Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 July 2016 - 02 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
21 September 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Diisopropylbenzene hydroperoxide
EC Number:
247-988-1
EC Name:
Diisopropylbenzene hydroperoxide
Cas Number:
26762-93-6
Molecular formula:
C12H18O2
IUPAC Name:
reaction mass of 1-(3-isopropylphenyl)-1-methylethyl hydroperoxide and 1-(4-isopropylphenyl)-1-methylethyl hydroperoxide
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: on the first day of treatment, the animals were 5 to 6 weeks old
- Mean body weight at study initiation: the males had a mean body weight of 240 g (range: 215 g to 269 g) and the females had a mean body weight of 189 g (range: 162 g to 211 g)
- Fasting period before study: no
- Housing: the animals were housed in twos or threes from the same sex and group, in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²)
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 11 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: 22 August 2016 to 02 January 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING FORMULATIONS:
- Emulsion in the vehicle
- Concentration in vehicle: 10, 55 and 100 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: Gas Chromatography with FID detection (GC-FID)
Test item concentrations: remained within an acceptable range of -1.7% to +3.8% when compared to the nominal values ( ± 15% of the nominal concentrations)
Homogeneity: The dose formulations containing the test item in corn oil at 2 and 200 mg/mL were found to be homogeneous. They are therefore considered to be suitable for routine administration in GLP Toxicological studies, based on a daily preparation.
Duration of treatment / exposure:
13 weeks followed by a 6-week treatment-free period
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
275 mg/kg bw/day (actual dose received)
Dose / conc.:
500 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 level selection:
The dose-levels were selected in agreement with the Sponsor, on the basis of the results of a previous toxicology study performed in the same species.
In this study, rats received the test item daily by gavage at dose-levels of 70, 230 or 700 mg/kg/day for 4 weeks. At 700 mg/kg/day, clinical signs were limited to ptyalism in both sexes. No test item-related effects were observed on body weight, food consumption or in laboratory parameters. Increases in the liver to body weight ratio were noted in males given 70 or 230 mg/kg/day and in males and females given 700 mg/kg/day, leading to a statistically significant increase in liver weight in males given 700 mg/kg/day. Histopathology findings consisted of yellow deposit on the forestomach of one male given 700 mg/kg/day; and non-adverse hepatocellular hypertrophy, acanthosis and hyperkeratosis in the forestomach, and hyaline droplets in the renal tubular epithelium (without tubular basophilia) in males and/or females at 70 or 230 mg/kg/day.
In males given 700 mg/kg/day, it was considered likely that the moderate acanthosis in the forestomach was an adverse effect. The No Observed Adverse Effect Level was considered to be 230 mg/kg/day in males (based on the moderate acanthosis in the forestomach observed at 700 mg/kg/day) and 700 mg/kg/day in females.

Thus, based on these available data, the selected dose-levels were 50, 275 and 500 mg/kg/day.

- Rationale for animal assignment: computerized randomization procedure.
Positive control:
no (not required)

Examinations

Observations and examinations performed and frequency:
MORTALITY/MORBIDITY:
- 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 treatment-free periods.

CLINICAL SIGNS:
- Time schedule: once a day.

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

NEUROBEHAVIOURAL EXAMINATION:
Functional Observation Battery (FOB)
This evaluation included a detailed clinical examination, the assessment of reactivity to manipulation and different stimuli, and motor activity.
The animals were randomized and all animals were observed in the cage, in the hand and in the standard arena.

Motor activity
For each animal, motor activity was measured by automated infra-red sensor equipment over a 60-minute period.

- Time schedule: all main animals were evaluated once in Week 12 (before the daily treatment).

BODY WEIGHT:
- Time schedule: the body weight of each animal was recorded once before the beginning of the treatment period, then 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 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 control- and high-dose animals on one occasion at the end of the treatment period.

HAEMATOLOGY:
- Time schedule for peripheral blood: the parameters were determined for all surviving animals sacrificed at the end of the treatment period.
- Time schedule for bone marrow: two bone marrow smears were prepared from the femoral bone (at necropsy) of each animal euthanized on completion of the treatment period or treatment-free period
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes
- How many animals: all surviving animals sacrificed at the end of the treatment period
- Parameters checked in table [No. 1] were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: the parameters were determined for all surviving animals sacrificed at the end of the treatment period. In view of the findings observed at the end of the treatment period, these examinations were also carried out at the end of the treatment-free period.
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table [No. 2] were examined.

