Registration Dossier

Administrative data

Description of key information

OECD 408 study

The potential toxicity of Luperox TBEC was evaluated following daily oral administration (gavage) to rats for 13 weeks (Papineau, 2018). 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 macroscopicpost-mortemexamination 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.

OECD 407 study

The potential toxicity of TBEC was evaluated in a repeated dose study performed following to OECD Guideline 407 (July 27th1995) (Cerven, 1999). TBEC was administered once daily by gavage to Sprague-Dawley rats (5 Males and 5 Females), at the dose-levels of 0, 150, 550 and 1000 mg/kg/d during 28 days (but animals were not dosed the 20 th day). Body weights were recorded pretest, weekly, at death and at termination in the survivors. The animals were observed once daily for toxicity and pharmacological effects and twice daily for morbidity and mortality. Food consumption was calculated weekly. A Functional Observational Battery examination, designed to assess specific neurotoxicity and behavioral changes, was conducted on days 23 and 26. On day 29, all surviving animals were anesthetized with ether and exsanguinated. Whole blood, plasma and serum were analyzed for selected hematology and clinical chemistry parameters. All animals were examined for gross pathology. All the tissues were preserved in 10% Neutral Buffered Formalin.

Two females dosed at 550 mg/kg and two females dosed at 1000 mg/kg died during the study. Pathological examination revealed abnormalities consistent with inadvertent gavage accidents and were not attributed to any toxic effect of the test article. Instances of abnormal systemic signs were minimal. There were no significant differences in mean body weights, food consumption, functional observational battery parameters, and organ weights. Instances of significantly different mean hematology, clinical chemistry and organ/body weight ratios were noted, but there was no evidence of dose-response effects and these instances are not considered biologically significant. Only the mean blood albumin was significantly less in the control group than in mid and high dose group. This was considered biologically unremarkable, since there were no related findings in any other liver or kidney enzymes nor any histological changes. Necropsy results of surviving animals were generally normal. Microscopic examination revealed no treatment related microscopic changes in any of the animals dosed at 150 mg/kg/day. Treatment-related changes were observed in kidneys of male rats in the high and mid dose groups and in the stomach of male and female rats in the high and mid dose groups. The incidence and severity of the treatment-related changes occurred in a dose-related manner. The kidney pathology noted in the male rats was not considered to be relevant to human exposure since the enzyme system responsible for the hyaline droplet formation are unique to the male rat.

In conclusion, the 28 day repeated oral administration (27 doses) of Luperox TBEC in rats produced no clinical effects but did produce microscopic changes at doses of 550 and 1000 mg/kg/day. The observed pathologic changes were noted in the stomach of male and female rats and in the kidneys of male rats only. The pathologic changes noted in the stomach are considered to be secondary to a local irritating effect and therefore of low relevance for hazard assessment. The kidney pathology noted in the male rats is not considered to be relevant to human exposure since the enzyme system responsible for the hyaline droplet formation are unique to the male rat. Therefore, the no observed effect level (NOEL) was estimated to be 150 mg/kg bw/day based on forestomach effects. In addition, the systemic NOAEL can be estimated at 1000 mg/kg bw/day in male and female rats.


Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Preservation of testes in formalin; animals not dosed on day 20 due to a lack of test material
GLP compliance:
yes (incl. certificate)
Remarks:
The range finding study was not GLP compliant.
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
not specified
Details on test animals and environmental conditions:
Wistar albino rats were received from Ace Animals, Boyertown. PA on 5/11/99.
The pretest body weight range for males was 176- 213 grams and for females 142- 165 grams
Housing: 1/cage in suspended stainless steel wire bottom cages and individually identification by a uniquely numbered eartag. The individual cages were identified with a cage card indicating the MB project number, test article, dose level, date of study initiation, animal number and sex.
Food and water were provided ad libitum.
Animal rool was temperature controlled (68- 72°F) with a humidity range of 53- 70%, and had a 12-hour light dark cycle.
Route of administration:
oral: gavage
Vehicle:
other: mineral oil (USP)
Remarks:
Lot #77H1380
Details on oral exposure:
2 mL/Kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A suspension/emulsion of the test article (TBEC) in mineral oil was prepared daily. Analyse of the samples: at least once per week, in another laboratory.
Duration of treatment / exposure:
28 days (excepting day 20: no treatment)
Frequency of treatment:
once daily
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
group 4
Dose / conc.:
550 mg/kg bw/day (actual dose received)
Remarks:
group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
group 2
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
The animals were dosed 27 days instead of 28 days because of material lacking (the animals were not dosed on day 20)

Positive control:
No
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
All animals were observed once daily for toxicity and pharmacological effects and twice daily for morbidity and mortality.

