Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
12 August 2015 - 28 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
other: Study synopsis
Title:
Unnamed
Year:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: on the first day of treatment, the animals were 9 weeks old.
- Weight at study initiation: the males had a mean body weight of 425 g (range: 383 g to 479 g) and the females had a mean body weight of 265 g (range: 234 g to 300 g)
- Fasting period before study: no
- Housing: the animals were housed in twos or threes of the same sex and in the same group, in polycarbonate cages with stainless steel lids
- Diet (e.g. ad libitum): SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): tap water (filtered with a 0.22 µm filter)
- Acclimation period: 35 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: From: 17 September 2015 To: 28 January 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other:
Remarks:
Drinking water treated by reverse osmosis
Details on oral exposure:
TYPE OF FORMULATION:
solution in the vehicle.

PREPARATION OF DOSING SOLUTIONS:
- Preparation procedure:
The test item was weighed and mixed with the required quantity of vehicle. The pH of the dose formulation was adjusted to 8.0 (± 0.5) with a solution of hydrochloric acid (HCl 5N, VWR Prolabo®); the volume of HCl added was recorded for each concentration. The final pH was checked and recorded by using a pH-meter.
- Frequency of preparation and storage conditions:
Fresh dose formulations were prepared on the day of administration and kept at room temperature prior to administration.

VEHICLE
- Concentration in vehicle: 10, 50 and 150 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:
Gas Chromatography with FID detection (GC-FID).
Duration of treatment / exposure:
13 weeks.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Groups 1 and 4: 15 males and 15 females
Groups 2 and 3: 10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected based on the results of a previous toxicology study performed in the same species. Male and female Sprague-Dawley rats received Diethylaminopropylamine (as pH-neutralized dose formulations) daily, by gavage, at dose-levels of 100, 300 or 1000 mg/kg/day for 2 weeks. At 1000 mg/kg/day, in-life observations were limited to ptyalism in both sexes, and lower body weight gain at treatment initiation resulted in a lower final body weight in males. Histopathology findings consisted of minimal to slight hyperplasia of squamous cells, associated with minimal to slight hyperkeratosis within the forestomach in all males and females given 1000 mg/kg/day.Thus, based on these available data, the following dose-levels were selected for the present study: 50, 250 and 750 mg/kg/day.

- Post-exposure recovery period in satellite groups: 6 weeks

Examinations

Observations and examinations performed and frequency:
MORBIDITY AND MORTALITY: Yes
- 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, including weekends and public holidays.

CLINICAL OBSERVATIONS: Yes
- Time schedule: each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL EXAMINATION: Yes
- 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: Yes
- Time schedule: all main animals were evaluated once in Week 13 (before the daily treatment). As no relevant changes were observed at the end of the treatment period, FOB examinations were not carried out at the end of the treatment-free period.

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

BODY WEIGHT: Yes
- Time schedule for examinations: 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: Yes
- Time schedule: The quantity of food consumed by the animals in each cage was recorded at least once a week, over a 6 or 7-day period, during the study.Food consumption was calculated per animal and per day.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examinations were performed on all animals, before the beginning of the treatment period, on all main animals on one occasion at the end of the treatment period.As no relevant changes were detected in the treated animals at the end of the treatment period, the recovery animals were not examined at the end of the treatment-free period.

MONITORING OF ESTROUS CYCLE: Yes
The estrous cycle stage was determined for each female sacrificed at the end of the treatment period, from a fresh daily vaginal lavage (stained with methylene blue) 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 daily for 5 consecutive days before the end of the treatment-free period.

HAEMATOLOGY: Yes
- 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 all animals sacrificed on completion of the treatment or treatment-free 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.

CLINICAL CHEMISTRY: Yes
- Time schedule: 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.

URINALYSIS: Yes
- Time schedule: 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.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: all main animals were evaluated once in Week 13 (before the daily treatment).
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity.

SEMINOLOGY: Yes
- Time schedule: before sacrifice at the end of the treatment period, each male was anesthetized by an intraperitoneal injection of sodium pentobarbital.As no relevant treatment-related 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
Sperm from the cauda epididymis was sampled for motility and morphology investigations.
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. The number of motile and immotile spermatozoa from a sample of 200 spermatozoa was evaluated under a microscope using a 40-fold magnification. Results were 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. 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 sampled and the number of spermatozoa was counted in a microscope slide counting chamber.
Results were expressed as the number of spermatozoa per cauda and per gram of cauda.
- Testicular sperm
At necropsy, the left testis was sampled and frozen at -20°C for further sperm count investigation. After thawing, the left testis was weighed (without the albuginea) 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 a number of sperm heads per gram of testis and the daily sperm production rate was calculated (a time divisor of 6.10 which represents the duration of spermatogenic cycle of homogenization-resistant testicular spermatids).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
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.

ORGAN WEIGHTS: Yes
The body weight of each animal was recorded before sacrifice. 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 sacrifice) was calculated.