THYROID HORMONES:
- Time schedule: an additional blood sample was taken from each surviving animal sacrificed at the end of the treatment or treatment-free period.

The blood was centrifuged within 2 hours after collection. The plasma was divided into two aliquots of at least 150 µL and then stored at -80°C pending possible analysis. The thyroid hormone (T3 and T4, determination by LC MS/MS method) and thyroid stimulating hormone (TSH, determination by Luminex® method) levels were not determined as there was no indication of an effect on the pituitary-thyroid axis.
- Animals fasted: Yes

URINALYSIS:
- Time schedule for collection of urine: the parameters were determined for all surviving animals sacrificed at the end of the treatment period. In view of the findings observed at the end of the treatment period, these examinations were also carried out at the end of the treatment-free period.
- Parameters checked in table [No. 3] were examined.
Sacrifice and pathology:
ORGAN WEIGHTS: see table 4
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. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.


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 Harderian glands, and the testes and epididymides which were fixed in Modified Davidson's Fixative).
Tissues intended for immunohistochemistry were kept for no longer than 96 hours in formalin (main and recovery males).
Two bone marrow smears for potential determination of the bone marrow differential cell count 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 four microns and stained with hematoxylin-eosin (except for the testes and epididymides which were stained with hematoxylin/PAS).

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 all animals that died or were euthanized prematurely,
- for all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) euthanized on completion of the treatment period.

According to the microscopic results of the high-dose group animals, a microscopic examination was performed as follows:
- liver, stomach (with forestomach), ovaries and vagina from low- and intermediate-dose main animals (groups 2 and 3) and from recovery animals (groups 1 and 4),
- kidneys from low- and intermediate-dose main males (groups 2 and 3) and from recovery males (groups 1 and 4).

In order to characterize the tubular hyaline droplets, immunostained kidney slides from control and high dose males (groups 1 and 4) sacrificed at the end of the treatment period were examined.

In addition, testicular staging was performed for control- and high-dose males (main animals from groups 1 and 4). A detailed examination of the testes was performed, using a thorough understanding of tubule development through the different stages of the spermatogenic cycle. Transverse sections of the testes were stained with hematoxylin-PAS in order to detect retained spermatids, missing germ cell layers, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.
Other examinations:
MONITORING OF ESTROUS CYCLE:
- Time schedule: the estrous cycle stage was determined for each female sacrificed 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, these examinations were carried out on all females daily for 14 consecutive days before the end of the treatment-free period.

SEMINOLOGY:
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 epididymis weight, sperm from the cauda of the left epididymis was sampled 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 needed. The number of motile and immotile spermatozoa from a sample of 200 spermatozoa was evaluated under a microscope using a 40 fold magnification. Results are expressed as the proportion of motile and non-motile spermatozoa.

Epididymal sperm morphology
The morphology was determined from a sperm smear, after eosin staining and counting of 100 spermatozoa per slide. This was evaluated in the first instance on groups 1 and 4.
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 and recovery animals.
Results are 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 sampled and the number of spermatozoa was counted in a microscope slide counting chamber.
Results are expressed as the number of spermatozoa per cauda and per gram of cauda.

Testicular sperm
The left testis was sampled and frozen 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 are expressed as a number of sperm heads per gram of testis and the daily sperm production rate was calculated (using a time divisor of 6.10).
Statistics:
Citox software was used to perform the statistical analyses of body weight, food consumption, sperm parameters, hematology, blood biochemistry and urinalysis data.
PathData software was used to perform the statistical analysis of organ weight data.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See Table 5.
At 500 mg/kg/day, 3/14 males had piloerection in Weeks 6, 8, 9 or 13 for few days, loud breathing on Day 41 (Week 6), hypoactivity on Day 56 (Week 8) and/or hunched posture from Day 90 (Week 13).

At 275 mg/kg/day, 1/10 males showed piloerection from Day 90 (Week 13).
These non-adverse clinical signs were attributed to the test item treatment.

Ptyalism was transiently observed in control males and in all test item-treated groups in a dose-related manner, from the first week of treatment in group 4 animals. This non-adverse finding is commonly reported when oily formulations are administered by gavage.

Reflux at administration was noted in 1/10 males given 50 mg/kg/day and in 1/14 males given 500 mg/kg/day on one to two occasions. This sign, commonly observed when a test item is administered by gavage, was considered to be of no toxicological importance.

A palpable mass in the mammary region was observed in 1/15 females given 500 mg/kg/day from Week 19. The palpable masse observed during the study did not show any indication of a treatment relationship, as the incidence recorded was consistent with those commonly noted in rats of this age and strain.