BODY WEIGHT: Yes
Body weights were recorded on the day following receipt, at the end of the first week of equilibration, immediately pretest, weekly, at death and at termination in the survivors.

FOOD CONSUMPTION :
Measured amounts of diet were presented weekly. The amount consumed was calculated each week.

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 29
- Anaesthetic used for blood collection: Yes (ether + exsanguination)
- How many animals: all surviving animals
- Parameters checked:hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration, white blood cell count, differential leukocyte count, platelet count and reticulocyte count

CLINICAL CHEMISTRY: Yes (all surviving animals)
Parameters checked: magnesium, sodium, alanine aminotransferase, sorbitol dehydrogenase, urea nitrogen, total bilirubin, calcium, phosphorous, aspartate aminotransferase, albumin, globulin, total cholesterol, triglycerides, chloride, potassium, alkaline phosphatase, gamma glutamyl transpeptidase, creatinine, glucose (fasting), total protein.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
On days 23 and 26, a Functional Observational Battery test, designed to assess specifie neurotoxicity and neurobehavioral changes, was conducted in the surviving animals.
Sacrifice and pathology:
On day 29, all animals were humanely sacrificed using ether and exsanguination. Each animal underwent a gross necropsy which included examination of the external surfaces of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
GROSS PATHOLOGY: Yes

The following tissues and organs were preserved in 10% neutral buffered formalin:
Digestive System: Esophagus, Stomach, Duodenum, Jejunum, Ileum,Cecum, Colon, Liver
Glandular System: Adrenais Thyroids/Parathyroids Pancreas
Respiratory System:Lung & Trachea
Nervous System: Brain (multiple sections), Pituitary, Peripheral Nerves(s), Spinal cord (3 levels)
Cardiovascular/hematopoietic System: Aorta (thoracic): Heart, Bone marrow (sternal), Lymph nodes, Spleen, Thymus
Urogenital System: Kidneys, Urinary bladder, Testes and accessory sex organs, Uterus, Ovaries Prostate
Other: All gross lesions and masses, Animal Eartag, Skin

All preserved tissues from the animals in control group and high dose, and tissues from the two animals of medium dose group which died during the study, were examined microscopically

HISTOPATHOLOGY: Yes
The preserved tissues were routinely processed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for the microscopic evaluation. The tissues specified for microscopic evaluation from all male and female rats of the control (Group 1) and high dose (Group 2) groups and nonsurviving rats of the intermediate groups included: adrenal glands, aorta, bane marrow (sternum), brain (three sections), cecum, colon, duodenum, epididymides, oesophagus, heart, ileum, jejunum, kidneys, liver, lung, mesenteric lymph node, sciatic nerve, ovaries, pancreas, parathyroid, pituitary, prostate, seminal vesicles, skin, spinal cord, (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus, and other tissues with gross lesions. Microscopic examination of the surviving rats of the intermediate dosage groups was limited to the stomach (male and female rats) and kidneys (male rats only). ln addition, sections of the kidneys stained with the Mallory-Heidenhain technique for the identification of hyaline droplets were prepared and examined from all male rats of all groups.

Statistics:
An Analysis of Variance (ANOVA) was performed on absolute organ weights, body weights and organ/body weight ratios. Parametric data was analyzed using ANOVA techniques with the Tukey-Kramer post hoc test. Non-parametric data was analyzed using Kruskai-Wallis analysis of variance with Dunn's post hoc test. lnstat® Version 2.0 software was used for statistical analysis.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The mean hemoglobin concentration of males in high dosed group2 was significantly less than the controls (-7%). There were no significant differences noted between the dosed groups and controls in all other hematological parameters.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
stomach (both sexes); kidney (males only)
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
One female (#B9661) dosed at 1000 mg/kg died on day 8 with predeath physical signs of chromorhinorrhea, dyspnea and wetness of the nose/mouth area. Another female (#B9657) dosed at 1000 mg/kg died on day 22 with predeath physical signs of lethargy, dyspnea, emaciation, wetness of the nose/mouth area, yellow staining of the anogenital area and brown staining of the nose/mouth area. One female (#B9670) dosed at 550 mg/kg died on day 10 with no abnormal predeath physical signs and another female (#B9671) dosed at 550 mg/kg died on day 23 with predeath physical signs of few feces, piloerection, lethargy, sagging eyelids, soiling of the anogenital area, flaccid muscle tone and dyspnea.
Histopathology showed that the premature deaths in all of these animals is attributed to inadvertent gavage dosing accidents.