HISTOPATHOLOGY: Yes
A microscopic examination was performed on:
- control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period and one prematurely sacrificed female (group 4),
- all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period. According to microscopic findings observed in the control and high-dose animals (groups 1 and 4), the following tissues from low- and intermediate-dose animals (groups 2 and 3) and from the recovery animals (groups 1 and 4) were examined: brain, forestomach, gut-associated lymphoid tissue, kidneys, mesenteric lymph nodes (males only), pituitary gland, spleen and thymus.
Statistics:
Citox software was used to perform the statistical analyses of body weight, food consumption, hematology, blood biochemistry and urinalysis data. PathData software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg/day, signs of poor clinical condition (thin appearance, hunched posture, piloerection, bent head, loud and/or abdominal breathing, and/or dyspnea) were noted in 1/15 males for few days and 3/14 females from Week 11. Thin appearance, hunched posture, dyspnea, abdominal, loud breathing and/or bent head were considered to be adverse at this dose-level in 3/14 females (one out of three females also showing a large chronic hematoma in the cranium at microscopic examination) as these signs were not just transient in contrast to males. Red discoloration of urine or vagina was transiently noted in 1/15 males and 2/14 females together with soiled urogenital region in one female.At 250 mg/kg/day, loud breathing (in 2/10 males) and red discoloration of the vagina (in 1/10 females) were observed.At 50 mg/kg/day, hunched posture and piloerection were noted in 1/10 females for 2 days in Week 13.These signs were not dose-related. They were considered to be of minor toxicological significance as they were sporadically observed at these dose-levels.Ptyalism was transiently observed in 12/15 males and 14/14 females given 750 mg/kg/day (generally from Week 2 or 3) and in 1/15 males and 2/15 females given 250 mg/kg/day (between Weeks 11 and 13). Reflux at dosing noted in 2/15 males and 2/15 females on one occasion at the high dose-level was considered to be test item treatment-related.These signs, commonly observed when a test item is administered by gavage, were considered not to represent an adverse effect.The other clinical signs recorded during the study, i.e. alopecia, scabs, soiled nose and mouth, soiled head and neck, wound, thinning of hair, opacity of eyes, bent tail, nodosities, chromodacryorrhea and/or chromorhynorrhea were observed both in control and test item-treated animals with no dose-relationship. They were therefore considered to be unrelated to the test item treatment.Test item treatment-related clinical signs were no longer observed over the treatment-free period.In follow-up to a wound on the neck region of one group 2 male, an antiseptic solution (povidone iodine, Vetedine®), was applied once or twice a day for 12 days. See table 1.
Mortality:
mortality observed, treatment-related
Description (incidence):
One femake given 750 mg/kg/day was sacrificed for ethical reasons on Day 72 (Week 11). Prior to sacrifice, thin appearance, piloerection, hunched posture, ptyalism, loud breathing, hypoactivity and coldness to the touch were observed. A body weight loss (-53 g, i.e. -20% of its Day 1 body weight) was recorded between Days 1 and 71, along with a low mean food consumption in the cage (up to -28% when compared to controls).At necropsy, the spleen was enlarged, the stomach was distended with food and the stomach wall showed a red discoloration of approximately 0.2 cm in diameter. The thymus showed red discoloration of up to 0.5 cm in diameter. Test item-related microscopic vacuoles were observed in the white matter from the brain (hippocampus, cingulum, cerebral peduncle and cerebellum), in the pars nervosa of the pituitary gland, in the renal tubules, in the choroid plexus, in the spleen and in the GALT.These vacuoles in brain, kidneys and pars nervosa were considered to have probably contributed to the moribundity of this animal.In addition, the following lesions which may be related partly with stress were noted: atrophy of the ovaries, uterus and vagina, pancreas degranulation, increased adipose tissue in the bone marrow associated with decreased cellularity of the hematopoietic cells and thymus atrophy. This unscheduled death was considered to be probably test item treatment-related.No other unscheduled deaths were observed during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg/day in males, when compared with controls, lower mean body weight gain was recorded throughout the treatment period (-21% vs. controls). This was statistically significant during the first and the third months (p<0.01 and p<0.05, respectively), leading to a slightly, non-statistically significant, lower mean body weight on completion of the treatment period (-6% vs. controls). This effect was attributed to the test item treatment, but considered as non-adverse. During the recovery period, a higher mean body weight gain was observed in males previously treated with 750 mg/kg/day, leading to a terminal mean body weight similar to the control animals.At 50 and 250 mg/kg/day in males, no relevant effects were noted on body weight or body weight gain during the study period.At all dose-levels of the test item in females, some instances of lower mean body weight gain, statistically significant on few occasions, were reported compared with controls over the treatment or treatment-free period. As these differences were occasional, not dose-related and/or of minimal amplitude, and did not affect the mean body weight, they were considered to be of no toxicological importance. Slight mean body weight loss (-5 g vs. +3 g in controls) was also recorded in females previously given 750 mg/kg/day during the last 2 weeks of the treatment-free period. This was considered to be fortuitous. See table 2.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In females, transient statistically significant lower mean food consumption, when compared with controls, was noted in the fourth week of treatment at 250 mg/kg/day (-10%) and first week at 750 mg/kg/day (-15%). The opposite trend was noted in males given 750 mg/kg/day between weeks 6 and 8 (+8% to +9%; p<0.05). As these differences were of isolated occurrence, not dose-related and/or of opposite trends, they were considered to be of no toxicological importance.No relevant effects were observed on food consumption in the other test item-treated animals during the treatment period.Mean food consumption was slightly higher in previously test item-treated animals during the treatment-free period.See table 3.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
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, treatment-related
Description (incidence and severity):
At 750 mg/kg/day in males, lower red blood cell parameters (statistically significant for most of them), including mean red blood cell count, mean hemoglobin concentration and mean packed cell volume, were noted. Minimal increase in reticulocyte count was also noted and correlated to the slight decrease in red blood cell mass. However, these differences were mainly due to the contribution of one male (showing blood in urine and renal tubular vacuolation). These effects were considered to be test item treatment-related but of minor toxicological importance as values remained within or were close to the range of historical control values.At 750 mg/kg/day, lower mean eosinophil counts were observed in males and females, whereas higher neutrophil counts were recorded in some males and females suggesting a stress leukogram for 4/10 males and 2/9 females. Prolonged prothrombin time was recorded in males and to a lesser extent in females. In the absence of any other significant changes and since values remained within or were close to the range of historical control values, these differences were considered to be of minor toxicological importance.A decreasing tendency in hemoglobin concentration and packed cell volume was observed in males given 250 mg/kg/day.The other statistically significant differences between control and test item-treated animals, namely in monocyte counts (females given 50 mg/kg/day) were considered to be of no toxicological importance as they were of low magnitude and/or noted with no dose-relationship.No test item-related effects on the hematology parameters were observed at the end of the treatment-free period.See table 4.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