The other clinical signs recorded during the study, i.e. alopecia, scabs, thinning of hair, soiling around the mouth, nodosities, chromodacryorrhea, chromorhynorrhea and/or wounds were of isolated occurrence, observed both in control and test item treated animals, and/or with 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. The ptyalism noted on Day 92 (first day of the treatment-free period) in 5/5 males previously given 500 mg/kg/day was considered to be incidental.

As a follow-up to the wounds observed on the head or ears of one control male, one group 2 male and one group 3 male and one group 4 male, appropriate medical care, as defined by the Veterinarian, was given as follows:
- an antiseptic solution (povidone iodine, Vetedine®) was applied once for the male of group 2,
- an antiseptic and healing solution (Cothivet®) was applied once a day for 7 to 12 days to all animals.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related deaths occurred during the study.

At 500 mg/kg/day
One male was found dead on Day 49 (Week 7). Ptyalism was observed from Day 8. At necropsy, a perforation of the esophagus was observed, together with red discoloration of bronchi and lungs, and brown content of the thoracic cavity. The perforation was confirmed histologically, accompanied by acute inflammation in the nearby tissues. The cause of death was therefore mis-gavage, with no relationship to the test item.

One female was euthanized prematurely for humane reasons on Day 88 (Week 13). From Day 87 and prior to sacrifice, post-blood sampling ocular damage was noted in the right eye, leading to swelling of the right cheek on Day 88. Ptyalism was regularly noted from Day 8.
At necropsy, enlargement and redness of skeletal muscles of the head were also noted. Acute inflammation of anterior segments of the eye and hemorrhagic muscles were the histological correlate. The cause of moribundity was most probably traumatic, related to blood sampling. Other relevant microscopic findings were minimal hepatic centrilobular hypertrophy, minimal acanthosis/hyperkeratosis in the forestomach, mucification of the vaginal epithelium accompanied by increased corpora lutea in the ovary (pseudo-gestation). Given the proximity with Day 92, this rat was included with animal killed at the end of treatment period in incidence tables.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See Table 6.
At 500 mg/kg/day in males, when compared with controls, statistically significant, lower mean body weight gain was recorded throughout the treatment period (-22% vs. controls) leading to a statistically significant, lower mean body weight from Day 29 until the end of the treatment period (-14% vs. controls on Day 91). The differences from controls were particularly noticeable from the second month. This effect was attributed to the test item treatment. During the recovery period, a statistically significant, higher mean body weight gain was observed in males previously treated at 500 mg/kg/day, leading to a terminal mean body weight similar to that of the control animals (-5% vs. controls).
In females, statistically significant, higher mean body weight gain was observed, mainly during the first month (+24%), followed by a statistically significant, lower mean body weight gain during the third month (-33%). Consequently, the mean body weight was not affected at the end of the treatment period. The variations observed in the body weight evolution of females throughout the study period were considered to be of no toxicological importance.

At 275 mg/kg/day in males, when compared with controls, instances of lower mean body weight gain were observed throughout the treatment period (statistically significant in Weeks 4, 5 and 10: -21 to -32% vs. controls), the differences reaching statistically significance over the whole third month and leading to a statistically significant, lower mean body weight from Day 43 until the end of the treatment period (-10% vs. controls on Day 91).
In females, statistically significant, higher mean body weight gain was observed during the first month (+24%) without relevant effects on the terminal mean body weight. This difference was therefore considered to be of no toxicological importance.

At 50 mg/kg/day in males, when compared with controls, a trend to a slightly lower mean body weight gain was noted throughout the treatment period (with statistically significant lower mean values in Weeks 4 and 7, p<0.05). No relevant effects were noted on body weight or body weight gain in females during the study period.
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.

In females at 275 mg/kg/day and 500 mg/kg/day, statistically significant, higher mean food consumption, when compared with controls, was noted on rare occasions (i.e. Weeks 2 and/or 11). As these differences were of isolated occurrence, they were considered to be of no toxicological importance.
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):
No test item treatment-related effects on the hematology parameters were observed at the end of the treatment period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See Table 7.
At 500 mg/kg/day and when compared with mean control values, statistically significant blood biochemistry changes were observed:
- higher mean sodium level in males (+1%) and lower mean chloride level in females (-2%),
- higher mean urea level in males (+24%),
- higher mean protein and albumin levels in females (+5% and +6%, respectively), along with higher mean calcium level (+5%),
- lower (in males: -39%) or higher (in females: +56%) mean triglyceride levels.

