hyperkeratosis

 

. Minimal

0

1

1

0

0

1

4

3

. Slight

0

0

0

5

0

0

0

1

. Moderate

0

0

0

5

0

0

0

0

Vagina

 

Mucification, epithelium

 

. Minimal

-

-

-

-

0

0

1

0

. Moderate

-

-

-

-

0

1

0

1

. Marked

-

-

-

-

0

0

1

2

Table 10: Microscopic examination (end of recovery period)

Incidence and severity of selected microscopic findings

(End of recovery)

Gender

Males

Females

Group

1

2

3

4

1

2

3

4

Dose (mg/kg/day)

0

100

300

600

0

100

300

600

Concentration (mg/mL)

0

20

60

120

0

20

60

120

Number of animals

5M

-

-

5M

5F

-

-

5F

Kidney

 

Tubular basophilia

 

. Minimal

0

-

-

4

-

-

-

-

Increased hyaline droplets

 

. Minimal

1

-

-

0

-

-

-

-

Cast, hyaline

 

. Minimal

0

-

-

3

-

-

-

-

. Slight

0

-

-

1

-

-

-

-

Stomach

 

Acanthosis/hyperkeratosis

 

. Minimal

0

-

-

3

0

-

-

4

Conclusions:
The toxicity of the test item was evaluated after daily oral administration (gavage) to Sprague-Dawley rats at dose levels of 100, 300 or 600 mg/kg/day for 13 weeks followed by a 6-week treatment-free period.
Under the experimental conditions of the study, from the dose level of 300 mg/kg/day, adverse test item related effects were observed in the kidneys of male rats: a2u-globulin accumulation (as confirmed by immunohistochemistry). This is considered to be a rat specific effect with no relevance for human risk assessment. No other adverse effects were observed in the study.
Consequently, the NOAEL (No Observed Adverse Effect Level) was established at 600 mg/kg/day in females and males if we exclude the adverse lesions seen in male kidneys (a2u-globulin protein nephropathy-related findings) which are specific to male rats and have no relevance for human risk assessment.
Executive summary:

The potential toxicity of Luperox TBEC was evaluated following daily oral administration (gavage) to rats for 13 weeks. On completion of the treatment period, designated animals were held for a 6-week treatment-free period in order to evaluate the reversibility of any findings. This GLP study was carried out according to OECD test guideline No. 408 (21 September 1998).

One group of 15 males and 15 females Sprague-Dawley rats was treated daily by the oral route (gavage) with the test item, at the dose level of 600 mg/kg/day (group 4) for 13 weeks. Two other groups of 10 males and 10 females were treated with the test item at the dose level of 100 or 300 mg/kg/day (groups 2 and 3, respectively). One control group of 15 males and 15 females received the vehicle only (corn oil) under the same experimental conditions, and acted as a control group (group 1). A constant dosage volume of 5 mL/kg/day was used. At the end of the treatment period, the animals were euthanized, except for the first five group 1 and 4 animals per sex, which were kept for a 6-week treatment-free period. The actual test item concentrations in the dose formulations prepared for use in Weeks 1, 4, 8 and 13 were determined using a High Performance Liquid Chromatography with tandem Mass Spectrometry detection method (LC/MS-MS). The animals were checked at least once daily for mortality and clinical signs. Detailed clinical examinations were performed weekly and a Functional Observation Battery (FOB) was conducted in Week 12. Body weight was recorded pre-test, on the first day of treatment and then once a week. Food consumption was recorded weekly. Ophthalmological examinations were performed on all animals before the beginning of the treatment period and on control and high-dose animals at the end of the treatment period (Week 13). Hematology, blood biochemistry and urinary investigations were performed on all animals euthanized at the end of the treatment period (Week 13). Hematology (females) and blood biochemistry (both sexes) were also determined at the end of treatment-free period (Week 20). Additional blood samples were collected in Weeks 13 and 20 for possible analysis of thyroid hormone levels. The estrous cycle was determined over 21 or 14 consecutive days for all females at the end of the treatment or treatment-free period, respectively. At the end of the treatment period, seminological investigations (count and motility) were performed on all males, and sperm morphology was determined in control and high-dose males. On completion of the treatment or treatment-free period, the animals were euthanized and a full macroscopic post-mortem examination was performed. Designated organs were weighed and selected tissues were preserved. A microscopic examination (including a detailed examination of the testes) was performed on designated tissues from control and high-dose animals euthanized at the end of the treatment period and from animals that were euthanized prematurely, and on all macroscopic lesions from low- and intermediate-dose animals (groups 2 and 3) euthanized on completion of the treatment period. A microscopic examination was also performed on kidney slides (immunostained with an antibody for a2u-globulin protein) from all control and high-dose males euthanized at the end of the treatment period. The stomach with forestomach (both sexes), kidneys (males) and vagina (females) were also microscopically examined for low- and intermediate-dose animals (groups 2 and 3) euthanized at the end of the treatment period, and for the recovery animals (groups 1 and 4) as changes were noted in these organs at the end of the treatment period.

Actual concentrations of the test item in the dose formulations administered to the animals during the study remained within an acceptable range (-6.3% to +8.9%) compared to the nominal concentrations.