When compared with mean control values, statistically significant blood biochemistry changes were observed:- lower mean sodium levels (-1% in females at 250 mg/kg/day and in males and females at 750 mg/kg/day),- lower mean chloride level (-2% in males at 750 mg/kg/day), - higher mean inorganic phosphorus level (+8 and +15% at 250 mg/kg/day, and +15 and +12% at 750 mg/kg/day, in males and females, respectively),- lower mean protein and albumin levels (-5% in males at 750 mg/kg/day) along with lower mean creatinine level (-9% in males at 750 mg/kg/day),- higher triglyceride levels (+38 and +58% in females at 250 and 750 mg/kg/day, respectively),- higher aspartate aminotransferase activity in males at 750 mg/kg/day (+23%) and higher alanine aminotransferase activity in males and females at 750 mg/kg/day (+88 and +62%, respectively). Although changes in the markers and electrolytes of the renal function (Na+, Cl-, inorganic phosphorus, proteins and albumin) are not consistent with each other, a relationship to microscopic vaculoation of kidneys and/or to vasopressin vacuoles in the pituitary gland could not be excluded.At 750 mg/kg/day, high aspartate aminotransferase activity (> 100 U/L) in 4/10 males and 3/10 females was also associated with high alanine aminotransferase activity in males (> 75 U/L) and females (> 60 U/L) (as well as in 2/10 females at 50 mg/kg/day). These findings were not associated with histological changes in the liver.All the above described findings were considered to be of minor toxicological importance were of minimal magnitude.The other statistically significant differences observed between control and test item-treated animals, namely in the potassium and triglyceride levels (males at 50 mg/kg/day) and total bilirubin level (males at 250 mg/kg/day and males and females at 750 mg/kg/day with many values below the lower limit of quantification) were considered to be within the range of physiological values or incidental and not test item-related as they were of low magnitude, of opposite trend and/or noted with no dose-relationship. Test item-related effects on the blood biochemistry parameters were no longer observed at the end of the treatment-free period.See table 5.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg/day, significant hematuria was noted in 2/10 males and 2/10 females at the end of the treatment period. In the absence of hyperglycemia, glucosuria observed in 3 males could be indicative of a tubular resorption problem. Moreover, in one male, the presence of proteins (= 3 g/L), bilirubin (high-level), nitrites, urobilinogen (= 66 µmol/L) and marked turbid appearance of urine were noted. Higher mean urine volume in males given 250 mg/kg/day, was considered to be incidental and not test item-related as it was of low magnitude and not dose-related.At the end of the treatment-free period, the lower mean urine volumes in males and females previously treated with the high dose level were not considered to be treatment-related as they were not observed at the end of the treatment period and were not associated with any other changes in the urine parameters.See table 6.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg/day, higher, test item-related, mean number of horizontal movements and rearing was noted in males (+24% and +31% vs. controls, respectively) and females (+22% and +76% vs. controls, respectively). There were no correlating clinical signs during the study. This finding observed with a low incidence and mainly due to the contribution of one male and one female was therefore considered to be of minor toxicological importance.At 50 and 250 mg/kg/day, a higher mean number of rearing movements was noted in males and females (between +20 and +44% vs. controls). In view of the slight severity, and as there was no correlating higher mean number of horizontal movements, these findings, mainly due to the contribution of one male and one female at 50 mg/kg/day, were considered to be of no toxicological importance.Differences from controls in grooming and defecation were noted in isolated animals at all dose-levels. In view of the very slight severity and incidence, and in absence of correlating clinical signs during the study, these findings were considered to be unrelated to the test item treatment. Other changes, including alopecia, ptyalism and abdominal breathing, were observed in one or two animals given 50 or 750 mg/kg/day, correlating with clinical signs already observed. Lower mean landing foot splay values were noted in females given 750 mg/kg/day (85 mm vs. 105 mm in controls). These findings were considered to be incidental (alopecia) or were considered to be of minor toxicological importance as they were of isolated occurrence (ptyalism and abdominal breathing) or did not correlate with any other findings (landing foot splay).See table 7.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment periodThere were higher relative-to-body kidney weights in males and females treated at 750 mg/kg/day when compared with controls (+17%, p<0.01 and +14%, p < 0.05, respectively). These organ weight differences were partially related to a lower terminal body weights, with a statistically significant decrease in males. A relationship of these kidney weights changes to microscopic vacuolation of renal tubules could not be established.There were lower absolute and relative-to-body thymus weights in males and females treated at 750 mg/kg/day (up to -24%; statistical significance not reached). This organ weight difference was considered to be related to test item administration and/or stress and correlated with increased incidence and severity of microscopic lymphoid atrophy.All other weight differences were minimal, did not correlate with macroscopic or microscopic findings and were considered no to be test item treatment-related.At the end of the treatment-free periodThere were slightly higher absolute and/or relative-to-body kidney weights in males and females previously treated at 750 mg/kg/day (up to +14% in males; absolute weights; p<0.01). This organ weight difference was considered not to be related to previous test item administration in males in view of the low magnitude of these differences, of the absence of microscopic correlates and of the absence of kidney weight differences at the end of the treatment period. In females, the higher relative-to-body kidney weight difference was considered to be related with the lower terminal body weights There were lower absolute and relative-to-body thymus weights in females previously treated at 750 mg/kg/day (up to -56% in absolute weights; statistical significance not reached). This organ weight difference was considered to be related to test item administration and/or stress and correlated in part with the minimal increased severity of microscopic lymphoid atrophy in females previously treated at 750 mg/kg/day when compared with controls.Consequently, there was no reversibility of the difference in thymus weights in females. All other weight differences were minimal, did not correlate with macroscopic or microscopic findings and were considered no to be test item treatment-related. These included the higher uterus weights in females previously treated at 750 mg/kg/day probably related with the estrus cycle.See table 8.