A summary of the systemic signs noted in the survivors for each group are as follows:
Group 1: Six animals appeared normal throughout the study. One instance of dyspnea was noted in animal #89646-M on day 17 and one instance of chromorhinorrhea was noted in animal #B9644-M on day 27. Two instances of chromorhinorrhea were noted in animal #B9649-F on days 17 and 18. Additionally, animal #B9650-F appeared emaciated on days 17 and 18 and had dyspnea on day 17.

Group 2: Five surviving animals appeared normal throughout the study. One instance of diarrhea was noted in animal #89654-M on day 4 and in animal #B89659-F on day 8. Chromorhinorrhea was also noted in animal #B9659-F on day 17. Chromorhinorrhea, dyspnea and wetness of the nose/mouth area was noted in animal #B9660-F on day 17 and dyspnea was noted on day 18.

Group 3: Three surviving animals appeared normal throughout the study. One instance of chromorhinorrhea was noted in two animals #89663-M on day 9 and #B9666-M on day 22. Animal #B9662-M had instances of chromorhinorrhea on days 4 and 13 and animal #B9664-M had instances of chromorhinorrhea on days 16 and 17, as well as few feces on day 18. Dyspnea, lethargy and wetness of the nose/mouth area were noted in animal #B9667-F on day 23.

Group 4: Five animals appeared normal throughout the study. Instances of chromorhinorrhea were noted in three animals (#B9674-M on days 17, 18, 22, 23 & 27; #B9676-M on day 18, and #B9673-M on days 24 and 25). Diarrhea was noted in animal #B9675-M on day 23 only. Dyspnea and/or wetness of the nose/mouth was noted in animal #B9677-F on days 17 and 18.

BODY WEIGHT AND WEIGHT GAIN
There were no instances of weight loss noted in any surviving animal or in the three of the four animals which died during the study. Animal #B9657-F which died on day 22 showed weight loss prior to death.

FOOD CONSUMPTION
There were no significant differences in food consumption between groups

HAEMATOLOGY
The mean hemoglobin concentration of males in high dose group was significantly less (p<0.05) than the controls (Table 1). There were no significant differences noted between the dosed groups and controls in all other hematological parameters.

CLINICAL CHEMISTRY
There were significant differences noted between group means in the following clinical chemistry parameters (ANOVA with Dunn's Multiple Comparison Post Hoc Test):
Chloride, males, (control group vs low and medium dose group)
Glucose, females, (ANOVA significant but no significance with Dunn's Multiple Comparison)
Albumin, females, (control group medium and high dose group)
Total Protein, females, (control group vs medium dose group)
There were additional significant differences between dose groups which did not occur in a dose-related manner and were deemed statistically irrelevant.

NEUROBEHAVIOUR - FOB
There were no significant differences between group means in the Orientation/Sensory Responsiveness, Posture, Locomotive/Patterned Movement and Integrated Movements subtotals as well as the summed scores. lndividual parameters were analyzed using non-parametric statistics (Kruskal­ Wallis, non-parametric ANOVA); whereas the summed scores were analyzed using parametric ANOVA techniques.

ORGAN WEIGHTS
There were no significant differences in organ weights between groups. Significantly larger liver/body weight ratios were seen in high dose females when compared to control females, it is secondary to a lower terminal body weight. No other significant differences were found in organ weights or in organ/body weight ratios.

GROSS PATHOLOGY
Control: All males and three out of five females appeared normal at necropsy. Dark areas on the thymus were noted in two female animais (#B9649 and #B9650).