At 275 mg/kg/day and when compared with mean control values, statistically significant, higher mean sodium and/or calcium levels were also noted in males (+1% and +2%, respectively) and females (calcium: +4%), and a lower mean triglyceride level was observed in males (-32%).

All the above statistical 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 the Historical Control Data.

The other statistically significant differences observed between control and test item-treated animals, namely in the potassium, calcium and glucose levels and in the albumin to globulin ratio (males at 275 mg/kg/day), albumin levels (males at 50 and 275 mg/kg/day), protein level (males at 50 mg/kg/day) and the alkaline phosphatase and aspartate aminotransferase activity (females at 500 mg/kg/day) were considered to be within the range of physiological values, or incidental and not test item-related as they were noted with no dose-relationship or without any relationship to microscopic findings.

Test item-related effects on the blood biochemistry parameters were no longer observed at the end of the treatment-free period.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
See Table 8.
At 500 mg/kg/day and when compared with mean control values, statistically significant urinalysis changes were observed:
- lower mean pH values in males,
- higher mean volume in males and females. As this difference did not correlate with changes in specific gravity, it was considered to be of minor toxicological importance.

At 275 mg/kg/day and when compared with mean control values, a statistically significant, lower mean pH value was also noted in males.

Calcium oxalate crystals were noted in test item-treated females in a dose-related manner. At this did not correlate with any other findings, this was considered to be of no toxicological importance.

The higher mean specific gravity in males given 275 mg/kg/day (1047 vs. 1036 in controls) was considered to be of no biological significance and not test item treatment-related as it was of low magnitude and not dose-related.

Test item-related effects on the urinary parameters were no longer observed at the end of the treatment-free period.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on Functional Observation Battery tests or motor activity data in any group.

Lower mean numbers of horizontal movements and rearing noted in females at 500 mg/kg/day were considered to be of no toxicological importance as they were of minor magnitude and mainly due to the higher mean control values (resulting from one female: horizontal movements: 2026, rearing: 546).
No differences from controls were noted in the motor activity of test item-treated males.

Differences from controls in grooming were noted in isolated males at 500 mg/kg/day. In view of the very slight severity and incidence, and in the absence of correlating clinical signs during the study, this finding was considered to be unrelated to the test item treatment.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See Table 10.
End of treatment
Final body weights were decreased in a dose-related manner in all male treated groups, but not in females.

Many differences in organ weights were the direct consequence of these lower final body weights, and even though they reached on occasion statistical significance (for instance higher testicular weights) they were unrelated to treatment.

Nevertheless, there were some minimal but noteworthy differences between treated and control groups.

The relative kidney weights, but not the absolute ones, were increased in a dose related manner in all male treated groups. At the mid and high-doses, it was associated with increased hyaline droplets, focal tubular basophilia and hyaline casts at histological examination.

Liver weights were increased from the mid-dose in both sexes (relative to body weights values only in males). At the high-dose, it correlated histologically with centrilobular hypertrophy.

Higher adrenal weights in both sexes from the dose of 275 mg/kg/day and lower thymus weights in both sexes at the high-dose were considered related to treatment, although they did not have histological correlated. They likely represent a non-specific stress response to the test item, and are not considered adverse.

Higher adrenal weights in both sexes from the dose of 275 mg/kg/day, and lower thymus weights in both sexes at the high-dose did not have histological correlates. They were considered possibly related to a non-specific stress response to the test item. Given the low magnitude of these differences, they are considered of no toxicological significance and non- adverse.

End of recovery
At the end of the treatment-free period, final body weights were comparable between controls and rats previously treated at the high-dose.

Kidney weights were increased in animals males previously treated at the high-dose compared to controls (+19% and +24% in absolute and relative values, respectively, statistically significant at p = 0.05, Wilcoxon’s test). It correlated with increased hyaline droplets and focal tubular basophilia observed in a few rats.

Thymic weights were decreased in high-dose females (-13 and -16% in absolute and relative value, respectively, not statistically significant) but the difference was slightly lower than at the end of the treatment period. This was considered spurious.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See Table 11.
At the end of treatment period, there were observations on the forestomach from the dose of 275 mg/kg/day in males and at the high-dose in females.

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

There was no histologic correlate for the yellow discoloration, which is likely to be deposits of the test item.

All other macroscopic observations belong to the spectrum of spontaneous findings in rats of this age and strain. This includes a red mass in the pituitary of one high-dose male, correlated with an osteosarcoma (likely to have originated in the skull).