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Unscheduled death There were no test item-related gross findings in the prematurely sacrificed female.At the end of the treatment periodThere were no test item-related gross findings with the exception of the 1 cm in diameter tan discoloration of the right kidney from 1/10 males treated at 750 mg/kg/day which correlated with moderate renal tubular vacuolation, and the small thymus seen in 1/9 females treated at 750 mg/kg/day which correlated with marked lymphoid atrophy. This affected female had also a marked hematoma in the cranium (see microscopic section).The few other gross findings were considered to be unrelated to test item administration since they were seen also in control group, were not dose-related and/or were considered to be part of the normal background in the rats of these strain and age kept under laboratory condition. At the end of the treatment-free periodThere were no test item-related gross findings suggesting the reversibility or the previous gross findings.The few gross findings were considered to be unrelated to test item administration since they were seen also in control group and/or were considered to be part of the normal background in the rats of these strain and age kept under laboratory condition.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Unscheduled death One female treated at 750 mg/kg/day and sacrificed on Day 72 had several microscopic findings:- slight vacuolation of the white matter in the brain (hippocampus, cingulum, cerebral peduncle, cerebellum) and in the choroid plexus,- moderate vacuoles in the pars nervosa of the pituitary gland,- marked vacuoles in the renal tubules,- minimal vacuoles in the spleen and in the GALT.The vacuolation in the white matter from the brain was not seen in the test item-treated animals at the end of the treatment period. The vacuoles in the pituitary gland, kidneys, spleen and GALT were seen also in the test item-treated animals at the end of the treatment period at similar or lower severityThese findings were considered to be related with the test item administration and the vacuoles in the brain with extended distribution, kidneys and pars nervosa were considered to have contributed to the moribundity of this animal.In addition, the following lesions were noted:- slight to moderate atrophy of the ovaries, uterus and vagina,- minimal pancreas degranulation,- increased severity of the adipose tissue in the bone marrow associated with moderate decreased cellularity of the hematopoietic cells,- slight thymus atrophy.These findings may be related with the test item administration and/or stress. The moribundity of this female was considered to be probably related with the test item administration.At the end of the treatment periodVacuoles in several organsMinimal to marked vacuoles were seen in the kidneys (tubules and, to a lesser severity, glomeruli), brain (choroid plexus), pars nervosa (pituitary gland), spleen, mesenteric lymph node and GALT (Gut Associated Lymphoid Tissue) in males and females treated at 750 mg/kg/day and were related with the test item administration. It is noteworthy that these vacuoles were present in the choroid plexus from only 2/10 females treated at 250 mg/kg/day.These vacuoles were round, of moderate to large size (15-50 µm in diameter) and devoid of any staining except those recorded in the pituitary gland which were pale and eosinophilic. The empty vacuoles in brain, spleen, lymph node and GALT were scattered, multifocal while the vacuoles in the pars nervosa were diffuse. An immunohistochemistry staining of vasopressin was performed in the pituitary gland from one control male, two high-dose males and two high-dose females. In the control, there was a diffuse moderate staining of the pars nervosa (pituitary gland), with a more pronounced staining in the neuronal ends. This staining was seen with a higher severity (marked) in the test item treated males and females, with accentuated staining in the neuronal ends and vacuoles seen with hematoxylin/eosin. This suggested that the vacuoles contained vasopressin and may be consistent with enlarged neuronal ends containing increased amount of vasopressin.The pars nervosa from all animals was not present on the submitted slide. That is why the number of lesions out of the number of present pars nervosa was recorded in individual data.ForestomachDose-related, minimal to slight, focal or multifocal orthokeratotic hyperkeratosis was seen in males and females treated at 250 and 750 mg/kg/day and was related with the test item administration. ThymusIncidence of minimal lymphoid atrophy was seen in some males and females of all control and treated groups. A slight and a marked atrophy were observed in one male and one female treated at 750 mg/kg/day, respectively. This correlated with the lower weights of thymus of these two animals but was not associated with change in the white blood cells count and cellularity. The thymus effects were most probably related with stress.In addition, there was a minimal increased vacuolation in the cortex of the adrenal gland from one male treated at 750 mg/kg/day and in the liver (macrovacuoles; centrilobular location) from one female treated at 750 mg/kg/day. A relationship to test item administration could not be excluded. There were no test item-related findings in testes after a detailed examination of these organs.The few other microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature or their appearance was similar to changes found in controls.Specifically, in one male treated at 250 mg/kg/day, there was a fibrosarcoma in the skin which correlated with macroscopic 2 x 1.5 cm mass. This tumor is not rare in rats. Since it was seen in a single male from intermediate group, it was considered not to be related to the test item administration. One female treated at 750 mg/kg/day had a fibroadenoma in the mammary gland. This tumor is not rare in this strain and is known to occur in rats as young as 12-week old. This animal had also a decidual reaction (or deciduoma) associated with mucification of the vagina, blood and cell debris in the lumen from vagina and uterus. This condition is not rare in the female rats of these strain and age.A PAS staining of the uterus from this female confirm the diagnosis of decidual reaction in the uterus (PAS positivity for the granules in the large cells which suggested that they contain glycogen).One female treated at 750 mg/kg/day had a large chronic hematoma in the cranium (probably close to the nasal meatus) which correlated with white mass at gross examination. The cause of this hematoma was not clear. It was associated with moderate lymphoid atrophy in the spleen, in the mesenteric lymph node and with minimal to slight degranulation of the salivary gland and pancreas. These last findings were possibly related with stress subsequent to the hematoma in the skull.At the end of the treatment-free period- Vacuoles in several organsMinimal vacuoles were seen in the kidneys (tubules) of one female and in brain (choroid plexus) of two females previously treated at 750 mg/kg/day and was related with the test item administration. No vacuoles were seen in males.This suggested reversibility of these test item treatment-related findings.- ForestomachMinimal orthokeratotic hyperkeratosis was seen in 1/5 males and 1/5 females previously treated at 750 mg/kg/day but also in 1/5 control females. This suggested reversibility of these test item treatment-related finding.- ThymusMinimal or slight lymphoid atrophy was seen in males and females of the control and/or previously treated at 750 mg/kg/day. For one female treated at 750 mg/kg/day, the slight atrophy was associated with a low weight of thymus, but it was not the case for the other animals presenting a minimal or slight atrophy. These effects seem therefore of spontaneous occurrence.For all these organs, this suggested reversibility of these test item treatment-related finding.See tables 9 and 10.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
- Estrous cycle (see table 11): There were no statistically significant test item-related effects on mean estrous cycle length or mean number of cycles. However a trend towards an increase in mean estrous cycle length was observed in females given 250 or 750 mg/kg/day at the end of the treatment period. This was considered to be of no toxicological significance in the absence of statistical significance and of differences in estrous cycles at microscopic examination of the genital tract. Differences from controls were no longer observed at the end of the treatment-free period.