High dose group: All surviving animals appeared normal at necropsy. Animal #B9657-F which died on day 22 revealed abnormalities of the liver and kidneys, as well as brown staining of the nose/moth and anogenital area and soiling of the anogenital area. Animal #B9661-F which died on day 8 revealed abnormalities of the lungs, liver, intestines and pleural cavity, as well as wetness of the nose/mouth and anogenital areas.

Mid dose group: Seven of eight survivors appeared normal at necropsy. Red areas on the thymus were noted in one female #B9669. Animal #B9670-F which died on day 10 revealed abnormalities of the liver and peritoneal cavity, as well as wetness of the anogenital area. Animal #B9671-F which died on day 23 revealed abnormalities of the lungs, liver, kidneys, pleural cavity and gastrointestinal tract, as well as wetness of the nose/mouth and anogenital areas.

Low dose group: All animals appeared normal at necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related microscopie changes observed in any of the organs and tissues examined from the male and female rats at the 150 mg/kg level.

Treatment-related microscopic changes were observed in the stomach of male and female rats of high and mid dose groups (Table 2) and in the kidneys of male rats of high and mid dose group. Microscopically, the treatment-related change in the stomach consisted of moderate to marked thickening of the squamous mucosa of the nonglandular area of the stomach due to increased hyperplasia and hyperkeratosis of the epithelial mucosa. There was also an extensive acute inflammation involving both the mucosa and submucosa of the nonglandular area with edema and mostly polymorphonuclear inflammatory cell infiltrations. One high dose male rat also had edema/inflammation in the mucosa and submucosa of the glandular area. Focal necrosis (erosions) of the superficial epithelium in the nonglandular area also was observed.

The treatment-related effect in the kidney consisted of an increased amount of hyaline droplets in the cortical tubular epithelial cells. This increase was characterized by very large, dense hyaline droplets which stained strongly positive with the Mallory-Heidenhain stain. The intensity of this effect was most extensive in the high dose group animals and somewhat less in the mid dose group. The kidneys of the low dose group male rats were considered not to have been affected and were comparable to the controls. ln the hematoxyline and eosin-stained sections, foci of degeneration of cortical tubules were seen in a few animais in the high and mid dose groups, and granular casts were observed in the outer medullary tubules in a few of the high dose group animals. The degeneration and casts do often times accompany increased hyaline droplet accumulations.
Hyaline droplets occur normally in the kidneys of male rats due to a unique accumulation of alpha µ globulin in phagolysosomes of the tubular epithelium. There have been several compounds of various types that have caused an exacerbation or excessive accumulation of these hyaline droplets in the cortical tubular epithelium. Similar changes do not occur in female rats and, in this study, there was no treatment-related effect of any type on the kidneys of the female rats.
Dose descriptor:
NOEL
Remarks:
Stomach irritation
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No effect
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No relevant effects at this dose level, excluding stomach irritation and male rat specific hyaline droplets nephropathy
Critical effects observed:
no

Table 1

Hemoglobin (g/dL)

control

 150 mg/kg

550 mg/kg 1000 mg/kg

Mean

16.3

16.1

16.7

15.2* (-7%)

sd

0.6

0.4

0.4

0.2

n

5

5

5

5

*significantly different (p 0.05)

Table 2

Dose Group:  control 1000 mg/kg 550 mg/kg 150 mg/kg control 1000 mg/kg 550 mg/kg 150 mg/kg
Sex: M M M M F F F F
STOMACH:                
NO. EXAMINED 5 5 5 5 5 5 5 5
NO. NORMAL  4 0 1 5 5 0 2 5
dilatation, mucosal glands  /1/ /0/ /0/ /0/ /0/ /0/ /0/ /0/
minimal  1 0 0 0 0 0 0 0
edema/inflammation, mucosa/submucosa, glandular,                
acute /0/ /1/ /0/ /0/ /0/ /0/ /0/ /0/
moderate 0 1 0 0 0 0 0 0
                 
edema/inflammation, mucosa/submucosa                
nonglandular,acute  /0/ /4/ /4/ /0/ /0/ /4/ /2/ /0/
minimal 0 0 0 0 0 1 0 0
mild 0 2 3 0 0 1 0 0
moderate  0 2 1 0 0 1 1 0
marked 0 0 0 0 0 1 1 0
erosion(s), nonglandular mucosa /0/ /4/ /1/ /0/ /0/ /0/ /2/ /0/
minimal 0 1 0 0 0 0 0 0
mild 0 2 1 0 0 0 1 0
moderate 0 1 0 0 0 0 1 0
hyperplasia/hyperkeratosis, nonglandular mucosa /0/ /5/ /2/ /0/ /0/ /5/ /3/ /0/
minimal 0 0 1 0 0 1 1 0
mild 0 0 0 0 0 1 0 0
moderate  0 3 1 0 0 3 2 0
marked 0 2 0 0 0 0 0 0