End of recovery
At the end of recovery all macroscopic observations were spontaneous and bore no relationship to treatment.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See Tables 12 and 12 bis.
Minimal to moderate increases in hyaline droplets in the kidney was diagnosed in males from the dose of 275 mg/kg/day. At the low-dose, the incidence and severity of affected animals were lower, but still considered treatment-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 2.8, vs. 1.4 in controls, confirming the increase noted in hematoxylin and eosin-stained slides. Hyaline droplets were associated with granular casts (from 275 mg/kg/day) and focal tubular basophilia (all doses). Together, these findings, which 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 275 mg/kg/day.

Minimal centrilobular hypertrophy in the liver was restricted to the high-dose. Centrilobular hepatocellular hypertrophy in the liver is often due to induction of metabolic enzymes and as such it is a direct effect of the test item. It is well-established as an adaptive and non-adverse change in the absence (as in this study) of histologic or clinical pathology alterations indicative of liver degenerative changes.

Acanthosis/hyperkeratosis was observed in the forestomach from the dose of 275 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.

Likewise, mucification of vaginal mucosa with enlarged corpora lutea in the ovary in 3/10 high- and 1/10 mid-dose females are reported in stressful conditions. Other females in these groups displayed a normal distribution of estrous cycle. Therefore, this is considered 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
At the end of the treatment-free period, increased hyaline droplets and focal tubular basophilia were still observed in male rats previously treated at the high-dose. The incidence and severity were lower compared to end of treatment, indicating partial recovery.

Hepatic changes, as well as stress-related changes in the stomach and female genital track were not observed, indicating full recovery.
Histopathological findings: neoplastic:
effects observed, treatment-related
Other effects:
no effects observed
Description (incidence and severity):
Estrous cycle
See Table 13.
There were no statistically significant test item-related effects on the mean length of the estrous cycle or the mean number of cycles. A trend towards an increase in the mean length of diestrus was observed in females given 275 or 500 mg/kg/day at the end of the treatment period. This was due to three intermediate-dose females and five high-dose females for which the diestrus period represented 8 to 11 days.
This was considered to be of minor importance in the absence of statistical significance and as microscopic findings (mucification of vaginal mucosa with enlarged corpora lutea) were considered as an indirect non adverse effect of the test item and since these variations were no longer observed at the end of the treatment-free period.

Seminology
See Table 14.
No test item treatment-related effects were noted on testicular sperm count, or on epididymal sperm count, motility or morphology.
Concerning the slight decrease, not statistically significant, in the testicular sperm count recorded in males given 500 mg/kg/day, the difference was due to the low values of one animal, and was considered to be of no toxicological importance as most of individual values were within the standard deviation of the control-dose groups.

Effect levels

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Excluding the male rat-specific hyaline droplets nephropathy and the local irritation of the forestomach

Target system / organ toxicity

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

Any other information on results incl. tables

Table 5: Clinical signs

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

50

275

500

0

50

275

500

Hunched posture

-

-

-

1

-

-

-

-

Hypoactivity

-

-

-

1

-

-

-

-

Piloerection

-

-

1

3

-

-

-

-

Loud breathing

-

-

-

1

-

-

-

-

Total affected animals

0/15

0/10

1/10

3/14

0/15

0/10

0/10

0/14

Ptyalism

5

9

10

14

0

5

10

14

Reflux at administration

-

1

-

1

-

-

-

-

Total affected animals

5/15

9/10

10/10

14/14

0/15

5/10

10/10

14/14

-: no clinical signs.

Table 6: Body weight

 

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

50

275

500

0

50

275

500

 

Treatment period

 

Mean BW gain Days 1/29

+235

+215

+207

+203*

+66

+70

+82*

+82*

 

Mean BW gain Days 29/57

+108

+97

+93

+82**

+44

+41

+43

+39

 

Mean BW gain Days 57/91

+66

+55

+47**

+39**

+21

+22

+21

+14*

 

Mean BW gain Days 1/91

+409

+367

+347**

+320**

+131

+133

+146

+136

 

% from controls

-

-10

-15

-22

-

+2

+11

+4

 

Mean body weight on Day 1

242

238

238

241

187

191

189

189

 

Mean body weight on Day 91

651

605

585**

559**

319

324

334

324

 

% from controls

-

-7

-10

-14

-

+2

+5

+2

 

Treatment-free period

 

Mean BW gain Days 91/133

+20

-

-

+47*

+11

-

-

+15

 