- Seminology (no effects observed)
No test item-related effects were noted on the epididymal sperm motility or morphology.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 1: Clinical signs (surviving animals)

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

50

250

750

0

50

250

750

Thin appearance

-

-

-

1

-

-

-

1

Hunched posture

-

-

-

-

-

1

-

2

Piloerection

-

-

-

-

-

1

-

1

Bent/tilted head

-

-

-

-

-

-

-

1

Abnormal reddish color

. urine

. vagina

-

-

-

1

-

-

-

-

 

-

1

 

1

1

Soiled urogenital region

-

-

-

-

-

-

-

1

Loud breathing

-

-

2

1

-

-

-

1

Abdominal breathing

-

-

-

-

-

-

-

1

Dyspnea

-

-

-

-

-

-

-

1

Total affected animals

0/15

0/10

2/10

2/15

0/15

1/10

1/10

5/14

Ptyalism

-

-

1

12

-

-

2

14

Reflux at dosing

-

-

-

2

-

-

-

2

Total affected animals

0/15

0/10

1/10

12/15

0/15

1/10

2/10

14/14

-: no clinical signs.

Table 2: Body weight

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

50

250

750

0

50

250

750

Treatment period

Mean BW gain Days 1/29

+102

+110

+101

+72**

+42

+35

+30**

+32

Mean BW gain Days 29/57

+56

+62

+60

+62

+19

+20

+22

+22

Mean BW gain Days 57/92

+35

+31

+31

+20*

+15

+10

+11

+4

Mean BW gain Days 1/92

+194

+202

+191

+154**

+76

+66

+63

+65

% from controls

-

+4

-2

-21

-

-13

-17

-14

Mean body weight on Day 1

418

428

438*

421

264

264

265

267

Mean body weight on Day 92

612

631

629

575

341

330

328

332

% from controls

-

+3

+3

-6

-

-3

-4

-3

Treatment-free period

Mean BW gain Days 92/133

+33

-

-

+58**

+15

-

-

+6*

Mean BW on Day 133

622

-

-

628

366

-

-

348

% from controls

-

-

-

+1

-

-

-

-5

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

Table 3: Food consumption

Sex

Male

Female

Dose-level (mg/kg/day)

0

50

250

750

0

50

250

750

Treatment period

Days 1 to 29

32.1

33.4

33.9

32.2

22.7

21.4

21.4

21.3

Days 29 to 57

30.8

32.5

32.5

33.1

22.3

21.9

21.3

21.8

Days 57 to 92

30.4

31.5

31.1

31.0

21.4

21.1

20.2

20.5

Days 1 to 92

31.0

32.4

32.4

32.0

22.0

21.5

20.9

21.1

% from controls

-

+5

+5

+3

-

-2

-5

-5

Treatment-free period

Days 92 to 133

28.0

-

-

29.8

19.8

-

-

20.7

% from controls

-

-

-

+6

-

-

-

+5

No statistics.

-: not applicable.