/Total incidence of specified lesion, all grades./

Conclusions:
The 28 day repeated oral administration (27 doses) of Luperox TBEC in rats produced no clinical effects but did produce microscopic changes at doses of 550 and 1000 mg/kg/day. The observed pathologic changes were noted in the stomach of male and female rats and in the kidneys of male rats only. The pathologic changes noted in the stomach are considered to be secondary to a local irritating effect and therefore of low relevance for hazard assessment. The kidney pathology noted in the male rats is not considered to be relevant to human exposure since the enzyme system responsible for the hyaline droplet formation are unique to the male rat.
Executive summary:

The potential toxicity of TBEC was evaluated in a repeated dose study performed following to OECD Guideline 407 (July 27th1995). TBEC was administered once daily by gavage to Sprague-Dawley rats (5 Males and 5 Females), at the dose-levels of 0, 150, 550 and 1000 mg/kg/d during 28 days (but animals were not dosed the 20 th day). Body weights were recorded pretest, weekly, at death and at termination in the survivors. The animals were observed once daily for toxicity and pharmacological effects and twice daily for morbidity and mortality. Food consumption was calculated weekly. A Functional Observational Battery examination, designed to assess specific neurotoxicity and behavioral changes, was conducted on days 23 and 26. On day 29, all surviving animals were anesthetized with ether and exsanguinated. Whole blood, plasma and serum were analyzed for selected hematology and clinical chemistry parameters. All animals were examined for gross pathology. All the tissues were preserved in 10% Neutral Buffered Formalin. 

Two females dosed at 550 mg/kg and two females dosed at 1000 mg/kg died during the study. Pathological examination revealed abnormalities consistent with inadvertent gavage accidents and were not attributed to any toxic effect of the test article. Instances of abnormal systemic signs were minimal. There were no significant differences in mean body weights, food consumption, functional observational battery parameters, and organ weights. Instances of significantly different mean hematology, clinical chemistry and organ/body weight ratios were noted, but there was no evidence of dose-response effects and these instances are not considered biologically significant. Only the mean blood albumin was significantly less in the control group than in mid and high dose group. This was considered biologically unremarkable, since there were no related findings in any other liver or kidney enzymes nor any histological changes. Necropsy results of surviving animals were generally normal. Microscopic examination revealed no treatment related microscopic changes in any of the animals dosed at 150 mg/kg/day. Treatment-related changes were observed in kidneys of male rats in the high and mid dose groups and in the stomach of male and female rats in the high and mid dose groups. The incidence and severity of the treatment-related changes occurred in a dose-related manner. The kidney pathology noted in the male rats was not considered to be relevant to human exposure since the enzyme system responsible for the hyaline droplet formation are unique to the male rat.  

In conclusion, the 28 day repeated oral administration (27 doses) of Luperox TBEC in rats produced no clinical effects but did produce microscopic changes at doses of 550 and 1000 mg/kg/day. The observed pathologic changes were noted in the stomach of male and female rats and in the kidneys of male rats only. The pathologic changes noted in the stomach are considered to be secondary to a local irritating effect and therefore of low relevance for hazard assessment. The kidney pathology noted in the male rats is not considered to be relevant to human exposure since the enzyme system responsible for the hyaline droplet formation are unique to the male rat. Therefore, the no observed effect level (NOEL) was estimated to be 150 mg/kg bw/day based on forestomach effects. In addition, the systemic NOAEL can be estimated at 1000 mg/kg bw/day in male and female rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
600 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
7
Species:
rat
Quality of whole database:
Klimisch 1 study, GLP compliant

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

According to EU Regulation (EC) N0. 1272/2008 (CLP), TBEC is not classified for repeated dose toxicity.