Mean BW on Day 133

655

-

-

622

330

-

-

337

 

% from controls

-

-

-

-5

-

-

-

+2

 

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

Table 7: Blood biochemistry

 

Sex

Male

Dose-level (mg/kg/day)

0

50

275

500

End of treatment period

Sodium (mmol/L)

142.5
(142.8-146.7)

143.1

144.1**

143.6*

Urea (mmol/L)

3.7

(2.6-5.2)

3.9

4.6**

4.6**

Triglycerides (mmol/L)

1.22

(0.22-2.62)

1.00

0.83**

0.74**

End of treatment-free period

 

 

 

 

Sodium (mmol/L)

143.6

/

/

143.2

Urea (mmol/L)

5.5

/

/

5.4

Triglycerides (mmol/L)

0.84

/

/

1.06

/    : not applicable; statistically significantfrom controls:*: p < 0.05 and**: p < 0.01.

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

Table 7 (continued)

 

Sex

Female

Dose-level (mg/kg/day)

0

50

275

500

End of treatment period

Chloride (mmol/L)

106.8

(99.8-108.4)

107.2

106.6

105.0**

Calcium (mmol/L)

2.55

(2.42-2.82)

2.57

2.66*

2.69**

Proteins (g/L)

58.4

(56.2-80.1)

57.4

60.8

61.5*

Albumin (g/L)

34

(33-50)

34

36

36*

Triglycerides (mmol/L)

0.39

(0.21-1.02)

0.35

0.45

0.61**

End of treatment-free period

 

 

 

 

Chloride (mmol/L)

107.1

/

/

107.2

Calcium (mmol/L)

2.77

/

/

2.79

Proteins (g/L)

68.5

/

/

69.4

Albumin (g/L)

40

/

/

42

Triglycerides (mmol/L)

0.67

/

/

0.76

/    : not applicable; statistically significantfrom controls:*: p < 0.05 and**: p < 0.01.

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

Table 8: Urinary parameters

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

50

275

500

0

50

275

500

End of treatment period

Volume (mL)

9

8

8

14

6

8

9

14*

pH

6.9
(6.0-8.0)

6.8

6.1**

6.1**

6.2
(6.0-7.0)

6.1

6.0

5.9

Calcium oxalate crystals (number of concerned animals)

 

 

 

 

 

 

 

 

. None observed

10

10

10

10

10

6

4

3

. Few in some fields

 

 

 

 

 

2

3

3

. Few in all fields

 

 

 

 

 

2

0

4

. Several in all fields

 

 

 

 

 

 

3

 

End of treatment-free period

Volume (mL)

11

/

/

15

10

/

/

9

pH

7.3

/

/

7.4

6.6

/

/

6.3

Calcium oxalate crystals

 

 

 

 

 

 

 

 

. None observed

5

/

/

5

5

/

/

5

/    : not applicable; statistically significantfrom controls: *: p < 0.05 and**: p < 0.01.

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

Table 9: Motor activity

 

Sex

Male

Female

Dose-level (mg/kg/day)

     0

       50

     275

     500

       0

      50

   275

     500

Horizontal movements

  515

     547

     438

     516

   935

    889

   993

     766

% from controls

-

       +6

      -15

        0

-

      -5

    +6

      -18

Rearing

  142

     147

     160

     124

   225

    286

   230

     199

% from controls

-

       +4

     +13

     -13

-

    +27

    +2

      -12

-: not applicable.

Table 10: Organ weights

End of treatment period

 

Gender

Male

Female

Group

2

3

4

2

3

4

Dose-level (mg/kg/day)

50

275

500

50

275

500

Concentration (mg/mL)

10

55

100

10

55

100

Number of animals

10M

10M

9M

10F

10F

9F

Final body weight

-8*

-12**

-18**

+1

+4

+2

Adrenal glands

 

Absolute (%)

+4

+1

+12

-

+14

+15

Relative to body weight (%)

+12

+15*

+37**

-2

+9

+13

Kidney

 

Absolute (%)

+1

+5

+4

-4

-3

+1

Relative to body weight (%)

+10*

+20**

+27**

-5

-6

-1

Liver

 

Absolute (%)

-6

+5

+1

-

+18**

+30**

Relative to body weight (%)

+3

+20**

+24**

-1

+14**

+28**

Thymus

 

Absolute (%)

-15

-18

-28**

-10

+2

-17*

Relative to body weight (%)

-8

-8

-14

-10

-2

-18**

Statistically significant from controls: *: p = 0.05; ** p = 0.01 (Student's test).