Table 4: Hematology

 

Sex

Male

Dose-level (mg/kg/day)

0

50

250

750

End of treatment period

Red blood cell count (T/L)

9.57

(7.96-10.67)

9.28

9.18

8.40**

(8.85)a

Hemoglobin (g/dL)

16.1

(13.3-17.1)

15.5

15.4*

14.4**

(15.1)a

Packed cell volume (L/L)

0.50

(0.41-0.55)

0.48

0.47*

0.44**

(0.45)a

Reticulocytes (%)

2.45

(1.47-3.54)

2.83

2.95

5.37*

(3.08)a

Reticulocytes (T/L)

0.23

0.25

0.27

0.36

(0.27)a

Neutrophils (G/L)

2.36

(0.80-5.80)

2.15

2.50

2.73

(2.87)a

Eosinophils (G/L)

0.20

(0.04-0.36)

0.20

0.18

0.02**

(0.02)a

Prothrombin time (s)

22.0

(18.6-23.8)

22.3

22.6

24.4**

(24.7)a

End of treatment-free period

 

 

 

 

Red blood cell count (T/L)

9.84

/

/

9.71

Hemoglobin (g/dL)

16.0

/

/

16.2

Packed cell volume (L/L)

0.48

/

/

0.49

Reticulocytes (%)

2.09

/

/

2.17

Reticulocytes (T/L)

0.21

/

/

0.20

Neutrophils (G/L)

3.39

/

/

2.70

Eosinophils (G/L)

0.21

/

/

0.19

Prothrombin time (s)

21.2

/

/

19.8

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

( ): minimum-maximum values from historical control data (HCD).

( )a: excluding animal E25833 from the mean calculation. No statistics.

 

Sex

Female

Dose-level (mg/kg/day)

0

50

250

750

End of treatment period

Red blood cell count (T/L)

8.60

(7.16-9.51)

8.66

8.63

8.33

Hemoglobin (g/dL)

15.4

(13.2-17.0)

15.5

15.4

15.1

Packed cell volume (L/L)

0.46

(0.38-0.51)

0.46

0.46

0.44

Reticulocytes (%)

2.29

(1.15-2.84)

2.25

2.18

3.07

Reticulocytes (T/L)

0.20

0.20

0.19

0.25

Neutrophils (G/L)

1.54

(0.40-2.92)

1.36

1.67

2.28*

Eosinophils (G/L)

0.15

(0.06-0.25)

0.15

0.10

0.02**

Prothrombin time (s)

22.4

(16.5-23.9)

22.4

22.8

23.8

End of treatment-free period

 

 

 

Red blood cell count (T/L)

8.62

/

/

8.90

Hemoglobin (g/dL)

15.5

/

/

15.8

Packed cell volume (L/L)

0.45

/

/

0.46

Reticulocytes (%)

1.78

/

/

1.55

Reticulocytes (T/L)

0.16

/

/

0.14

Neutrophils (G/L)

1.47

/

/

0.94

Eosinophils (G/L)

0.13

/

/

0.11

Prothrombin time (s)

22.9

/

/

23.9

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

( ): minimum-maximum values from historical control data (HCD).

Table 5: Blood biochemistry

 

Sex

Male

Dose-level (mg/kg/day)

0

50

250

750

End of treatment period

Sodium (mmol/L)

144.0

143.9

143.3

142.7**

Chloride (mmol/L)

105.4

105.4

104.4

103.5**

Inorganic phosphorus (mmol/L)

2.09

2.08

2.25*

2.40**

Creatinine (µmol/L)

38.22

36.59

36.14

34.70*

Proteins (g/L)

64.1

64.5

63.3

61.0*

Albumin (g/L)

37

37

36

35**

Triglyceride (mmol/L)

1.03

1.47*

0.89

1.12

Aspartate aminotransferase (U/L)

87

77

87

107*

Alanine aminotransferase (U/L)

40

36

42

75**

End of treatment-free period

 

 

 

 

Sodium (mmol/L)

143.7

/

/

143.1

Chloride (mmol/L)

105.7

/

/

105.3

Inorganic phosphorus (mmol/L)

2.02

/

/

2.07

Creatinine (µmol/L)

37.10

/

/

36.05

Proteins (g/L)

66.2

/

/

67.2

Albumin (g/L)

37

/

/

37

Aspartate aminotransferase (U/L)

94

/

/

87

Alanine aminotransferase (U/L)

43

/

/

39

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

 

Sex

Female

Dose-level (mg/kg/day)

0

50

250

750

End of treatment period

Sodium (mmol/L)

143.0

143.1

141.9*

141.2**

Chloride (mmol/L)

105.1

106.1

105.1

103.8

Inorganic phosphorus (mmol/L)

1.78

1.65

2.04**

2.00*

Creatinine (µmol/L)

36.13

38.42

36.21

36.28

Proteins (g/L)

66.4

66.5

64.8

64.1

Albumin (g/L)

38

39

38

38

Triglyceride (mmol/L)

0.48

0.48

0.66*

0.76**

Aspartate aminotransferase (U/L)

80

98

73

82

Alanine aminotransferase (U/L)

37

47

35

60*

End of treatment-free period

 

 

 

 

Sodium (mmol/L)

142.1

/

/

142.1

Chloride (mmol/L)

105.6

/

/

106.5

Inorganic phosphorus (mmol/L)

1.58

/

/

1.44

Creatinine (µmol/L)

41.65

/

/

38.64

Proteins (g/L)

69.9

/

/

68.5

Albumin (g/L)

41

/

/

41

Aspartate aminotransferase (U/L)

90

/

/

75

Alanine aminotransferase (U/L)

45

/

/

30

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

 

Table 6: Urinalysis

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

50

250

750

0

50

250

750

End of treatment period

Presence of glucose

(approx. 5.5 mmol/L)

0

0

0

3

0

0

0

0

Presence of blood

 

 

 

 

 

 

 

 

. low

2

1

0

0

0

0

0

1

. moderate

0

0

0

0

0

1

0

1

. high

0

0

0

2

0

0

0

2

Red blood cells

1

4

2

6

0

0

1

3

. 1 - few in some fields

1

4

2

4

0

0

1

1

. 2 - few in all fields

0

0

0

0

0

0

0

1

. 3 - several in all fields

0

0

0

1

0

0

0

1

. 4 - large number in all fields

0

0

0

1

0

0

0

0

End of treatment-free period

 

 

 

 

 

 

 

 

Presence of glucose

(approx. 5.5 mmol/L)

0

na

na

0

0

na

na

0

Presence of blood

0

na

na

0

0

na

na

0

Red blood cells

0

na

na

0

0

na

na

0

na: not applicable.