There were no test item-related unscheduled deaths in any group. Ptyalism was observed in controls and at all dose levels with a dose-related incidence. This sign was considered to be non-adverse The FOB results were unaffected by the test item treatment. Lower body weight gain was recorded throughout the treatment period in males given 600 mg/kg/day (-10% vs. controls) leading to a minimally lower body weight on completion of the treatment period (-8% vs. controls). As these differences were of low magnitude, not associated with any other relevant findings and reversible at the end of the treatment-free period, they were considered to be non-adverse. Food consumption was not affected by the test item treatment. No ophthalmological findings were observed at the end of the treatment period. A non-adverse slight increase in the length of diestrus was observed in females given 600 mg/kg/day at the end of the treatment period. No variations were observed at the end of the treatment-free period. The epididymal sperm motility and morphology, and the testicular and epididymal spermatozoa count were unaffected by the test item treatment. At hematology and blood biochemistry investigations, all changes were considered to be of no toxicological importance. At urinary investigations, no test item-related effects were observed at the end of the treatment period. At pathology investigations, the alpha 2u-globulin nephropathy observed in male rats at all dose levels was considered adverse from 300 mg/kg/day, but specific to male rats and irrelevant for human risk assessment. In addition, there was non-adverse acanthosis/hyperkeratosis in the forestomach (both sexes, all doses), possibly related to a local irritant effect of the test item, and vaginal mucification in a few high-dose females which was considered possibly test item-related but non-adverse effect.

At the end of the 6-week treatment-free period, full recovery was observed for vaginal mucification, and ongoing recovery was observed for the alpha 2u-globulin nephropathy in males and for acanthosis/hyperkeratosis of the stomach. 

The toxicity of Luperox TBEC was evaluated after daily oral administration (gavage) to Sprague-Dawley rats at dose levels of 100, 300 or 600 mg/kg/day for 13 weeks followed by a 6-week treatment-free period. Under the experimental conditions of the study, from the dose level of 300 mg/kg/day, adverse test item-related effects were observed in the kidneys of male rats: a2u-globulin accumulation (as confirmed by immunohistochemistry). This is considered to be a rat specific effect with no relevance for human risk assessment. No other adverse effects were observed in the study.

Consequently, the NOAEL (No Observed Adverse Effect Level) was established at 600 mg/kg/day in females and males if we exclude the adverse lesions seen in male kidneys (a2u-globulin protein nephropathy-related findings) which are specific to male rats and have no relevance for human risk assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Type of method:
in vivo

Test material

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

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Control animals:
yes, concurrent vehicle
Details on study design:
SPERM PARAMETERS:
Before euthanasia at the end of the treatment period, each male was anesthetized by an intraperitoneal injection of sodium pentobarbital.
As no relevant changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.

Epididymal sperm
Under deep anesthesia and after weighing the epididymis, sperm from the cauda of the left epididymis was collected for motility and morphology investigations. Animals were then euthanized.
The cauda of the left epididymis was separated from the corpus using a scalpel and subsequently kept at 20°C pending further investigation.

Epididymal sperm motility
The sperm was evaluated on a slide, after appropriate dilution if required. The numbers of motile and immotile spermatozoa in a sample of 200 spermatozoa were evaluated under a microscope using a 40 fold magnification. Results were expressed as the proportions of motile and non-motile spermatozoa.

Epididymal sperm morphology
Morphology was determined from a sperm smear, after eosin staining and counting of 100 spermatozoa per slide. This was evaluated for groups 1 and 4 in the first instance.
In view of the findings observed in these groups at the end of the treatment period, this determination was not extended to groups 2 and 3 or recovery animals.

Results were expressed as the proportion of spermatozoa in each of the following categories:
- normal,
- normally shaped head separated from flagellum,
- abnormal head separated from flagellum,
- abnormal head with normal flagellum,
- abnormal head with abnormal flagellum,
- normally shaped head with abnormal flagellum.

Epididymal sperm count
After thawing, the left cauda epididymis was weighed, minced and homogenized in a saline-triton solution using a Polytron.
An aliquot of the suspension was collected and the number of spermatozoa was counted in a microscope slide counting chamber.
Results were expressed as the numbers of spermatozoa per cauda and per gram of cauda.

Testicular sperm
The left testis was collected and kept at -20°C for further sperm count investigation. After thawing, the left testis was weighed and ground. The resulting preparation was diluted and sperm heads resistant to homogeneization (i.e. elongated spermatids and mature spermatozoa) were counted in a microscope slide counting chamber.
Results were expressed as the number of sperm heads per gram of testis, and the daily sperm production rate was calculated (using a time divisor of 6.10).

MONITORING OF ESTROUS CYCLE:
The estrus cycle stage was determined for each female euthanized at the end of the treatment period, from a fresh vaginal lavage (stained with methylene blue), daily for 21 consecutive days before the end of the treatment period.
In view of the findings observed at the end of the treatment period, this examination was carried out daily for 14 consecutive days before the end of the treatment-free period.

ORGAN WEIGHTS:
Epididymides, Ovaries (including oviducts), Testes, Uterus (horns and cervix).

HISTOPATHOLOGY:
Epididymides, Ovaries (including oviducts), Prostate (dorso-lateral, and ventral), Seminal vesicles (including coagulation gland), Testes, Uterus (horns and cervix), Vagina.

Results and discussion

Effect levels

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

Applicant's summary and conclusion

Executive summary:

In the OECD 408 study (Papineau, 2018), the estrous cycle was determined over 21 or 14 consecutive days for all females at the end of the treatment or treatment-free period, respectively. At the end of the treatment period, seminological investigations (count and motility) were performed on all males, and sperm morphology was determined in control and high-dose males. On completion of the treatment, epididymides, ovaries (including oviducts), testes and uterus (horns and cervix) were weighed. A microscopic examination was performed on epididymides, ovaries, prostate , seminal vesicles (including coagulation gland), testes, uterus and vagina from control and high-dose animals euthanized at the end of the treatment period.

A non-adverse slight increase in the length of diestrus was observed in females given 600 mg/kg/day at the end of the treatment period. No variations were observed at the end of the treatment-free period. The epididymal sperm motility and morphology, and the testicular and epididymal spermatozoa count were unaffected by the test item treatment. At pathology investigations, there was no adverse effect on the male and female reproductive organs.