Table 11: Macroscopic examination

End of treatment period

Gender

Male

Female

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

50

275

500

0

50

275

500

Concentration (mg/mL)

0

10

55

100

0

10

55

100

Number of animals

10M

10M

10M

9M

10F

10F

10F

9F

Forestomach

 

Red discoloration

-

-

-

1

-

-

-

-

Thickened

-

-

-

1

-

-

-

1

White discoloration

-

-

2

1

-

-

-

2

Yellow discoloration

-

-

1

2

-

-

-

-

 

Table 12: Microscopic examination

End of treatment 

 

Gender

Males

Females

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

50

275

500

0

50

275

500

Concentration (mg/mL)

0

10

55

100

0

10

55

100

Number of animals

10M

10M

10M

9M

10F

10F

10F

10F

Kidney

 

Tubular basophilia, focal

 

. Minimal

1

5

5

4

0

-

-

0

. Slight

0

1

4

5

0

-

-

0

. Moderate

0

0

1

0

0

-

-

0

Increased hyaline droplets

 

. Minimal

2

3

1

0

0

-

-

0

. Slight

0

1

8

7

0

-

-

0

. Moderate

0

0

1

1

0

-

-

0

Hyaline casts

 

. Minimal

1

0

5

5

0

-

-

0

. Slight

0

0

0

1

0

-

-

0

Liver

 

Centrilobular hypertrophy

 

. Minimal

0

0

0

4

0

0

0

6

Ovaries

 

Enlargedcorpora lutea

 

. Present

-

-

-

-

0

0

1

3

Stomach

 

Acanthosis/hyperkeratosis

 

. Minimal

0

0

6

0

0

0

6

1

. Slight

0

0

2

9

0

0

0

8

. Moderate

0

0

0

0

0

0

0

1

Vagina

 

Mucification, epithelium

 

. Minimal

-

-

-

-

0

0

1

0

. Slight

-

-

-

-

0

0

0

1

. Moderate

-

-

-

-

0

0

0

2

Table 12bis: Microscopic examination

End of recovery

 

Gender

Male

Female

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

50

275

500

0

50

275

500

Concentration (mg/mL)

0

10

55

100

0

10

55

100

Number of animals

5M

-

-

5M

5F

-

-

5F

Kidney

 

Tubular basophilia, focal

 

. Slight

0

-

-

2

-

-

-

-

Increased hyaline droplets

 

. Minimal

0

-

-

2

-

-

-

-

Table 13: Estrous cycle

 

Dose-level (mg/kg/day)

         0

    50

    275

      500

Treatment period

 

 

 

 

Number of cycles

         3.9

      3.7

        4.1

         3.6

Cycle length (days)

         4.3

      5.7

        4.0

         4.5

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

         8

      7

        9

         7

Number of days of diestrus

         5.6

      5.9

        7.0

         7.1

Treatment-free period

 

 

 

 

Number of cycles

         3

/

/

         2.6

Cycle length (days)

         3.7

/

/

         4.6

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

         3

/

/

         4

Number of days of diestrus

         4.4

/

/

         4.6

/: not applicable.

Table 14: Seminology

 

Dose-level (mg/kg/day)

       0a

   50

  275

  500

% of motile epididymal sperm

     99.7

   99.0

    98.1

    98.6

% of morphologically normal epididymal sperm

     96.9

/

/

    96.3

Mean number of epididymal sperm (106/cauda)

   128.5

(142.8)

  130.1

  153.5

  143.4

Mean number of epididymal sperm (106/g cauda)

   464.4

(516.0)

  464.1

  541.3

  515.7

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

   109.8

(120.2)

  115.1

  114.5

  102.0

[106.3]

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

     18.0

(19.7)

   18.9

    18.8

    16.7

[17.4]

/    : not applicable.

a       : small epididymis and no seminal liquid for one male.

( )  : the mean was calculated without the values of one male .

[ ] : the mean was calculated without the values of one male.