Table 7: Motor activity

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

50

250

750

0

50

250

750

Horizontal movements

373

400

371

461

522

590

548

635

% from controls

-

+7

-1

+24

-

+13

+5

+22

Rearing

108

137

130

142

144

207

206

254

% from controls

-

+27

+20

+31

-

+44

+43

+76

-: not applicable.

Table 8: Organ weights

Main organ weight differences in male groups (express in percentage, when compared to controls)

 

Sex

Male

Group

2

3

4

Dose-level (mg/kg/day)

50

250

750

Number of examined animals

10

10

10

Body weight at sacrifice

+1

0

-9*

- Kidneys 

.absolute

+8

+6

+6

.relative-to-body

+6

+6

+17**

- Thymus 

.absolute

+4

+7

-16

.relative-to-body

+2

+6

-7

Statistically significant from controls: *: p<0.05, **: p<0.01.

The significance concerned the organ weights values and not the percentages.

 

Main organ weight differences in female groups (express in percentage, when compared to controls)
at the end of the treatment period

 

Sex

Female

Group

2

3

4

Dose-level (mg/kg/day)

50

250

750

Number of examined animals

10

10

9

Body weight at sacrifice

-1

-2

-4

- Kidneys 

.absolute

0

-1

+8

.relative-to-body

+1

+1

+14*

- Thymus 

.absolute

+8

+5

-24

.relative-to-body

+9

+7

-24

Statistically significant from controls: *: p<0.05.

The significance concerned the organ weights values and not the percentages.

Table 9: Microscopic findings at tne end of the treatment period

 

Males

Females

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

50

250

750

0

50

250

750

Number of examined individuals

10

10

10

10

10

10

10

9

Kidney; vacuoles; tubules

 

 

 

 

 

 

 

 

Grade 1

1

-

-

2

-

-

-

3

Grade 2

-

-

-

-

-

-

-

2

Grade 3

-

-

-

1

-

-

-

-

Grade 4

-

-

-

-

-

-

-

1

Kidney; vacuoles; glomerulus

 

 

 

 

 

 

 

 

Grade 1

-

-

-

4

-

-

-

-

Grade 2

-

-

-

-

-

-

-

1

Brain; vacuoles; choroid plexus

 

 

 

 

 

 

 

 

Grade 1

-

-

-

9

-

-

2

5

Grade 2

-

-

-

1

-

-

-

3

Pituitary gland*; vacuoles

 

 

 

 

 

 

 

 

Grade 1

0/8

1/7

0/6

6/9

0/10

0/9

0/7

3/6

Grade 2

0/8

0/7

0/6

1/9

0/10

0/9

0/7

1/6

Spleen; vacuoles

 

 

 

 

 

 

 

 

Grade 1

-

-

-

7

-

-

-

2

Mesenteric lymph node; vacuoles

 

 

 

 

 

 

 

 

Grade 1

-

-

-

3

-

na

na

-

GALT; vacuoles

 

 

 

 

 

 

 

 

Grade 1

-

-

-

3

-

-

-

2

Thymus; lymphoid atrophy

 

 

 

 

 

 

 

 

Grade 1

2

5

5

4

2

1

2

2

Grade 2

-

-

-

1

-

-

-

-

Grade 4

-

-

-

-

-

-

-

1

Forestomach; hyperkeratosis

 

 

 

 

 

 

 

 

Grade 1

-

-

1

5

-

-

3

6

Grade 2

-

-

-

-

-

-

-

1

-: not seen in this group.

na: not applicable.

*out of the number of present pars nervosa.

Table 10: Microscopic findings at the end of the treatment-free period 

 

Males

Females

Group

1

 

 

4

1

 

 

4

Dose-level (mg/kg/day)

0

 

 

750

0

 

 

750

Number of examined individuals

5

 

 

5

5

 

 

5

Kidney; vacuoles; tubules

 

 

 

 

 

 

 

 

Grade 1

-

 

 

-

-

 

 

1

Brain; vacuoles; choroid plexus

 

 

 

 

 

 

 

 

Grade 1

-

 

 

-

-

 

 

2

Thymus; lymphoid atrophy

 

 

 

 

 

 

 

 

Grade 1

-

 

 

2

3

 

 

2

Grade 2

1

 

 

-

-

 

 

1

Forestomach; hyperkeratosis

 

 

 

 

 

 

 

 

Grade 1

-

 

 

1

1

 

 

1

-: not seen in this group.

 

Table 11: Estrous cycle

 

Dose-level (mg/kg/day)

0

50

250

750

Treatment period

 

 

 

 

Number of cycles

4.2

3.9

3.8

3.2

Cycle length (days)

4.2

4.3

5.1

5.9

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

7

6

5

4

Treatment-free period

 

 

 

 

Number of cycles

1.0

na

na

1.0

Cycle length (days)

4.0

na

na

4.0

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

2

na

na

1

na: not applicable. 

 

Applicant's summary and conclusion

Conclusions:
The toxicity of the test item, Diethylaminopropylamine (as pH-neutralized dose formulations), was evaluated after daily oral administration (gavage) to Sprague-Dawley rats at dose-levels of 50, 250 or 750 mg/kg/day for 13 weeks followed by a 6-week treatment-free period. Under the experimental conditions of the study, clinical signs of poor condition were observed at the dose level of 750 mg/kg/day in four females, which induced the premature sacrifice of one of them more severely affected.No other adverse effects were observed in the study.Consequently, the NOAEL (No Observed Adverse Effect Level) was established at 750 mg/kg/day in males and 250 mg/kg/day in females.
Executive summary:

The potential toxicity of Diethylaminopropylamine (as pH-neutralized dose formulations) 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). Sprague-Dawley rats were treated daily by the oral route (gavage) with the test item, Diethylaminopropylamine, for 13 weeks as follows: one group of 15 males and 15 females at the dose-level of 750 mg/kg/day (group 4) and two other groups of 10 males and 10 females at dose-levels of 50 (group 2) or 250 (group 3) mg/kg/day. One group of 15 males and 15 females received the vehicle only (drinking water treated by reverse osmosis) 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 agas chromatography with flame ionization detection analytical method. 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 13. 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 test item-treated animals at the end of the treatment period (Week 13). Hematology, blood biochemistry and urinary investigations were performed at the end of the treatment and treatment-free periods (Weeks 13 and 20). Additional blood samples were collected in Weeks 13 and 20 for possible analysis of thyroid hormones levels. The estrous cycle was determined for all females over 21 or 5 consecutive days at the end of the treatment or treatment-free period, respectively. Seminology investigations (count, motility and morphology) were performed on all males at sacrifice at the end of the treatment period. 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 died prematurely and on all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period. PAS staining in the uterus in one high-dose female and immunohistochemistry for vasopressin in the pituitary gland on one control male and two high-dose males and females were examined. The brain, forestomach, gut-associated lymphoid tissue, kidneys, mesenteric lymph nodes (males only), pituitary gland, spleen and thymus of the low- and intermediate-dose animals (groups 2 and 3) sacrificed at the end of the treatment period and of the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment-free period were also microscopically examined as changes were revealed in these organs at the end of the treatment period.

Actual concentrations of Diethylaminopropylamine in the dose formulations administered to the animals during the study remained within an acceptable range (-6.5% to +9.2%) compared to the nominal concentrations. During the treatment period, three females given 750 mg/kg/day showed clinical signs which were considered to be adverse (i.e.hunched posture, dyspnea, abdominal, loud breathing and/or bent head). Hunched posture and piloerection were transiently noted in one female given 50 mg/kg/day. Ptyalism was observed with a dose-related incidence at 250 and 750 mg/kg/day and reflux at dosing was noted in few males and females given 750 mg/kg/day on one occasion.These signscommonly observed when a test item is administered by gavage, were considered not to be an adverse effect. One female given 750 mg/kg/day more severely affected, showed signs of poor clinical condition and therefore was sacrificed in Week 11. Microscopic findings were noted (i.e. vacuoles in the pars nervosa of the pituitary gland, in the renal tubules, in the white matter from the brain, in the choroid plexus, in the spleen and in the GALT). At Functional Observation Battery examination, slightly higher incidences of horizontal movements and rearing were recorded in males and females given 750 mg/kg/day. Body weight gain was slightly lower in males given 750 mg/kg/day during the first and the third months of the treatment period, leading to a slightly lower body weight on completion of the treatment period (-6% vs. controls). Food consumption was not affected by the test item treatment. No ophthalmology findings were observedat the end of the treatment period. Estrous cycle was not altered by the test item treatment. The epididymal sperm motility and morphology and the spermatozoa count were unaffected by the test item treatment. At hematology investigations, minimally to slightly lower hemoglobin concentration and packed cell volume were noted in males from 250 mg/kg/day as well as lower red blood cell count in males given 750 mg/kg/day. This was accompanied with slight increase in reticulocyte count at 750 mg/kg/day. There were also non-adverse lower eosinophil count and prolonged prothrombin time in males and females treated at 750 mg/kg/day.These variations were considered to be of minor toxicological importance. At blood biochemistry investigations, changes in the markers of the renal function (lower sodium and chloride levels, higher inorganic phosphorus levels and lower protein and/or albumin levels) observed in males and/or females from 250 mg/kg/day could be secondary to the electrolytes imbalance induced by the intake of chloride ions used to neutralize the test item. A minimal increase of higher aspartate aminotransferase and alanine aminotransferase activity was observed in males and females given 750 mg/kg/day. All these changes were considered to be of minor importance. At urinary investigations, hematuria in males and females given 750 mg/kg/day along with glucosuria in males were suggestive of a tubular resorption problem. Reversibility of these laboratory findings was noted at the end of the treatment-free period. At the end of the treatment period, microscopic vacuoles were seen in the kidneys (correlated with tan discoloration), brain (in choroid plexus), pars nervosa (pituitary gland), spleen, mesenteric lymph node and/or GALT in males and females treated at 750 mg/kg/day and at a lesser extent, at 250 mg/kg/day in isolated females. There was also a non-adverse orthokeratotic hyperkeratosis in the forestomach from males and females treated at 750 mg/kg/day and increased severity and incidence of lymphoid atrophy in the thymus from males and surviving females treated at 750 mg/kg/day which correlated with small thymus and lower weights and may be related in part with stress.  

Under the experimental conditions of the study, clinical signs of poor condition were observed at the dose-level of 750 mg/kg/day in four females, which induced the premature sacrifice of one of them more severely affected. No other adverse effects were observed in the study.

Consequently, the NOAEL (No Observed Adverse Effect Level) was established at 750 mg/kg/day in males and 250 mg/kg/day in females.