 

Applicant's summary and conclusion

Conclusions:
The toxicity of Luperox DH was evaluated after daily oral administration (gavage) to Sprague-Dawley rats at dose-levels of 50, 275 or 500 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 275 mg/kg/day, adverse test item related effects were observed in the kidneys of male rats: the hyaline droplets in renal tubular epithelium were associated with a2u-globulin accumulation as confirmed by immunohistochemistry and with increased tubular basophilia. It 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 500 mg/kg/day in females and males excluding the adverse lesions seen in male kidneys (a2u-globulin protein nephropathy-related findings) which are specific to male rats and with no relevance for human risk assessment.
Executive summary:

The potential toxicity of the Luperox DH 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 male and 15 female Sprague-Dawley rats was treated daily by the oral route (gavage) with the test item at the dose-level of 500 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 50 or 275 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 sacrificed, 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 Gas Chromatography with Flame Ionization Detection method (GC-FID). The animals were checked 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 study and on control and high-dose animals at the end of the treatment period (Week 13). Hematology, blood biochemistry and urinary investigations were performed at the end of the treatment period (Week 13). Blood biochemistry and urinalysis parameters were also determined at the end of treatment-free period (Week 19). Additional blood samples were collected in Weeks 13 and 19 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, seminology 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 sacrificed and a full macroscopic post-mortem examination was performed. Designated organs were weighed and selected tissues were preserved. A microscopic examination (including testicular staging) was performed on designated tissues from control and high-dose animals sacrificed at the end of the treatment period and from animals that were euthanized prematurely, and on all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period. A microscopic examination was also performed on kidney slides immunostained with an antibody for alpha 2u globulin protein from all control and high-dose males sacrificed at the end of the treatment period. The liver, stomach (with forestomach), ovaries and vagina from low- and intermediate-dose animals euthanized at the end of the treatment period and from recovery animals, and the kidneys from the low- and intermediate-males euthanized at the end of the treatment period and from recovery males were also microscopically examined, 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 (-1.7% to +3.8%) compared to the nominal concentrations. There were no test item-related unscheduled deaths in any group. Non-adverse clinical signs were noted for a few days during the treatment period in 3/14 males given 500 mg/kg/day (i.e. hunched posture, hypoactivity, piloerection and/or loud breathing) and in 1/10 males given 275 mg/kg/day (piloerection). Ptyalism was observed with a dose-related incidence at all dose-levels. This sign,commonly observed when oily formulations are administered by gavage, was considered to be non-adverse. The FOB results were unaffected by the test item treatment. Lower body weight gain was occasionally recorded in males given 275 mg/kg/day (-21 to -32% vs. controls) and throughout the treatment period in males given 500 mg/kg/day (-22% vs. controls), leading to minimally to moderately lower body weights on completion of the treatment period (-10% and -14%v s. controls, respectively). As these differences were not associated with any other relevant findings, they were considered to be as non-adverse. Reversibility of these differences was noted at the end of the treatment-free period. Food consumption was not affected by the test item treatment. No ophthalmology findings were observed at the end of the treatment period. A slight increase in the length of diestrus was observed in females given 275 or 500 mg/kg/day at the end of the treatment period. Variations were no longer observed at the end of the treatment-free period. The epididymal sperm motility and morphology, and the spermatozoa count were unaffected by the test item treatment. At hematology investigations, no test item-related effects were observed at the end of the treatment period. At blood biochemistry investigations, all 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 the Historical Control Data. At urinary investigations, non-adverse changes were observed (i.e. lower pH values in males from 275 mg/kg/day and higher volume in males and females at 500 mg/kg/day). Reversibility of these urinalysis differences was noted at the end of the treatment-free period. At pathology investigations, the oral administration of the test item produced a2u-globulin nephropathy in male rats at all doses, which was considered adverse from the dose of 275 mg/kg/day, but is specific to male rats and irrelevant for human risk assessment. It also induced minimal hepatic centrilobular hypertrophy in both sexes at the high-dose only, which was considered adaptive and non-adverse. In addition, there were decreased thymic weights (both sexes at the high dose), increased adrenal weights (both sexes from the dose of 275 mg/kg/day), acanthosis/hyperkeratosis in the forestomach (both sexes from the dose of 275 mg/kg/day) and vaginal mucification with increased size of corpora lutea (from the dose of 275 mg/kg/day) which were secondary effects of treatment, stress-related and non-adverse. At the end of the 6-week treatment-free period, none of these changes were observed, indicating full recovery, with the exception of a2u-globulin nephropathy in males, for which there was evidence of ongoing recovery.

The toxicity of Luperox DH was evaluated after daily oral administration (gavage) to Sprague-Dawley rats at dose-levels of 50, 275 or 500 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 275 mg/kg/day, adverse test item-related effects were observed in the kidneys of male rats: the hyaline droplets in renal tubular epithelium were associated with a2u-globulin accumulation as confirmed by immunohistochemistry and with increased tubular basophilia. It 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 500 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 with no relevance for human risk assessment.